BIOL 200 Learning Objectives

Unit 1

1.1 Eukaryotic Cells & Organelles

Critically discuss classical definitions of cells and organelles

-eukaryotic cells

• membrane bound unit capable of carrying out essential life processes

-plasma membrane

• lipid bilayer that controls what enters and exits the cell

-cell wall

• rigid structure around plasma membrane that provides shape and support

-nucleus

• membrane bound organelle containing DNA and controls cell functions

-nuclear envelope

• double membrane surrounding nucleus, has pores that regulate molecule transport in and out of nucleus

-nucleolus

• region inside nucleus where ribosomes are made

-chromatin

• DNA and protein complex within nucleus → condenses into chromosomes during cell division

-mitochondria

• generates ATP

-chloroplast

• performs photosynthesis to convert sunlight to energy

-endoplasmic reticulum (ER)

• protein synthesis

-rough er

• has ribosomes; synthesizes and processes proteins

-smooth er

• lacks ribosomes; synthesizes lipids

-golgi apparatus

• modifies, sorts and packages proteins and lipids for transport

-vesicle

• small membrane bound sac that transports materials in cell

-lysosome

• contains digestive enzymes to break down waste

-peroxisome

• breaks down fatty acids and detoxifies harmful substances

-ribosome

• synthesizes proteins using mRNA instructions

-cytoskeleton

• network of protein filaments providing structural support and helps with movement

• microtubules

  • hollow tubes that maintain cell shape and aid in cell division (spidle fibers)

• microfilaments

  • filaments involved in movement and support

-vacuole

• stores water, nutrients, waste; helps maintain cell structure (plant cells)

Compare and contrast major characteristics of bacteria and eukaryotes

1.2 Microscopy

Distinguish between 4 major classes of microscopy: brightfield light microscopy, fluorescence light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM)

Discuss the major advantages and limitations of each of the 4 major classes of microscopy

Understand the difference between magnification and resolution and how each can be used to identify the type of microscopy used

-magnification

• enlarges image

-resolution

• determines clarity

• high resolution → clear more details

• low resolution → blurry, less details, things mushed together

Recognize the major organelles in light and electron micrographs

Predict the type of microscopy that can be used to detect and study cellular components based on their size and functional aspect being studied

Interpret the results in micrographs based on scale, magnification, resolution and plane of section

Unit 2

2.0 Macromolecules

-Describe the terms redundant and ambiguous in terms of genetic code

• redundant

  • multiple codons can code for same amino acid

  • ensures that genetic code is robust against mutations

  • if mutations occur in 3rd position of codon → may still result in same amino acid being made into protein

• ambiguous

  • genetic code isn’t ambiguous

  • single codon always codes for same amino acid

  • no one codon can specify more than one amino acid

-What machinery is involved in tRNA activation and why are tRNA molecules considered the translators of genetic code

• tRNA activation is where amino acids are attached to corresponding tRNAs

• aminoacyl-tRNA synthetase

  • ensure correct amino acid attached to right tRNA

• tRNA, ATP used in tRNA activation

• tRNA molecules interprets genetic information encoded in mRNA and translates it into corresponding sequence of amino acids during protein synthesis

-How is it possible that mRNA can be simultaneously producing several copies when there is only one start codon

• multiple ribosomes simultaneously translate a single mRNA molecule

• multiple ribosomes can bind to same mRNA and translate it simultaneously

Types of non-covalent bonds

-ionic bonds

• atoms with opposite charges

-van der waals

• dipoles formed when electrons move around atoms

Types of covalent bonds

-polar bonds

• electrons unequally shared between atoms with different electronegativities

-non-polar bonds

• electrons equally shared between atoms with similar electronegativities

What is the monomer of a protein, nucleic acid, and carbohydrate?

-protein

• amino acids

-nucleic acid

• nucleotides

-carbohydrates

• monosaccharides

How are lipids different than other macromolecules when considering polymers?

-lipids don’t form true polymers like proteins, nucleic acids and carbohydrates do

-lipids aren’t made from repeating monomers

• they are small nonpolar molecules that don’t form long chains through polymerization

• aggregate into structures like membranes or fat deposits

What type of reaction brings monomers together?

-condensation reaction

• 2 monomers are joined together by covalent bond and a molecule of water is released as a byproduct

• removal of H from one monomer & OH from other monomer → combines to form H₂O

What is amino acid residue?

-individual amino acid incorporated into a protein after water is removed during formation of peptide bond

2.1 Biological Membranes

Discuss the structural and functional features of biological membranes and relate how the different macromolecular components (lipids, proteins, carbohydrates) interact to form biological membranes

-molecular components of membranes

• lipids → phospholipids, sterols, glycolipids

• proteins found on both side of membrane

• carbohydrates

  • found outside (non-cytosolic) of membrane; not cytosol side

  • glycocalyx

    • sugar coat on plasma membrane

    • protects cell

    • acts in cell communication/identification

-lipids form the basic structural framework of the membrane (phospholipids)

• in charge of the barrier, fluidity, ins and outs of the membrane

-proteins are embedded in or attached

• in charge of transport, signaling, cell recognition

• amphipathic nature of lipid bilayer allows a hydrophilic head to face extracellular environment and cytoplasm

  • allows protein transports to be spread around bilayer → selective transport of the efflux and influx of molecules

  • allows for the maintenance of ion concentrations to maintain necessary osmotic pressure of the cell

-carbohydrates attached to proteins or lipids on outer surface

• in charge of cell recognition, communication, glycocalyx formation

-membrane features

• bilayer

• organized but fluid

• different permeabilities for different molecules

• asymmetric

-membrane functions

• barrier to outside environment

  • protects cells from outer attacks

• form cellular compartments

  • ionically distinct from extracellular space

  • allows for different environment compared to outside the cell

• controls transport process

  • pumps & pores in membranes

  • controls what comes in & out of cell

• localize reactions

  • digestion in lysosomes can’t occur if digestive enzymes loose in cytoplasm

• regulate cell communication

  • receptors embedded in membranes

    • signals cross barriers

-membrane is selectively permeable

• some molecules can pass through while others can’t

• gases, hydrophobic, small nonpolar molecules cross lipid bilayer easily

  • oxygen, CO2, NO

• large, uncharged polar molecules can’t cross bilayer due to size

  • amino acids, nucleotides, glucose

• small, uncharged, polar molecules cross bilayers at slower rates

  • H2O

• charged ions can’t diffuse across lipid bilayer

-thermodynamics of hydrophobic effect → forms bilayer in water

• most stable form of phospholipids

• minimizing exposure of hydrophobic groups to water

• phospholipids spontaneously form bilayer because it’s most stable

  • most energetically favourable

Describe the chemical properties of phospholipids, cholesterol, and glycolipids and how these properties contribute to the lipid bilayer structure and function

-phospholipids

• amphipathic

  • hydrophilic head (phosphate)

    • polar

    • interacts with water

  • hydrophobic tail (fatty acid chains)

    • nonpolar

    • avoids water

• in aqueous solution → bilayer formation

  • hydrophilic heads face outward → interacts with aqueous environment

  • hydrophobic tails face inward → avoid water

  • allows fluidity → movement of molecules across membrane

  • gives membrane self-sealing & self-healing property

    • if hole in membrane → lipids heal and seal hole because its more favourable and stable

-cholesterol

• amphipathic

  • polar hydroxyl group (-OH) at one end → slightly hydrophilic

  • hydrocarbon ring structure → mostly hydrophobic

• in aqueous solution → -OH polar groups orients itself to be in water

-glycolipids

• lipids with carbohydrate attached via glycosidic bond

• amphipathic

  • hydrophilic head (carbohydrate)

    • interact with water

  • hydrophobic tails (fatty acid chains)

    • avoids water

• located on outer leaflet of lipid bilayer → contributes to glycocalyx

-fatty acids in aqueous environment → micelle

-triacylglycerols in aqueous environment → oil droplets

Recognize the 20 amino acids according to their chemical properties and their 3-letter and 1-letter abbreviations

Differentiate between the primary, secondary, tertiary, quaternary levels of protein structure

-domain — secondary structure fold into domains within a tertiary structure

List and discuss the role of non-covalent bonds in protein folding and stabilization of protein structure

Illustrate how the primary sequence and environment of a protein influenceits final 3D structure

-primary sequence of a protein provides the initial blueprint for its structure

• environmental factors fine tune the final 3D shape

  • pH

  • temperature

  • ionic strength & salts

-interactions between amino acid residues within protein and surrounding environment results in protein’s 3D conformation

2.2 The Lipid Bilayer

Describe factors affecting or regulating the fluidity of lipid bilayer

-better to have ↓ van der waals, ↑ fluid

-lipid composition

• degree of unsaturation of lipids

• ↑ saturated lipids, ↓ fluid, ↑ van der waals

  • tightly packed

• ↑ unsaturated lipids, ↑ fluid, ↓ van der waals

  • more kinks in fatty acid tail

-sterol content (cholesterol — animal, phytosterol — plant)

• low temperature, ↓ fluid, no sterol

  • add sterol → ↑ fluidity

  • prevents tight packing of fatty acids → ↓ van der waals

• high temperature, ↑ fluid, no sterol

  • add sterol → ↓ fluidity

  • sterol ring structure stiffens cell membrane →↑ van der waals

-fatty acid tail length

• ↓ tail length, ↑ fluid, ↓ van der waals

  • less surface area

  • < 18 carbons

• ↑ tail length, ↓ fluid, ↑ van der waals

  • more surface area

  • 14-24 carbons (~18-20)

-temperature

• cold temperature

  • ↓ fluid, ↑ van der waal

  • membrane bilayer freeze

  • crystalline-like structure → very fragile

  • phospholipids stuck together

    • trapped by van der waals

Predict the relative fluidity of membrane bilayers based on their lipid composition

-membrane fluidity

• essential for cell movement, membrane fusion and activity of membrane proteins

• how easily molecules within a cell membrane can move around

• fluid membrane

  • lipids and proteins in membrane move around easily

• rigid membrane

  • lipids and proteins packed tightly together → hard to move around

• membranes can self-heal if they are punctured

  • self-seal when in close proximity to a second membrane

Explain how Fluorescence Recovery After Photobleaching (FRAP) works and interpret results of FRAP experiments

-green fluorescent proteins (GFP) bleached via laser

• no more fluorescence

• black patch of proteins as a result

-tagged proteins diffuse into the area

-untagged proteins diffuse out of area

-over time → area becomes fluorescent again

-proteins moving because it’s fluid → if tagged proteins don’t diffuse back into area → membrane not fluid

-fluorescence fully recovers to pre-bleached intensity → molecules are mobile and can diffuse quickly into bleached region → high diffusion & little immobility

-fluorescence partially recovers → molecules have some degree of mobility, constrained by various factors (molecules may be immobile due to being bound in clusters, complexes, macromolecular assemblies)

• due to adhesion to proteins inside cell/ECM/neighbouring cells

-no fluorescence recovery → molecules in bleached region are immobile (trapped or tightly bound within structure)

• due to adhesion to proteins inside cell/ECM/neighbouring cells

Describe membrane asymmetry and explain the origin of the asymmetric distribution of membrane lipids

-membrane is asymmetric

• arrangement of components is not the same on 2 sides of bimolecular leaflet

  • always an inside (cytoplasmic) and outside (external)

  • non-cytosolic side faces extracellular side

  • cytosolic side faces intracellular space

• carbohydrates are major contributors to membrane asymmetry

• 2 halves of membrane are different from each other

• orientation of membrane remains constant — even throughout transport

• asymmetry established by lipid flipping enzymes

  • flippases

    • enzymes in membrane that moves phospholipids from one leaflet to another

    • uni-directional movement

  • floppases

    • uni-directional movement

  • scramblases

    • bi-directional movement

-lipid rafts

• microdomains in plasma membrane rich in specific types of lipids

  • sphingomyelin, cholesterol

• more ordered than surrounding membrane

• thicker than other regions of cell membrane

-attachment to structures inside/outside cell

• adhesion to proteins inside cell

  • inside cell cortex

  • RBCs → membrane proteins attached to cell cortex

  • plasma membrane reinforced by association of membrane proteins with cell cortex

• adhesion to extracellular matrix (ECM)

  • integrin proteins connected to ECM

• adhesion to neighbouring cells

  • cell to cell adhesion molecules linking plasma membranes of neuronal cells

  • cells attached to each other so don’t flow away

• barriers to diffusion

  • tight junctions

    • adhesions between neighbouring epithelial cells forming attachment points between 2 cells so nothing leaks between cels

    • segregates tops and sides/bottoms of cells to distinct membrane domains

*important for FRAP

2.3 Membrane Proteins

Distinguish between integral and peripheral proteins with regard to their solubility, structure and manner of attachment to membranes

-integral proteins

• proteins directly attached to membrane

• amphipathic

• monomeric

  • one polypeptide

• multimeric

  • multiply polypeptide

• can’t be removed without destroying membrane completely

• insoluble

• hydrophobic regions; embedded in bilayer

• amino acids found in transmembrane (hydrophobic) region

  • nonpolar amino acids (Leu, Iso, Val, Phe, Ala, Met, Try)

  • hydrophobic amino acids interacts with lipid tails of bilayer → stabilizes protein within membrane

• amino acids found on extracellular/cytoplasmic surface (hydrophilic)

  • charged polar amino acids (Asp, Arg, Glu, Lys, His)

  • uncharged polar amino acids (Ser, Thr, Gln, Tyr)

1. transmembrane proteins

   • proteins that pass through lipid bilayer

   • uses secondary structures to pass through membranes (alpha helices, beta sheets)

     • alpha helices

       • 1+ alpha helical transmembrane domains

         • domains - stretches of 20+ amino acids with hydrophobic side chains

       • can form pores & channels but several alpha helices required

     • beta-sheets

       • beta strand/flat beta sheet not stable enough to be used in transmembrane protein

         • edges of sheet has exposed backbone that doesn’t interact with hydrophobic core of bilayer

       • beta sheets transform in to beta-barrels

         • hydrogen bonds between beta sheets form a cylindrical structure

   • backbone of polypeptide chain always polar

   • transmembrane domain

     • part of membrane protein that passes through bilayer

     • mostly alpha-helices; some beta-barrels

     • need multiple alpha helices/beta-barrels to make a pore

2. monolayer-associated proteins

   • only attached to one side of membrane

-peripheral proteins

• bound to membrane surfaces via non-covalent association with other membrane proteins

• not attached to membranes → attached to something attached to membrane

• can be removed using mild salt washes

• soluble in water

• hydrophilic regions; loosely associated with membrane

• amino acids found in peripheral membrane proteins (hydrophilic)

  • hydrophilic amino acids (Glu, Asp, Lys, Arg, His, Ser, Thr, Glu, Asp)

  • charged polar amino acids help peripheral proteins bind to hydrophilic heads of membrane lipids

• no amino acids found in membrane → proteins not embedded in membrane

1. lipid-linked proteins

   • proteins covalently bonded to lipid that is attached to membrane

-plasma membrane polysaccharides

• glycocalyx

  • glycolipids

    • integral parts of membrane

    • fatty acids hold glycolipid firmly in membrane

    • synthesized on cytosolic side of ER (like other membrane lipids)

  • glycoproteins

    • proteins that carry attached polysaccharides

    • integral membrane proteins that extend through or into membrane

    • attached to exterior part of protein

Interpret results from SDS-PAGE, fluorescence and glycoprotein staining experiments to determine orientation and location of proteins and glycoproteins in a membrane

-SDS-PAGE

• treatments

  • beta mercaptoethanol

    • breaks disulfide bonds within proteins

  • salt solution

    • breaks ionic interactions of peripheral proteins and structures they are bound to

  • brief detergent

    • pokes holes in plasma membrane by solubilizing phospholipids

  • digestive enzyme

    • digests parts of proteins accessible

    • if combined with detergent → digestion of proteins on cytosolic side of membrane

1. protein isolated from membrane (SDS)

   • proteins treated with protease enzyme

     • enzyme that break peptide bonds, can’t cross lipid bilayer

   • peripheral proteins treated with high salt concentrations to wean interactions

   • integral proteins treated with harsh treatments (detergents) to solubilize proteins

2. proteins are now separated from membrane

3. filter proteins through polyacrylamide gel matrix (PAGE)

   -polyacrylamide gel matrix → gel electrophoresis separates proteins based on size

   • large proteins = top

   • small proteins = bottom

   • more proteins = thick bands

   • less proteins = thin bands

-fluorescence staining

• fluorescent dyes used to tag membrane proteins

  • dye only tags protein on outer membrane (dye can’t cross lipid bilayer)

    • poke holes in membrane → dye can enter

• comparing staining before and after permeabilization → can tell which proteins are outside vs inside cell

• fluorescence microscope can be used to see where proteins are located in cells

  • fluorescence only on cell surface → protein is outer membrane protein

  • fluorescence inside cell (after permeabilization) → protein on inner leaflet or inside organelle

-glycoproteins staining

• glycoproteins (proteins with sugars attached) usually found on outer leaflet of membrane

• special stains that bind to sugars are applied to cells

• if stain binds membrane without needing permeabilization → glycoproteins facing outwards

  • confirms which proteins are exposed on outer cell surface

Predict the hydropathy profile for a given protein given its amino acid sequence or for a given hydropathy plot, predict the amino acid composition of the protein domains within a membrane protein.

-bioinformatics (hydropathy plots)

• only works on alpha-helices

• doesn’t work on beta barrels

  • amino acid sequence of beta-barrels have alternating hydrophobic and hydrophilic R groups facing in and out of the pore → hydropathy plot ends up oscillating between hydrophobic & hydrophilic regions

  • shows no significant peaks → near zero hydropathy plot

• determines whether a protein is a transmembrane protein, how many transmembrane regions (helices) it has, orientation of protein (which parts are inside vs outside membrane)

• can predict if protein is embedded in cell membrane

• based on properties of amino acids in peptide sequence

• hydrophobic regions → positive peaks → membrane spanning alpha helices (proteins embedded within bilayer — interacts with tails) → line above 0

• hydrophilic regions → negative peaks → extracellular or cytoplasmic alpha helices (proteins on outer side of head) → line below 0

• if first region is hydrophilic → N terminus in cytoplasm or extracellular space

• if last region hydrophilic → C terminus in cytoplasm or extracellular space

-Fibronectin is a large glycoprotein present in extracellular matrix (ECM), if a cell contains a mutation that leads to a loss of fibronectin, how would plasma membrane attach to cytoskeleton and is the ECM affected?

• plasma membrane’s attachment to cytoskeleton and ECM affected

• cell has trouble holding its shape, moving or responding to signals from environment

• cells have a harder time sticking to ECM → weaker tissue structure → ECM disrupted

Unit 3

3.1 Nuclear Structure & Protein Import

Describe the structure of an interphase nucleus and recognize structural elements in different kinds of microscopy

-nuclear envelope

• double membrane with perinuclear space in between

• surrounded by 2 lipid bilayers

• outer nuclear membrane

  • continuous with rough ER

  • ribosomes attached

• inner nuclear membrane

• cytoplasm and nucleoplasm connected through nuclear pores

-nucleolus

• not membrane bound

• site of rRNA synthesis

• assembly site of ribosomal subunits made from rRNA and ribosomal proteins

• 1-10 nucleoli in nucleus depending on cell cycle

• disassembled during mitosis so chromosomes can be condensed

• ribosomes

  • made of protein and RNA

  • ribosomal proteins made in cytosol

    • then imported to nucleus and assembled with rRNA molecules in nucleolus

-nuclear lamina

• provides structural support

• important role in DNA replication, transcription, gene regulation

• made of strong nuclear lamin proteins

  • proteins in inner nuclear membrane can be anchored to it

• aids in disassembly and reassembly of nuclear envelope during cell division

  • disassemble → phosphorylation

  • reassemble → dephosphorylation

Describe nuclear pore complex (NPC) and explain the different types of nuclear transport mechanisms

-nuclear pore complex (NPC)

• nuclear pores — very small

• protein subunit complex that controls what enters and leaves nucleus

• pore complex ~90-120 nm

  • import in (proteins required for)

    • DNA replication, transcription, gene regulation, mRNA processing, DNA structure, ribosomal subunit assembly

• cytosolic fibrils

  • guide molecules in and out of nucleus

  • export out

    • assembled ribosomal subunits

    • mRNA

    • tRNA

Explain how proteins are transported into and out of the nucleus, including the roles of nuclear localization signal (NLS), nuclear transport receptors and NPC itself

-nuclear transport

• bidirectional movement

• free diffusion

  • ions & small molecules can pass (~9 nm) freely through NPC

  • non selective import via NPC

• active transport

  • energy required → hydrolysis of GTP

  • transport molecules (>9 nm) in/out of nucleus

  • NLS binds to NIR

  • proteins without localization signal in primary sequence will end up localized in cytosol because no signal to enter nucleus

  1. NLS of protein binds to NIR

  2. complex binds to cytosolic fibril on annular ring → guides protein into pore

  3. protein moved through pore

     • protein to target location (KKKRK)

       • signal sequence in transported protein → protein that wants to enter nucleus must have targeting signal in primary amino acid sequence

       • nuclear transport receptor → protein receptor that recognizes protein’s signal

-components for import/export

• cargo proteins

  • signal sequence NLS = KKKRK

• energy (GTP)

  • required for import/export of large molecules

• signals

  • import → nuclear localization signal (NLS)

    • must be accessible on surface of protein to be recognized

    • stays on protein (doesn’t dissociate and return to cytosol)

    • NLS necessary & sufficient for protein import

  • export → nuclear export signal (NES)

• receptors— recognizes signal sequence & binds cargo protein

  • import → nuclear import receptors (NIR)

    • once inside nucleus → NIR dissociates from protein and returns to cytosol

    • binds to nuclear signal sequence (NLS)

  • export → nuclear export receptors (NER)

    • NES in protein bound by protein receptor

-mature mRNA for export must be bound by proteins or else it can’t leave nucleus

• finds a protein with a signal that is leaving the nucleus → hops onto protein and gets a ride out of nucleus

-protein targeting

• targeting signals

  • encoded within proteins

  • direct protein to specific organelle

  • must be present for protein to leave cytosol

-loss of function

• removing something from system to see if removed component is necessary

• necessary — required, essential

• system can’t function without it → necessary

• eg. mutation of NLS on nuclear protein is no longer found in nucleus → NLS necessary for nuclear import of that protein

-gain of function

• adding something not normally present to see if sufficient

• sufficient — enough, adequate

• system can function with or without it → sufficient

• eg: addition of NLS to a cytosolic protein → protein found in nucleus → NLS sufficient for nuclear import of that protein

Analyze experimental evidence from fluorescence microscopy and explain how it provides evidence that the primary sequence of a protein contains all of the necessary information to determine whether a protein is imported into the nucleus

-How is the nuclear envelope’s double membrane different than a plasma membrane?

-How is the structural integrity of nucleus maintained? How does this facilitate the breakdown and reformation of nucleus during mitosis?

-What are the macromolecular components of the nucleus organizer regions (NORs)?

-What kinds of molecules are required for transport into/out of nucleus?

-What major factor determines whether a molecule requires active or passive transport through nuclear pore?

-Why do proteins require a targeting signal to enter nucleus? When do proteins acquire the targeting signal?

-What is the difference between something being necessary vs something being sufficient for a process?

-Why is it important for NLS to remain part of the protein after it has entered the nucleus

-is export from nucleus controlled in same way as nuclear import?

3.2 Chromatin & Chromosomes

Discuss how proteins and DNA interact to form chromosomes

-primary structure — DNA sequence

• nucleotides

• nitrogenous bases

-secondary structure — double helix, base pairs

-tertiary structure — DNA packs into chromatin

• nucleosomes

  • DNA wraps around histone proteins (H2A, H2B, H3, H4) to form nucleosomes

  • beads on a string

    • 10 or 11 nm fiber

• chromatin

  • nucleosomes further organized into chromatin

  • H1 interacts with nucleosome by binding to outside of nucleosome and pulls adjacent nucleosomes together

  • can be relaxed or packed state

    • heterochromatin

    • euchromatin

• 30 nm fiber

  • nucleosomes form 30 nm fibers

  • compact chromatin

  • stabilized by H1 linker histones

-quaternary structure — higher order chromatin organization

• chromatin loops

  • 30 nm fiber further organized into loops

  • anchored to scaffold of non-histone proteins

  • essential for organizing genome in nucleus

  • allows for gene regulation

Explain how primary, secondary, tertiary and quaternary levels of histone structure contribute to nucleosome assembly

-histones

• basic proteins that interact with DNA

• important for regulation of chromosome structure

• 5 histones used to pack DNA and produce chromatin

  • core histones (H2A, H2B, H3, H4)

    • interacts with each other and DNA to form nucleosome

    • 4 pairs of H2A, H2B, H3, H4

    • DNA & core histones = nucleosome core particle

    • in between each nucleosome core particle = linker DNA

    • nucleosome core particle + linker DNA = nucleosome

  • linker histones (H1)

    • binds to outside of nucleosome

    • packs nucleosome together

    • tightly packs DNA to 30 nm fiber

Interpret experimental results providing quantitative information about spacing of nucleosomes and amount of DNA associated with these structures

-Discuss the general structure of core histones (H2A, H2B, H3, H4) and their role they have in histone protein structure & chromatin assembly

-What is the purpose of core histone tails

-How does histone H1 fit into packaging of DNA

-How does 11 nm fiber form and how is it different from 30 nm fiber

-What is the level of packaging of DNA in interphase nucleus

-Difference between gel electrophoresis of DNA vs membrane proteins

-What is linker DNA

-What do nucleases do and what is its substrate and how are they useful in determining chromatin structure and nucleosome size

-How does micrococcal nuclease work and what does the time length of exposure of chromatin to micrococcal nuclease change results of the experiment?

Nuclease Digestion Experiment

-separating DNA and histone proteins

• via ionic salt washes

• multiple washes at increasing concentrations

-breaks down chromatin into its components

Summary of Nuclease Digestion Experiment

1. lightly digest chromatin with nuclease

2. remove all associated proteins

3. separate DNA molecules by size using gel electrophoresis

-lightly digest chromatin with enzyme that selectively digests exposed DNA only

-enzyme used for this is from bacteria → micrococcal nuclease

• enzyme will cut exposed DNA but can’t reach DNA sequences covered by other molecules

-if chromatin is digested for a short time → a few cuts in DNA will form but not enough time for all exposed DNA to be degraded

• some nucleosome would still be joined by uncut linker DNA and others would not be → partial nuclease digestion of chromatin

Micrococcal Nuclease

• enzyme that can cut DNA that is not tightly bound to proteins

• can’t properly bind and cut DNA that is tightly bound to surface of proteins

• can be used to determine what DNA is tightly bound to proteins

  • protected from cutting by nuclease

• all unprotected DNA eventually destroyed if nuclease is left to digest for long enough

3.3 Regulation of Gene Expression

Discuss the different types of DNA and histone modifications and their roles in the regulation of gene expression

-euchromatin

• lighter regions on TEM

• lightly packed chromosomes (less dense)

• interphase chromatin can form higher-ordered loops of 30nm fiber

• chromatin fibers can form chromosomes

• attached to nuclear envelope via nuclear lamina

• genes are transcriptionally active

-heterochromatin

• darker regions on tEM

• densely packed chromosomes

• not many active genes

• genes are transcriptionally inactive

• ~10% of interphase chromosome

• loops of 30 nm chromatin

• formed by non histone chromatin proteins that form a scaffold

-chromosome territories

• interphase chromosomes spatially organized

• specific regions of chromosomes attached to nuclear envelope or nuclear lamins

Gene Regulation

-histone tail modifications

• interphase chromatin regulated by chemical modifications of histones

• acetylation, methylation, phosphorylation

  • addition or removal of acetyl, methyl, phosphate

  • gene activation → histone acetylation relaxes chromatin structure → increases access to genes

  • gene repression → histone methylation compacts chromatin structure → blocks access to DNA

• histone tails are short, flexible extensions of histone proteins that stick out from nucleosome

  • controls chromatin structure (affects gene expression)

• loosen or tighten chromosomes

  • loosened chromatin (euchromatin) → DNA is accessible to RNA polymerase and transcription factors

    • genes active and transcription can occur

  • tightened chromatin (heterochromatin) → DNA is packed tightly and inaccessible

    • genes inactive

-chromatin remodeling complex

• large protein complexes that alter nucleosome structures

• makes chromatin more or less accessible for transcription

• complexes binds to histones/DNA to slide nucleosomes along DNA to expose DNA for transcription

• depends on ATP

• can be recruited by transcription regulators

-transcription regulators

• inactive genes → nucleosomes packed tightly

• active genes → nucleosomes destabilized

• enhance or inhibit transcription by destabilizing nucleosomes and opening DNA regions

• regulatory transcription factors

  • bind to specific DNA sequences to activate or repress transcription

  • control gene expression in response to signals

  • activators

    • bind to enhancer regions to recruit RNA polymerase and increase transcription — boosts gene expression

    • bring co-activators that help RNA polymerase bind to DNA

  • repressors

    • bind to silencer sequences to block RNA polymerase and decrease transcription — reduces gene expression

    • recruit proteins that block RNA polymerase

    • tightens chromatins via histone modifications

  
  

  • competitive DNA binding

    • repressor that competes with activator for activation binding site

    • repressor binds to site where activator normally binds → activator can’t do its job

    → parking spot gets taken so you can’t park there

  • masking activation site

    • repressor binds to activator instead of allowing activator to bind to general transcription factors

    • repressor blocks activation sites so activator can’t bind to it

    → if repressor covers keyhole (activation site) the activator can’t fit its key in → no transcription

  • direct interaction with general transcription factors

    • repressor binds to general transcription factors so activators can’t bind to it

    → manager (repressors) tell workers to not do their job so nothing gets done

Transcription & Translation

-Where does the transcription initiation complex bind the DNA and where is this site relative to the transcription start site and what order is the complex assembled

• transcription initiation complex binds the promoter region of gene

-what direction does the transcription complex move on the DNA and how does the transcription complex recognize which orientation to bind the DNA

• template DNA

  • read in 3’ to 5’

  • strand that RNA polymerase uses to synthesize mRNA

  • complementary strand

• coding strand/non-template strand

  • read in 5’ to 3’

  • same sequence as mRNA

• RNA polymerase moves along template strand in 3’ to 5’ direction

• adds RNA nucleotides to growing mRNA strand in 5’ to 3’ direction

• orientation of promoter region (TATA box) and specific transcription factors ensure that transcription complex binds the DNA in correct direction to allow RNA polymerase to synthesize mRNA in 5’ to 3’ direction

• key DNA sequences in transcription unit

  • 5’ flanking sequence

    • DNA found upstream of transcribed region

    • regulatory sequences found here

    • transcription factors bind to regulatory sequences in this region

  • 3’ flanking sequence

    • downstream from end of transcription

  • regulatory regions (DNA sequences)

    • provides binding sites for transcription factors (3.2)

    • enhancer region

      • located thousands of base pairs away either upstream/downstream of transcription unit

      • can bind with transcription factors and increase levels of transcription of related gene

    • promoter region (TATA box)

      • binding site for RNA polymerase

      • adjacent to transcription start site (5’ area)

-general transcription factors

• binds to promoter region (TATA box)

• help RNA polymerase bind to DNA at start site of transcription

• essential for all genes

Types of RNA polymerases in nucleus

1. polymerase I

   • transcribes majority of rRNA genes

2. polymerase II

   • transcribes mRNA and genes that code for non-coding RNAs

3. polymerase III

   • transcribes small stable RNAs

Distinguish between the different types of transcription factors/regulators and explain how their interaction with specific regulatory regions of DNA regulate transcription

-transcription factors are proteins that turn genes on or off by binding DNA and recruiting or blocking RNA polymerase

• controls when and how transcription happens

Discuss how mRNA processing events (cap, tail) can regulate overall gene expression

RNA Processing

• takes place in nucleus before mRNA is transported to cytoplasm for translation

1. 5’ capping — methylated guanine added to 5’ end

   • added after transcription starts

   • protects mRNA from degradation by nucleases (stabilizes mRNA)

   • helps with ribosome recognition & binding (translation initiation)

   • assists in nuclear export of mRNA to nucleus

2. splicing

   • introns (non-coding regions) always removed

   • exons (coding regions) joined together

     • destined for export to cytoplasm

   • ensure only correct and proper protein-coding sequence is translated

   • 75-80% initial primary mRNA transcript lost as result of splicing

   • exon skipping

     • entire exon may be skipped → leaving out part of gene sequence

   • snRNPs

     • small nuclear ribonucleoproteins

     • bind to splice sites and forms spliceosome

       1. recognition & binding

          • spliceosome recognizes splice sites by binding to conserved sequences

       2. intron looping

       • intron folds and brings splice sites together

       • branch point A attacks 5’ splice site and cuts mRNA to form lariat loop

       • exon joining and intron removal

         • 3’ splice site is cut and 2 exons are joined

         • intron released as lariat structure

   • alternate splicing

     • different combinations of exons can be spliced together/genes spliced in different ways → one gene can code for multiple proteins/produce variants of same protein

     • occurs in nucleus before mRNA transported to cytoplasm for translation and occurs at different time points

     • reduces number of genes needed to express different proteins

     • splice donor site (5’ end of intron) to splice acceptor site (3’ end of intron)

3. 3’ polyadenylation — poly-A tail added to 3’ end

   • added shortly after transcription starts

   • protects mRNA from degradation by nucleases (stabilizes mRNA)

   • helps with mRNA export to cytoplasm

   • helps with translation termination

Unit 4 Mitochondria & Chloroplasts

4.1 Introduction & Protein Import

Explain the unique characteristics of mitochondria and chloroplasts that are the result of their evolutionary origin as bacterial endosymbionts

-endosymbiont theory based on structural & genetic similarities between bacteria and mitochondria & chloroplasts

• evolved from theory that mitochondria and chloroplasts are able to divide themselves via binary fission

• circular genomes

• ribosomes that produce proteins

• mitochondria: reduction in size of organelle genome by

  • gene loss of unrequired genes

  • transfer of genes to nucleus (genes became nuclear coded)

-evidence suggests that mitochondria & chloroplasts originate from other prokaryotic cells via phagocytosis

-bacteria → mitochondria → chloroplasts

-new mitochondria & chloroplasts can only be produced via binary fission

• if cell’s mitochondria or chloroplasts are removed or destroyed → cell can’t create new ones

-mitochondria & chloroplasts generate energy like bacteria

• mitochondria uses ETC to produce ATP

• chloroplasts use ETC in thylakoid membrane to produce energy via photosynthesis

-structural similarities

• chemical components found in mitochondria and/or chloroplasts that are only found in bacteria but not other eukaryotes

  • cardiolipin (membrane lipid) found in inner mitochondrial membrane & bacterial cell membranes

  • proteins translated by mitochondria & chloroplasts use N-frmylmethionine as initiating amino acid (eukaryotes use unmodified form of methionine)

• double membrane

  • inner membrane from engulfed bacterial ancestor

  • outer membrane from engulfing eukaryotic cell

• porins (transport proteins) found in outer membranes of mitochondria and chloroplasts also found in bacterial cell membrane

• ribosomes (70S) similar to bacterial ribosomes (eukaryotic ribosomes 80S)

-genetic similarities

• carry circular DNA molecules similar to bacterial DNA

• genome and protein synthesizing systems

• synthesize components of ETC & photosynthetic systems

• mitochondrial genome

  • small (16.5 kb - 2500 kb)

  • genes encode for parts of

    • transcription/translation machinery (tRNAs, ribosomal proteins, RNA)

    • mitochondrial ETC

    • ATP synthase complex

  • proteins encoded in genome

    • hydrophobic subunits of membrane proteins

    • proteins translated by mitochondrial ribosomes

    • remain in mitochondria

• chloroplast genome

  • circular(~120 kb - 160 kb)

  • gene encode for parts of

    • transcription/translation machinery (tRNAs, ribosomal proteins, rRNA)

    • ATP synthase complex

    • large subunit of RuBisCo (enzyme in carbon fixation)

    • some of the 20 components of chloroplast ETC

-almost all proteins in mitochondria/chloroplasts are synthesized on free ribosomes in cytosol then targeted to move to mitochondria/chloroplasts

Describe the post-transitional targeting of proteins to mitochondrial and chloroplast depending on specific domains in its primary sequence and its final destination within these organelles

-specific targeting signal located at N-terminal end of proteins destined for mitochondria/chloroplasts

• signal sequence always found at N-terminus of proteins

-chloroplast & mitochondria have different sequences

-mitochondrial matrix targeting

• one way movement → sequence is cleaved after it enters mitochondria

1. protein with signal sequence synthesized in cytoplasm

2. signal sequence binds to receptor on outer mitochondrial membrane

   • chaperones help maintain protein in unfolded state

     • pulls protein through channel then refolds protein on inside

3. receptor-protein complex diffuses within membrane to a contact site

   • 2 membranes close in proximity

   • translocation channel present

4. protein moves across membrane

   • energy required

     • ATP hydrolysis and harnessing energy stored in proton gradient across inner mitochondrial membrane drives protein import

   • carried out by translocation channel (protein complex)

     • channel has 2 major components

       • outer membrane (TOM - transporter outer mitochondrial membrane)

       • inner membrane (TIM - transporter inner mitochondrial membrane)

5. signal sequence is first part of protein to enter matrix

   • signal sequence cleaved off by specific peptidase once inside

   • proteins refold with help of chaperones

-chloroplast matrix targeting

• plants must send different proteins to mitochondria & chloroplasts → localization sequences needs to be different

  • precise targeting sequence that fits into a pocket in membrane receptor

• one way movement → sequence is cleaved once it enters chloroplast

• stromal proteins fed across a translocation channel and passes through inner & outer membrane at the same time

  • channel has 2 major components

    • outer membrane (TOC - transporter outer chloroplast membrane)

    • inner membrane (TIC - transporter inner chloroplast membrane)

  • proteins destined for thylakoid lumen ends up in stroma first

    • after second sequence revealed when initial chloroplast targeting sequence is cleaved → directs protein to thylakoid lumen

• energy required

  • ATP (not proton gradient)

• protein meant for stroma → TIC complex

• protein meant for thylakoid membrane → second targeting pathway within stroma

• sequence cleaved and protein folds into proper shape once it reaches final location

Compare and contrast protein targeting to mitochondria and chloroplasts with targeting to other organelles/destinations already described

-similarities

• use targeting signals

  • special sequence of amino acids that tells cells where to send protein

• start in cytoplasm

  • proteins made by ribosomes in cytoplasm before sent to destination

• use protein transport complex

  • mitochondria: TOM/TIM complex

  • chloroplast: TOC/TIC complex

  • nucleus: nuclear pore complex

-differences

• mitochondria/chloroplast: proteins unfolded until inside

• nucleus: proteins can be fully folded when entering

• mitochondria/chloroplast sequence: only at N-terminal

• nucleus sequence: anywhere on protein

• mitochondria destination: OM, IM, intermembrane space, matrix

• chloroplast destination: OM, IM, intermembrane space, stroma, thylakoid

• nucleus destination: inside nucleus

• mitochondria/chloroplast reversibility: one way (can’t leave)

• nucleus reversibility: two way (can exit if have export signal)

4.2 Mitochondria

Explain and interpret micrographs of mitochondria in cells

Relate mitochondrial structure to the chemiosmotic coupling of proton pumping and ATP formation in mitochondria during the process of oxidative phosphorylation

-outer membrane

• controls what enters and exits mitochondrion

-inner membrane

• contains ETC & ATP synthase

• folded into cristae → increases SA for more ATP production

-intermembrane space

• area between inner and outer membrane

• fills with protons (H⁺) during energy production

  • stored with protons to create proton gradient

-matrix

• citric acid cycle occurs here

• molecules that feed the ETC

-substrate level phosphorylation

• produces ATP y combining substrate with phosphate group

• doesn’t produce enough ATP to meet energy needs of complex organisms

• all ATP produced by glycolysis

-chemiosmotic coupling (oxidative phosphorylation)

• produces enough energy to meet energy needs of complex organisms

• proton motive force (pmf)

  • usage of H⁺ protons

  • actively being transported against their gradient so that they can flow back down their gradient to do work

• ATP synthase

  • aids with production of ATP from ADP

  • large protein complex

  • same enzyme used by bacteria to produce ATP on plasma membrane

  • portion is embedded in membrane

1. electron transport & proton pumping (electron donors drive ETC)

   • occurs in inner membrane

   • electrons energized with food enters ETC

   • electrons move through ETC → energy is released and used to pump protons into intermembrane space

   • proton gradient formed (more H⁺/protons on outside than inside)

2. proton flow drives ATP formation (proton gradient drives ATP production)

   • energy in electrochemical gradient drives photophosphorylation of ADP to produce ATP

   • proton gradient stores potential energy

   • protons pass through ATP synthase complex and turns ADP → ATP

3. oxygen is the TEA

   • O₂ combines with electrons and H⁺ to form H₂O

1. electrons from food → ETC (inner membrane)

2. energy from electron pumps H⁺ into intermembrane space (proton gradient)

3. O₂ as TEA forms water

4. H⁺ flows back to matrix via ATP synthase where ADP → ATP

4.3 Chloroplasts

Recognize chloroplasts and its various compartments on electron micrographs

Discuss the chloroplast structure and its relevance to the process of photophosphorylation and photosynthesis

-inner and outer membrane

-thylakoid membrane

• third internal membrane system not present in mitochondria

• flattened sac-like structures where photosynthesis occurs

• has proteins that help in electron transport → H⁺ protons pumped into lumen to create high concentration of protons → protons rush back via ATP synthase → ATP used in Calvin cycle

-plastids

• family of organelles in plants that chloroplast is part of

-lumen

• space inside of thylakoid

-production of

• purines, pyrimidines for nucleic acids

• complex lipids (all fatty acids)

• most amino acids for plant cells

  • except cysteine, methionine, sulfur containing AA

• stress response chemical involved in triggering plant immune response

-chemiosmotic coupling (photophosphorylation)

• occurs in thylakoids

• sunlight required and energy captured by chlorophyll

• electrons with energy moves through ETC → powers ATP synthesis → water split to O₂, H⁺ and electrons → NADPH + ATP made

• ATP made in chloroplast stays in chloroplast

  • used in Calvin cycle

-Calvin cycle

• light not required

• ATP & NADPH from photophosphorylation converts CO₂ into glucose → for plant growth & energy storage

• -inputs H₂O & CO₂ → outputs O₂ & glucose

1. electrons energized from sun enters ETC (thylakoid membrane)

2. energy allows H⁺ pumped into thylakoid lumen

3. H₂O split to release more H⁺ and O₂

4. H⁺ flows back into stroma via ATP synthase where ADP → ATP

5. ATP powers Calvin cycle to make glucose

Compare and contrast chemiosmotic coupling of proton pumping and ATP formation in mitochondria and chloroplasts

-similarities

• use ETC

  • electrons move through proteins in

    • mitochondria: inner mitochondrial membrane

    • chloroplasts: thylakoid membrane

  • powers proton pumping to create H⁺ gradient

• create a proton gradient

  • higher proton concentration on one side

• ATP synthase complex used

  • protons flow back to low concentration via ATP synthase

  • converts ADP + phosphate → ATP

• membrane to separate protons

  • mitochondria: inner membrane

  • chloroplast: thylakoid membrane

-differences

• mitochondria energy: food

• chloroplast energy: light

• mitochondria electron source: NADH + FADH₂

• chloroplast electron source: H₂O

• mitochondria TEA: O₂ to form H₂O

• chloroplast TEA: NADP⁺ to form NADPH

• mitochondria proton pump: matrix to intermembrane space

• chloroplast proton pump: stroma to thylakoid lumen

• mitochondria ATP production: matrix

• chloroplast ATP production: stroma

• mitochondria ATP usage: cellular work (muscle movement, metabolism, etc)

• chloroplast ATP usage: making glucose for Calvin cycle

Explain the relationship between mitochondria and chloroplasts in plant cells

-mitochondria and chloroplasts work together

-plants remove CO₂ in atmosphere and put it back into biosphere

• helps combat climate changes, CO₂ accumulation

• humans rely on plants

-sugars produced by chloroplast in plants are used by mitochondria to produce ATP required for cellular function

-mitochondria → oxidative phosphorylation

-chloroplast → photophosphorylation

Unit 5 Endomembrane System

5.1 Introduction & Protein Transport

Explain the structural & functional relationships between the different compartments of the endomembrane system and identify them on micrographs

-endomembrane system

• membrane limited compartments involved in processing & movement of proteins and membrane

• consists of ER, Golgi apparatus, lysosomes, endosomes, secretory vesicles

• involved in

  • processing of proteins for export from cell

  • proteins destined for lysosomes

  • proteins entering cell from surface

• 3 major subdivisions

  1. secretory pathway

  2. lysosomal pathway

  3. endocytic pathway

• 3 types of proteins enter endomembrane system after translation

  1. proteins destined for secretion

  2. plasma membrane proteins

  3. proteins destined to be endomembrane resident proteins

     • lives in Golgi apparatus or ER

• only points of entry into endomembrane system are ER and plasma membrane

  • proteins destined for secretory/lysosomal pathways must enter at ER

  • proteins for endocytic pathway must enter via plasma membrane

• proteins that enter endomembrane never leave pathway planned for them until final destination has been reached

  • ONLY return to cytosol if they are misfolded (refolded or destroyed)

-endoplasmic reticulum (ER)

• structure: network of membrane-bound tubules & sacs

  • rough ER: covered with ribosomes

  • smooth ER: no ribosomes

• function

  • rough ER: synthesizes proteins and sends them to Golgi in vesicles

  • smooth ER: makes lipids & steroids; doesn’t participate in protein synthesis

• relationship to endomembrane system: ER packages proteins and lipids into vesicles → Golgi

-golgi apparatus

• structure: flattened membrane sacs

• function: modifies, sorts, packages proteins & lipids before sending them to their destination

  • proteins enter from cis to trans

• relationship to endomembrane system: receives vesicles from ER, modifies and packs protein into new vesicles → plasma membrane or lysosome or other parts of cell

-lysosomes

• structure: small, membrane-bound organelles filled with digestive enzymes

• function: break down worn-out cell parts, food particles, foreign invaders

• relationship to endomembrane system: made by Golgi and contain enzymes that help break down waste materials inside vesicles

-endosomes

• structure: vesicle-like membrane bound compartments; have acidic interior

• function: sort and transport materials inside the cell (particularly those taken in from outside cell via endocytosis); decides whether materials should be sent to lysosomes, recycled, sent to other areas of cell

• relationship to endomembrane system: connects plasma membrane, Golgi, lysosomes and vesicles

-vesicles

• structure: small, membrane-enclosed sac

• function: transports proteins, lipids, waste materials between organelles

• relationship to endomembrane system: vesicles move between ER, Golgi, lysosomes, plasma membrane

-plasma membrane

• structure: lipid bilayer that surrounds the cell

• function: controls what enters and exits the cell

• relationship to endomembrane system: vesicles from Golgi fuse with plasma membrane to release proteins outside the cell (exocytosis) or bring materials inside (endocytosis)

-protein processing

• all proteins are processed after translation

  • folding

    • supported by disulfide bridges between cysteine residues

      • bonds formed as protein assumes its folded structure

    • disulfide isomerase in ER facilitates formation of disulfide bridges

      • disulfide bridges not common in cytosolic proteins

  • removal of first methionine (carried by first tRNA)

  • methylation

  • phosphorylation

  • acetylation

  • formation of disulfide bridges

  • glycosylation

• new polypeptides from ribosomes fold immediately based on primary sequence

  • spontaneous

  • lowest possible energy conformation

  • proteins fold in different ways → different path each time

Describe how proteins are targeted and imported into the ER and compare these mechanisms to protein targeting and import into the nucleus

-endoplasmic reticulum

• flattened membrane cisternae and tubules

• continuous with nuclear envelope

• controls calcium levels in cytoplasm (calcium store)

  • cell signaling, muscle contraction

-rough ER

• cytosolic surface of membranes have attached ribosomes synthesizing proteins for import into ER

• site of synthesis for proteins destined for secretion, lysosomes, membranes

• quality control → checks for defective proteins

-smooth ER

• continuous with rough ER

• site of lipid and steroid synthesis

• doesn’t participate in protein synthesis

-transitional ER

• site where both ERs meet

• vesicles form and newly synthesized proteins exit ER to be able to move to next destination

-protein targeting

• proteins going to a specific destination containing a sorting/targeting signal in their sequence

-entry/exit from ER

• cells control what is allowed to enter/exit er

• only proteins with proper signals are allowed to enter

  • proteins are either

    • ER residents

      • proteins that live in ER

      • carries ER retention signal (KDEL) at carboxyl ends

      • if no KDEL → protein will travel through endomembrane system and secreted out of cell

    • passing along through to other destinations

• once proteins enter ER → never return to cytosol (unless sent to proteasome)

• proteins that get exported from ER must be properly folded by chaperone proteins, packaged into vesicles and moved into next organelle (usually Golgi)

-proteins enter ER co-translationally

• ribosome that is translating proteins destined for ER becomes attached to surface of ER during translation

-soluble proteins entering ER

• translocated into lumen of ER

• need only a single transfer sequence (N-terminus)

• peptides move through translocation channel into ER lumen

• signal sequence remains embedded in membrane

  • cleaved by signal peptidase → protein free in lumen of ER

-membrane proteins entering ER

• partially located in ER

• stuck with part of it embedded inside membrane

• proteins can be destined for

  • ER

  • resident proteins in Golgi

  • plasma membrane

    • once inserted into membrane → can’t be removed

Predict the signal sequence required to insert a protein into the ER membrane in any orientation and predict protein topology from a corresponding domain map

-2 things required for protein to be targeted to ER

1. signal encoded within protein

2. receptor that recognizes & binds signal

   Signal sequences are necessary & sufficient to direct proteins to ER

   • necessary (lof): remove signal sequence from ER protein to see if it stays in cytoplasm — ER protein doesn’t enter ER

   • sufficient (gof): add signal sequence to cytosolic protein to see if it ends up in ER — supposed to be in cytoplasm but has sequence so it enters ER

-ER signal sequence

• always at least 8-10 nonpolar amino acids in a row

• N terminal start transfer sequence

• internal start transfer sequence

• stop sequence

-signal recognition particle (SRP)

• binds to exposed N-terminal signal sequence and ribosome

-start transfer sequences

• N-terminal start transfer sequence

  • ~18 hydrophobic amino acids at N-terminal

  • always cleaved off by signal peptidase

  • initiates transfer of protein across ER membrane

  • if present → N-terminal end of protein in ER lumen

• internal start transfer sequence

  • located internally

  • binds to SRP

  • initiates transfer of protein across ER membrane

  • membrane crossing domain

  • not cleaved after transfer of protein

  • if starts with internal start sequence and not N-terminal start sequence → N-terminal end of protein in cytosol

*can have N-terminal start and internal start transfer sequence BUT is separated by a stop sequence

-stop transfer signal

• ~18 hydrophobic amino acid residues

• found internally

• only occurs after N-terminal start sequence or internal start transfer sequence

• stops transfer of protein across ER membrane

• membrane crossing domain

• remains in membrane

• not cleaved

1. ER signal sequence translated as part of primary sequence

   • recognized by ribonucleic protein

2. SRP binds to ER signal sequence → inhibits translation

3. ribosome + mRNA + partially translated protein complex → ER → binds to SRP receptor protein in ER membrane

4. ribosome attaches to translocation channel for newly synthesized polypeptide

   • attachment facilitated by SRP receptor; energy required (GTP)

5. SRP removed and translation resumes

   • new protein pushed through translocation channel into ER lumen

• C - cytosol; L = lumen

• A: protein inserted in ER lumen

• B: vesicle with protein buds off from ER

  • cytosolic portion of protein still in cytosol

• C: vesicle travels to plasma membrane and starts fusing with membrane

• vesicle successfully fuses with membrane → protein now part of membrane

  • cytosolic portion still in cytosol

-domain maps

• protein 1: ends up in plasma membrane via ER to be a integral membrane protein

  • protein has NLS (but signal is only recognized after protein has been completely translated & folded in cytoplasm)

  • internal start transfer signal sequence is recognized first

  • protein has

    • N-terminus in cytoplasm

    • transmembrane domain in ER membrane

    • C-terminus in ER lumen

• protein 2: forms a soluble protein in extracellular space

  • has 1 hydrophobic sequence → N-terminal signal sequence → protein is co-translated into ER lumen → N-terminal signal sequence is cleaved → soluble protein in ER lumen BUT because no KDEL → moves to membrane

  • protein processed → vesicles → vesicle fusion with membrane → protein excreted as extracellular soluble protein

• protein 3: ends up as ER resident protein (soluble)

  • has 1 hydrophobic sequence → N-terminal signal sequence → protein is co-translated into ER lumen → N-terminal signal sequence is cleaved → soluble protein in ER BUT because KDEL retention sequence → kept in ER lumen

• protein 4: becomes integral protein in plasma membrane

  • has 2 hydrophobic sequences

    • N-terminal signal sequence

    • internal stop transfer signal sequence

  • protein has

    • N-terminus in ER lumen

    • transmembrane domain in ER membrane

    • C-terminus in cytosol

  • becomes an integral protein in plasma membrane after processing

  • 
    

    

-hydropathy plots

• shows protein sequence before N-terminal signal sequence is cleaved off

• produced based on original protein sequence

  • not what they look like after protein processing

Discuss the role of chaperones in protein folding and role of proteasomes in quality control of misfolded proteins in the secretory pathway

-if cells make mistake in protein assembly → ER ensures properly folded proteins move on

• misfolded proteins → refolded with chaperone proteins

-chaperones

• facilitate and aid with proper polypeptide folding

• binds specific regions of polypeptide

• provides a protected space away from rest of cytosol so protein can fold in peace (in ER)

• BiP (binding protein)

  • prevents hydrophobic domains of proteins from aggregating and promotes promotes proper folding

-proteasome (only time protein is sent back to cytosol after entering ER)

• found in cytosol and nucleus

• misfolded proteins destroyed

  • amino acids recycled and incorporated into new proteins

• degrades proteins tagged for destruction with small polypeptide ubiquitin

Describe and interpret the results of the different experimental tools studying the secretory pathway

-microscopy

• live cell imaging of GFP-tagged protein

5.2 Vesicle Transport

List the 4 stages of vesicle transport and list the protein machinery involved at each step

-vesicles

• moves proteins (cargo) around the cell

  • from membrane-bound organelles to another compartment or plasma membrane of endomembrane system

• produced by all organelles of endomembrane system

1. formation

   • regulated by GTPase

     • bound to GTP: active

       • allows adaptors and coat proteins to bind

       • assembles membrane coat

   • GTP hydrolyzes to GTP: inactive

     • GTPases, coat proteins & adaptors dissociate from vesicle

   • flippase — generated asymmetry

     • 
       

       

   • vesicle formation & fusion relies on same membrane thermodynamics

   • coat proteins

     • coats have to be shed off → naked vesicle for transportation

     • 2 primary functions

       1. force membrane to curve and form bud

       2. capture cargo molecules for transport

     • COP → coatamer proteins

     • 
       

       

   • clathrin-coated vesicle

     • used for

       • endocytosis

       • traffic to lysosome

       • receptor recycling

       • between trans Golgi network (TGN)

       • endosomes/lysosomes

       • plasma membrane

     • requires

       • cargo receptor

       • adaptin

       • coat protein

       • dynamin

     • uptake of extracellular

   • COPI-Coated Vesicles

     • involved in moving cargo from Golgi → ER traffic (retrograde)

     • dynamin protein not required

   • COPII-Coated Vesicles

     • involved in moving cargo from ER → Golgi traffic (anterograde)

     • dynamin protein not required

     • cargo can things that were secreted

     

2. transport

   • short distance vesicles move via diffusion

   • long distance vesicles move along

     • cytoskeletal tracts

       • microtubules

     • motor proteins

       • kinesins/dynein for microtubules

       • myosin for actin

3. docking

   • vesicles MUST dock before fusing (vesicles directed to correct place for fusion)

   • dock via Rabs & Tethers

     • proteins that direct vesicles to the right place for docking

     • active Rab-GTPases are present on vesicular membranes

     • if right tethering (Rab-binding) → protein is present on target membrane → vesicle will tether

     • brings vesicle in close enough proximity to begin membrane fusion

   • Rabs

     • small GTPases that sit on vesicle surface when activated

     • identifies vesicle targeting to particular membrane

   • Tethers

     • binds to vesicle Rab and brings in vesicle from cytosol

   • 
     

     

4. fusion

   • fuse via SNAREs

     • proteins that mediate vesicle fusion with membrane

     • alpha-helix structures

     • vesicle-SNAREs (v-SNAREs)

       • on vesicle membrane

     • target-SNAREs (t-SNAREs)

       • on target membrane

   • must have at least

     • 1 SNARE on each membrane

     • 4 distinct SNARE coils to mediate fusion

   • orientation of membrane constant

     • cytosolic face of vesicles remains cytosol facing throughout vesicle transport

     • what happens in cytosol stays in cytosol

   • 
     

     

Explain how vesicle coats facilitate cargo loading and vesicle budding

Discuss the importance of maintaining specificity in both docking and fusion of vesicles at their target compartments, and how Rabs, tethers and SNAREs facilitate this process

Discuss and illustrate how the orientation of membrane proteins in the lipid bilayer is maintained during transport and after fusion of vesicles with target membranes

5.3 Golgi & Protein Processing

Discuss the functional compartmentalization of the Golgi structure and recognize the Golgi apparatus using different types of microscopy

-Golgi apparatus

• center of endomembrane system

• receives cargo from

  • ER, lysosome, plasma membrane, endosomes

• receives cargo → modifies → repacks into new vesicles → sent to new destination

• site of all major cellular work involving polysaccharides

• site of all known glycosyltransferases

  • glycosyltransferase — enzymes capable of attaching sugars to each other

  • important for production of

    • glycoproteins

    • cell walls of plants and fungi

• structure

  • varies between different kingdoms/species

  • polar

    • cargo enters from one end

      • cis face for newly synthesized proteins

    • cargo exists from other end

      • trans face

  • cisterna

    • each pancake of Golgi

    • series of flattened stacks

    • fluid containing sac

  • lumen

    • interior space of each cisterna

-Golgi resident proteins

• distributed in Golgi

• cis to trans

• full time jobs in Golgi

• different resident proteins present in different cisternae

• resident proteins have retention signal so they know to stay in Golgi

-transient/cargo proteins

• moves through golgi (cis → trans) to get to where they need to (final destination)

• just passing through golgi

• moving on to other destinations of endomembrane system

• cis cisterna → medial cisterna → trans cisterna → into TGN to be packed into vesicles → sent to next destination

-trans golgi network (TGN)

• protein sorting center

• vesicles leave TGN for different destinations

  1. maturing lysosome

  2. condensing secretory granules released through regulated secretory pathway

  3. plasma membrane

     • vesicles containing membrane and proteins are released to surface via constitutive secretory pathway

-golgi aids in production of cell wall

• rosettes (proteins) too large for vesicle but has to be delivered to plant plasma membrane

  • rosettes must follow same path as a regular newly synthesized protein destined for plasma membrane

  • rosettes synthesized one sugar at a time as molecules move from cis to trans in Golgi

    • packaged into vesicles in TGN → plasma membrane → secreted and incorporated into plant cell wall

• 
  

  

Describe the sequence of events occurring during protein glycosylation in the ER and Golgi

-glycosylation

• post-translationational modifications of proteins

• addition of polysaccharides

  • adding sugar molecules (glycans) to proteins

  • helps proteins fold properly, become more stable

• enzymes that carry out reactions are located in lumen of ER and Golgi

• 2 types of glycosylation

  • N-linked glycosylation

    • occurs in ER & golgi

    • attaches sugars to N of Asn amino acid

    • starts in rER → Golgi

      1. large sugar group built on lipid molecule (dolichol) in ER

      2. sugar transferred to specific Asn-X-Ser/Thr) site on a protein

      3. protein checked for proper folding

      4. Golgi trims and modifies sugar to make mature glycoprotein

  • O-linked glycosylation

    • occurs in golgi AFTER protein has folded

    • attaches sugars to O of Ser/Thr amino acids

      1. single sugar (N-acetylgalactosamine) added first

      2. more sugars added in step-by-step manner

      3. final glycoprotein sent to destination

-oligosaccharides assembled one sugar at a time

• specific enzymes found in each compartment of Golgi stack that does its job

-secretion of glycoprotein

1. ribosomes binds to mRNA in cytoplasm

2. co-translational insertion of protein into ER

3. enzyme transfers oligosaccharide core from dolichol to protein

4. oligosaccharide has sugars trimmed and added by glycosyl transferases

5. protein packaged in trans-Golgi into secretory vesicle

Use experimental evidence to determine how membrane and cargo are transported through the Golgi

-vesicle transport model (lecture 25)

• cisternae stationary

• resident proteins living in cisternae

• cargo proteins moved from one cisterna to next via transport vesicles

  • vesicles need to be formed every time from cis to medial to trans

  • vesicles fuse every time they enter a cisterna

• transient proteins in vesicles (vesicle formation via COPII coat proteins) → bud from periphery of Golgi cisterna → fuses with target cisterna

• new vesicles formed every time

• vesicle fuses with cis membrane → proteins enter cis cisterna→ protein forms new vesicle to exit cis → vesicle fuses with medial membrane → proteins enters medial cisternae → protein forms new vesicle to exit medial → vesicle fuses with trans membrane→ proteins enter trans → proteins sorted into new vesicles (in TGN) for final destination

  *sugars added at every cisternae

  *transient proteins moving forwards via vesicles; cisterna stationary

  *resident proteins stay in place

• 
  

  

-cisternal maturation model

• cisternae moves

• resident proteins move backwards to the original cisterna location

• new cis-cisterna forms → old cis-cisterna becomes medial-cisterna (and so on)

  • eventually trans-cisterna broken down to form vesicles

• transient protein with vesicle fuses with cis membrane → transient proteins enter cis cisterna → cisternae shifts (cis → medial, medial → trans, trans → broken down, new cis comes in) → resident proteins move backwards to the original location of cisterna via vesicles (fuse) & transient proteins don’t move via vesicles, but they move with the cisternae → when transient proteins reach cisternae that has matured into trans cisternae → packaged into new vesicles (in TGN) for final destination

  *resident proteins moving backwards via vesicles; cisternae not stationary

  *transient proteins stay in place

• 
  

  

5.4 Post-Golgi Traffic

Describe the secretory, lysosomal, endocytic pathways and list all components of these pathways

-secretory pathway (exocytosis)

• proteins carry information needed to determine which pathway they belong within their primary amino acid sequence

• constitutive secretion

  • occurring continuously

  • vesicles are continuously forming

  • vesicles continuously carrying proteins from Golgi to cell surface

  • leads to plasma membrane

  • supplies continuous stream of vesicles to plasma membrane for release outside of cell

  • vesicles containing lipids, proteins, polysaccharides

    • supplies plasma membrane with these

    • refreshes old lipids

    • contributes to extracellular matrix

  • no signal required for vesicle release

  • vesicle formation doesn’t require coat proteins

• regulated secretion

  • vesicles only released when specific signal is received

    • binding of hormone

  • vesicles may or may not require coat proteins

    • pH may play a role in formation of vesicles when no coat used

      • proteins aggregates at low pH

      • aggregation allows vesicles to be highly condensed

  • proteins consolidated into vesicles that are stored in cell until secreted

  • leads to plasma membrane

  • release of vesicles with cargo requires signal from cell before final fusion with membrane

  • vesicle formation aided by proteins destined for this pathway

    • proteins clump in acidic conditions (aggregation)

      • helpful since Golgi is acidic as it moves from cis to trans)

      • TGN → large protein aggregates can push up against membrane and help to promote curvature

-lysosomal pathway

• directs newly synthesized lysosomal proteins to lysosomes via endosomes

• sends lysosomal proteins to late endosome

  • acidic enough to allow separation of enzyme from receptor that carried it there

• late endosome can become a hybrid compartment before maturation to lysosome

-endocytic pathway

Compare and contrast constitutive and regulated secretion and explain the important of each

-secretory mutants

• proteins needed for secretion

• temperature-sensitive mutants are observed only at higher temperatures

  • proteins function normally at lower temperatures

• secretory proteins with GFP can be tracked in live cells

• 
  

  

• wild type: secretory proteins in vesicles, ER, Golgi

• sec mutant left: secretory proteins in cytoplasm

  • transport of proteins into ER disrupted

  • mutation → components for ER targeting/co-translation

    • ER signal sequence & SRP amy be specific mutated components

• sec mutant right: secretory proteins in ER

  • transport of proteins from ER → Golgi disrupted

  • mutation → vesicle formation error

    • COPII-vesicle formation (COPII coat, adaptor protein, cargo receptors)

    *mutation due to ER retention signal is unlikely

Discuss the structure and function of lysosomes and explain how they mature from late endosomes

-lysosome

• secretory vesicles that contain digestive enzymes

• site of intracellular digestion

• contains mixture of ~40 different types of digestive enzymes

  • degrades nucleic acids, proteins, lipids

• membrane bound

• very acidic (pH ~4-5)

  • maintained by proton pumps embedded in membrane

  • membrane of lysosome resistant to action of its own digestive enzymes

    • due to extensive glycosylation of proteins on inside of membrane

• facilitate digestion of intracellular structure that cell needs to dispose

  • large polymers brought in from exterior via endocytic pathway

• breakdown of bacteria engulfed by cell during phagocytosis

Trace the path of a newly synthesized lysosomal protein to its final destination and compare this to the path of proteins to all other destinations within the endomembrane path

-for protein to be directed to lysosome → specific sorting signal required

• signal within chemical structure of that protein cell can recognize

1. targeting sequence (M6P - mannose-6-phohsphate)

   • M6P is built from oligosaccharide tree added to asparagine in protein in ER based on its amino sequence

   • in cis Golgi → phosphate added to 6th carbon of specific mannose in tree

     • 2 fold result

     • M6P tag formed used later by receptor

     • added as result of stopping other glycosyltransferases in Golgi from being able to further modify sugar tree

2. signal recognized by M6P receptors in TGN

3. packaged into clathrin-coated at TGN → vesicles sent to endosome

4. lysosomal proteins separated from their receptors due to increased acidity in endosome

5. lysosomal proteins travel with endosome to lysosome and receptors are returned to TGN

5.5 Endocytosis

Discuss the structure and function of endosomes and differentiate between early and late endosomes

-endosomes

• sorting center

• junction between lysosomal pathway and endocytic pathway

  • differentiates between cargo going to lysosome and receptors that has to be returned to TGN/pM

• cargo fate compared to receptor is dependent on acidic environment of endosome

  • cargo is one that gets released by acidic endosomal environment → cargo in lysosome & receptor free to be recycled

• receives cargo from plasma membrane destined for different places

  1. receptors returned to PM domain from where they came from while their cargo is passed onto lysosome

  2. some receptors not recycled but sent to lysosome to be degraded

     • done with or without cargo

  3. some receptors along with their cargo transferred to another plasma membrane domain

     • allows specific cargo to be passed from one side of cell to another (transcytosis)

• pH

  • endomembrane more acidic than cytosol

  • increasingly acidic moving from point of origin to endpoint

  • culminates in lysosome (as acidic as stomac acid)

  • specific pH causes change in certain proteins

    • gives cell control over how and when certain events happen

  • proper functioning of TGN requires it to maintain appropriate pH

    • different from cytosol pH and nearby surroundings

-early endosome

• first compartments that form when material from outside cell is engulfed into cell via endocytosis

• material brought to cell in vesicle and fuses with early endosome

• sorts internalized materials

  • decide whether substances should be sent back to cell surface, go to lysosomes (degradation), stored for future use

• slightly acidic (6.0-6.5)

-late endosome

• later stage of endocytic pathway

• early endosomes mature into late endosomes after they undergo acidification and internal sorting

• involved in preparing cargo for degradation in lysosomes

• more acidic (5.0-5.5)

  • activates enzymes needed to break down internalized cargo before it transfers to lysosome for complete digestion

Compare and contrast between constitutive and receptor-mediated endocytosis

-constitutive endocytosis = pinocytosis

• continuous process where cells remove excess membrane added by exocytosis

• allowing recycling of plasma membrane

• non-selective process

• fluid & macromolecules from extracellular region taken up without regard to type of concentration of molecules present

• no specific receptors for uptake of macromolecules

• may or may not use coated vesicles

• advantages

  • doesn’t require receptor proteins

  • vesicles may not be required → coat proteins, adaptors not required → less energy required

  • lower energy cost

• disadvantages

  • accidentally bring in unwanted items into cells

  • cell membrane gets bigger

-receptor mediated endocytosis

• receptors used to collect specific extracellular compounds

• clathrin used for vesicle formation

• endosomes act as sorting centers for endocytic pathways (from TGN & plasma membrane)

  • helps sort

    1. proteins/materials enter cell that are destined for

       • lysosome

       • cell surface

       • other endomembrane compartments

    2. proteins that leave TGN destined for lysosomes

• early endosomes mature into late endosomes → form lysosomes or fuse with existing lysosomes

Trace a molecule from the exterior of the cell to the lysosome along the endocytic pathway

1. uptake via endocytosis

   • molecule from outside of cell binds to receptor in cell membrane

   • vesicle created that engulfs molecule (early endocytic vesicle) via clathrin protein

2. early endosome

   • vesicle formed from membrane fuses with early endosome inside cell

   • slightly acidic pH dissociates cargo from receptors

   • early endosome sorts material and decides where it should go

3. maturation into late endosome

   • undergoes acidification

     • internal environment is more acidic → activates enzymes to break down internalized material

4. lysosome fusion

   • late endosome fuses with lysosome

   • lysosome contains hydrolytic enzymes that degrade wastes

   • fusion creates mature endolysosome

     • material is broken down into small components by enzyme in lysosome

5. degradation and recycling

   • simpler molecules recycled within cell

   • pathogen/damaged cellular material is ingested by lysosome → resulting molecule used/expelled by cell

Explain with examples how receptor proteins facilitate endocytosis

-low-density lipoprotein (LDL) receptor

• complex that makes lipids/cholesterol water soluble so they can be carried in bloodstreop

• receptors used to bring LDL into cell

• receptor: LDL receptor on cell membrane recognizes and binds LDL (carries cholesterol in bloodstream)

1. binding: LDL molecule binds to LDL receptor on cell surface

2. clathrin coat formation

   • binding triggers clathrin formation

3. internalization

   • clathrin-coated vesicle buds off from plasma membrane

   • carrying LDL and its receptor inside the cell

4. uncoating

   • clathrin coat removed once inside cell

   • vesicle → early endosome

5. sorting

   • acidic environment of early endosome sorts molecule

   • causes LDL to dissociate from receptor

   • receptor recycled back to plasma membrane

   • LDL directed to late endosomes → lysosome for processing

Compare and contrast receptor recycling in the lysosomal and endocytic pathways

Unit 6 Cytoskeleton

6.1 Overview of Cytoskeleton: Intermediate Filaments

Compare and contrast the structure and function of three types of cytoskeletal elements: actin filaments, intermediate filaments and microtubules

-cytoskeleton

• gives cell its shape

• resists mechanical stresses

• cells aren’t static → change shapes all the time

  • cytoskeleton coordinates the change while maintaining tissue integrity

• acts as an anchor for many proteins and organelles

• provides tracks for transport of vesicles, organelles, other cargo

• helps with division of both cytoplasm and DNA during mitosis

• 3 major types of filaments in most cells

  • actin filaments

  • intermediate filaments

  • microtubules

-dynamic instability

• actin filaments, microtubules are dynamically unstable

• grow, pause, shrink in the span of a few seconds

• unstable, constant state of influx

-actin filaments (AF)

• smallest of the three (~ 7 nm in diameter)

• polymer (f-actin/filamentous actin/microfilaments) of repeating subunits (g-actin/globular actin)

• undergoes dynamic instability

• attaches to plasma membrane proteins

  • shapes cell

  • respond to cellular environment

• animal cells

  • involved in cellular locomotion, cellular adhesion & shape, cytokinesis, cargo trafficking, muscle contraction

  • close to membrane

    • cortical arrangement

• plant cells

  • cellular arrangement is different

  • found throughout cytoplasm

    • not just underneath plasma membrane

  • involved in cargo trafficking and cell shape

• actin binding proteins (APBs) (MARCH 26 LECTURE FOR FULL DEPTH OF WHAT APBS DO)

  • uses proteins to control when and where actin filaments form

  • allow cell to control how actin filament grows

  • nucleating protein allows cell to control where actin grows

  • severing proteins cut actins

  • capping proteins (plus end) allows actin to stay the same length

  • side-binding proteins stabilize actins

  • bundle proteins form actin bundles

  • work with actin to form different actin networks in cell

    1. parallel bundles

       • filaments closely spaced and have some polarity

       • ABPs (formin & profilin) build array

    2. contractile bundles

       • in which filaments are arranged anti-parallel and cross-linked by stabilizing proteins

       • molecular motors drive contraction of these arrays

    3. cross-linked gel

       • random orientation of fibers, linked at crossing by filamin

       • actin gel major component of cell cortex of most cell types

       • plays role in locomotion

       • ARP 2/3 used to build filaments in these arrays

         • additional proteins used to crosslink and stabilize

• can be controlled by

  • controlling where an actin filament forms

    • provide nucleation sites

    • Arp 2/3 binds to side of a pre-existing actin filament and makes a branching network

    • formin & profilin work together to make parallel networks

  • stabilizing actin filaments using protein caps

    • structures formed by actin are required long term

-intermediate filaments

• middle size of the three (~10 nm in diameter)

• only found in animal cells

• very strong

• family of proteins classified into 5 major types

  • keratins, vimentin, neurofilaments, nuclear lamins

    • only 1 or 2 expressed in each cell types

    • nuclear lamins important in maintenance and organization of cell envelope

    • single filament always made from same type of subunit

  • different cell types express different intermediate filament proteins

• doesn’t undergo dynamic instability (actin & microtubules do)

• can be assembled and disassembled as needed

• helps with anchoring non-motile cells

• resists mechanical strain in tissues

• vital to maintenance of cell shape

• long, twisted strands that are fibrous

• structure depends on twisting of coiled-coils via hydrophobic amino acids

  • twisting due to van der waal interactions

  • intertwined alpha-helices form coiled-coil dimers

  • whatever is structurally stable/favoured

-microtubules

• largest of the three (~25 nm in diameter)

• hollow tubes made of repeating subunits (tubulin)

  • tubulin made of 2 smaller subunits (monomers) that carries a GTP molecule

    • alpha-tubulin

      • carries GTP

      • exposed at minus end

      • doesn’t hydrolyze GTP → GDP

    • beta-tubulin

      • hydrolyzes GTP → GDP

      • exposed at plus end

• have polarity → ends are different from each other

• undergoes dynamic instability

  • uses GTP as its energy source

  • constantly growing/shrinking

    • growing via addition of new monomers at the end of existing microtubule

    • addition of tubulin dimers is faster at plus (beta) end than the minus (alpha)

      • more growth on plus end vs the minus end

• if microtubule required for a long period of time in same place → must be stabilized

  • dynamic instability reduced or eliminated

  • gamma-tubulin acts as a cap that stops microtubule from being able to depolymerize from minus end

  • protein cap can be added to microtubule at plus end so that it can’t deploymerize

  • microtubules that are capped at both ends are stable

    • remain in place for as long as cells need them

• involved in mitosis in all cells, cargo trafficking, maintenance of cell shape

  • pathways where vesicles and cargo travel on

  • controls and maintains specific cellular shapes and location

  • provides sites to anchor complexes and proteins in specific regions of a membrane

  • cell controls conditions in order to promote growth, maintenance or shrinkage as required

Identify the different types of cytoskeletal elements on different type of micrographs

Discuss different types of intermediate filaments and correlate their structure with the strength of the assembled, functional cytoskeletal elements

-intermediate filaments

• only found in animal cells (vertebrates)

• within one filament → single kind of subunit

• keratin

  • protein found in epithelial cells and their derivatives

  • hold skin cells to each other and to underlying membrane

  • provides resistance against mechanical tension

• vimentin

  • found in intracellular fibers in connective tissue cells

    • including muscles

  • helps to hold tissues together

  • provides strength

• neurofilaments

  • found in neurons (expresses neurofilaments)

    • neurons have a distinct shape

  • helps to form and support characteristic shape of nerve cell

  • long axon projecting out one side of cell

    • longest axon in human body (sciatic nerve — base of spinal cord → big toe)

  • if neurofilaments or microtubules become disrupted → axon loses its shape, retracts and dies

    • dementia as a result

• lamins

  • components of nuclear lamina

    • in interphase, assembled nuclear lamins forms a lattice work under nuclear envelope

    • provides structure to nuclear envelope

    • aids with chromatin organization

  • nuclear lamina

    • maintains attachments to cytoskeleton on exterior of nucleus

    • during mitosis

      • destabilization of nuclear lamina (via phosphorylation of lamins) drives disassembly of nuclear envelope

    • after mitosis

      • lamins dephosphorylated and lamins reforms

      • since its attached to membrane and chromosomes → everything ends up back where it’s supposed to be

  • form a meshwork beneath nuclear envelope

  • maintain integrity of nuclear envelope

-all intermediate filament subunits undergo self-assembly to spontaneously form final filament

• energy not required

-amino acid sequence itself promotes formation of alpha-helix that makes up bulk of structure

• formation of first dimer facilitated by this process

-chemical analysis of alpha-helix shows that they have a line of non-polar amino acids on one side of alpha-helix

-central helical domain is highly conserved among the different intermediate filaments

• N & C terminal domains on either side of central domain can differ in size, sequence & function

• differences in N & C terminal allows for diversity of functionality amongst the 3 different intermediate filaments

-in order for skin to resist everything it gets put through

• cells need to be stronger than plasma membrane

• keratin (proteins) provides protection in skin

• keratin ropes pass from one side of cell to other and bind to desmosomes in plasma membrane

  • keratin + desmosome complex transmits mechanical stress along ropes instead of through membrane

-desmosomes & hemidesmosomes

• cell junctions that connect cells to each other or to the extracellular matrix

• anchored to intermediate filaments

• strengthen tissues

• desmosomes

  • connect adjacent cells together

    • cell-to-cell connection

  • provides mechanical strength to tissues under stress

  • binds to membrane layer under skin (hemidesmosome)

  • cadherins involved

    • adhesion molecules

• hemidesmosomes

  • attaches cells to extracellular matrix

    • cell-to-matrix connection

  • anchors epithelial layers

    • anchors cells to basal lamina

  • integrins involved

    • adhesion molecules

• diseases → mutations in intermediate filaments affect cell’s ability to resist tensile strength

  • epidermolysis bullosa (EB)

    • skin blisters and tears at the smallest touch

    • skin extremely fragile

6.2 Microtubules

Compare in vivo (in cells) and in vitro (in test tube) microtubule polymerization

-in vitro (test tubes)

• tubulin removed from cells to see how it works

• tubulin dimers have affinity for each other → over time → polymerize on their own into polymer

  • polymers form via non-covalent interactions

• initiating cytoskeleton polymerization (nucleation) to build microtubule or actin polymers is a slow process in vitro

• active tubulin dimers

  • GTP bound β-tubulin

  • higher affinity for microtubules

• inactive tubulin dimers

  • GDP bound β-tubulin

  • can bind to microtubules, but less efficiency than active tubulin dimers

• once bound to microtubule, over time GTP β-tubulin hydrolyzed into GDP β-tubulin

• kinetics of spontaneous microtubule assembly in vitro

  • no enzymes required

• rate of polymerization dependent on location

  • slow growing end — (-) end — higher critical concentration (Cc)

    • doesn’t easily bind β-tubulin in an incoming dimer → not the right conformation

    • needs more tubulin for it to grow

  • fast growing end — (+) end — lower critical concentration (Cc)

    • adding new subunits causes a conformational change in β-tubulin that increases binding for more subunits

    • doesn’t need as much tubulin for it to grow

• lag phase is slowest

  • initial nucleation dependent on molecules running into each other via random motion in aqueous solution

• if concentration of tubulin is low → hard for tubulin subunits to find each other in solution to start the process

  • when concentration is so low that process doesn’t start → lag phase only → no polymerization

• critical concentration (Cc) → concentration of tubulin required for polymerization to occur

  • minimum concentration of monomers required for polymer formation to begin

  • concentration of monomers that is maintained when the reaction is at an equilibrium

  • concentration of monomers where the microtubule switches from shrinkage to growth

  • growth rate = disassembly rate

• rate of assembly increases with increasing concentration of tubulin subunits in solution

  • difference in both critical concentration AND rate of assembly at plus and minus end

  • plus end starts growing more easily than minus end (grows at a faster rate)

• when bound as part of a microtubule

  • beta-subunit of tubulin dimer hydrolyzes its GTP

  • hydrolysis of GTP → GDP reduces affinity of binding of tubulin dimers to each other within microtubule

  • over time, affinity of tubulin dimers for each other decreases

• formation of GTP cap

  • encourages microtubule growth

  • microtubule assembly happening faster than rate of GTP hydrolysis

  • occurs when high enough [free tubulin] is available

  • 
    

    

• loss of GTP cap → rapid shrinkage of molecule

  • lower affinity

  • if polymerization slows down → GTP hydrolysis catches up and is now converted to GDP → GTP cap disappears

  • if there is high enough local concentration of activated tubulin → microtubule can be rescued and start growing again

• GTP hydrolysis changes subunit confirmation forcing the protofilament into a curved shape

  • destabilization of microtubules through GTP hydrolysis results in instability in microtubule structure

  • 
    

    

  • destabilization of microtubules via GTP hydrolysis results in unstable microtubule structure

  • 
    

    

    • catastrophe

      • when GDP tubulin units accumulates too much → microtubule undergoes catastrophe

-in vivo (cells)

• polymerization of microtubules influenced by environment

• local tubulin concentrations may go up or down as nearby microtubules grow and shrink

  • other kinds of proteins bind to both monomers and polymers to influence their behaviour

• microtubule organizing centers (MTOCs) in cells provide right conditions for rapid nucleation of microtubules

  • grow from centrosome

    • sites where microtubules growth is promoted via gamma-tubulin

  • gamma-tubulin

    • promotes microtubule growth

    • provides pre-made template for tubulin subunits to bind to

    • reduces lag phase of growth curve

    • controls exactly where microtubule is grown

    • side effect: bound to minus end of microtubule

      • minus end no longer free for growth or shrinkage

      • only plus end (beta) undergoing dynamic instability

  • microtubules can be stabilized via binding to proteins (microtubule associated protein — MAPs)

    • some MAPs bind microtubules to neighbouring microtubules but can still grow

    • establishes a network of microtubules going in the same direction

    • eventually microtubules can be capped (capping proteins) at the growing end by different types of MAPs

      • microtubules not growing or shrinking

  • specialized MTOC

    • spindle poles/fibers found in all cells during mitosis

    • cilia & flagella found in specialized cell types from different kingdoms

    • basal bodies are the MTOC

  • centralized MTOC

    • during mitosis → all eukaryotic cells have 2 centralized MTOCs (spindle poles)

      • coordinate proper placement of microtubules for separation of chromosomes

    • interphase → only some cell types have centralized MTOC

      • mammalian cells — centrosome (pair of centrioles and associated proteins

    • eukaryotic cells with cilia and/or flagella have additional MTOC (basal bodies)

      • located at base of each flagellum or cilium

  • decentralized MTOC

    • in plants and fungi → don’t have centralized MTOC in interphase

    • gamma-tubulin rings carried along pre-existing microtubules is motor proteins

    • during mitosis → cells collect all gamma-tubulin into 2 MTOCs

      • acts as spindle poles for mitotic spindle

• GTP-bound tubulin dimers are in activated form

  • assembles at a faster rate compared to GDP-bound tubulin

• GTP is in excess in cytosol

  • GDP-bound tubulin dimers that are bound in microtubules are reactivated by replacing GDP in beta-subunit with GTP

  • concentration of free-floating inactive tubulin is low in cell

    → only consider GTP-bound tubulin during microtubule assembly

Interpret in vitro microtubule polymerization curves in terms of microtubule dynamics

Explain the process of dynamic instability, focusing on how tubulin dimers (alpha/beta) interact non-covalently to make microtubules with a ‘plus’ and ‘minus’ end

-dynamic instability

• process where cells are constantly growing and shrinking

• crucial for cell shape, movement and division

  • moving cargo around

  • helps cells divide by pulling chromosomes apart

  • allows cells to change shape and move

-microtubules made of tubulin dimers

• alpha-tubulin

• beta-tubulin

-microtubules have 2 ends

• plus (+) end

  • grows and shrinks quickly

• minus (-) end

  • more stable

  • usually anchored inside cell at MTOC

-growing microtubules (polymerization)

• tubulin dimers add onto the plus end in a head-to-tail fashion

• each beta-tubulin carries GTP

  • when GTP-bound tubulin dimer attaches → stabilizes the growing microtubule

Interpret live-cell images of microtubules undergoing dynamic instability and explain the current model of how GTP binding to tubulin can bring about this dynamic behaviour

Explain how motor proteins work and how their movement relates to the polarity of the microtubule

-motor proteins

• able to bind to microtubule and move along it in a specific direction

  • energy required; ATP

  • microtubule has directionality (plus and minus end)

    

  • bind to microtubule in an orientation so that it moves cargo toward

    • minus → plus end (kinesins)

      • transports vesicles, organelles, proteins toward edges of cell

      • closer to cell

      • maintains structure of ER

    • plus → minus end (dyneins)

      • bringing cargo/materials back toward cell center/nucleus

      • moves structures (cilia, flagella)

      • farther from cell

      • maintains structure of Golgi

      → kinesis & dyneins are protein complexes with multiple chains

      • globular heads that attach to microtubule

      • tail region attached to cargo

      • two light chains part of molecule

        • located away from region of molecule that interacts with microtubule

      • powered by ATP

      • stepping cycline linked to ATP hydrolysis

• may transport

  • vesicles

  • proteins and/or protein complexes

  • protein-mRNA complexes

    • RNP particles

  • membrane vesicles and organelles

  • other microtubules

    • flagella, cilia, other motile microtubule arrays

Provide examples of how proteins can interact with microtubules or tubulin to influence their structure, which in turn, will influence function

6.3 Actin Filaments

-polymerization of g-actin requires energy

• g-actin subunit carries a single molecule of ATP in its core → hydrolyzed slowly once activated monomer gets added to polymer

• ATP-cap found when polymer is adding g-actin at high rate

• 
  

  

-dynamic instability

• in vitro (test tube)

  • plus end has higher affinity for monomers than minus end

    • lower critical concentration

    • increased rate of assembly once polymerization starts

• in vivo (cell)

  • cell has high concentration of ATP in cytosol

  • easy to keep unpolymerized g-actin in cell in activated states

Describe in vitro (in test tube) actin filament structure and polymerization and the role of monomer concentration and ATP in dynamic instability

-in vitro (test tube)

• actin monomers (G-actin) assemble into actin filaments (F-actin)

• kinetics of spontaneous actin filament assembly in vitro

  • same polymer assembly kinetics as microtubules

  • 
    

    

  • monomer accumulation → increases then plateaus → monomers being used to form filaments

  • filament starts low but as monomers start forming filaments → filament mass starts increasing

  • filament mass not present until actin concentration reaches point where monomer mass stops increasing and stays constant

    • filament mass increases linearly with actin concentration

  • critical concentration found at ______ LECTURE 32 MARCH 26th

Correlate in vivo (in cells) actin filament polymerization and organization at the cell cortex with its function of deforming the plasma membrane and regulating cell shape

-in vivo (cell)

• free actin is 50-200 um → cC is < 1 because proteins bind to free actin subunits

• f-actin uses nucleating sites in cell

  • no MTOC equivalent to organize actin like microtubules have

  • actin has to be nucleated at multiple sites

• various APBs control behaviour of F-actin polymers in cells

  • refer to top part about what APBs do

• ARP 2/3 complex nucleates branched actin arrays

  • attaches at minus end

• nucleases at 70° and branches (branching network/effect)

  • 
    

    

    • animal cells: actin filaments have different arrangements in different parts of the cell that performs different functions

    • formation of actin network at plasma membrane pushes membrane forward & allows cells to move

    • 
      

      

    • formation of actin network at plasma membrane pushes membrane forward allows cells to move

    • 
      

      

Relate how actin binding proteins influence actin filament polymerization and organization to regulate microfilament function (eg. cell motility)

-cellular locomotion

• ABPs required

• forming protrusion via formation of lamellipodium requires actin polymerization directly underneath plasma membrane

  • membrane at leading edge can be pushed forward

• actin must be depolymerized as membrane moves forward near rear of network

• ensures cell continues to have access to free actin for polymerization

• actin polymerization in cell cortex results in cell movement

• 
  

  

Interpret results from experiments using drugs that disrupt actin filaments and compare with equivalent experiments that disrupt microtubules

Explain the function of myosin motors and their role in contractile bundles as well as in other cellular contexts such as cytoplasmic streaming

Unit 7 Cell Cycle

7.1 Cell Cycle & Checkpoints

Review the overall organization of the cell cycle and its relation to cell division (mitosis and cytokinesis)

-cell cycle

• divided into 4 stages

• interphase consists of G1, S, G2

• discrete process

  • events that only occur at certain points in the cycle

• continuous process

  • occurs throughout the cell cycle

    eg. nutrient assimilation, cell growth

-gap 1 (G₁) phase

• gap between end of cytokinesis and start of DNA synthesis

• major period of cell growth → longest phase

• duplication of organelles, volume of cytoplasm increases

• cell gets bigger

• ends with G₁/S checkpoint

-synthesis (S) phase

• initiation and termination of DNA synthesis

• DNA replication → DNA doubled → sister chromatids formed

• S-cyclin/CDKs active

• ends at the end of replication

-gap 2 (G₂) phase

• second gap phase lasts from end of DNA synthesis to onset of mitosis

• cell doubles in size in preparation for mitosis

• S-cyclin/CDKs active

• ends with G₂/M checkpoint → commitment to divide

  • once cell enters mitosis → can’t go back

  • cell must be sure it can undergo mitosis properly

  • managed by M-CDKs

-mitosis (M) phase

• division occurs

• cell divides in 2

• division of parental cell in 2 genetically identical cells

• nuclear division which chromosomes condense & separate

• cytokinesis

  • cytoplasm division during which cell actively divides in 2

• M-CDK/M-cyclins active in first half of mitosis

  • to allow cells to exit mitosis

• M-cyclin targeted for destruction by anaphase promoting complex (APC)

-cell moves past G₁ into the next stage of cell cycle if and only if division is the eventual goal → cell must divide if they move to next phase

-growth & division requires a lot of resources → cell won’t unnecessarily enter into these cycles unless required

-there must be provision for cells to get out of cell cycle and into another developmental pathway (mating, meiosis, differentiation) or to arrest

• necessary for these cells to get from these alternate fates back into cell cycle

-controls placed on the progression of cell cycle where the cell is required to meet certain criteria before it’s allowed to proceed

• each checkpoint is controlled by gatekeeper protein that respond to cellular conditions → only allows cell to move forward into next phase of cell cycle if certain conditions are met

• checkpoints help cell to ensure certain conditions are met before cell cycle is allowed to continue

Use experimental evidence to distinguish what phase of the cell cycle that a cell is in (ie. G1, S, G2, M phase)

-chemically synchronizing cells in a population

• block stalls the cell cycle at a known location

• cells continue to process through cell cycle until they hit a block where they can go no further

-fluorescence activated cell sorting (FACs)

• used to measure DNA content of a cell

• cells stained with fluorescent dye upon binding DNA

  • amount of DNA is proportional to amount of fluorescent emitted

• using fluorescently labeled cell

• flow cytometer measures the fluorescence using a laser and separates a mixed population of cells

  • separates cells based on amount of fluorescence cell is emitting

• result is two-fold

  1. synchronized population of cells without chemical inhibitors or temperature-sensitive mutations → used for further experiments

     • G₁ phase = 1 full set of DNA

     • S phase = more than 1 set but less than 2 sets of DNA

       • replication started but not complete

       • in between peaks

     • G₂/M phase = 2 full sets of DNA

     • cells in both peaks have the same number of chromosomes → chromatids is different

  2. flow cytometer produces a graphical readout that summarizes how many cells were found with each amount of fluorescent material (DNA)

• [x-axis]: peaks represent separate groups of cells

  • peak 1: 1 full set of DNA (G₁ phase — after cytokinesis & before S phase)

  • peak 2: 2 full sets of DNA (after S phase & before cytokinesis → before cytokinesis since 2 full sets of DNA → if it’s after cytokinesis it would only be 1 full set of DNA)

• [y-axis]: size of peaks represent amount of cells in each grou

  • peak 1: more cells

  • peak 2: less cells

• ex (above): FACs readout if chemically stopped cells at the G₂ phase

• interpreting FACs readouts (frequency histogram)

  • X-axis measures amount of DNA

  • Y-axis represents number of cells counted at each point on the X-axis

  • because amount of DNA correlates to phase of cell cycle → determine how many cells are in each stage of cell cycle at the moment at which they were measured

• 
  

• second peak = replicated DNA

  • 2 phases that both have replicated DNA (G2 & M)

  • FACs can’t differentiate these → lumped together in graph

• dip/section in between the peaks is important

Explain the concept at checkpoint control of cell cycle and give examples of what types of conditions should be met before S and M phases proceed

-cell cycle checkpoints

• 2 checkpoints

  • G₁/S checkpoint

  • G₂/M checkpoint

• coordinates continuous processes (ie growth) with discrete processes (ie DNA replication) so that cell size is maintained

• cells don’t move through stages of cell cycle on their own → signal always required

• transition from one stage to next is controlled by proteins in cytoplasm

  • when no proteins present → cells stays in G₁

  • proteins required to turn on and promote cells to next stage

-G₁/S checkpoint

• transition from G₁ to S phase

• restricts cells from beginning DNA synthesis before cell is ready

• conditions that need to be met

  • nutrients (carbon source, energy source, Pi, S, N, vitamins) must be present at specific concentrations

  • chromatid separation (from previous mitosis) must be complete

  • no detectable DNA damage

  • cells must have reached a critical threshold size

  • external factors must be appropriate

-G₀ phase

• when cells aren’t ready to be divided → stalled in G₀ phase for extended periods of time

• cells remove themselves from cell cycle → arrested

  • senescent (permanent arrest)

    • cell to stay the same size and don’t divide

  • quiescent (temporary arrest)

• long term deprivation of nutrients or other resources required for cell division → G₀ phase allows cell to stay alive

-G₂/M checkpoint

• stops cell from entering mitosis before it’s ready

• commitment to division (cell is 100% ready to divide) → cell can’t stop the process of mitosis once it has started

  • checkpoint controls transition from G₂ to M phase

• conditions that need to be met

  • DNA replication must be complete

  • DNA must be undamaged

  • cell must have reached a certain minimum size

• cells can only move beyond checkpoint and into next stage of cell cycle when they have met required conditions

-xenopus cells demonstrated that something in cytoplasm controls entry into M phase

• MPF (positive regulator that makes mitosis happen) → found to contain a kinase (enzyme that phosphorylates proteins using ATP bound)

• MPF later became M-CDK (target protein involved in mitosis)

• (cyclin-dependent) kinases activates/inactives target proteins by phosphorylating them and causing a conformational change → regulates progression into mitosis phase

7.2 CDK-Cyclin Regulation

Describe how specific protein modifications (eg. phosphorylation and ubiquitination) result in activation/reactivation of cyclin-CDK complexes to regulate cell cycle checkpoints

-cyclin-CDK complex work together to control the cell cycle

• made of

  • cyclin (protein)

    • interacts with CDK

    • acts as a regulatory unit

  • cyclin-dependent kinase - CDK (protein)

    • regulates activity of a large number of other proteins required for cell cycle progression by phosphorylating them

    • only performs its function as a kinase when cyclin is bound

    • if cyclin removed from CDK → CDK inactivated

  • 
    

    

• phosphorylation controls cyclin-CDK enzymatic activity

  • cyclin binding to CDK not sufficient on its own

  • cyclin-CDK complex gets phosphorylated by other kinases

  • 
    

    

• ex: addition of phosphate group to M-cyclin by Wee1 (kinase/protein) → inhibition of complex

  • Wee1 responds to intracellular cues to ensure cell doesn’t go to G₂/M phase checkpoint until big enough to divide

  • after cell is big enough → Wee1 degraded → can’t phosphorylate M-CDK → CDC25 (protein) removes original phosphate from M-cyclin-CDK complex → activates complex

• ex: in yeast → Wee1 & CDC25 are key regulators of M-CDK

  • increasing gene dosage (gof) → increases enzyme concentration

  • decreasing gene dosage (lof) → decreases enzyme concentration

  • 
    

    

-anaphase promoting complex

• signal that pushes cell from metaphase → anaphase during mitosis

• large protein complex

• functions as E3 ubiquitin ligase

  • adds ubiquitin tags to proteins to tag them for destruction by proteasome

• triggers separation of sister chromatids

  • degrades cohesin proteins that binds sister chromatids together → chromatids pulled apart → anaphase begins

• helps exit mitosis

  • activates ubiquitin that tags M-cyclin for degradation → deactivates M-CDK

  • causes M-CDK activity to drop → cell exits cytosis → cell returns to low-CDK state in G₁

  • 
    

    

• blocking APC function blocks its ability to activate proteins used to degrade cohesins

  • checkpoint in mitosis

  • protein inactive (securin)

  • active enzyme (separase) → not functional until activated

    • cuts cohesins so sister chromatids can separate from metaphase → anaphase

  • 
    

    

Explain how the activation of the cyclin-CDK complexes results in the start of the next phase of the cell cycle. Use examples of M-CDK complex and S-CDK complex to explain this

-cycling of cyclin concentration and CDK activity allows the cell to progress through checkpoints and the cell cycle

-cyclin-CDK activity must hit threshold before checkpoint is passed

-S cyclin

• activated at start of S-phase

• directly induces DNA replication

• concentration of cyclins remains high through to M phase

• activity of CDK activated at one checkpoint and stays high throughout cycle

-M cyclins

• controls the G₂/M checkpoint

• active in first half of mitosis until destruction signalled by anaphase promoting complex (APC)

• activity of CDK decreases rapidly not long after checkpoint has been passed

-G₁ phase → S phase (G₁/S checkpoint)

• starts DNA replication

• controlled by S-cyclin-CDK2 complex

• forms toward the end of G1

• remains active right until start of M phase despite S-phase being long over before that

• once active → phosphorylates proteins involved in DNA replication so cells can enter S phase and begin DNA replication

• S-CDK phosphorylates proteins at origins of replication

• in order for cells to enter S phase (to replicate DNA) → must pass through G₁/S checkpoint

  • no DNA damage

  • p53 (transcription regulator) protein that checks for damage

    • when DNA damage detected → p53 becomes phosphorylated → stops degradation → binds to promoter sequence for CDK inhibitor (p21) → activates transcription and subsequent translation (blocks S-phase entry)

    • p21 blocks cyclin-CDK complex activity & S phase entry

    • 
      

      

• once checkpoint passes → G₁/S cyclin initiates S phase via phosphorylation of replication machinery

  • G₁/S cyclin degraded and S cyclin takes over

• S-cyclin-CDK complex activates DNA helicase → promotes assembly of replication machinery

• prevents replication from being able to happen more than once a cell cycle

  • via phosphorylating key enzymes in replication (Cdc6)

  • phosphorylation of Cdc6 → tagged for degradation → without Cdc6 → no replication

  • 
    

    

-chromosomes before replication

• single (double stranded) DNA molecule (chromatid)

-chromosomes after replication

• consist of 2 DNA molecules (sister chromatids)

  • connected at centromere (bound by cohesins - protein)

    • cohesins must be degraded prior to chromatid separation during anaphase

• complete with full complement of histones and any other proteins required for chromosome packing

-G₂ phase → M phase (G₂/M checkpoint)

• starts mitosis

• shuts down transcription & translation

• preparing organelles for separation → shut down

• completely rearranges cytoskeleton

• controlled by Mitotic-cyclin-CDK1 complex

  • cyclin accumulates during interphase

    • reaches maximum in mitosis then rapidly declines at the end of mitosis

  • forms in G₂ but stays inactive until its needed

  • inactive at first because of inhibitory phosphate added by Wee1 (kinase)

  • Cdc25 (phosphatase - enzyme) removes inhibitory phosphate to activate M-cyclin-CDK complex

    • controls when M-CDK becomes active via dephosphorylation

      • removing inhibitory phosphate → activates M-CDK complex

  • positive feedback loop

    • phosphorylates and activates more Cdc25

    • inhibits Wee1

    • mitosis becomes fast and irreversible

    • ex: Wee1 puts lock on; Cdc25 unlocks → once running → engine powers itself by activating more keys (Cdc25) and disabling more locks (Wee1)

  • 
    

    

    • inhibitory phosphates added by Wee1 (inhibitory kinase)

    • Cdc25 removes inhibiting phosphate to activate M-CDK

  • complex activation of CDKs allow cells to ensure activation occurs under ideal cellular conditions

    • once cyclin bound to CDK → phosphated added/removed based on current cellular conditions

    • when all checkpoint conditions met → correct arrangement of phosphate groups added/removed from CDK so that it becomes active

    • 
      

      

    • 
      

      

• once active → triggers chromosome condensation (chromosomes become visible), breaks down nuclear envelope, activates spindle apparatus to divide the chromosomes

• targets of activated M-cyclin-CDK complex (proteins phosphorylated by M-CDKs)

  • histone H1

    • changes in chromatin configuration → chromatin condensation

  • condensins

    • DNA binding proteins that binds to chromatins

    • aids with chromosome condensation

  • nuclear lamins

    • fall apart because phosphorylated lamins have lower affinity for each other

    • nuclear lamina disassembly → breaks up nuclear envelope into ER

  • nucleolus structural proteins (nucleolin)

    • dispersion of nucleolar proteins

    • nucleolus disintegrates

    • vital for chromosomal condensation

  • protein kinases that regulate the cytoskeleton

    • arrangement of cytoskeleton as the cell disassembles the interphase microtubule network and forms the mitotic spindle

  • Cdc5 - activating phosphatase

    • CDKs activate phosphatases by phosphorylating them

      • positive feedback loop - further activation of CDK

    • required for rapid rise in CDK activity after cell division commitment

  • anaphase promoting complex (APC)

    • degrades cohesin proteins that are keeping sister chromatids together → progeny cell chromosomes released

    • tags and deactivates M-CDK-cyclin

-cyclin-CDK complex deactivation

• transcription of cyclin genes shut down

• degrade cyclin proteins that already exist

• negative feedback loops built so CDKs can be shut down

• APC triggers ubiquitin to tag M-CDK for degradation by proteasome

  • cyclin concentration drops rapidly

  • cyclin degraded → CDK deactivated → next step of cell cycle begins

  • APC gets deactivated in G₁

Interpret experimental results of experiments on cyclin-CDK regulation

-fission yeast studies

• 2 lof mutants with different sized cells after division

• lof mutants

  • lof Cdc25 (knockout Cdc25)

    • results in big cells → Cdc25 mutants divide later than wild-type

    • wild-type allele of this gene advances cell into cell division

    • 
      

      

    • not as many activating phosphates added

    • when its added → cells are bigger → grows bigger than wild-type

  • lof Wee1 (knockout Wee1)

    • results in small cells → Wee1 mutants divide sooner than wild-type

    • wild-type allele of this gene slows entry into cell division

    • 
      

      

    • no inhibitory phosphates → G₂ shortened → cells smaller and doesn’t grow as big as wild-type

7.3 Mitosis and Cell Division

Review the stages of mitosis and relate how the process of mitosis is driven by the activation of proteins by the mitotic CDK-cyclin complex

-M phase consists of

• mitosis phase (nucleus division and its contents)

  • interphase

  • prophase

  • prometaphase

  • metaphase

  • anaphase

  • telophase

• cytokinesis phase (rest of the cytoplasm gets divided)

-prophase

• first stage of mitosis

• prophase entry triggered by M-CDK activation by Cdc25

  • cell has passed G₂/M checkpoint (M-CDK fully activated)

• M-CDK phosphorylates

  • histone H1

    • serine residues at the ends of H1 histones are phosphorylated by M-CDK

      • adds negative groups to positive groups

      • decreases strength of H1 ends’ interaction with DNA

      • results in strong interactions between H1 molecules → pulls them together

      • chromatin fiber condenses → becomes shorter and fatter

  • condensin

    • helps with further packaging of interphase chromatin fiber

    • holds sister chromatids together (center point)

  • MT-associated proteins

  • nucleolin

• for ^ those events to happen → 2 centralized MTOCs in cell must be produced

  • become the opposite poles of the spindle

  • 
    

    

• mitotic spindle formation

  • mitotic spindle formed by selective stabilization of interacting microtubules

    • dynamic instability at the free plus-ends

    • interpolar microtubules that are interacting with other microtubules

    • motor proteins and other microtubule-associated proteins (MAPs) help stabilize the microtubules

  • relies on dynamic instability of microtubules

  • microtubules grow outwards from spindle pole → search throughout cytosol for things to grab onto

    • if it doesn’t find what its looking for in the cytosol → will depolymerize

• nucleoli disappears

  • nucleolin

    • structural protein of the nucleous

      • M-CDK target

      • phosphorylation results in dispersion of the nucleolar proteins and disintegration of the nucleolus

-prometaphase

• nuclear envelope breaks down

  • broken down by phosphorylation of nuclear lamins via activated M-CDK

• nucleolus has disappeared

• chromosomes now fully condensed and kinetochores formed at centromeres

  • spindle poles free to associate with chromosomes

• by the end of

• mitotic spindle formation during prometaphase

  • made up of

    • astral microtubules (green)

      • microtubules anchored at the plasma membrane

    • kinetochore microtubules (blue)

      • form when they attach to a kinetochore during random growth and shrinkage in the cytoplasm

      • kinetochore

        • specialized region of chromosome associated with centromere

        • plaque or button shaped structure composed of proteins

        • microtubules become inserted into kinetochore on each side of chromosome

        • one kinetochore associated with each chromatid

          • faces in opposite directions on the 2 sides of chromosome

        • 
          

          

    • interpolar microtubules (red)

      • forms when 2 of the microtubules from opposite poles interact with each other

      • stabilizes each other via motor proteins

      • forms a bridge between 2 MTOCs

    • 
      

      

    • XKCM1 is necessary for proper microtubule depolymerization since microtubule depolymerization is required for proper spindle assembly

  • formed via selective stabilization of interacting microtubules — motors play important role

• kinetochore microtubules from both poles attach to sister chromatids

  • mitotic chromosomes can be pushed and pulled into place

    • kinetochore attachment via random growth/shrinkage of microtubules

    • 
      

      

-metaphase

• chromosomal spindle fibers form after nuclear envelope breaks down

• chromosomes aligned at equator of spindle/metaphase plate

  • after being simultaneously pulled toward both spindle poles by kinetochore microtubules

  • tension on microtubules and kinetochores provides even tension from both sides

• small amounts of GFP-tubulin added to cell undergoing mitosis → GDP-tubulin becomes incorporated into spindle

  • fluorescence moves toward spindle pole

    • even though chromosomal fiber length remains the same

  • plus end has polymerization occuring at the same rate as tubulin addition as the other end so they remain the same length

    • mitotic spindle constantly assembling and disassembling

    • 
      

      

-anaphase

• APC initiates anaphase

  • APC tags securin (regulatory protein) → releases separase (enzyme) → cleaves/degrades cohesins that allow sister chromatids to stay together → sister chromatids separate

• sister chromatids synchronously separate

  • separates at the same time with same tension

• each sister chromatid pulled toward opposite spindle pole → chromosome segregation

  • movement of kinetochores takes place via motor proteins (same motor proteins that aid with spindle assembly is now separating sister chromatids)

  • microtubules depolymerizing rapidly from plus ends

  • 2 forces work together to separate sister chromatids and move them to opposite sides

• kinetochore microtubules shortening (Anaphase A)

• interpolar microtubules growing, pushing poles apart (Anaphase B)

• 
  

  

• anaphase A

  • initial part of anaphase

  • chromosomes pulled poleward (centromere/kinetochore first)

  • kinetochore microtubules shorten & move chromosomes toward opposite poles

    • depolymerization of kinetochore microtubules required

    • motor proteins at kinetochore allow chromosomes to stay connected to shrinking microtubule

  • Taxol effect

    • microtubules still growing; stabilizes microtubules

    • prevents depolymerization of kinetochores

• anaphase B

  • poles pushed and pulled apart

  • interpolar microtubules lengthen

  • astral microtubules shorten

  • kinetochore microtubule length remains mostly constant

  • 3 separate forces

    • tubulin subunits added to plus ends of interpolar microtubules (longer tubulin subunits)

    • motor proteins move overlapping microtubules from 2 poles (longer spindle)

    • other motor proteins move astral microtubules (animal cells) against the cell cortex (peripheral cytoskeleton

      • shorter distance between boles of spindle and plasma membrane

  • Taxol effect

    • microtubules still growing; stabilizes microtubules

    • interpolar microtubules can still lengthen (but wouldn’t be able to disassemble once this phase passes); astral microtubules can’t depolymerize

-depolymerizing tubulin pulls things apart

• kinetochore-attached fibrils bind to chromosome & to protofilament (PF)

  • protofilament undergoing depolymerization at plus end

    • gives ability to be pulled

• protofilament bending will pull on fibrils & associated chromosome (attached via kinetochores)

• affinity for fibril to soluble tubulin is low and allows for tubulin dimer from bending protofilament to be released

• fibrils re-engage with protofilaments that are less bent or just about to bend, providing for a continuous pull

• 
  

  

-telophase

• division of nuclear content is complete

• nuclear envelope reassembles around each set of chromosomes - 2 nuclei

• spindle fibers disappear

• division of cytoplasm

• controlled by destruction of M-cyclin

• shut down mitosis

  • end of M phase → M-CDK needs to be shut down

    • APC inactivates M-CDK via activating ubiquitin to tag M-cyclin for destruction by proteasome

    • 
      

      

• prepare for entry into G₁

  • chromatin decondensation

  • reformation of nuclear lamina (and nuclear envelope) around chromosomes

    • centromeres and telomeres retain connection to nuclear lamina throughout cell cycle

  • reformation of nucleolus

  • disintegration of spindle and reformation of peripheral cytoskeleton

• new nuclear envelope re-assembles around each chromosome set

  • lamin proteins dephosphorylate

  • vesicles of nuclear membrane first bind to chromosome

  • nuclear pores associate

  • membrane fuse together to form nuclear envelope (inner and outer)

  • 
    

    

-cytokinesis

• cytoplasm divides in 2

• cell divides into 2 progeny cells

• cytokinesis in animal cells

  • division via myosin/actin contractile ring that pulls plasma membrane inwards → splits cells in 2

    • contractile ring always forms around mid-zone of mitotic spindle

  • actin & myosin filaments pinch cell to divide cell into 2 daughter cells

  • dividing cytoplasm also divides organelles

  • vesicles fuse rapidly with plasma membrane → ensure there is enough plasma membrane to fit each progeny cell

  • 
    

    

• cytokinesis in plant cells (watch recording from april 4th)

  • new cell wall must be produced between 2 daughter cells

  • Golgi must remain functional during cell division

    • secretes cell wall compounds (via vesicles) rapidly to center

    • actin plays a role ensuring vesicles are correctly targeted to cell plate

      • cell plate grows from center of cell towards outer walls → fuses with walls of dividing cell

  • new cell formation uses interpolar microtubules to help guide this process via formation of a phragmoplast in the center of the cell

  • phragmoplast complex

    • comprise of

      • microtubules

      • actin filaments

      • membrane components

      • other proteins

  • new progeny cells fully formed with full complement of organelles and DNA

    

Illustrate how the process of mitosis is driven by the activation of proteins by the mitotic CDK-cyclin complex

-M-CDK-cyclin complex

• condensin proteins

  • helps chromosomes tighten

• nuclear envelope proteins

  • breaks down nucleolus

• microtubule associated proteins

  • builds the mitotic spindle that pulls chromosome apart

• cohesin regulators

  • activates APC to regulate cohesin to drive M phase

Explain the role of the cytoskeleton (and associated motor proteins) during mitosis including how dynamic instability of microtubules contributes to the formation and function of the mitotic spindle, and the role of the actin cytoskeleton during cytokinesis

-cytoskeleton (microtubules — tubulin, actin filaments — actin)

• microtubules reorganize to form the mitotic spindle → pulls chromosomes apart

  • guided by centrosomes (MTOCs)

  • dynamic instability aids in formation

  • kinesins & dyneins (motor proteins) walk along microtubules, carrying cargo or sliding microtubules against each other

  • kinesins

    • moves toward plus end

    • push centrosomes apart

  • dyneins

    • moves toward minus end

    • pull spindle poles toward edges of cell

• actin filaments form a contractile ring around middle of cell

  • pinches cell in 2 during cytokinesis

Explain how proteins and DNA interact to further condense the DNA to a mitotic chromosome

Describe how the regulation of cyclin via ubiquitination results in the end of M-phase and a transition to the next phase of the cell cycle

• shut down mitosis

  • end of M phase → M-CDK needs to be shut down

    • APC inactivates M-CDK via activating ubiquitin to tag M-cyclin for destruction by proteasome