BICD 110 midterm 1

LECA

last eukaryote common ancestor

1.5-2 billion years ago

mechanistic cell biology

biochemistry, cell biology, molecular biology/genetics

golden age of cell bio as a prerequisite to understand disease

discovery of cells

problem of scale thru developing tech

electron microscope increased scale and resolution

Hooke

discovered plant cells

created compound microscope, 50x magnification into visible light (photons)

saw cells in cork micrograph drawing

Leeuwenhoek

discovered microorganisms (protists, bacteria), RB cells, cell theory

created single lens microscope, 200-300x magnification

saw mobile animacules in gum swabs

Schleiden

every part of plant is made up of cells

cells made from crystallization process

Schwann

plants and animals are composed of cells and their products

Virchow

1. all living organisms compared of one or more cells

2. cell is basic life unit

3. all cells arise from pre-existing cells

Brown and Flemming

using brown stains, they discovered nucleus, chromosomes, different stages of cell division

black reaction

staining in neurons allowed Golgi to find Golgi apparatus

modern light microscope

advanced optics, only limited by 100 nm resolution of light and advanced contrasting methods

DIC: differential interference contrast (destructive vs constructive interference of shifting light waves)

electron microscopy

superior resolution

much smaller wavelength than photons (2000 A) for high resolution

different types of electron microscopy

TEM, SEM, cryo-EM

TEM

transmission electron microscopy, shine electrons thru sample

thin samples: stained with heavy metals that refract electrons, metal binds to proteins and membranes

thick samples: fixed and dehydrated with heavy metals

SEM

scanning electron microscopy

surface of sample is metal shadowed

cryo-EM

cryogenic electron microscopy

unstained samples frozen, leads to non-crystalline ice

high sensitivity without metal, still measures direct e- interact

can also measure tomography of cells: 3D computer reconstructions

Palade

changed field of cell biology

thru his images of the cell that we’re still attempting to understand under mechanistic biology

different components of mechanistic cell bio?

biochemistry: modification, manufacture

cell bio: anatomy, behavior

molecular bio / genetics: inventory, hierarchy

identification and localization in organism of interest

some organisms (ex jellyfish) produce fluorescent proteins (FPs) that can be fused to gene of interest

prod recombinant fluorescent protein to be expressed in organism / cell line of interest

lower wavelength = more photon energy, purple on color spectrum

different strategies to deliver recombinant DNA to cells

transfection / transduction of cells results in expression of ectopical protein (out of place, ex: myosin w GFP)

over expression of protein (endogenous + exogenous protein ) (normal vs foreign to cell)

fluorescence microscopy

can visualize with GFPs where different compartments in a cell are spatially organized in their proteins

immunolabeling

use of antibodies to detect specific protein

1. generate antibodies to specific protein of interest: antibodies produced in response to infection

2. antibodies generated by injecting a model animal with antigen (interest) OR antibodies from a cell line (monoclonal antibodies) to be purified

immunolabeling process

1. immobilized antigen A of mouse

2. antigen injected in rabbit, produces primary antibody against antigen A, binds to protein of tissue

3. secondary antibodies, marker-coupled with GFP against rabbit antibodies, locates protein to amplify and visualize

4. brightens image to be visualized

parameters for immunolabeling

• fixed with fixative such as formaldehyde or glutaraldehyde, to cross-link amino groups onto molecules. tissue embedded in paraffin for sectioning

• permeabilized with non-ionic detergent to make plasma membrane permeable to reagents (ex: antibodies), dead tissue fixed in time

• stained with marker (FPs) to covalently attach to specific antibodies with heavy metals (like gold) that create contrast. small fluorescent dyes can also bind to membranes or DNA

confocal microscopy to improve resolution

increases resolution by filtering out focus light

capture thinner focal plane using a pin hole

allows a larger imaged volume

4 key concepts of chemistry

1. molecular complementarity: mcs are 3D and interact in space / chemically, + - reactions

2. polymerization: from nucleotides to macromolecule DNA

3. chemical equilibrium: reversible reactions, dif temps, form / break bonds

4. energy: high energy bonds in ATP

energies of covalent bonds and noncovalent interactions

bond strength / energy is energy required to break / make bonds

covalent bonds are stronger and stable than noncovalent interactions (C=C > van den waals)

multiple noncovalent interactions combine to form strong associations

weaker when bond energy is close to thermal E, K_B (van der waals, H bonds)

noncovalent interactions

additive to increase stability

binding disassociation constant K_d is a measure of affinity

conformational selectivity

induced fit: binding of one molecule changes conformation (3D structure) of other, increases molecular complementarity

K_d

low Kd means lower amount (concentration, M) for binding partner to form interaction

lower Kd = stronger interaction

hydrophobic effect

association depends on solvation, H2O

H2O is 70% of cell mass

driven by thermodynamics, increase entropy / disorder and decrease total surface area

macromolecules

chemical building blocks

universal building blocks polymerize / assemble to form essential macromolecules

goal is to understand how chemical properties give rise to macromolecule function

HOH present in all

lipids

amphipathic

hydrophilic head

hydrophobic fatty acyl tails

= makes phospholipid

proteins

monomers - amino acid

polymers - peptide bonds to form polypeptide

nucleic acids

nucleotides make nucleic acids with phosphodiester bonds

carbohydrates

monosaccharides and polysaccharides formed from glycosidic bonds

emergent properties of macromolecules

property that an individual subunit does not have, but arises from collective/complex system

RNA world theory

most ancient bio mc

starts from nucleotides to form a short to long oligomer pool

folding and emergence of ribozymes, catalytically active RNA

how to build a cell

step 1: creating a barrier

cell barrier functions

separates in and out (endogenous and exogenous)

interior biochemical environment can differ from the exterior

protection of endogenous macromolecules and processes from outside

allows to catalyze certain rxns that wouldn’t happen spontaneously

bilayer structure advantages

self assembly: driven by hydrophobic effect, 2 phospholipids tg

fluidity: lipids can move into bilayer plane

barrier is semi permeable

molecular shape of membranes

geometry matters

micelle: cone shape of molecule, packs into single layer

lipid bilayer: cylindrical, packs into bilayer

bilayer compartments

thermodynamically prone to form

energetically unfavorable: planar phospholipid bilayer has edges exposed to H2O

energetically favorable: sealed compartment of a barrier

bilayer fluidity

controlled by temperature

gel like to fluid like by adding heat

melting temperature Tm = temp at which 50% of lipids are fluid

fatty acid composition

control of bilayer fluidity

C=C bonds aren’t rotatable, cis bond (most naturally occurring) introduces rigid kink that prevents tight packing into bilayer

cis > trans via artificial desaturation processes can make trans fats

trans fats are unsaturated and more fluid, ways to modulate

membrane lipid diversity

three groups: phosholipids (phospholipid head group), glycolipids (sugar heads), sterols (cholesterol

know the names and general features

homeoviscous adaptation

cells dynamically change their membrane lipid composition to control membrane fluidity

VdW forces btwn lipid tails determine fluidity

decreasing transition temp: unsaturated bonds (kinks), shorter acyl chains (less tail interaction)

increasing transition temp: saturated bonds (straight tails = packing), longer acyl chains (more VdW interactions)

cholesterol

special fluidity regulator

lowers membrane permeability (tight acyl chain packing)

adjusts membrane fluidity:

• low temp - increase fluidity (prevents tight acyl chain packing, ex: decreasing butter solidifying)

• high temp - decreases fluidity (rigid structure, ex: increase butter melting)

both are present at all temps, but dominating in dif temp ranges

polymers of amino acids

unbranched polymers: out of 20 AA, R side chains

side chains determine properties

polypeptide: linear polymer has free amino end and carboxyl end

protein function: derived from 3D structure, AA sequence and intramolecular noncovalent interactions

special amino acids

cysteine, glycine, proline

cysteine

can form disulfide bonds

-SH SH- to -S-S-

glycine

only has a proton as a side chain

allows for tight packing within polypeptide and facilitates interactions

proline

cyclized side chain

introduces kinks that can undergo cis-trans isomerization

phosphorylation of AA side chains

most abundant post-translational protein modification in eukaryotes

kinases catalyze transfer of phosphoryl group to AA side chain from ATP

reverse rxn: dephosphorylation, phosphatases

major eukaryotic phosphorylation sites: Ser, Thr, Tyr with an OH

prokaryote phosphorylation: on His, Glu, Asp

functions: enz regulation, protein-protein interactions, signaling networks, cell cycle

acetyl lysine

epigenetic control

cytoskeleton dynamics

3-hydroxyproline

cross linking of collagens for stability

requires vitamin C

3-methylhistidine

molecular function largely unknown

urinary excretion index of muscle protein breakdown

gamma-carboxylglutamate

high affinity binding of Ca ions

enriches in GLA domains like coagulation factors

O-GlcNAc-threonine

placeholder for S/T phosphorylation sites

preference for intrinsically disordered domains with defects that impact cellular processes

proteins in human genome

20000 to 23000 protein encoding genes thru alternative splicing of mRNAs + post translational mods for distinct protein activities

protein hierarchical structure

primary: linear seq of AA linked by peptide bonds

secondary: local A helixes / beta sheets

tertiary: 3D peptide shape with a / B

quaternary: btwn multipeptide complexes

supramolecular complexes: very large, consisting of tens to hundreds of subunits

protein structure

protein seq specifies folding into secondary / tertiary structures that are functional units OR interact with other peptides to form quaternary units

intrinsically disordered proteins (w/o 3D structure) have conformational flexibilities for multiple functions

polypeptides with dissimilar seq can also fold into similar 3D-structures

homologous proteins evolved from a common ancestor

protein functions

structure: organizing genome, organelles, cytoplasm, protein, etc in 3D space

regulation: control protein activity

signaling: monitor enviro and transmit info

transport: move small mc and ions across membranes

enz activity

motors: generating force for movement, ex: myosin

a-helix

secondary structure

stable arrangements of polypeptide chain (held together by backbone H bonds)

3.6 AA per turn

R groups project outward from surface of helix

prolines can’t participate in H bonding, excluded in helix

B-sheet

three stranded B sheet: antiparallel stands with connecting loops stabilized by H bonds btwn O and H atoms of AA on dif strands

a carbon bond angles prod pleated polypeptide backbone contour

alternative R groups project above and below plane of sheet

parallel B strand sheet: same N-to-C strand orientations with connecting loops

coiled-coil motif

two-a helices wound around each other

a-helix-heptad repeat seq with hydrophobic residue at positions 1 and 4

ex: motor proteins are mediated by coiled-coil formation to form stable dimers

oil drop model of protein folding

in tertiary structures

unfolded protein is folded to bury hydrophobic residues to the core

expose hydrophilic residues to surrounding aqueous environment

mitofusion 2

quaternary structure

heterozygous disease caused by a dimer formed btwn normal and mutant structure

mutant structure dominates and inactivates

complexity of eukaryotic cells

much larger than prokaryotic cells, “bag of bags”

eukaryotic cells have endomembrane system comprised of membrane-enclosed compartments (=organelles)

strong diversification of cell function, needed in a specific enviro

cytosol

matrix / fluid around organelles

cytoplasm

matrix including the organelles EXCEPT the nucleus

animal cell membrane compartments

• ER (endoplasmic reticulum)

• ER-Golgi intermediate compartment (ERGIC)

• golgi apparatus

• trans-golgi network (TGN)

• plasma membrane (PM)

• endo-lysosomal system

• nucleus

• mitochondria

• lipid droplets (LD)

• peroxisomes

animals vs plant cells

• microvilli in animal cells increase SA for absorption of nutrients

• cell wall (made of cellulose) maintains cell shape and protects against stress

• vacuole (plant) maintains turgor osmotic pressure

• chloroplasts for photosynthesis

• plasmodesmata: for membrane transport, connect cytoplasms of adjacent plant cells

amount of lipid layers in membrane compartments

single bilayer: ER, ERGIC, golgi, TGN, PM, lysosomes, peroxisomes

two bilayers: mitochondria / chloroplasts, nucleus

monolayer: lipid droplets

classical endosymbiosis

possible evolutionary scenario for origin of eukaryotic cells

1. ancestral prokaryote

2. membrane tubules isolate / wrap around DNA with a single membrane

3. nuclear envelope around DNA + nucleus makes two bilayers

4. “outside in” model for mitochondria, mitochondria engulfed by outer membrane = two membranes

inside-out evolutionary theory

different components are exchanges over time, fuse into each other for a gradual growth of membrane structures

lumen (including ER) is topologically equivalent to endomembrane

archaeal cells (ancient) have blebs that trap bacterial symbionts (like mitochondria)

membrane donor to membrane acceptor compartment (inside-out theory)

single bilayer-enclosed organelles exchange contents

shapes: tubular, spherical, sheet-like

lumen (inside) is topologically identical to cell exterior (EC space) as part of a continuous, enclosed pathway separated from the cytoplasm

dynamic equilibrium: compartments are not static and depend on flux of material, specific seq of transport btwn compartments

organelle biogenesis and inheritance

stochastic and ordered inheritance

stochastic inheritance

every organelle comes from a parent organelle after random mixing / cell division

causes inheritance of dif compositions

in mitochondria, ER, golgi

ordered inheritance

occurs in chromosomes

nucleus

mitochondria dynamics

actin causes sorting during mitochondria division (fission - fusion)

actin waves (mixing) accelerates during mitosis, by comet tail formed during mitosis

• can also be done during parasitic invasion, similar machinery utilized for actin

mixing NOT required for symmetrical inheritance of mitochondrial mass

purpose of actin wave

comet tails shuffle mitochondrial positions within cytoplasm

unmixed: less homogenous mixture of damaged vs healthy mitochondria to pass to daughter cells

want to pass down healthy mito to future generations

total cell membrane percentages

ER = highest lipid composition, includes rough and smooth

inner mitochondrial membrane is 2nd

80% of total cell membrane

individual organelles to total cell volume

mitochondria has higher vol than ER

cytosol has highest (50-60%)

space between membrane organelles

in cytosol, extremely crowded

debated of whether there’s enough space for 70% of our cells to contain H2O

endoplasmic reticulum

“mother” of all organelles, touches and interacts with 97%

rough: perinuclear region, near Golgi for protein processing

smooth: exterior of ER, tubules

ER regulation of mitochondrial fission

mitochondria constriction facilitated by ER prior to division (fission)

ER-mediated actin interactions at ER-organelle contact sites

*ER also controls fission of organelles other than mitochondria

ER regulation of mitochondrial fusion

mito/ER associated actin accumulates during fusion of mitochondria

rough ER function

ribosomes (black densities discovered by Pallade)

> 10 mil ribosomes bound to rER bc of high EC space

sheets have a stacked appearance that secrete high lvls of protein - secretory pathway

near nucleus and Golgi

protein folding and post-translational mods (N-glycans, S-S bonds)

protein quality control

why are so many ribosomes attached to the ER?

lumen of the ER is topologically equivalent to the EC space

if a protein is made that needs to go to EC space, it is co-translationally inserted into the ER lumen and anchored

smooth ER function

evenly distributed thru cytoplasm

tubular networks with 3-way junctions

responsible for lipid synthesis: steroid hormone synthesis (adrenal cortex, endocrine glands)

cellular detox center

calcium-ion storage (ex: control contraction of muscle cells)

ERGIC morphology

ER-golgi intermediate compartment

ER exit sites (ERES) > ERGIC > Golgi stack (cis, medial, trans)

ERGIC is an intermediate between exit and Golgi as a filter to proofread proteins

ERGIC function

vesicular tubular clusters (VTC) that are adjacent to ERES for export from ER

100-200 ERGICs/cell

COPI and COPII

only 50% of ERES are adjacent to Golgi membranes, but most ERES have ERGICs

COPI

dependent for sorting of retrograde cargo from ERGIC

shipped back to ER

COPII

concentration / sorting of biosynthetic cargo

shipped toward Golgi

golgi apparatus morphology

cis face towards rER, trans face towards plasma membrane (PM)

present as ribbons in animals

golgi function

sorting hub (secretion out of cell, transport to other organelles)

enzymes are transmembrane proteins

lipid synthesis / transport

remodeling of N-glycans (tree-like) / adding O-glycans (linear)

lipidation of proteins as a post translational mod.

perinuclear region (adjacent to nucleus)

3-8 disc-like cisternae (diameter 1 um) stack to form ribbons of Golgi stack (100 stacks / cell) for distinct faces (cis medial trans) and stack polarity

• stepwise mod of cargo

• each cisternae has unique biochem comp / function

TGN function

trans-golgi network

sorting hub to receive cargo from endosomal system and golgi

• transmembrane proteins + soluble lysosomal hydrolases, clathrin coated vesicles

• large extracellular matrix proteins (ex: collagens)

• protein complexes like dense core secretory granules

• calcium-dependent sorting of sphingolipid-enriched carriers

plasma membrane morphology

membrane protrusions

most rigid membrane

contains transmembrane proteins (ex: glycoprotein facing EC fluid)

hydrophilic heads on the outside from lipid bilayer

filaments of cytoskeleton face cytoplasm

signal transduction thru proteins

plasma membrane function

physical barrier

selective permeability (impermeable to ions and polar mcs)

transport of solutes and macromolecules thru active transport and facilitated diffusion

endocytosis / phagocytosis / exocytosis

cell signaling

interactions w neighboring cells in tissues

fluid mosaic model: PM is a 2D liquid composed of discontinuous phospholipid bilayer with patches of embedded protein complexes (hence mosaic)

endo-lysosomal system morphology

endosomes can become lysosomes thru acidification, or can be recycled as multivesicular bodies

EML