Cell Biology

For Professor Manor's BICD 110 Class

Last Eukaryote Common Ancestor

1.5-2 billion years ago

Hooke

Discovered (plant) cells

Leeuwenhoek

• Discovered

  • Microorganisms (protists, bacteria, etc.),

  • Red blood cells (saw nucleus)

• Contributed to cell theory by disproving spontaneous generation of cells

  • Observed sperm fertilization of oocyte

Schleiden

• Cell is the basic building block of life

  • Discovered that every part of a plant is made up of cells

• Cells made from a “crystallization” process

Schwann

• Contributed to cell theory

  • Both plants and animals are composed of cells

Virchow

• Formally proposed Cell Theory with three basic tenets:

  • All cells arise from pre-existing cells

  • The cell is the basic building block of life

  • All plants and animals are composed of cells

Palade

Saw subcellular components

Visible Light Spectrum

• Shorter Wavelength = Higher Energy

• Red: 650 nm

• Orange: 600 nm

• Yellow: 580 nm

• Green: 500 nm

• Blue: 450 nm

• Violet: 400 nm

Electron Microscopy: Fixation

• Use Glutaraldehyde or Formaldehyde to cross-link amino groups between molecules

• Embed tissue in paraffin

Electron Microscopy: Permeabilization

Non-Ionic detergent makes cells permeable to reagents (ie immunolabeling or antibodies)

Electron Microscopy: Scanning Electron Microscopy (SEM)

Surface of sample is metal-shadowed

Electron Microscopy: Transmission Electron Microscopy (TEM)

• Thin Samples: Stained/shadowed with heavy metals

• Thick Samples: Fixed, dehydrated, embedded in resin, sectioned, and stained with heavy metals

Fluorophores in GFP Live Microscopy

• Used to detect certain molecules and cells

  • Recombinant proteins are made by fusing FP genes (fluorescent protein) with gene of interest to produce recombinant fluorescent protein

• Fluorescent proteins used to conduct live cell imaging

  • Transfection/Transduction of cells leads to overexpression of a protein (ectopical protein)

Identification and Localization of Proteins via “Immuno-Labeling”

• Probe is covalently attached to antibodies that are encoded to specific protein of interest

• Inject animal with protein of interest with specific antigen to generate monoclonal antibodies from cell line

• Immunolabeled secondary antibodies are attracted to primary antibodies made by animal

Antibody Labels

• Stained with a “marker” attached to heavy metals, like gold or osmium, to create contrast

• Small fluorescent dyes that bind to membranes, DNA, or other structures

Chemistry of Life

• 4 Key Concepts:

  • Molecular Complementarity

  • Polymerization

  • Chemical Equilibrium

  • Energy

Energy

• Relative energies of covalent bonds and non-covalent interactions

  • Covalent bonds are stronger than non-covalent interactions

    • C=C > C-C > Hydrogen > Van der Waals > Thermal Energy

    • Weak (non-covalent) interactions are additive

The Hydrophobic Effect

Driven by need for higher entropy, since hydrophobic aggregation leads to a “cage” formation with water molecules

Amphipathic Molecules

• Compose membranes (phospholipid bilayer)

• Phospholipids:

  • Polar (hydrophilic) head: Glycerol, Phosphate, Polar Group

  • Hydrophobic Tail: Fatty Acyl Tails

• Semi-permeable

Melting Temperature of Lipids

Temperature where ~50% of lipids are fluid

Lipid Classes: Glycerophospholipids

• PC

• PS

• PE

• PI

• PG

Lipid Classes: Sphingolipids

SM

Lipid Classes: Sterols

Cholesterol

Homeoviscous Adaptation

• Cells dynamically change their membrane lipid composition to control membrane fluidity

• Decrease Transition Temp:

  • Unsaturated Bonds (kinks, defects in packing)

  • Shorter acyl chains

• Increase Transition Temp.:

  • Saturated Bonds (Straight Tails = Tighter Packing)

  • Longer acyl chains (more VdW interactions)

Protein Hierarchical Structure

• 5 Layers:

  • Primary Structure: Linear sequence of amino acids linked by peptide bonds

  • Secondary Structure: Local α-helices or β sheets

  • Tertiary Structure: Peptide three-dimensional shape

  • Quaternary Structure: Association between multipeptide complexes

  • Supramolecular Complexes: Can be very large, consisting of tens to thousands of subunits

Coiled-Coil Proteins

• Motif for α-helices

• Two α-helices wound around each other

• Have a heptad repeat w/ hydrophobic residues at positions 1 and 4

Anatomical Differences Between Plant and Animal Cells

• Animal Cells have Microvilli

  • Increase surface area for nutrient absorption

• Plant Cells have..

  • Vacuoles

  • Cell Walls

  • Chloroplasts

  • Plasmodesmata: For intercellular transport

Membrane-Enclosed Organelles

• # of lipid layers around membrane compartments differ

  • Monolayer: Lipid Droplets (LD) with lipids inside

  • Single Bilayer: ER, ER-Golgi Intermediate Compartments (ERGIC), Golgi, Plasma Membrane, Lysosomes, Peroxisomes, Trans-Golgi Network (TGN)

  • Double Bilayer: Mitochondria/Chloroplasts, Nucleus

Two Pathways for How the First Membranous Organelles Emerged

• Classical Endosymbiosis: Cell engulfs smaller prokaryotes/cyanobacteria

• Inside-Out Theory: Interactions between prokaryotes, slowly form endomembrane machinery that bond/bind them together

Inheritance of Membranous Organelles

• Stochastic Inheritance: Random sorting of organelles between daughter cells

  • Every cell stems from another (same is true for organelles)

Contribution of Individual Organelle to Total Cell Membrane Depends on the Cell Type

• ER and inner mitochondrial membrane represent 80% of the total cell membrane

  • Rough ER and Smooth ER

    • Liver: 35% and 16%

    • Pancreatic: 60% and <1%

  • Mitochondria Outer and Mitochondria Inner

    • Liver: 7% and 32%

    • Pancreatic: 4% and 17%

Endoplasmic Reticulum (ER)

• Morphology: Tubules on outside, sheets in inner area

• Function: Chief Operating Officer of the cell

  • Protein folding and post-translational control

  • Lipid Synthesis, Steroid Hormone Synthesis

  • Detox Center

  • Calcium Ion Storage

ER-Golgi Intermediate Compartment (ERGIC)

• Morphology: Tubular; has vesicular-tubular clusters, adjacent to ER exit sites

• Functions: Decides what goes to golgi

  • Sorting and concentration of biosynthetic and retrograde cargo

Golgi Apparatus (GA)

• Morphology: Cis-face towards nucleus (perinuclear region), trans-face where proteins move out of it (pancake stack structure)

• Function: Sorting hub

  • Lipid synthesis/transport

  • Remodeling of N-glycans, addition of O-Glycans

  • Lipidation of proteins

  • Stepwise modification of cargo

  • Individual stacks linked to Golgi

Trans-Golgi Network (TGN)

• Morphology: Vesicular-tubular structure at trans-face of Golgi, has dense core secretory granules

• Function: Sorting hub (golgi and endomembrane system)

  • Calcium-dependent sorting

  • Sphingolipid-enriched carriers

Plasma Membrane

• Morphology: Has microvilli protrusions (sense environment, produce forces), Fluid Mosaic (not just lipids, has proteins that undergo modification @ Golgi)

• Function: Physical barrier with selective permeability

  • Endocytosis: Regions of PM invaginate, coated vesicles pinch off; maturation from early into late endosomes

  • Phagocytosis: Uptake of large, insoluble extracellular material

  • Exocytosis

  • Cell Signaling: Proteins send signals due to environmental change

  • Transport of solutes/macromolecules

  • Interactions with neighboring cells

Endo-Lysosomal System (Endosomes)

• Morphology: Come from plasma membrane, network of constantly changing endosomes/lysosomes

• Functions: Molecular switches (GTPases) trigger endosome conversions

  • Directions of endocytic pathway

  • Interconnected network

  • Internalization and sorting of transmembrane proteins, receptors and ligands

  • Degradation in lysosome

  • Endocytosis/Phagocytosis

  • Autophagy: ER engulfs damaged organelles

  • Lysosomes: pH drop to 4.5-5 as they contain enzymes that degrade polymers

Nucleus

• Morphology: Have two bilayers, nuclear envelope supported by Lamins (polymer filament), with a nucleolus

• Function: Defines the eukaryotic cell (storage of blueprints)

  • Separates transcription from translation

  • Continuous with the ER

  • Nuclear pore complexes provide “good” access to nucleus

Mitochondria

• Morphology: Double bilayer

  • Very dynamic, can be long/tubulated or small/fragmented vesicles based on nutrient environment

• Function: Production of ATP, 2000/cell

  • Mitochondria have their own genome

  • Outer membrane: Large pores allow molecules to enter from cytosol

  • Intermembrane Space: Between inner/outer

  • Inner Membrane: Imaginations (“cristae”) drastically increase surface area where ATP production occurs primarily

  • Matrix: 2/3 of total proteins, oxidation of pyruvate and fatty acids

Lipid Droplets (LD)

• Morphology: Monolayer of phospholipids, highly regulated and dynamic organelles found in most cells

• Function: Energy Storage

  • Neutral lipids and cholesterol-esters

  • Biogenesis within ER bilayer

  • Size is dynamically controlled

Peroxisomes

• Morphology: Matrix-like granule, maybe born from mitochondria

• Function: Generating/scavenging of reactive oxygen species

  • Catabolism of long-chain lipids

  • Biosynthesis of special-membrane lipids (ether phospholipids)

  • Contribute to bile acid synthesis

Auto-Radiography of Proteins Separated with SDS Polyacrylamide-Gel Electrophoresis (SDS-PAGE)

• Denature proteins w/ ionic detergent

• Acrylamide acts as a “sieve,” proteins move towards anode when current is applied

• Smaller proteins migrate faster through the sieve than large proteins

• Detection of radioactivity by film exposure

Palade’s Pulse-Chase Experiment

• Hypothesis: Segregation step occurs at ER

• Radio-labeled cells with a short “pulse” of amino acid and see where newly synthesized proteins go

• Radio-labeled proteins moved from the ER to the Golgi and ended up in secretory granules at different timepoints!

  • Secretory Pathway!

Pulse-Chase Experiments w/ Purified ER membranes

• Secretory proteins enter the ER

  • Incubate for a brief time w/ radiolabeled amino acids

  • Homogenize cells from microsomes

  • Isolate microsomes with bound ribosomes

  • Treat purified microsomes with protease

• (-) detergent: Protein in ER are protected from digestion

• (+) detergent: Dissolves ER membrane, proteins not protected, digested

• Conclusion: Newly made proteins are inside the microsomes

Signal-Sequence Hypothesis

• Proposed by Guenter Blobel

• Proteins contain signal sequences that can direct them to the rER

Co-Translational Insertion of Secretory Proteins into the ER

• Signal Sequence (SS): Can be cut or cleaved off if short, or kept if longer (as an anchor)

  • Signal Peptidase: Cleaves Signal Sequence

• Signal Recognition Particle (SRP): A protein RNA complex that recognizes and binds the hydrophobic SS

  • Arrests protein synthesis

• Translocon + SRP Receptor: A pore complex that recognizes SRP

  • Translocon (Sec61): Preserves integrity of the ER membrane by three gating mechanisms

Translocon Sec61 Anatomy

• Hourglass-shaped channel

• (-) peptide: Channel closed by “plug” (gate 1)

• (+) peptide: Ring of isoleucine residues forms “gasket” (gate 2)

• Lateral Exit: For the signal sequence, and for (hydrophobic) transmembrane domains (gate 3)

Classes of Membrane Proteins: Type I

• Classified by orientation in the membrane

  • Type I: Single-spanning membrane protein

    • C-terminus faces cytosol with one cleaved signal sequence at N-Terminus

Classes of Membrane Proteins: Type II/III

• Type II/III: Single-spanning membrane protein

  • No N-Terminal SS

  • Single internal hydrophobic signal-anchor sequence

  • Positive inside rule: Flip orientation if signal-anchor sequence-flanking positively charged residues are mutated

Topogenic Sequences Determine the Orientation of ER Membrane Proteins

• Type II-IV: Positive inside (cytosol) rule

• Hydropathy Profiles: Predict likely topogenic sequences in integral membrane proteins

Folding of Proteins in the ER Lumen: BiP

• Chaperone protein that binds to exposed hydrophobic regions of the nascent chain and stabilizes them until the protein is folded correctly (aids protein folding in ER)

• Example: Hemagglutinin (Influenza A)

Folding of Proteins in the ER Lumen: CNX/CRT

• The Calnexin and Calreticulin Cycle: Quality Control

  • Bind to N-linked oligosaccharides add glucose to make Glc1Man9(GlcNAc)2

  • Retain protein in ER

  • Undergo Mannose Trimming to form Man5–6(GlcNAc)2

  • OS-9 binds and guides misfolded protein out of the ER and facilitates degradation by proteasomes

Protein Disulfide Isomerase (PDI)

Catalyzes disulfide bond formation

N-Glycosylation

• N-Glycans are added to proteins in the lumen of the ER

• Promote protein folding!

  • N-glycans are attached to asparagine residues

  • Consensus sequence Asn-X-Ser/Thr (N-X-S/T): N-Linked Glycosylation site

  • Pre-formed N-linked oligosaccharide is added to many proteins in the rER

  • N-Glycan precursor is synthesized at the ER membrane on a special lipid (dolichol-phosphate)

Synthesis of N-Glycan Precursor

• Steps 1-3: Two N-acetylglucosamine (GlcNAc) and five mannose residues are added on ER membrane cytosolic face

• Step 4: Dolichol pyrophosporyl intermediate is flipped

• Steps 5-6: Additional sugars added by ER enzymes to complete N-Glycan Precursor

Addition of N-Glycans to Target Proteins and Initial Processing

• Step 1: Glc3Man9(GlcNAc)2 precursor is transferred to asparagine residues in a tripeptide

• Step 2: Glucose residues are removed

• Step 3: Addition and removal cycles of Glucose residue (plays a role in protein folding)

• Step 4: Removal of one mannose residue

Secretory Proteins

• Fraction of total Proteome: 30% of all proteins are encoded in the genome

  • 50% of ER volume exported every 40 minutes

  • 90% of membrane leaving the ER is recycled

Ts Mutant Genes

• Novick and Schekman used budding yeast to create a library of Temperature-sensitive (Ts) mutant genes:

  • Permissive temperature (24°C): Protein folds normally

  • Restrictive temperature (37°C): Protein does not fold, function impaired

• Several mutant classes identified (secretory cargo stuck in different places of the cell)

Basic Components of Vesicular Transport

• “From a membrane - to a membrane (using a membrane carrier)”

  • From rER to ERGIC

• Requirements:

  • Machinery to attract coat

    • Can only form at specific sites, selects cargo

  • Coat proteins

    • Deform membrane, stabilize carrier

  • Uncoating of carrier

    • To allow for fusion

  • Machinery for fusion

    • Needs specificity for target membrane

    • Pulls carrier and target membrane together

Sar1

GTPase that regulates COPII coat assembly

COPII Vesicle Biogenesis (Anterograde)

• Cycle of GEF-activated GTP Binding and GAP-activated GTP hydrolysis control coat assembly/disassembly

• COPII carriers form at ER exit sites

• Steps:

  • Sar1-GDP interacts with ER membrane protein Sec12 → Sar1-GTP

  • Sar1-GTP recruits Sec23/Sec24 coat protein complex

    • Sec13/Sec31 complex finishes coat assembly

  • Sec23 GAP activity stimulates Sar1-GTP hydrolysis

  • Release of Sar1-GDP causes coat disassembly

Cytoplasmic Signals: COPII

• Di-Acidic Signals:

  • DXE

  • EXE

  • DXD

• Di-Hydrophobic Signals:

  • FF

  • YY

  • FY

ERGIC-53

• A lectin protein and Type I Transmembrane protein with a dihydrophobic motif

• Binds specifically to high-Mannose N-Glycans on cargo proteins made in the ER, clusters limnal cargo into COPII carriers

  • Binding depends on pH: Cargo released at low (acidic) pH at ERGIC

Rabs

GTPases that regulate vesicle targeting and fusion

Docking and Fusion of Transport Vesicles with Their Target Membrane (SNAREs)

• Rab1 protein on COPII vesicles interact with Rab effectors (rod-like golgin proteins) to melt through the “cocoon” around the ERGIC

• v-SNARE protein on vesicle forms coiled-coil interaction with the t-SNARE on the target membrane

  • SNARE 4-Helix Bundle: Zippers vesicle and target membranes together

• Disassembled by NSF ATPase

COPI Vesicle Biogenesis (Retrograde)

• Assembly and disassembly of COPI coats

• Steps:

  • Arf1-GDP interacts with p23/p24 membrane proteins

    • GBF1 GEF causes GTP exchange

  • Arf1-GTP recruits heptameric coatomer coat protein complex → Makes coat

  • ArfGAP activity stimulates Arf1 GTP hydrolysis

  • Release of Arf1-GDP from the vesicle membrane causes disassembly of the coat

Cytoplasmic Signals: COPI

• Basic Signals:

  • KKXX

  • K/HDEL

  • RXR

  • RRR

Organization of Golgi

• Cis → Medial → Trans

  • Perinuclear region → Plasma Membrane

Structure of Golgi

• Human cells have “ribbons” in Golgi; interconnected stacks

  • Flat Centers: Golgi enzymes/proteins

  • Fenestrated Zones (Curved Regions): Full of cargo, near transport

Transport from Cis to Trans-Golgi: Cisternal Maturation Model

• Golgi cisternae are dynamic compartments

  • “Mature” (convert) from cis to trans

  • COPI vesicles move enzymes to favor cisternae to convert into new cisterna

  • Cargo stays put

Transport from Cis to Trans-Golgi: Vesicular Transport Model

• Golgi cisternae are static compartments

  • Each cisternae has a specific set of resident enzymes, which stays put

  • Cargo is transported via tubular or vesicular carriers from one cisterna to the next

Processing of N-Glycans

Follows a specific sequence and occurs in different cisternae of the Golgi

Processing of N-Glycans: Cis-Golgi

Mannosidases (ManI and II) remove three mannose residues

Processing of N-Glycans: Medial-Golgi

• One N-acetylglucosamine (GlcNAc) added from UDP-GlcNAc

• Two more Mannose residues removed

• Two N-acetylglucosamine (GlcNAc) and one fucose residue added

Processing of N-Glycans: Trans-Golgi

• Add:

  • Three Galactose (Gal) residues

  • Three N-acetylneuraminic acid (NANA) residues

O-Glycosylation

• Synthesis in the Cis-Golgi

  • Glycans are added to the Hydroxyl group of Serine and Threonine

  • Linear Glycans

Temperature-Sensitive VSV G

• Jim Rothman’s Golgi Purification Experiment: Biochemically measuring protein transport kinetics

  • Restrictive Temp. (40°C): Misfolded protein retained in ER

  • Permissive Temp. (32°C): Protein folds and move through secretory pathway

    • Led to identification of Arf1 coatomer, the GTP dependency, etc.

Gradient of Lipids in the Secretory Pathway

• Membranes are not well packed in the ER, and the lipid concentration is dominated by PC (Phosphotidylcholine)

• Approaching Trans-Golgi, sphingomyelin, sphingolipids, and cholesterol increases (produced there)

  • Gives greater rigidity near plasma membrane

The Trans-Golgi Network (TGN): Sorting Station

• Five destinations

  • Retrograde transport of Golgi enzymes to the Trans-Golgi

  • Transport of lysosome enzymes directly to the lysosomes

  • Transport of lysosomal enzymes to late endosomes (exocytosis)

  • Constitutive Secretory Vesicles:

    • Transport constitutively secreted proteins and PM residents

    • Cargo proteins

  • Regulated Secretory Vesicles:

    • Store and process secreted proteins until signaled to fuse with the plasma membrane

Clathrin-Coated Vesicles (CCV)

• Bud from the trans-Golgi and PM

• Have Triskelion structure, 36 form the CCV

  • Each Triskelion has 3 heavy chains and 3 light chains

CCV Formation: Arf1 Recruits Adapter Protein (AP) Complexes

• GTP-bound, membrane-associated Arf1 recruits cytosolic AP-1 to TGN

  • Conformational change induced in AP-1

  • Exposes cargo sorting motifs

Adapter proteins (AP Complexes)

Help select cargo and recruit coat proteins

CCV Formation: AP Complexes Recruit Clathrin

• Intrinsic curvature bends membrane, forming bud

  • GTPase Dynamin pinches off the bud

    • GTP hydrolysis is required for this pinching to occur due to increased rigidity near plasma membrane

Dynamin

GTPase that mediates CCV fission

CCV Formation: Uncoating of CCVs by the Chaperone Hsc70 and Auxilin

Break down the coat

Hsc70 and Auxilin

Involved in clathrin coat disassembly

Mannose-6-Phosphate (M6P) Signal Sorts Soluble Lysosomal Enzymes from the TGN to the Lysosome

Luminal cargo directed to lysosome/endosomes

M6P: Step 1

• Cis-Golgi-localized N-acetylglucosamine (GlcNAc) Phosphotransferase

  • Recognizes Q-S/H-E-Y sequences in newly synthesized lysosomal enzymes

• Transfers a phosphorylated GlcNAc group to one or more mannose residues

N-Acetylglucosamine Phosphotransferase

Adds M6P to lysosomal enzymes

M6P: Step 2

• Phosphodiesterase removes the GlcNAc group

• Final Product: 6-phosphorylated mannose residues on the lysosomal enzyme

Phosphodiesterase

Removes GlcNAc from M6P precursor

M6P Receptor

• In TGN, it binds M6P-tagged lysosomal enzymes causing vesicle formation to transport cargo to lysosomes

• Drop in pH releases cargo

Proteolytic Processing of Proproteins at the TGN

• Constitutive Secretory Pathway:

  • Furin Endoprotease

    • Cleaves peptide at the C-Terminal end of two consecutive amino acids

• Regulated Secretory Pathway:

  • PC2 and PC3 Endoproteases

    • Cleave central region of insulin

  • Carboxypeptidase

    • Sequentially cleaves two C-Terminal basic amino acid residues

Furin Endoprotease

Cleaves proproteins in the constitutive secretory pathway

PC2 and PC3 Endoproteases

Cleave proproteins in the regulated secretory pathway

Carboxypeptidase

Removes C-Terminal basic amino acid residues from processed peptides

COPI and GOLPH3-Mediated Retrograde Transport of Golgi Residents

• Different “flavors” of COPI vesicles are made

  • GOLPH3: Cargo adaptor that confers sorting of Golgi-resident proteins into COPI vesicles

GOLPH3

Cargo adaptor for COPI vesicles

Ca2+-Dependent Sorting at the TGN

• Regulated Secretion: Chromogranin

• Constitutive Secretion: Cab45

  • Scaffold protein that clusters with cargo/clients selectively with exposure to Ca2+

Cab45

Calcium-binding protein involved in regulated secretion