Monday, April 8, 2019

Bio130

Review
- Animal cell specific
• ECM
• Lysosome
- Plant cell specific
• Vacuole
• Chloroplast
• Cell wall
- Found in both
• ER
• Nucleus
• Golgi
• Plasma membrane
• Peroxisome
• Mitochondria
- Cytoplasm- everything in the cell except the nucleus
- Cytosol- aqueous part of cell; no organelles but we have ribosomes and cytoskeleton
- Lumen- everything inside the organelle
- Functions of Membrane
• Compartmentalization
• Responds to external signalling
• Selectively permeable barrier
• Scaffold for biochemical reactions
• Transporting solutes

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• Cell-cell interactions
- Membrane bilayer- form 2 layers in water
• Amphipathic bc of phospholipids
- Hydrophobic tails
- Hydrophilic polar head groups
• Fluid mosaic model- fluid bc of lipid mobility and some proteins and mosaic bc its a
mix of different proteins and lipids
- Phosphoglycerides- type of phospholipid
- it has one polar head group and 2 tails- one is uns
- Glycolipids and glycproteins are found on the ectoplasmic leaflet
• Addition of sugar protects the membrane from harsh environments
- Types of Membrane proteins
• Integral/transmembrane- inserts itself into the membrane
- Ampipathic- hydrophilic part outside and hydrophobic part inside
• hydrophobic transmembrane domain- alpha helix- hydrophobic
- Single pass- one alpha helix
• Acts as a receptor (MULTIPASS CAN BE RECEPTORS TOO)
- Multipass- many alpha helices
• Acts as ion channels- undergo conformational changes to regulate
permeability

- Beta barrel- rolled beta sheet

• Found in bacteria mitochondrionria and chloroplasts
- Makes a pore
- Pore is made up of hydrophilic AAs
- rigid cant undergo conformation change
- Pore allows transport

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- Cell membrane proteins on ONE SIDE- i.e. cytosolic face- amphipathic alpha
helix

- How are membrane protein structures identified?

• Xray crystallography- identifies overall 3D structure
• Hydrophobicity plot- 20-30 hydrophobic AAs find the transmembrane
domain/helix of the protein
- Odd number- N and C on opposite side
- Even number- N and C on the same side
• Peripheral- associates with membrane proteins/lipids via weak non covalent
interactions

• Lipid anchored proteins

- GPI-anchored- Exoplasmic face
• made in ER lumen and ends up on cell surface- N terminal sequence
- Other lipid anchor like fatty acids and prenyl
• Found on cytosolic face
• enzymes add this
• How do we extract membrane proteins?
- Transmembrane/Integral membrane- embedded in the bilayer- need to
destroy bilayer

• Us detergent- one head and one tail

- Peripheral- not embedded- loosely associated- gentle extraction- doesn't
destroy lipid bilayer

• How would you study lateral diffusion of membrane proteins?

- FRAP- fluorescent recovery after photobleaching
• tells you how fast and how mobile these proteins are

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• Label proteins with fluorescent dye- then photo bleach a certain area (destroys
fluorescent molecules)- and then measure how long it takes for fluorescent
molecules to move into the photobleached area
- Electrochemical gradient= conc. gradient+ membrane potential (electrical gradient)
• Electrical gradient doesn't affect UNCHARGED molecules

- Transport proteins
• Channel proteins- ion channels- multipass
- only PASSIVE transport
• forms a hydrophilic pore across membrane
• Weak interactions between solute
• no conformational changes
• FASTEST TRANSPORT
- Two types
• Gated- requires a stimuli i.e. chemical or electrical signal to open the channel
- Voltage gated- change in voltage across membrane
- Mechanically gated- requires a mechanical stimuli/stress i.e. stretching
plasma membrane opens this
- Ligand gated- INTRACELLULAR- ions, nucleotides
- Ligand gated- EXTRACELLULAR- NTs
• Non gated- always open i.e. K leak channel
- TransportER/Carrier proteins
• Binds a specific solute strongly and undergoes conformational change to
transport molecule
- Passive
• Uniporter- 1 molecule passes through down its electrochem. gradient
- Direction of transport is REVERSIBLE depending on the gradient

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- I.e. GLUT transporter- takes glucose from blood lumen and dumps it into the
cytosol
- FACILITATED DIFFUSION- passive transport using a tranportER
- Active- needs ENERGY NOT ATP to move solutes AGAINST the gradient
• Cotransporter/ Secondary active transport
• USES MEMBRANE POTENTIAL TO CARRY THIS OUT
- Symporter: two molecules moving in same direction
• i.e. Na/Glucose symporter- glucose from extra and dumping it into the
intra
- Antiporter: two molecules moving in the opposite direction
• Na/H exchanger- regulates cytosolic pH- kicks H out of cytosol
• ATP drive pumps/ATPases/Pumps
- Needs ATP
• P type- pee themselves- phosphorylate themselves to transport molecules
- Na/K pump- maintains the Na gradient
• BOTH Na and K moved against gradient
- 3 Na out
- 2K in
• F/V type
- F- ATP synthase- uses H+ gradient to make ATP- not a transport
protein
- V- acidify the lumen!
• uses ATP to pump H from the cytosol (low) into lysosome (high)
• plant vacuole and lysosomes
• Light driven pump
- light energy used to move solute against its gradient
• Bacteria

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- Transport glucose form the intestinal lumen to the blood
• Na/K pump- lateral side- maintaining the Na gradient we need
• GLUT2 uniporter- basal side
- Cytosol of epithelial (high conc.) to the ecf/blood (low conc)
• Na/Glu symporter- apical side
- From lumen (low) to the epithelial cytosol (high)
- Tight junctions make sure proteins stay on the right side- regulate and restrict
this
- In a hepatocyte- liver cell- 50% of cell volume is occupied by the cystol
• Mitochondria—> 22%
- Centrosomes and nucleolus have no membranes
- Mitochondria chloroplasts (plastids)- post translational sorting
• Made completely in cytosol before getting sorted
• Mitochondria and plastids of chloro- protein has to stay UNFOLDED (Hsp70
Chaperon protein ensure this) so it can enter it

- How can we see path of newly made proteins? i.e. digestive enzymes
• Pulse chase exp
- Pulse cells with radioactive AA and follow their path
- Peroxisomes- long chain fatty acid degradation
- Actin filaments- need ATP to form
• Polar
• Smallest diameter
• made up of actin monomers that make up protofilaments
- 2 protofilaments twist into a helix
• Involved with crawling of neutrophils
- Disassembly and assembly- needs to be dynamic

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• Have myosin- pushes actin filaments closer together during muscle contraction-
plus end directed

• Treadmilling- to see this-pulse of radioactive actin monomers

• Cytokinesis
- Actin and myosin form the contractile ring
• disassemble at the start of mitosis
• Reform during anaphase
- Intermediate filaments
• Medium diameter
• made up intermediate filament proteins
• coiled coil dimer—> antiparallel tetramers that form rope like filaments
• not polar
• no motor proteins
• i.e keratin
- anchored at sites of cell-cell contact by desmosomes to give mechnical strength
• Desmoglein and desmocollin- cadherin family—> adherin proteins
• Plakoglobin and desmoplakin—> anchor proteins links adhesion protein to
intermediate filaments

• Plectin- anchor link cytoskeleton filaments to transmembrane proteins like BP180/

integrin

• Keratin in cell is linked to the laminin in ECM via integrin

- Microtubules- need GTP to form
• polar- plus end= beta and minus end= alpha
- minus end is stable
- plus is dynamic- addition=T FORM and subtraction= D FORM
• Largest diameter
• made up of tubular heterodimers- beta and alpha tubulin

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- 13 parallel protofilaments make up the hollow cylinder
- bonds between individual subunits- STRONGEST NONCOVALENT
- bonds between two heterodimers- weak
- bonds between protofilaments- weakest
• Form the mitotic spindle
• Also involved with moving vesicles
- Motor proteins- use ATP
• Dyneins- go towards minus- cell body
• KInesins- go towards plus- axon
• Microtubules also anchor organelles and the motor proteins POSITION
organelles
- How do we visualize these structures?
• Immunofluorescence
- Tight junctions
• Prevent extracellular environment from mixing
• Prevent membrane proteins from mixing
• Occludin and claudin- proteins that form the tight junctions
- Homotypic- occluding only interacts only with occluding- same thing fro claudin
- Tight junctions need adherent junctions to form properly
• Junctional complex- desmosomes adherent and tight
- Anchoring junctions
• Desmosmes- cell-cell anchoring
• Hemidesmosomes- cell to ECM anchoring
• Adherens- cell-cell anchoring

- Anchoring junctions and adherens- link cytoskeletons of neighbouring cells

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• Adhesion proteins- transmembrane proteins- internal signal sequence
- interacts with extracellular matrix- its like grabbing the other adhesion from
outside

• Anchor proteins- cystosolic p- interacts with the intracellular domain (holding onto
cytoskeleton on one side and the and links the adhesion protein to the cytoskelton

- Adheren junctions- link actins! homotypic junctions as well

• Cadherin protein (adherin protein)—> gets linked to actin by anchor proteins
- Gap junctions- channel forming junctions
• Connexin- subunit
• 6 connexions make up one connexon- hemichannel that is closed until it binds at
least one more
- homotypic channels
• increase in Ca closes the channel bc its membrane damage and to protect
metabolite leakage
- forms plaques
- Freeze fracture shows this
- allows CAMP, NTs, AAs and glucose to pass; less than 1000 daltons
- macromolecules proteins and nucleic acids dont pass
• Plants produce and deposit cell wall
- More rigid than ECM in animals
- Composed of
• Cellulose
• Pectin
- Desmotubule- continuous with ER- tubule of ER
- Annulus- ring of cytosol- 2 plant cells sharing cell membrane
- Plasmodesmata
• Junctions between cells

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• Cytoplasmic channels passes through cell walls
• kinda like gap junction
- cell-cell
- Collagen—> triple helix
- Fibronectin binds collagen and cell surface receptors
• Dimer linked by dilsufide bonds
• RGD- cell binding site
• Fibronectin + laminin= cell migration
• Laminin+integrin= cell organization
- Prophase
• Replicated chromosomes condense
• Mitotic spindle assembly
- Requires microtubule dynamics- assemble and disassemble
- Microtubule motor protein activity- move DNA out of the way to form spindle
properly

- Centrosome duplication—> S phase

- Bipolar spindle assembly starts in M phase- prophase
• Disassembly and reassembly
• Assembly- centrosome copied 1—>2 - Reassemble
- Centrosome MTOC
• A pair of centrioles
- Organized at right angles to each other
- Composed of 9 fibrils of 3 microtubules each (9+3)
- surrounded by pericentriolar material (protein cloud)
• this is where gamma tubulin ring complexes- y-TuRCs
• on the cloud not on microtubule

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• one centrosome- pair of centrioles
• Nuclear envelope breakdown- between prophase and pro metaphase
- Lamins- class of intermediate filaments that form a 2D lattice on the inner
membrane- forms nuclear lamina
- Lamins get phosphorylated using kinases- nuclear lamina disintegrates
- Prometaphase
• mitotic spindle assembly complete
• chromosomes attach to spindle microtubules
• chromosome movement begins
- Astral spindle- positions mitotic spindle
- Chromosomal/kintechore spindle- 20-30 MTs attach to kinetochore/centromere
- Polar spindle- spindles coming from both sides and intertwine
- Metaphase
• All chromosomes aligned on the metaphase plate
• microtubules from opp. poles attach to the kinetochores (at centromere)

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