BIO230 2nd half

Big questions — cancer

Cell polarization is essential for multicellular organisms

Polar = different at either end

• apical domain (top)

• basolateral domain (bottom and sides)

Polarized cells can:

• have different functions at different cell regions

• define inside vs outside

• transmit signals from one end to the other

How does membrane trafficking send different proteins to different domains?

1. exocytosis directly to target domain

2. exocytosis to ANY domain then endocytosis followed by recycling to target domain

Membrane trafficking determines where proteins end up

• some trafficking routes are polarized

• protein are organized at sorting stations

• different routes are balanced by retreival pathway

Constitutive secretion is the “default pathway”

• most cargo move through constitutive secretion

• no specific signal needed (ER still needed)

• cell membrane gets more lipids

• ALL eukaryotic cells

Regulated secretion can release material in response to a signal

• regulated secretion vesicles are fully-formed but do not fuse with plasma membrane until signal is recieved

• cell membrane also gets more lipids

• in specialized eukaryotic cells

Regulated secretion can deliver extra membrane material

For:

• cytokinesis

• phagocytosis

• plasma membrane repair

Secretion can release concentrated cargo

• clathrin-coated vesicles can return membrane back to golgi

• shrinks vesicle and makes cargo more concentrated

Endocytosed proteins have 3 options

1. recycling to same domain of plasma membrane

2. transcytosis to other domain (side) of plasma membrane

3. degradation in lysosome

Cholesterol uptake as an example of endocytosis

1. low density lipoprotein (LDL) with cholesterol attaches to LDL receptor on clathrin coat

2. uncoating of clathrin

3. fusion with early endosome, recycling of LDL receptors to plasma membrane

4. maturation into late endosome

5. arrival of hydrolytic enzymes

6. degradation of cholesterol

Three types of membrane changes during vesicle trafficking

1. forms from donor membrane into cytoplasm (extracellular or lumen)

2. fusion: vesicle merges with target membrane (extraceullar or lumen)

3. forms from donor membrane away from cytoplasm (extraceullar ONLY)

   • viruses or RNA/proteins

1) Vesicles coats are selective of cargo and specifically targeted

2) SNARE proteins help mediate vesicle fusion

• both t-SNARE and v-SNAREs required

• t-SNARE and v-SNARES must be on opposite membranes

3) ESCRT proteins can form vesicles away from cytsoplasm

• form away from cytoplasm into lumen or extracellular space

• vesicle formation machinery in cytoplasm

ESCRT proteins can form vesicles away from the cytoplasm

1. ESCRT-0 binds to signalling receptor with ligand attached (has multi-ubiquiation)

2. passes to ESCRT-1, passes to ESCRT-2

3. ESCRT-3 builds up, helps with budding

4. vesicle buds off with receptors into lumen or extracellular

Phosphoinositides “label” different membrane domains

• Different membrane domains and compartments contain different lipids

• Different types of phosphoinositides (PIPs) are found at different subcellular locations

All phosphoinositides (PIPs) share the same basic structure

Head group:

• inositol sugar

• phosphate group

Glycerophospholipid

• glycerol

• lipids

Phosphoinositides can be phosphorylated on the inositol sugar

Carbon attached to phosphate = carbon 1

Structural phosphate always part of PIPs

Count counterclockwise — carbons can be additionally phosphorylated

Phosphoinositides are named by their phosphorylated groups

PI(phosphorylation site positions)P(total number of phosphorylation sites

• PI(3,4)P₂

Phosphoinositides are interconverted by kinases & phosphatases

• specific kinases and phosphotases for specific positions adding/removing

• not every kinase and phosphotase exsists — have to reconvert

Different proteins bind to different PIPs

PI(4,5)P₂ targets clathrin coat assembly

Adaptor proteins bind PI(4,5)P₂, cargo, and clathrin

• helps the bnding of clathrin inner coat and cargo receptors

Rab GTPases are molecular switches that can direct vesicles

Rab GTPases can act like molecular switches

• GEF — exchanges GDP for GTP

• GAP — activates GTPase

Rabs and PIPs combine to give membranes different identities

1. Rab5-GDP —→ Rab5-GTP by Rab5GEF

2. Inserts into membrane

3. Recruits PI 3-kinase

4. Phosphorylates PI —→ PI(3)P

5. PI(3)P recruits Rab5GEF

6. Positive feedback loop

7. Activates tethering proteins

Rabs and SNAREs work together in vesicle targeting & fusion

Polarized cells often have polar cytoskeleton organization

• polar microtubules can transport vesicles and proteins to different ends of the cell

• polarized actin can define cell shape and behaviours

• intermediate filaments also contribute to cell polarity

The cytoskeleton undergoes dynamic rearrangements

Interphase crawling/migrating cell

• microtubules radiate from cell centre

• actin enriched at cell cortex

.

Mitosis

• microtubule form mitotic spindle

• actin at cell cortex disassembles

.

Cytokinesis

• microtubules keep cell components separate

• actin forms the contractile ring

Polar tubulin dimers form polar microtubules

• monomeric protein a-tubulin and b-tubulin form dimers

• tubulins can bind and hydrolyze GTP

• tubulin heterodimers assembles head-to-tail to polarized protofilaments

Polar tubulin dimers form polar microtubules

• a-tubulin is found at the minus-end

• b-tubulin defines the plus-end

• 13 protofilaments associate to form a hollow microtubule

Microtubules exist in a state of dynamic instability

Tubulin dimers hydrolyze GTP after a while

• T-form at end = growing (GTP cap)

• D-form at end = shrinking

Microtubules exist in a state of dynamic instability

Depolymerization is ~100X faster at an exposed D-from heterodimer

• growth with GTP-cap

• loss of GTP-cap (catastrophe)

• rapid shrinkage

• regain of GTP-cap (rescue)

• rapid growth with GTP-capped end

𝛾-tubulin helps to nucleate microtubules

• y-tubulin (gamma) interacts with a-tubulin at minus-end

• y-tubulin nucleates or stablizes the minus-end

• y-tubulin protects microtubules from depolymerization at the minus end

• plus-ends grow away from the nucleation site

Different patterns of nucleation can create microtubule patterns

In many animal cells, 𝛾-tubulin is found near centrioles

Microtubules growing from y-tubulin ring complexes of centrosome

• protein complex around the centrioles — pericentriolar meterial

• has nucleating sites (y-tubulin ring complexes)

In many plant cells 𝛾-tubulin is found on other microtubules

Branch off of other microtubules

Vesicles and organelles can be transported along microtubules

MAPs = microtubule-associated proteins

Kinesins: walk towards plus-end

Dyneins: move towards minus-end

• both can hold onto vesiles or organelles with (tail domain)

• walk with head domain

• both use ATP

Vesicles and organelles can be transported along microtubules — Tilapia example

• fish such as tilapia can change colour using microtubule motors

• dark: kinesins & dyneins compete for pigment-containing vesicles (melanosomes)

• light: kinesins are inhibited, vesicles moved to minus ends near centrioles

Polar actin monomers form polar actin filaments

• actin monomers are asymmetric (polar)

• actin monomers can bind and hydrolyze ATP — cuts after a while

• monomers assemble into polarized actin filaments, usually 2 strands twisted together

Actin filaments undergo treadmilling

• Soluble subunits in T-form

• Polymers mixture of T-form and D-form

• Depolymerization is MUCH faster at an exposed D-form monomer

• Rate of addition on plus end can equal depolymerization on the minus end = treadmilling

The ARP2/3 complex helps nucleate actin filaments

• ARP2 & ARP3 have very simila structure to actin monomers

• ARP2/3 complex nucleates the minus end of actin filaments and protects them from depolymerization

• Plus-ends grow away from ARP2/3 complex

• NPF activates ARP2/3

The ARP2/3 complex helps nucleate actin filaments

• ARP2/3 can nucleate actin filaments on pre-existing filaments

• Whole network can undergo treadmilling

• Proteins (e.g. cofilin) sever minus of early actin treadmills for growing end of network to release them from ARP2/3 (more to use)

• Proteins cap the plus ends to shape the network

• POLAR network — for cell crawling

Actin treadmilling and adhesions help cells crawl

• growing actin network pushes the cell leading edge forward

• actin and myosin contract to bring lagging edge forward

• has to grip on lamellipodium to move

Integrins anchor actin filaments to the extracellular matrix

• Integrin heterodimers directly bind extracellular matrix proteins

• Integrins indirectly interact with actin filaments

• Actin-integrin-extracellular matrix interactions can provide the adhesion necessary for cell migration

Actin & myosins can generate contractile forces

• There are many types of myosins

• Myosins motor domains use ATP hydrolysis for energy

• Myosins can hold onto vesicles or organelles with their other domain or myosins can help cells contract

Actin & myosins can generate contractile forces

• Some myosins can “walk” towards the plus end of actin filaments

• Actin and myosin work together to generate force (e.g. cell migration or muscle contraction)

Rho family small GTPases can influence actin organization

• Rho family GTPases can act like molecular switches

• Rho family GTPases (Rho, Rac, and Cdc42) can influence actin organization

• Actin organization affects cell shape, cell polarity, and cell behaviour

Activation of Rho family GTPases can dramatically change actin

Healthy cells have a balance of active Rho family members: Rho, Rac, and Cdc42

Over-activation of different Rho family members leads to different actin organization patterns

Actin organization can influence cell shape & behaviour

Rac-GTP activation dominates at the leading tip to explore and to push the cell forward

Rho-GTP activation dominates at the back to pull the back of the cell

Local cytoskeletal organization can define cell movement

1. Bacterium releases chemoattractant

2. Binds to receptor

3. Most receptor binding = most of amount RAC

4. Least receptor binding = most of amount of RHO

5. Mutual inhibition

Anterior vs Posterior vs Dorsal vs Ventral

Front vs back vs back vs belly

Cytoskeletal organization can define cell polarity — c.elegans

Symmetry breaking defines anterior vs posterior

• In C. elegans, sperm entry defines the posterior

• Triggers cytoskeleton polarization in the fertilized egg

  • actin filaments are organized by a gradient of Rho-GTP activity

  • microtubules organized by centrosomes near where the sperm entered