Membrane_dynamics

Question 1

Three aspects of membrane dynamics

Answer 1

Membrane fusion, fission, and curvature

Question 2

Three classes of membrane fusion and an example of each

Answer 2

Cell-cell fusion (sperm-egg), host-pathogen (viral infection) and intracellular fusion (neurosecretion/exo-endo cytosis)

Question 3

Steps in membrane fusion (4)

Answer 3

1. Adhesion (targeting)
2. Stalk intermediate
3. Hemifusion
4. Fusion pore (opening)

Question 4

How are different stages of membrane fusion assayed? (2)

Answer 4

Content dye, lipid markers

Question 5

What dictates specificity during membrane fusion?

Answer 5

For viruses- HA1, binds to sialic acid on host cell

For vesicles - Rab GTPases, tethers, SNAREs

Question 6

Methods to study fusion

Answer 6

1. Mixing of fluorescent dyes

2.

Question 7

How is the repulsive force of the two bilayers overcome?

Answer 7

1. By the force of fusogenic proteins and their interacting factors (ie SNAREs and NSF/SM/SNAP)
2. Charge gradient?

Question 8

What is the nature of the hole that opens between the two bilayers?

Answer 8

Up for debate - whether it is formed by lipids only, lipids+SNAREs, more study needed

Question 9

How is membrane fusion regulated?

Answer 9

In neurosecretion - Ca and Ca binding proteins, activates docked synaptic vesicles
Could also be fusion protein modification, synthesis or degradation, or protein targeting

Question 10

What defines influenza virus subtype?

Answer 10

HA fusion protein

Question 11

Is pH within endosomes very high or very low, or in between?

Answer 11

Very low

Question 12

What environmental factor triggers HA conformational change?

Answer 12

pH change, allows for extension of coiled coil structure

Question 13

HIV entry mechanism

Answer 13

Attaches to CD4, binds chemokine receptor, membrane insertion, conformational change, fusion, and entry of viral nucleocapsid

Question 14

Antiviral drug strategy for HIV (enfuvirtide)

Answer 14

Hairpin inhibitor that prevents conformational change of fusion proteins

Question 15

Two types of SNARES and an example of each

Answer 15

T-SNARES (target membrane)
- syntaxin
V-SNARES (vesicle)
- VAMP

Question 16

Proteins that help SNAREs drive membrane fusion

Answer 16

NSF (chaperone), SNAP (soluble NSF attachment, (SM)

Question 17

What structure do SNAREs form between vesicle and target membrane

Answer 17

stable 4-helix bundles (parallel coiled coil)

Question 18

How was it shown that SNAREs are sufficient to drive fusion?

Answer 18

reconstitute SNAREs into liposomes and examine fusion by de-quenching

Question 19

Issue with in-vitro SNARE reconstitution

Answer 19

Needed a super high concentration in order to do anything, which is how people identified a positive regulator of SNARE action - SM proteins

Question 20

Mechanism of SM action

Answer 20

Form stable complex with SNARE complex, push reaction forward to drive membrane fusion even at high concentration

Question 21

Difference between cis and trans-SNARE complexes

Answer 21

cis-SNARE conformation exists after membrane fusion, no force applied
trans-SNARE exert inward force in order to create pore

Question 22

Recycling factors for future rounds of membrane fusion

Answer 22

NSF / SNAP (? - double check this)

Question 23

hemifusion

Answer 23

lipid mixing but not content mixing

Question 24

Function of NSF chaperone and SNAP

Answer 24

disassembly of SNARE complex

Question 25

What are diff types of membrane division? (4)

Answer 25

Cell division, intracellular vesicles pinching off, organelle division, formation of multi-vesicular bodies

Question 26

Two classes of membrane fission mechanisms and an example of when you see them

Answer 26

Pinch from outside (dynamin-related GTPase driven during mito division) Pull from inside (actin/myosin filaments of contractile ring in cytokinesis)

Question 27

What is the role of dynamin in endocytosis

Answer 27

Assembles onto neck of fusion pore created by plasma membrane invaginations, squeezes (powered by GTPase) to release vesicle

Question 28

Does dynamin function as a "pinchase" or a "poppase"

Answer 28

who knows

Question 29

What protein group mediates division/fusion of mitos

Answer 29

Dynamin-related GTPases (DRPs)

Question 30

ESCRT

Answer 30

- membrane budding and fission complex, important with multi-vesicular body formation, cytokinesis, and viral budding - sufficient for formation of intraluminal vesciles

Question 31

Three ways in which vesicles can be formed

Answer 31

Filament assembly Protein conf change GTP hydrolysis

Question 32

Different phospholipid shapes largely driven by (3)

Answer 32

- Size of head group - Number of fatty acid chains - Shape of fatty acid chains (dependent on saturation state)

Question 33

Negative curvature dominated by what shaped lipids

Answer 33

cone-shaped

Question 34

Enzymes that can change shape of lipids

Answer 34

phospholipase (alter), acyl transferases (invert)

Question 35

Ways to curve a membrane (5)

Answer 35

1. Insert small protein in one side of membrane 2. Scaffold membrane 3. Lipid composition 4. Transmembrane proteins 5. Cytoskeleton