Lecture #6 - Membrane Dynamics

Question 1

What do membrane dynamics include

Answer 1

1. Membrane fusion
2. Membrane Division (Fission)
3. Membrane curvature

Question 2

Different types of membrane fusion

Answer 2

1. Cell-Cell fusion (Ex sperm-egg fusion + Skeletal muscle cell formation/repair + placenta synctium)
2. Host Pathogen interaction during viral infection (viral membrane fusion)
3. Intracellular fusion (Ex. nueroscreton + edocytosis/exocytosis + mitocondrial fusion)
   
   • Have regulated membrane fusion (Ex. nueroscretion)

Question 3

What are most biological membranes

Answer 3

Most biological membranes are bilayers (have outter leaflet and inner leaflet)

In membrane fusion – two membranes get close together and start to merge the membrane BUT have two types of merging

Question 4

Membrane fusion Process

Answer 4

Image - two synthetic liposomes with green in one and red other

Two types of merging - First have mixing of outer leaflet THEN have mixing of inner leaflet and eventually you create a fusion pore
- The membranes to come together in order to fuse
- First have mixing of lipids (vesicles mix membranes) BUT at this point the content is not mixed
- OLY when have fusion pore do you have mixing of contents

Can define distict steps in membrane fusion by looking at membrane and contents

Question 5

Steps in membrane fusion

Answer 5

1. Adhesion (taregting)
   
   • I THINK – vescles come close together and bend
2. B1 – Stalk intermediate
   
   • I THINK – outter leavflet lipids mix
3. B2 – Hemifusion –> lipid mix but not the content
   
   • I THINK – inner leaflet lipids mixes
4. C – fusion pore (opening) –> NOW have mixing of contents
   
   • Before only the mixed lipids in the membranes BUT the contents were separated but once have fusion pore you mix the contents

Question 6

Controling membrane fusion

Answer 6

Want to control membrane fusion/have specific membrane fusion because dont want random fusion

Example – virus recognizes specifc host cells or don;t want mitocondia to fuse to peroxisomes

Question 7

Key for membrane fusion

Answer 7

The key for membrane fusion is to force the bilayers together so that the lipids can interact with each other and merge

Have conformational changes of fusogenic proteins that help brings the bilayers together so that the lipids will interact
- Have different types of fusoenic proteins in different types of membrane fusion

Question 8

Liposome

Answer 8

Synthetic vesicle

Question 9

Methods to study fusion - Mixing of flouresnce dyes (in vitro)

Answer 9

Overall - Looks to see if lipids mix AND if content mix
- This is used to distinguish full fusion and hemifusion

Process – Have 2 types of liposomes (1 has nothing in it – is just membrane lipids ; 1 liposome has florurensct dye with red membrane dye and green content dye) –>
- IF fusion happens then you have mixing of contents and lipids –> IF have fusion – the green dye will be surrounded by red membrane
- IF you only fuse the lipids (mix lipids) BUT not the contents = have only red dye in membrane–> means have hemifusion (see two vesicles that are attached with a red ring (both have red ring but one has green dye and one has no color)

Question 10

Hemifusion

Answer 10

Have mixing of lipids but not contents

Question 11

Methods to study fusion - dequencing of floruesnce

Answer 11

More qunatitative + more used + simple
- Use – measure membrane fusion events based on flouresnece
- Con – can’t distinguish if the contents are fused because there is no content dye

Have 2 types have flourescetly labled lipids –> makes a flourencent liposome:
1. NBD-PE with green dye attached to PE (lipid dye)
2. Rhodamine-PC with red dye

Using FRET – Know if green and red are close if you excite the green and get floruensce form the red because the green gave the energy to the red (Flourence from green is tranfsered to red dye)

Question 12

Methods to study fusion - dequencing of floruesnce Process

Answer 12

Process – make a flourence liposomes with labeled lipds and a non-floruencent liposome and mix the liposomes

IF the green and red lipids are close -When exite the green dye you get flournece from the red lipid
- Green and red stay close together IF the lipids do not mix (If have no fusion = see more red (when ecxite green it passes the energy to red and excutes red because green and red are close together)

If green and red are far apart (have fusion) = get less red florusnece and more green florusnece
- IF the vesciles mix and fuse then increase the distane between the two lipids (and therefor the distance between teh dyes) = efficeint of FRET is lower = at first se red dye but then see less red floruesnce and increase in green floruensce

Question 13

Floresinece before and after quenching in FRET

Answer 13

Quenched (low flournece) before fusion and de quench (high flourence) after membrane fusion
- Quenched = low florunce at the start (Eitehr the are quenching or because only have red floruence because green and red are close)
- Dequench = high flournece (either because allowed for flrounce OT becasuse now see green because gfreen and red are far apart

Question 14

Method to study fusion - TIRF

Answer 14

Can see exocytosis in live cells + Use – Can count invidual membrane fusion process

Process – put cells in microscope ; cells have vesicles that are labled with YFP –> look at the bottom of the cells using TIRF –> can see individual exocytic vesiclee (over time see fusion events)
- Fusion of vesicles = detected by flashes
- Flashes = fusion of vesciles with the plasma mebrane (get flash because when the vescile fuses with the plasma membrane the because dye diffuses out)

Question 15

Exocytosis

Answer 15

Exocytosis = process small vesicles fuse with the plasma membrane (Ex. Insulin secretion)

Question 16

Method to study fusion - In vivo fusion assays

Answer 16

Example - mitocondrial fusion (can look in live cell)

Process:
1. 1 - Make two types of cells
- One cell expresses GFP in the mitocondria matrix ; One cell expressed RFP in the mitochondria matrix
2. Chemically fuse the cells using PEG (drive cell to cell fusion)

IF the cells fuse and mitocindiral fuses then mitocidniral contents are mixed = see yellow mitochondria

IF the cells don’t fuse mitocondria = see green and red seperately

Question 17

How do you get mitocondria to express GFP in the matrix

Answer 17

We know the targeting signal to drive a protein into the matrix of the mitcondria (can add the signal to GFP to drive GFP expression in the matrix of mitocondria)

Question 18

Method to study fusion - In vitro fusion assay

Answer 18

Fusion of isolated mitochondria

Use – Separates outer and inner membrane fusion

Process – use subceular fractionaton to isolate mitochondria from both cells –> mix the mitocondira together in vitro
- Have two types of mitocondira (Cell that has mitocondria express RFP in the matrix and BFP in the outer membrane ; Cell that has mitocindra that expressed GFP in the matrix and nothing in the membrane)

IF the mitocondria fuse –> Green and red mix = have yellow matrix and is surounded by blue outer mmebrane
IF only the outer membrane fused BUT not the inner membrane = have 2 separate matrix

Question 19

What dictates fusion specifcity during membrane fusion in viruses

Answer 19

Fir viruses – have specific interaction between the viral particle and the host receptor (each virus uses a different combination)

Examples:
1. Influenza HA1 viral proteins binds to surgar (sialic acids) on surface of host cell
2. HIV gp120 proetin binds to CD4 and chemokine recpetor on host cell
3. Sars-CoV2 stoke 1 protein binds to ACE2 on host cell to recognize specific target

Question 20

Differences in HA1 recognition

Answer 20

HA1 = not protein protein interacton have protein recognzing a sugar

Compared to HIV and Sars using protein-protein interaction (between viral protein and host receptor)

Question 21

What dictates fusion specifcity during membrane fusion in intracellular membrane fusion vesciles

Answer 21

For Intracellular membrane vesicles specificty uses Rab GTPases + tethers + Snares

Question 22

How are the two bilayers brought close together in viral enter - Overall

Answer 22

Flu –> first internalized to endocytic vesicle (endocytosis) THEN virus will fuse with endosome membrane

HIV/Sars DO NOT use endosomes –> instead fuse with the plasma membrane

For both Flu and HIV/Sars –> fusion drives putting the genome into the host cytoplasm where DNA replicaton machinery is available for viruses

Question 23

Key molecule for fusion in Flu

Answer 23

Key molecule for fusion = HA1 and HA2
- HA1 and HA2 = different proteins BUT they are coded for in 1 gene (single peptide gets cleaved during budding from parent host= get HA1 and HA2)

HA1 and HA2 form a protein complex that embeds into the viral membrane

Question 24

HA1 vs. HA2

Answer 24

HA1 and HA2 have different functions in membrane fusion:

HA1 = used for binding to host cell –> HA1 binds to sialic acids on host cell (specificty factor to identify the traget)

HA2 = drives fusion reaction –> HA2 is fusogenic because HA2 has fusion peptide domain
- HA2 has transmembrane domain and anchors to the viral membrane

Image – shows HA protein on viral particle

Question 25

HA2

Answer 25

HA2 - flu fusion protein HA2 has 3 domains: 1. Transmembrane segment/domain (integral membrane protein) 2. Coil-coil domain 3. Hidden fusion peptide (FP) - Fusion peptide is hidden at the beginning because you don’t want to virus to be randomly fused --> when activated the Fusion peptide is exposed to the surface

Question 26

Where are HA proteins + importance of location

Answer 26

HA proteins need to be on the surface of the viral particle HA is important for generating the vaccine because the protein is on the viral particle surface = makes it a good target for vaccines - Target the flu with vaccine that targets different types of HA proteins

Question 27

Membrane fusion in Flu

Answer 27

Flu recognizes Sialic acid on the surface of host cells/target cells and are internalized by endocytosis and then virus will fuse with the endocytic vescile to release viral contents HA1 acts at the plasma membrane for the virus to recognzie sialic acid on host cell THEN the vrius is inertnilzied in the endocytic vescile --> right away HA2 is still masked BUT Then it will be revealed after internilizaion when HA1 dissociates in the endosome so that HA2 can fuse the membranes

Question 28

HA2 mediated fusion

Answer 28

NOW the virus is internilized in cell BUT it is in endocytic vescile ; Viral membtane has HA2 When interilzied – HA2 is masked (fusion protein can’t reach the endosome membrane) --> change in pH stimuates the change in confirmation in HA protein --> confirmation change allows the fusion protein to extend and extend all the way to the host endocytic embrane --> insertion of fusion protein into the endosome mebrane induces a second confrimation chnage of the protein = get hairpin strcuture --> THIS creates force to bring the two membranes together so the membranes can fuse

Question 29

pH decreasing during interilziation of Flu

Answer 29

Have Fusion protein in the pocket first BUT then when expose to lower pH the fuson protein is extded ad can extend to the endosome membrane As endosome matures the pH continues to decreases --> Low pH = causes confirmation change that stimulates the extension of coil-coil domain = get straight shape = the fusion protein can insert into with the host endosome membrane

Question 30

2nd confimration chnage in HA2

Answer 30

insertion of fusion protein into the endosome mebrane indices a second confrimation chnage of the protein = get hairpin strcuture = creates force to bring the two membrane together so the membranes can fuse

Question 31

OVERAL FLU - just make sure know

Answer 31

HA1 recignizes sialic acid on surface of host cell --> virus undergoes endocytosis and enters a endocytic vescile --> After internilziatiion into the endocytic vesicle pH stiumlates confirmational chage in HA protein that extends the coil coil region and inserts the Fusion protein to the endosome membrane --> NOW have a bridge to the endosome membrane --> bridge incudes 2nd confirmation chnage to get hairpin structure --> hairpin brings the two membranes together closley = induces merger of the outer leaflet THEN the inner leaflet then fusion pore formation completes fusion (mix contents) - Have intermediate with hemifusion --> membranes are mixed BUT not the contents THEN have final viral fusion step

Question 32

HIV entry

Answer 32

HIV entry – fusion with plasma membrane (NO endocytic pathway)

Question 33

HIV recognition of host + fusion

Answer 33

Use gp120 and gp41 - Gp120 = specificty factor that recognize CD4 on host - Gp41 = fusio peptide 1 gene codes for both proteins --> peptide is cleaved into 2 peptides = forms protein complex on surface of viral particle

Question 34

HIV fusion process

Answer 34

gp120 binds to CD4 on host --> removes gp120 from the viral particle --> THIS exposes fusion petide gp41 --> gp41 insertes into the plasma membrane --> after insertion gp41 has confirmation chnage to form hairpin structure --> hairpin structure brings the viral membrane and host membrane together and drives membrane fusion - gp120 masks gp41 = when gp120 is removed you expose FP to Plasma membrane (NO confirmation chnage to get to membrane just reveal gp41)

Question 35

Proteins used in Sar-Cov2 entry

Answer 35

Spike 1 = confers specifcity Spike 2 = fusion factor Spike 1 and 2 = encoded in 1 gene

Question 36

Sars CoV2 process

Answer 36

Spike 1 recognizes ACE2 receptor on host membrane --> spike 1 gets removed once it binds to ACE2 --> removal of spike 1 exposes spike 2 fusion peptde --> spike 2 fusion peptide is inserted into the host membrane --> insertion in host mebrane causes confirmation chnage to hair pin structure --> hair pin dirves membrane fusion

Question 37

Fusion + vaccines

Answer 37

Fusion mechanism = important for vaccines Vaccine against Sars targets Spike1/2 complex before the confirmation chnage occurs (profusion state) - Antibody in vaccine holds spike 1/2 complex = can’t have confirmation chnage = can’t fuse with host membrane (blocks the first step in infection)

Question 38

Fusions in cells

Answer 38

Have exocytosis and endocytosis (SNAREs = used for endocytosis and exocytosis) Fusion in cells = used for constitutive membrane traficking and regulated traficking - Example of fusion in cells = nuerotransmitter release (regulated) Regulators of fusion = NSF + SNAP + SM protein

Question 39

SNAREs overall + Types of Snares

Answer 39

Snare = fusion proteins that drive membrane fusion - Used in exocytosis and Snare endocytic pathway (used for intracellular membrane fusion) - Function is similar to the viral fusion peptide Have 2 types of snare fusogenic protein: 1. V-snare (Vesicle snare on vescile) 2. T-Snare (traget membrane Snare - Ex. Located at PM for exoytsois because vesicle targets the plamsa membrane in exocytosis

Question 40

Snare complex

Answer 40

Snares = form 4 helix bundles (parallel coil-coil) between the Vescile membrane and the traget membrane V and T Snare intercat during fusion --> interaction makes coil-coil structure (mkae hairpin) --> hair pin brings the mebranes together and drives membrane fusion

Question 41

Snare Vs. viral fusion peptides

Answer 41

Snare and viral fusion peptides make hairpin structure and coil-coil structure Snare = makes coil-coil/ hairpin using 2 protein (V and T Snare have protein-protein interactions) Bacteria that use 1 protein to make hairpin/coil-coil structure Suggests a common mechanism for fusion proteins - in both the hairpin brings the membranes together to drive membrane fusion

Question 42

Experiment – Are Snares sufficinet to drive membrane fusion (in vitro study)

Answer 42

Overall – reconstitute Snares into liposomes and examine fusion by de-quenching (FRET experiment) Have 2 membrane dyes ; mkae vescile with green and red lipids and one vesicle with no floruencent lipids --> mix vesciles - One vesicle ALSO has T snare and the other vesicle has V snare Results - Showed that Snares are fusogenic protein - I you only have V snare on 1 vescile and T snare on the other = drivs membrane fusion of vesicles = shows the proteins are sufficinet to fuse membranes in vitro

Question 43

Issue in FRET Snare experiment

Answer 43

The experiment working required a high conetration of V snares and T snares on the vesciles (higher concetration compated to physiologic) --> thought only got fusion because used higher concenrration BUT this would not actually happen in vivo - Need lower concentration and have more fusion in cells because in cells have other factors

Question 44

SM proteins

Answer 44

SM proteins = increase the efficiency of Snare mediated fusion SM proteins bind to v-tsnare complex and help pull the two bilayers togther ('clasping') - SM stabilizes the Snare complex and helps the Snares drive membrane fusion efficiently

Question 45

Why did they need a high concetration of Snares in Vitro

Answer 45

Needed a high concentration of proteins in in vitro reaction because there is another protein that helps membrane fusion in cells ( I vitro did not have SM = needed a higehr concertaion of Snares to drive membrane fusion) OTHER protein = SM SM = not fusiogenic itself BUT it helps increase the efficiney of snares/membrane fusion Can use a lower concertation of Snare to drive membrane fusion in vitro when SM is added

Question 46

SM proteins and Snare in exocytosis

Answer 46

SM proteins and snare = important for exocytosis in cell IF you Knockdown or Knockout genes in cells = block exocytosis in cells

Question 47

Mechanism for SM proteins

Answer 47

V snare and T snare forms protein complex (NOT very stable) --> when add SM it binds to Snare complex and stabilizes it (makes it stronger) --> stabilization helps the recation move fowards instead of reversing back = get membrane fusion

Question 48

Cis Vs. Trans Snare complexes

Answer 48

Trans snare complex = complex before the membrane fuses - Seen in image (A) - V and T snare form complex to start fusing membrane BUT the membrane has not fused yet (proteins are on separate membranes) - Trans = exerts inward force Cis snare complex = after fusion the snare are on the same membrane and maintain the complex - Cis Snare complexes must be disassembled into individual Snare SU for the next rounds of fusion

Question 49

What drives membrane fusion intracelluar membrane fusion

Answer 49

Membrane fusion is driven by protein protein complex formation (energetically stabel) = hard to disaseble after the cis-snare complex once it is formed = cells need anotehr factor to disasselble - Use NSF and SNAP to disable the stable Cis-snare complex Solution - NSF and SNAP disassemble Cis snare complex after fusion - NSF = AAA + chaparone - SNAPE = soluble NSF attached protein

Question 50

Energy in Cis-Snare disassmbly

Answer 50

Need energy for process NSF ATPase = hydrolyzes ATP to generate energy to disassemble V snare and T snare - Membrane fusion itself does not need ATP BUT need ATP to disable the Snare complex NSF NOT fusogenic BUT it is important for rounds of membrane fusion - To have roudns of membrane fusion you need to release SU from the Cis-Snare complex

Question 51

Regulated membrane fusion

Answer 51

Example - Regulated fusion of secretory granuals in nerosecretion (synapses at the end of axons with snyamptic vesciles that contain nuerotransmitters) - Example of regulated exocytosis When have action potential --> AP stimulates nuerons to release nuerotransimitters via exocytosis Membrane fusion at synpase = faster membrane fusion copmared to other membrane fusion Uses calcium + Complexin + synaptagmain

Question 52

What regulates fusion in NT secretion

Answer 52

Calcium regulates secretroy graniuals in nuerosecretion

Question 53

How is membrane fusion faster at synapse

Answer 53

Have synpatic vesciles at nueronal synpase that are already attached/docked to plasma membrane - Synpatic vesciles are docked by forming loss trans snare complex (in synpase the snare complex is aready formed) Snare complex is formed BUT the membranes don’t fuse because you also have complexin - Complexin = inhibitory protein

Question 54

Why are the docked secretry vesciles not secreted in nuerons

Answer 54

Snare complex is formed BUT the membranes don’t fuse because you also have complexin - Complexin = inhibitory protein At synapse - Complexin binds to the trans snare complex and prevents the reaction from moving forward = Snare can't chnage the snare confirmation to get membrane fusion - Vesciles are docked and form trans snare complex BUT the vesicles are inhibited from moving foward with fusiion by complexin

Question 55

What stimulats the release of doscked vesciles at the synapse

Answer 55

WHEN the nuerons are stimulated (have AP) --> synaptagmain is stimulated - When have AP to stimulate nuerons – calcium from outseide the cell goes inside the cell and Ca binds to synaptagmin = activates synaptagmin When activated synaptamiagin binds complexin --> removes complexin --> NOW the preformed trans snare complex can move foward and drive membrane fusion and release neurotransmitters Because had docked vesicles with preformed trans snare complex = just need to have calcium to remove compelxin and drive membrane fusion = make fast Nuerotranismitter release (get exocytosis in nueorns)

Question 56

Other ways to regulate fusion

Answer 56

1. Fusion protein modifctaion 2. Fusion protein synethsis or degedation 3. Fusion protein localization

Question 57

Answer 57

Answer - C FOR snare assmeble protein complex assmebly is important + protein protein interaction is important ; ATP hydrilsysis is not used for membrane fusion but need to disaseble teh Snare complex

Question 58

Answer 58

Answer – B What to distiguish lipid mixing vs. Content fusion = need two reporters B vs. C - B has lipid dye to measure membrane mixing and content dye to measure matrix mixing - C – FRET based which only looks ay lipid mixing

Question 59

Types of membrane division (fission)

Answer 59

1. Cell division (cytokensis) 2. Pinching off of intraceular vesciles (Ex. During endocytosis) 3. Division of orgneleles (Ex. Mitochondria) 4. Fomration of multi-vescular bodies (used in endocytosis and lysosomes) 5. Pathogen budding (Ex viruses leaving)

Question 60

Two main ways to cut membranes

Answer 60

1. Pinch from outside to drive division (Ex. DRP1 in Mitocondria divison) - Division machinery goes to target --> makes division machinery on target --> squeeze and cut from the outside pushing towards the inside 2. Pull from inside (Ex. cytokensis making contractile ring) - Creates fission machinery inside the cell and pulls the plasma membrane and then cut cell

Question 61

Example of membrane division (fission)

Answer 61

Example - Endocytosis Endocytosis = process of making small vesicles from the plasma membrane Endocytosis = uses Dynamin

Question 62

Dynamin

Answer 62

Used in endocytic membrane division Dynamic = GTPase that cutting the membrane in the final stage so you can make seperate vesciles - Hydrolyses GTP to make energy to cut membranes

Question 63

How do you show that a protein is the main driver of a reaction

Answer 63

To show that a protein is the main dirver of a reaction - purify the protein --> reconstitute the process in vitro without any other protein to show that the protein is sufficient Knockdown/Kockout that shows if a protein is necessary

Question 64

Is dynamin sufficient to drive divsion process

Answer 64

People have purified Dynamin --> used purified Dynmain to show that Dynamin is a fission/division factor and is sufficient to drive the division Process - Purify dynamic and mix with liposomes (add dynamin to liposomes THEN add GTP) - When add dynamin --> liposomes make tubes THEN when add GTP to the liposomes make small vesciles - No GTP then lipsoome stays as a tubulate ; add GTP the dynamin causes tubule to fragment Shows Dynamin cuts the membrane into small pieces using GTP hydrolysis = dynamin is sufficient to drive membrane fission

Question 65

Dynamin shape

Answer 65

Dynamin forms spirals (olgiomers) around membrane tubules Dynamin = GTPase – has different cofirmations in GTP vs GDP state

Question 66

Dynamin Superfamily (dynamin related GTPAse)

Answer 66

Have many types of Dynamin that are invided in different types of membrane fission Ex – DRP1 = on the surface of mitochondria or peroxisomes --> cuts mitochondria or perozisomes into smaller structures

Question 67

Mitocondira fission

Answer 67

DRP1 assembles on the surface of mitocodnria --> DRP1 then uses GTP hydrolysis to cut the outer membrane and inner mebrane and make 2 mitochondria - Uses GTP hydrolysis just like Dynamin - DRP1 is recruited to mitocondira outer membrane bevause Mfn1 (dynamin receptor protein) is on the sruface of mitochondria (means protein-protein interaction makes specificity of membrane division/fission)

Question 68

Block Mitocondria division vs. Block fusion

Answer 68

Block division (KO DRP1) = get elongated mitocondria + decrease the amount of mitocondria because keep fusing without division Block fusion = then keep dividing and you keep making small mitochondria

Question 69

Membrane budding and fission by ESCRT (Endosomal sorting complex required for transport)

Answer 69

2nd mechanism for membrane division that is driven by ESCRT ESCRT functions in different fission reactions: 1. Cytokensis 2. MVB formation (NO Dyanamin ONLY ESCRT) 3. Viral budding to leave host membrane

Question 70

Multi-vesicular body (MVB)

Answer 70

Multi-vesicular body (MVB) = part of endocytosis and lysosomal degredation pathways - Have many vesicles inside the biger vesciles MVB = takes up cytosilic components into big vesciles and then fuses with the lysosome to degrade those components inside

Question 71

Is ESCRT sufficient to make intraluminal vesciles

Answer 71

ESCRT is sufficinet to drive fission for the formation of intraluminal vesciles (MVB) in vitro Isolated ESCRT complex --> mixed ESCRT with floruence labled vesciles - When add ESCRT the membrane is internalized and makes intrealuminal vesciles

Question 72

A model for ESCRT - proposed mechanism of ESCRT making MVB

Answer 72

ESCRT binds to surface of vesicles --> ESCRT assembles into snake like filaments (polymerization) --> polymerization push part of the membrane towards inside and eventually cut sand makes a Intraluminal vesicle Reaction does NOT need ATP BUT when finish reaction need ATP to disassemble ESCRT complex

Question 73

Mechansims for membrane division

Answer 73

1. Dynamin GTPase (Inlcude DRP1) - Used for endocytosis or mitocondria/peroxisiome division - Rely on dynamin.dynamin related GTpases (DO NOT need ESCRT) 2. ESCRT – MVB + Cytokensis + viral budding

Question 74

Answer 74

Answer – ALL of the above Different membrane fusions use different mechanisms: 1. Filemnet assmebley for DRP2 and ESCRT 2. Protein confimrstino hange in dynamin (dynamin chnages conformation after hydrolysis to cut the membrane) 3. Have GTP hydrolysis in DYnamin and DRP1

Question 75

Answer 75

Question is syaing that dynmain would not be needed in nuerons that are specilaied for nuerosecretion (exicystsis) because dynamin is used for endocytosis Answer – False - Because synpatic vesicles need to be recycled after fusion with Plasma membrane using endocytosis = dynamin is important for recycling of synaptic vesciles - Exocytiosis and endocytosis are almost always coupled

Question 76

Fission Vs. Fusion

Answer 76

Fussion = two membranes get close --> get intermediate fusion process with lipid mixing but not contents Fission = just reverse of fusion - Endocytosis coated pit --> then dynamin cutes the neck and then eventually get two membranes In fusion and fission pathays = see bending of the membrane (protein/ lipids regulate the bending process to increase efficiency of fission and fusion)

Question 77

Different phospholipid shapes

Answer 77

Combination of head group + acyl chain = determines the shape of the lipid On phospholipid you can chnage: 1. Size of head group 2. Number of fatty acid chains - Ex. removing acyl chain =makes inverted cone shape 3. Shape of fatty Acid chain - Ex. saturated fatty acid = staight tail Vs. Unsatutaed fatty acid (add double bond) = makes a kink (curved) tail (Unsaturated = has cone structure) Shape of Lipid = affects the curvature of the membrane that they are in

Question 78

Shapes of lipids

Answer 78

Shapes of lipids: 1. Cylinder 2. Inverted cone (Big head and small tail) 3. Cone (Small head and big tail) Exmaple: PC (major lipid in cells) = cylindrical shape PE = has smaller head = makes cone shape If have many PC = makes flat membrane Vs. If have many PE = make curved membrane

Question 79

Negative Vs. Positive Curve in membrane

Answer 79

Negative curvature = inside layer of lipids (cone shaped lipids) Positive side/positive curvatore = outside layer (inverted cone shape lipid) Lipid shape stabilizes intermediate structures in membrane fusion and fission

Question 80

Regulation of lipids

Answer 80

Regulation of lipids = enzymes control process (Enzymes control lipids = control fusion and fission events) 1. Phospholipase take up a chain from Phosphaatidly choline (PC) and make Liso-PC = make confirmation of lipid from cylinder to inverted cone 2. Add Acyle chain to lysophosphatadic acid = get phosphatic acid --> chnage the lipid shape from inverted cone to cone shape by adding unsaturated acyle chain

Question 81

Organelle membrane curvature

Answer 81

Organelles in cells have curvature: 1. ER = flat surface BUT at the end they have a curved region 2. Golgi = have flat part and a highly curved region

Question 82

Ways to curve a membrane

Answer 82

1. Helix insertion to bend membrane 2 Scafolfing 3. Lipid composition 4. Cytoskelaton affects the membrane curvature

Question 83

Examlple Scafold

Answer 83

Endophillin and other BAR domain proteins play a scaffold role in membrane curvatures - Bar domain = banana shaped domain curve If have protein with Bar domain attached to membrane then you curve the membrane because the protein shape is curved - Can assemble Bar domain proteins into oligomers and enhance the activity to curve membrane 1 type of endophilin is found at the neck of the endocytic coated pit --> helps with endocytosis when the membrane is cut

Question 84

ER Shape

Answer 84

RER (has ribosomes) = flat sheet structure Other part of ER = forms tubules (Tubules are dynamic --> interacts with other organelles such as mitocondria

Question 85

How do you make 2 morphologys in 1 organelle

Answer 85

Uses Reticulon and Sheet forming protein (ex. Climp63) Having both climp63 and reticulum = allows you to control the shape of ER - Change ratio between tubulue forming proteins and sheet proteins = chnages the ratio of tubule vs. Sheet

Question 86

Reticulon

Answer 86

Reticulon = wedge shaped protein located in the ER tubules - Curves to bend of membrane to make ER tubules When isolate reticulon and mix with membrane vescile --> they do make tubules similar to ER tubules Think that insert reticulon to the outer leaflet of the ER and the bends it - If have many reticulon proteins bind to each other and can make a frame of tubulues that supports tubulues structure

Question 87

ER sheet structure

Answer 87

Hypothesized that cell types with ER sheet structure (Ex. Acinar Cells) might express a sheet forming protein Did a proteomyic anlaysis to see proteins expressed in these cells with lots of sheet structures - Top hit = ribosome protein (because acinar cells make a lot of digestive enzymes) BUT the second hit was a sheet protein called climp63

Question 88

Climp63

Answer 88

Climp63 = flatens the membrane by using luminal bridge Climp63 proteins have luminal domain and transmembrane domain - Luminal domains bind to each other -> when bind to each other they bind to proteins on oppsote membrane = flattens the membrane and makes a ER sheet

Question 89

Answer 89

Need inverted cone Negative side has cone shape ; positive has inverted cone

Question 90

Answer 90

Answer - All of the Above

Question 91

Summary

Answer 91