Lecture #6 - Membrane Dynamics Flashcards
What do membrane dynamics include
- Membrane fusion
- Membrane Division (Fission)
- Membrane curvature
Different types of membrane fusion
- Cell-Cell fusion (Ex sperm-egg fusion + Skeletal muscle cell formation/repair + placenta synctium)
- Host Pathogen interaction during viral infection (viral membrane fusion)
- Intracellular fusion (Ex. nueroscreton + edocytosis/exocytosis + mitocondrial fusion)
- Have regulated membrane fusion (Ex. nueroscretion)
What are most biological membranes
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
Membrane fusion Process
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
Steps in membrane fusion
- Adhesion (taregting)
- I THINK – vescles come close together and bend
- B1 – Stalk intermediate
- I THINK – outter leavflet lipids mix
- B2 – Hemifusion –> lipid mix but not the content
- I THINK – inner leaflet lipids mixes
- 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
Controling membrane fusion
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
Key for membrane fusion
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
Liposome
Synthetic vesicle
Methods to study fusion - Mixing of flouresnce dyes (in vitro)
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)
Hemifusion
Have mixing of lipids but not contents
Methods to study fusion - dequencing of floruesnce
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)
Methods to study fusion - dequencing of floruesnce Process
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
Floresinece before and after quenching in FRET
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
Method to study fusion - TIRF
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)
Exocytosis
Exocytosis = process small vesicles fuse with the plasma membrane (Ex. Insulin secretion)
Method to study fusion - In vivo fusion assays
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
How do you get mitocondria to express GFP in the matrix
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)
Method to study fusion - In vitro fusion assay
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
What dictates fusion specifcity during membrane fusion in viruses
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
Differences in HA1 recognition
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)
What dictates fusion specifcity during membrane fusion in intracellular membrane fusion vesciles
For Intracellular membrane vesicles specificty uses Rab GTPases + tethers + Snares
How are the two bilayers brought close together in viral enter - Overall
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
Key molecule for fusion in Flu
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
HA1 vs. HA2
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
HA2
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
Where are HA proteins + importance of location
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
Membrane fusion in Flu
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
HA2 mediated fusion
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
pH decreasing during interilziation of Flu
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
2nd confimration chnage in HA2
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
OVERAL FLU - just make sure know
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
HIV entry
HIV entry – fusion with plasma membrane (NO endocytic pathway)
HIV recognition of host + fusion
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
HIV fusion process
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)
Proteins used in Sar-Cov2 entry
Spike 1 = confers specifcity
Spike 2 = fusion factor
Spike 1 and 2 = encoded in 1 gene
Sars CoV2 process
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
Fusion + vaccines
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)
Fusions in cells
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
SNAREs overall + Types of Snares
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
Snare complex
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
Snare Vs. viral fusion peptides
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
Experiment – Are Snares sufficinet to drive membrane fusion (in vitro study)
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
SM proteins
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
Mechanism for SM proteins
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
Cis Vs. Trans Snare complexes
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
Energy in Cis-Snare disassmbly
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
Regulated membrane fusion
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
How is membrane fusion faster at synapse
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
Why are the docked secretry vesciles not secreted in nuerons
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
What stimulats the release of doscked vesciles at the synapse
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)
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
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
Two main ways to cut membranes
- 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
- Pull from inside (Ex. cytokensis making contractile ring)
- Creates fission machinery inside the cell and pulls the plasma membrane and then cut cell
Example of membrane division (fission)
Example - Endocytosis
Endocytosis = process of making small vesicles from the plasma membrane
Endocytosis = uses Dynamin
Dynamin Superfamily (dynamin related GTPAse)
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
Mitocondira fission
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)
Membrane budding and fission by ESCRT (Endosomal sorting complex required for transport)
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
Multi-vesicular body (MVB)
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
Is ESCRT sufficient to make intraluminal vesciles
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
A model for ESCRT - proposed mechanism of ESCRT making MVB
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
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 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
Fission Vs. Fusion
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)
Shapes of lipids
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
Negative Vs. Positive Curve in membrane
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
Regulation of lipids
Regulation of lipids = enzymes control process (Enzymes control lipids = control fusion and fission events)
- Phospholipase take up a chain from Phosphaatidly choline (PC) and make Liso-PC = make confirmation of lipid from cylinder to inverted cone
- Add Acyle chain to lysophosphatadic acid = get phosphatic acid –> chnage the lipid shape from inverted cone to cone shape by adding unsaturated acyle chain
Organelle membrane curvature
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
Ways to curve a membrane
- Helix insertion to bend membrane
2 Scafolfing - Lipid composition
- Cytoskelaton affects the membrane curvature
Examlple Scafold
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
ER Shape
RER (has ribosomes) = flat sheet structure
Other part of ER = forms tubules (Tubules are dynamic –> interacts with other organelles such as mitocondria
Reticulon
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
Climp63
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
Need inverted cone
Negative side has cone shape ; positive has inverted cone
Answer - All of the Above
Summary
