Membrane Dynamics Flashcards
membrane fusion
- cell-cell fusion- first step of life in mammalian cells is fusion between oocyte and sperm, formation of skeletal muscle
- host-pathogen- interaction during viral infection
- intracellular fusion- fusion happening in cells during neurosecretion, exocytosis, and other organelles
influenza virus
viral particle is first recognized by host receptor proteins and particle gets internalized through endocytosis –> viral membrane fuses with endocytic membranes
intracellular fusion
-exocytic-endocytic vesicles
-neurosecretion- rapid and highly regulated
steps in membrane fusion
-2 vesicles- synthetic liposomes made by defined sets of membranes and lipids
-these will have red or green dye inside
-2 membranes get together and start merging
-distinguish 2 types of merging- biological membranes mostly have bilayers with outer and inner lipids- mixing of outer leaflet with inner leaflet
-eventually you create a fusion pore
-first you see mixing of lipids –> make fusion pores –> start mixing up contents
membrane fusion
mechanism to drive this can be boiled down to forcing 2 bilayers together –> lipids can interact with each other and merge
assay 1 (in vitro): mixing of fluorescent dyes
-lipid and content mixing
-2 types of liposomes: one with nothing in it and the other has fluorescent dyes inside content is green and lipids is red
-if fusion happens you can see the mixing of contents and the lipid
-if you fuse only lipid or membrane but not content (intermediate stage) or hemi-fusion, you can see mixing of lipids but not contents
–> both of have red dyes but only one has green
–> helps distinguish full vs hemi-fusion
assay 2: ‘dequenching’ of fluorescence
-2 types of fluorescently labelled lipids: one is NBD-PE (green dye) attached to a phosphatidylethanolamine and other has rhodamine-PC (red dye) that is conjugated to PC –> make fluorescent liposomes and mix them together
-depending on distance- if you hit green fluorescent dye and generate fluorescence, it is absorbed by red dye and you see red color
–> process is fluorescence resonance energy transfer (FRET)
-if you mix these vesicles together and fuse distance between these dyes or increase, efficiency of red goes down
Ex. initially you see red dye then gradually you see loss of red dye and increase in green fluorescence- measure the many fusion events by measuring fluorescence
-only measures lipid mixing
assay 3 (in vivo): TIRF (total internal reflection fluorescence microscopy)
-exocytosis- process where small vesicles fuse with plasma membrane
-you put cells under microscope and label each vesicle using fluorescence like YFP and then looking at the bottom of the cells using TIRF- under microscope you can see individual vesicles
assay 4: in vivo fusion assays (eg. mammalian mitochondrial fusion)
-2 types of cells: one expressing GFP in the mitochondria and the other using RFP in the mitochondria
-chemically fuse cells using polyethylene glycol (PEG) and drive cell-cell fusion
-you see mitochondria fusion and contents of matrix mix –> yellow mitochondria
-when cells do not mix mitochondria, you see red and green mitochondria separately
assay 5: in vitro fusion assays (fusion of isolated mitochondria)
-expressing RFP in the matrix and BFP in the outer membrane and other cells express GFP in the matrix but not membrane
-mix mitochondria together in vitro –> if they fuse, you see yellow mitochondria surrounded by blue
-if you can fuse outer membrane but not inner membrane, mitochondria carry separate matrices with one green and the other red
–> separates IM fusion from OM fusion
fusion specificity
-for viruses, there are specific interactions between viral protein and host receptor protein
–> in influenze, beta protein called HA1 recognizes sugar molecule on surface of host cell sialic acid
–> in HIV, protein-protein interaction between gp120 to host CD4
–> in SARS, stoke 1 proteins interaction with ACE2 host proteins
-for intercellular vesicles, different factors like Rab GTPases, tethers, and SNAREs
viral entry
-for influenza, first internalized in endosytic vesicles and fuse with them
-for HIV, they do not use endosomes and instead fuse directly with plasma membrane
–> in both cases, fusion drives putting their contents into host cytoplasm where the DNA replication machinery is available for viruses
influenza virus HA (hemagglutinin) protein
-hemagglutinin- fusion protein found on the surface of influenza viruses
-key molecule for fusion: H1 and H2 hemagglutinin protein- different proteins that encoded in single gene with a fusion peptide
-after translation, single peptides get cleaved to create H1 and H2
-look at the structures of H1 and H2, they form protein complexes and then embed into the viral membrane
–> distinct functions in membrane fusion- H1 binds the host cell (sialic acid)- specificity factor to ID target and H2 is fusagenic because it has fusion peptide to drive fusion reaction
influenza HA protein (fusion protein)
-3 important domains in HA2: transmembrane, coiled-coil, ‘hidden’ fusion peptide
-another way to look at the HA protein- transmembrane domain fusion peptide
-at the beginning, fusion peptide is hidden/masked but when it’s activated, it’s exposed to the surface
-in addition to fusion, HA proteins are important for us to generate specific vaccines since it’s exposed on the cell surface
membrane fusion
influena virus is recognized –> recognize sialic acid on the surface of host cell –> gets internalized by endocytosis
HA2 trimers mediate fusion (in endosomes after internalization)
-after internalization, H1 proteins recognize host cells and are removed so that H2 can fuse membrane but not yet
-when it’s internalized, fusion peptide is still masked
-during internalization, pH goes down from the neutral pH outside to lower pH in endocytic pathways
–> change in pH stimulate conformation of HA protein- lower pH stimulate extension of coiled-coil domain alpha helix and makes straight shape to help fusion peptide interact with host endocytic membrane
HA2 trimers mediate fusion (in endosomes after internalization) part 2
-you have fusion peptide in pocket first –> expose to low pH then that brings them together –> insertion into the membrane induces a 2nd conformational change of this protein
–> 2nd conformational change is to create force to bring 2 membranes together and induce merging of lipids
HIV entry
-don’t use endocytic pathways directly- they directly fuse with the plasma membrane
-instead of H1 and H2, they have gp120 and gp41
-one gene encode two proteins and after translation, cuts into two peptides to form protein complex on surface of viral particle
-gp120 is a specificity factor that recognizes CD4
-first interaction removes gp120 from complex viral particle and exposes fusion peptide to gp41 –> fusion peptide is masked by gp120 –> when it’s removed, expose fusion peptide and host plasma membrane and insert it
-second process step is very similar after insertion- gp41 forms hairpin structures and brings viral membrane and host membrane together to drive membrane fusion
SARS-CoV-2 entry
-you have two peptides: spike one and spike two encoded by the same gene –> cleaved after translation
-spike one is specificity factor and spike two is fusion factor
-ACE2 is receptor on host surface for SARS
-spike one recognizes ACE2 –> gets removed and exposes fusion peptide –> inserted into host membrane –> see conformational change with hairpin structures to drive membrane fusion
-vaccine against SARS targets spike 1-2 complex before conformational change occurs
what is the NEM-sensitive factor?
NSF
nomenclature
-SNAREs- fusion protein in exocytosis and endocytic pathways in cells
-SNAP- soluble NSF attachment protein
-NSF- regulators
SNAREs (fusagenic protein that drives membrane fusion) forms a stable 4-helix bundles (parallel coiled-coil) between vesicle and target membrane
-form hairpin-like structures like in viral fusion
-instead of single protein, SNAREs create hairpin structures using more than one subunit
-in intercellular membrane fusion, we have two types of SNAREs- tSNAREs and vSNAREs
tSNAREs and vSNAREs
-tSNAREs are located at the plasma membrane and vSNAREs are exocytic vesicles- tSNAREs and vSNAREs interact during fusion process to create cortical structures
-in intracellular fusion, we create hairpin structures by tSNAREs and vSNAREs
how are viral hairpins and SNAREpins generated?
-viral hairpin is made by single peptide
-SNAREpin generated by more than one protein through protein-protein interaction
-hairpin structures bring membranes together to drive membrane fusion
experiment: SNAREs are sufficient to drive fusion (in vitro study)
-FRET assay with two dyes and quenching after membrane fusion
-test the role of SNAREs you have to put tSNAREs in one vesicle and vSNAREs in another
-vSNAREs mimicking vesicle and tSNAREs mimicking plasma membrane then mix them together
–> showed that vSNAREs in only one vesicle and tSNAREs in the other you can drive the membrane fusion of vesicles- sufficient to drive membrane fusion
-concentration of tSNAREs and vSNAREs in vesicles are extremely high compared to physiological concentrations
-need high [] of proteins for reaction since there’s another protein to help with function –> without this in vitro, you need higher [] of SNAREs to drive membrane fusion proteins
-if you add SM protein to SNARE complex, it stabilizes the complex and helps them drive fusion of membranes
-this protein and SNARE proteins are important for exocytosis in cells
SM proteins and SNAREs
SNAREs form protein complex that is not very stable but if you add SM protein, it binds to the SNARE complex and stabilizes it –> helps drag in one direction
trans- and cis-SNARE complexes
-trans-SNARE complex- complex before membrane fuses- proteins coming from 2 separate membranes
-after fusion event, they will be in the same membrane
-cis-SNARE complex- same membrane after fusion
-membrane fusion is driven by protein complex formation- stable energy
-cells need another factor to disassemble energetically stable protein complexes
NSF and SNAP disassemble cis SNARE complex after fusion
-trans-SNARE complex drive membrane fusion to become cis-SNARE complex, which is very stable
-using SNAP and NSF they disassemble into individual units
-NSF-ATPase which hydrolyzes ATP for energy
-membrane fusion process itself doesn’t need ATP
-NSF is important for many rounds of fusion but not the single fusion events by releasing individual subunits from the cis-SNARE complex
regulated fusion of secretory granules in neurosecretion
-when APs stimualte neurons, they release neurotransmitter s uisng exocytosis- fusion between synaptic vesicles with plasma membrane
-2 proteins: complexin and synaptotagmin plus Ca2+
-major difference in unregulated vs regulated exocytosis neuron is structure
Ca regulation of fusion (regulated secretory granules in neurosecretion)
-many synaptic vesicles are already attached by forming loose trans-SNARE complexes- in neurons they’re already formed but they don’t fuse since there’s another inhibitory protein called complexin
-at synapses, the complexin binds to the trans-SNARE complex and to prevent from changing conformation to drive membrane fusion
-vesicles are docked and form trans-SNARE complexes but inhibited by complexin
-neurons are stimulated –> synaptotagmin functions- Ca2+ from outside the cell goes into the cytoplasm of neurons and that synaptotagmin is Ca2+-binding protein that binds entering calcium in a synapse
-once Ca2+ binds to synaptotagmin, it binds to complexin and removes it so pre-formed trans-SNARE complex moves forward
what are other ways to regulate fusion?
-PTM to regulate activities- phosphorylation, ubiquitination, or other modifications
-change the amounts of proteins in cells by protein synthesis or degradation
-localization of proteins can affect the activities
which mechanism directly mediates viral membrane fusion?
protein conformational change (hairpin formation)
which experiment would you do to observe hemifusion?
liposome assay using lipid and content dye
what are the 2 major ways to perform membrane fission?
- pinch from the outside
- pull from inside
what is an example of membrane fission?
-endocytosis- create small vesicles from PM
-in endocytic membrane division process, Dynamin GTPase plas an important role at the final stage by cutting membranes to make separate vesicles
-dynamin GTPase hydrolyzes GTP to cut the membranes
-people purified dynamin GTPases and used it to demonstrate that dynamin is sufficient to drive membrane fission
experiment to demonstrate dynamin GTPase in membrane fission
purify the protein –> reconstitute in vitro without any other protein to say that the protein can drive reaction itself (sufficient) then use knockdown or KO to see the defect in the process (necessary)
what happens if you have dynamin in the liposomes?
they make tubules without GTP then if you add GTP, you create small vesicles –> dynamin cuts the membrane into small pieces using GTP hydrolysis
dynamin-related GTPases mediate mitochondrial division and fusion
-DRP1 assemble onto the surface of mitochondria and using GTP hydrolysis (like dynamin) cut outer membrane and inner membrane to create 2 separate mitochondria
-DRP1 is recruited to mitochondria since the dynamin receptor proteins like MMF is located on the surface- protein-protein interactions are important to create specificity for membrane fission
dynamin-related GTPases control division of mitochondria
-if you KO DRP1, the mitochondria elongate since they fuse mitochondria without division
-if you block mitochondria division, the number increases
-if you block mitochondria fusion, you keep dividing mitochondria and you create small mitochondria
–> in both cases you don’t want very small or large mitochondria
membrane budding and fission by ESCRT
ESCRT function in different processes like cytokinesis, viral budding, and multivesicular body formation
ESCRT is sufficient for the formation of intraluminal vesicles
-ESCRT contain many different subunits- isolated ESCRT complex and mixed with fluorescently-labelled vesicles –> see that if you add ESCRT, these membranes are internalized and create interluminal vesicles
-multi-vesicualr body- named this because they have many vesicles inside big vesicles –> MVB takes up cytosolic components into big vesicles and by fusing lysosomes you degrade these components
ESCRT model
ESCRT binds to the surface of vesicles and assembles into snake-like filaments –> push part of the membrane toward the inside –> cut and make small vesicles into luminal vesicles
what is the mechanism involved in membrane fission?
in different types of membrane fission, you need different mechanisms- filament assembly for ESCRT and DRP1 for mitochondrial division, dynamin conformational change after hydrolysis that will cut the membrane and GTP hydrolysis for DRP or dynamin proteins
membrane fusion/fission requires extreme bending
-fusion- 2 membranes get close together- intermediate fusion process where lipids mix but not contents and fusion pore
-fission- reverse reaction- dynamin cut and make 2 separate membranes
different phospholipid shapes
-PC- phosphocholine take cylindrical shape- many make flat membrane
-PE- has smaller head and creates cone shape- curved membrane
-combo of head group and aceyl chain can determine shape of the lipid- can overall affect curvature of membrane they’re in
-same head group but change aceyl chain part- if you have saturated fatty acid, you can make straight tail but if you un-saturate, you can create kink and curve the tail to create cone shaped structures
-another way is to remove one aceyl chain- lysil lipid that creates inverted cone shape
different lipids can alter membrane shape
-negative curvature on inside of membrane and positive curvature on the outside
-if you look at individual lipid, on the negative side you have cone shaped lipids and other side you have inverted cone shape- these lipids stabilize intermediate structures during fusion/fission processes
phospholipases and acyl transferases (LPAAT) can directly alter membrane shape
-phospholipases can take up one of two acigens from PC and create lysoPC and change conformation from cylinder to inverted cone shape
-add aceyl chain to lysophatidic acid, you can change lipid shape from inverted cone shape to cone shape
locations of membrane curvature
-if you look at organelles in cells, they have interesting curvature
-ER has flat surface then at the end highly curved region
ways to curve a membrane
helix insertion to bend a membrane and scaffolding
endophilin (and other BAR-domain proteins) play a scaffold role in membrane curvature
-BAR-domain has banana shape
-if you have this attached to membrane immediately curves the membrane
-one type is concentrated at the neck of pit and helps endocytosis when membranes are cut
rough ER is dynamic
-called rough since it has ribosomes that create many proteins and flat sheets
-another part form tubules that are dynamic
how do you create 2 distinct morphology in the same organelle?
proteins
reticulons (wedge-shaped proteins) form ER tubes
-wedge-shaped proteins located in ER tubules and curve membranes
-people isolated these with lipids and tested if they could make tubules
ER sheets
-people hypothesized that cell type, which have ER sheet structures may have highly expressed sheet-forming protein
Ex. pancreatic acinar cells have many of these- looked to see which proteins were highly expressed and it was ribosomal proteins then climp-63
climp-63 forms ER sheets
-flattens membrane through lipid bridge
-luminal domains bind to each other and flap the membrane
-both climp-63 and reticulon, you can control the shape of ER and change ratio between 2 sheet-forming proteins and tubule-forming proteins –> change the ratio of sheets to tubules
