Membrane Dynamics Flashcards
(55 cards)
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
