Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Membrane FusionChapter 8 of Yeagle.Majority of work is taken from papers by B. Lentz at UNCCH (Biochemistry)Membrane fusion is ubiquitous and critical process in biology:•Cellular trafficking and compartmentalization•Import of food•Export of waste•Intracellular communication•Sexual reproduction•Cell division•Viral fusionProtein MediatedtubesLipid segregationFission/fusionLipid mixingMechanisms of Membrane Fusion: Fusion Pore is made of (A) protein and lipid, (B) lipid only(B) Is the accepted pathway, this does not imply proteins are NOT involved. Proteins are involved, but they do not line the fusion PoreDetailed molecular mechanism is still unclear for fusion.Secretory Vesicles and Viral FusionToday: Focus just on the lipids and bilayer fusion without the aid of proteins.Research of the last 15 years has shown a close association between membrane fusion and inverted phase formation.Both TH and fusion can be affected by the physical properties of the lipids and the system:•Temp•pH•Lipid composition, lipids of negative or positive spontaneous curvature•Cation binding to lipidsFusion Mechanisms are believed to proceed though intermediate structures that are also observed in the L to HII phase transition“Stalk” HypothesisRelationship between Fusion and Inverted Phase FormationTMC – trans monolayer contactETMC – extended trans monolayer contactFusion is a necessary step in the lamellar to inverted phase transitionSo, if this is true, the physical properties that affected the phase transition should also affect fusion similarlyWhat are the 2 competing forces?Two processes occur during fusion:1) Lipid mixing of different leaflets2) Content MixingContent Mixing is true Fusion•If Stalks form, lipids can mix.•Contents only mix upon FP (Fusion Pore) formation2 competing forces: curvature energy,Interstitial chain packingSpontaneous curvature of a mixture of lipids is the mole-fraction weighted average of the spontaneous curvatures1997 modelLipid Effects.Negative Spontaneous Curvature – PE, anything that lowers TH also facilitates fusionPositive Spontaneous Curvature – LPC, anything that raises TH also inhibits fusionPhospholipase C can induce membrane fusion because it’s in vivo reaction is to produce DAG. DAG has small head group compared to chain, hence, negative spontaneous curvature.Conversely, production of LPC, lysophosphatidylcholine will inhibit fusion.Additionally, sphingomylenase action of sphingomyelin to produce ceremide can also affect fusion. Ceremide seems to induce leakage instead of fusion unless cholesterol is present.Arachidonic acid will also promote fusionAll of this work is done with PEG induced fusionPEG will force lipid bilayers together in close proximity to induce fusion. Factors that can be affected to induce fusion:•Bilayer dehydration•Imperfect lipid packing•Local alterations in bilayer curvature•Outer leaflet packing defects•Elastic free energy•Changes in membrane fluidity•Locally induced non-bilayer phasesAssays to monitor lipid mixing versus content mixing:FluorescenceHTPS at pH 8.0(8-hydroxy-pyrene-1,3,6-trisulfonic acid)Make other vessicle with pH 5.0,A decrease in fluorescence of HTPS indicates movement of protons, hence a small pore formed.Tb3+ - DPA fluorescence increases upon interaction with DPA. Here an increase indicates content
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