1. Membrane Fusion Chapter 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 fusion

3. Protein Mediated

4. tubes Lipid segregation Fission/fusion Lipid mixing

5. Mechanisms 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 Pore

6. Detailed molecular mechanism is still unclear for fusion. Secretory Vesicles and Viral Fusion Today: 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 T H 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 lipids Fusion Mechanisms are believed to proceed though intermediate structures that are also observed in the L  to H II phase transition

7. “Stalk” Hypothesis Relationship between Fusion and Inverted Phase Formation TMC – trans monolayer contact ETMC – extended trans monolayer contact

8. Fusion is a necessary step in the lamellar to inverted phase transition So, if this is true, the physical properties that affected the phase transition should also affect fusion similarly What are the 2 competing forces?

9. Two processes occur during fusion: 1)Lipid mixing of different leaflets 2)Content Mixing Content Mixing is true Fusion If Stalks form, lipids can mix. Contents only mix upon FP (Fusion Pore) formation 2 competing forces: curvature energy, Interstitial chain packing Spontaneous curvature of a mixture of lipids is the mole-fraction weighted average of the spontaneous curvatures 1997 model

10. Lipid Effects. Negative Spontaneous Curvature – PE, anything that lowers T H also facilitates fusion Positive Spontaneous Curvature – LPC, anything that raises T H also inhibits fusion Phospholipase 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 fusion

13. All of this work is done with PEG induced fusion PEG 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 phases

15. Assays to monitor lipid mixing versus content mixing: Fluorescence HTPS 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. Tb 3+ - DPA fluorescence increases upon interaction with DPA. Here an increase indicates content mixing.