1. Membrane Fusion

3. Introduction Ubiquitous cell biological process Part of many house keeping functions – Endocytosis – Constitutive secretion – Recycling Specialization of cells – Sperm-egg fusion – Exocytosis of hormones, neurotransmitters, enzymes

4. Introduction Role in disease – Giant cells during inflammatory response – Viral entry into host cells – Induced cell-cell fusion by HIV Several types of fusion events

5. Influenza HA-Mediated Membrane Fusion Reasons for being a model for study – Single gene product – Easily induced by pH change – Easily produced – X-ray structure known – Large database of cloned sequences – Numerous characterized mutants

6. Influenza HA-Mediated Membrane Fusion Hemagglutinin (HA) – Trimer of identical subunits – Conserved, hydrophobic fusion peptide in stem of each subunit Slight mutations in structure alter or abolish fusion capabilities – Receptor binding sites lie at distal tip of protein (globular head)

7. Influenza HA-Mediated Membrane Fusion Fusion Steps 1.Fusion peptides, induced by low pH, destabilize viral and target bilayers 2.Conformational changes in HA trimer (rotational and lateral movements) after initial binding to receptor 3.Hemifusion followed by full fusion to form fusion pore

8. Exocytosis of Mast Cell Granules Fusion of intracellular storage vesicle with the cytoplasmic face of the plasma membrane. The mast cell releases a mixture of compounds, including histamine, proteoglycans, serotonin, and serine proteases from its cytoplasmic granules. (inflammatory response to allergen)

9. Exocytosis of Mast Cell Granules Fusion Steps 1.Outward current transient (release of Ca 2+ )— discharge of vesicle membrane potential through nascent pore 2.Increase in cell membrane capacitance due to interaction with vesicle (directly proportional to surface area 3.Narrow conducting pore forms (2-2.5 nm)— width of 10 water molecules 4.Pore grows in concurrence with capacitance until it reaches a plateau level

10. Intra-Golgi Transport Membrane fusion essential for membrane trafficking of macromolecules Studied through careful application of inhibitors

11. Intra-Golgi Transport Steps in Transportation Between Cisterna 1.Nascent coated bud forms on the donor cisterna and detaches 2.Targeted and attaches to acceptor cisterna 3.Vesicle is uncoated and matures 4.Fuses with acceptor cisterna 5.Deposits contents

12. Intra-Golgi Transport Requirements – ATP and GTP for vesicle budding – GTP hydrolysis for uncoating – Fatty Acyl coA for budding and maturation – Ca2+ cofactor for late transport – GTPases (monomers and trimers) and phosphoproteins for fidelity, timing, and vector

13. Intra-Golgi Transport Fusion Machine – 3 Proteins Homotetramer NSF (NEM-sensitive factor) SNAP (Soluble NSF Attachment Protein) Integral Membrane Receptor (SNARE)

15. Membrane Fusion Still a lot to learn!!! Pores developed during influenza-mediated fusion and exocytosis of mast cell granules Complex fusion machine utilized during intra- Golgi transport (unknown if pore forms) Why is this complexity necessary for membrane fusion events to occur?? It is possible that the fusion pore is element that units all fusion events—viral and cell-cell fusion, regulated exocytosis, intra-Golgi transport, and other intracellular fusion events

16. For the exam… Identify when membrane fusion occurs in cells Why is it necessary?