Glut4

P13K

PIP3 PIP2

PDK1

Akt2/PKBβ

IRS1

GlucoseInsulin

IR

Glucose Transporter-4

Phospholipid membrane

Membrane fusion

• Can occur between cells, between intracellular compartments, between intracellular compartments and the plasma membrane, between lipid bound structures (such as viral particles) and cellular membranes

• High curvature of the membrane promotes fusion

Fusion

• Process by which 2 initially distinct lipid bilayers merge their hydrophobic cores resulting in one interconnected structure.

• Contents can mix• If only one leaflet from each bilayer is involved

in the fusion process the bilayer is hemifusion.-Inner layer remains distinct.

Hemifusion model• Believed that most if not all

biological fusion proceeds through a hemifusion intermediate

• Membrane fusion intermediates are regulated by proteins that bend and remodel membranes or by acting upstream to regulate the lipid or protein composition of the respective bilayers

Many membrane fusion events…

• Exocytosis• Endocytosis• Fusion of egg to sperm• Transport of waste to lysozome• Entry of pathogens• Formation of myotubules• Synaptic vesicles

Challenge!Energy barriers have to be overcome

• Bringing membranes in close proximity• Bringing together of repulsive membrane

charges• Energy barrier of curvature formation for both

hemifusion-stalk and fusion pore formation• Role of fusion proteins is to lower these

barriers

4 Steps need to happen for fusion to occur

1) Involved membranes must aggregate2) bilayer needs to partially dehydrate otherwise

they will repel each other3) A destabilization must develop at one point b/w

the bilayers, inducing a highly localized rearrangement of the two bilayers

4) This point defect grows and the components of the 2 bilayers mix and diffuse away from the site of contact

Divalent cations

• Divalent cations play a critical role by binding to negatively charged lipids such as phosphatidylserine, phosphatidylglycerol and cardiolipin

• One purpose of this is to shield negative charge on surface of bilayer and reduce electrostatic repulsion between bilayers

Fusion events need:

• Molecules to tether and dock membranes and bring them in close proximity.

• Molecules that locally disturb the lipid bilayers (ex. by inducing curvature)

• Molecules that give direction to the process

Other factors

• Lipid head group also affects dehydration• Independent of charge• PE binds water less tightly than PC• Size might also be a factor, according to the

stalk hypothesis, a highly curved bridge must form. PE has smaller head group and can form inverted micelle phases

Fusion proteins

• In vitro fusion is regulated by membrane associated proteins

• First to be studied is the viral fusion proteins which allow an enveloped virus to insert its genetic material into the host cell

2 classes of viral proteins

• Acidic and pH independent• pH independent fusion proteins can function

under neutral conditions and fuse with plasma membrane

• HIV, measles, mumps• Acidic fusion proteins such as in influenza are

only activated in low pH of acidic endosomes and must first be endocytosis.

Viral fusion1) Transmembrane viral fusion proteins are kept in an inactive state on the viral surface

2) following exposure to an appropriate trigger the viral fusion proteins undergo dramatic conformation changes exposing fusion peptides or loops which insert into target

3) either concurrently or subsequently the fusion proteins undergo an additional conformational change that brings the transmembrane domains in the viral envelop into close proximity with the viral fusion peptides that are embedded in the target

4) membrane fusion occurs as a consequence of close proximity and bilayer disturbance

Viral surface fusion proteins: 3 classes

• Class I mainly alpha helical• Class II mainly beta sheets • Class III are mixed secondary

Often disrupt one layer and induce curvature

Mitochondrial fusion• Fusion and fission are necessary for normal mitochondrial function• Process unclear!• Homotypic fusion is unusual because it involves both outer and

inner mitochondria• Dependent on dynamin superfamily GTPases, OPA1 (optin atrophy

protein-1) and mitofusion• Members of this family are large, self-oligomerizing GTPases that

mediate remodeling • Mitofusions can tether mitochondria to each other – mediated by

c-terminal alpha helices from opposing mitochondra together form an antiparallel coiled structure

Cell-cell fusion

• Essential during fertilization, development and immune responses

• Little conservation from yeast to nematodes to insects to mammals likely involved separately

• Lots unknown!

Assays to measure fusion:

• Measure either mixing of membrane lipids or mixing of aqueous contents

Lipids mixing• NBD-Rhodamine Energy Transfer: membrane labeled with both NBD and

Rhodamine combine with unlabeled membrane. When NBD and Rhodamine are within a certain distance, the Förster resonance energy transfer (FRET) happens. After fusion, resonance energy transfer (FRET) decreases when the average distance between probes increases, while NBD fluorescence increases.

• Pyrene Excimer Formation: emission wavelength of monomer of pyrene is 400nm. The eximer is 470nm. Membrane labeled with pyrene combines with unlabeled membrane. Before fusion, majority of emission is excimer, after the distance between probes

• Octadecyl Rhodamine B Self-Quenching:. Rhodamine dimers quench fluorescence. Fusion with unlabeled membranes resulting in dilution of the probe.

Contents mixing• Fluorescence quenching assays with ANTS/DPX: ANTS is a

polyanionic fluorophore, while DPX is a cationic quencher. The assay is based on the collisional quenching of them. Separate vesicle populations are loaded with ANTS or DPX, respectively.

• Fluorescence enhancement assays with Tb3+/DPA: This method is based on the fact that chelate of Tb3+/DPA is 10,000 times more fluorescent than Tb3+ alone.

Eukaryotic cells use different proteins

• Best studied are SNAREs• Direct all vesicular intracellular trafficking• Debate on whether SNAREs are involved in

early docking or participate late in the fusion process by facilitating hemifusion

• Enormous diversity of structure and function within these classes and very few themes are conserved.

SNARE-dependent

• SNARE motifs are the regions that contribute to the formation of a highly stable four-helix bundle called the SNARE complex

• Each SNARE contributes one helix• Helices are all aligned in parallel• The folding of this bundle is thought to drive the fusion reaction

Fertilization

• Few candidate protein for mediating fusion have been identified

• CD9, a multiple transmembrane –domain protein on the egg surface

• IZUMO, a single transmembrane protein with an exterior Ig-like domain on the sperm

• CD9 localize in areas of extreme curvature called microvilla

Endosomal fusion

• Endosomes fuse with late endosomes or lysosomes

• SNAREs are essential• Syntaxin-6, syntaxin-13,

VTI1A, VAMP4

Synaptic vesicleSynchronous release• Is the burst of small synaptic

vesicle exocytosis after depolarization

Asynchronous release

• Following synchronous release, isolated exocytic events occur

• Both are Ca2+ dependent• Molecules that are directly involved in fusion steps are synaptotagmins and SNAREs.• Accessory proteins are involved.