Lecture 7

Introduction to Vesicle Formation and Fusion

Vesicles are small, spherical, membrane-bound sacs essential for transport, storage, and secretion within cells. This process involves intricate mechanisms, starting with formation.

Vesicle Formation and Fusion Overview

• The process begins with the budding of specialized coat proteins on a donor membrane, which ensures trafficking specificity:

  • Clathrin: Involved in endocytosis and transport from the trans-Golgi to endosomes/lysosomes.

  • COPI: Mediates retrograde transport within the Golgi and from Golgi to ER.

  • COPIII: Facilitates anterograde transport from ER to Golgi.

• Cargo selection occurs as cargo molecules bind receptors, which are linked to coat proteins by adaptor proteins, initiating membrane curvature.

• Inner membrane proteins select cargo and target vesicles for fusion.

• G-proteins (GTPases) act as molecular switches, cycling between active (GTP-bound) and inactive (GDP-bound) states, regulating vesicle formation, budding, and scission. GEFs activate them by promoting GTP binding, while GAPs inactivate them via GTP hydrolysis.

GTPases and Shape Change in Vesicles

• GTP binding induces conformational changes critical for protein interactions and sequential steps of vesicle release and fusion.

• Specific GTPases recruit coat proteins, driving membrane curvature.

• Scission, the pinching off of the vesicle, is often facilitated by proteins like Dynamin (a large GTPase) for clathrin-coated vesicles.

The Role of SNARE Proteins

• SNAREs (V-SNARE on vesicle, T-SNARE on target) are integral membrane proteins crucial for specific and efficient membrane fusion.

  • Their interaction ensures vesicles fuse with the correct target membrane.

• Tethering factors and Rab GTPases initially dock vesicles to target membranes.

• During fusion, V-SNAREs and T-SNAREs twist into a four-helix bundle, drawing membranes together and facilitating their merging.

Cycling of GTPases

• After formation and scission, GTPases hydrolyze GTP to GDP, leading to coat disassembly. This uncoats the vesicle, exposing SNAREs and targeting machinery for docking and fusion, and recycles components.

Transport Mechanisms in Cells

• Motor proteins (ATP-dependent enzymes) actively transport vesicles along cytoskeletal tracks.

  • Myosin 5A: Travels along actin filaments (to '+ end') for vesicle and organelle transport.

  • Kinesin: Transports cargo along microtubules (generally to '+ end').

  • Dynein: Moves along microtubules (predominantly to '- end') for vesicle trafficking and organelle positioning.

• Cytoskeleton Components: Provide structural framework and tracks:

  • Microtubules: Largest fibers ($25 \, \text{nm}$ diameter), polymers of $\alpha$- and eta-tubulin; polar (+ and - ends).

  • Actin Filaments (Microfilaments): Smallest fibers ($7 \, \text{nm}$ diameter), two intertwined helical strands of actin monomers; polar (+ and - ends).

  • Intermediate Filaments: ($10 \, \text{nm}$ diameter), rope-like, provide mechanical strength, generally non-polar and stable.

Dynamic Nature of the Cytoskeleton

• Actin filaments and microtubules are dynamic, undergoing rapid polymerization (assembly, to '+ end', ATP for actin, GTP for microtubules) and depolymerization (disassembly).

• Treadmilling: Subunits are added to one end and removed from the other, maintaining length.

• Dynamic Instability: (Microtubules only) Rapid cycles of growth ('rescue') and shrinkage ('catastrophe') at '+ ends', driven by GTP hydrolysis and a 'GTP cap'.

• Cytoskeletal dynamics are regulated by accessory proteins (e.g., nucleating, capping, severing proteins).

Endocytosis and Exocytosis

• Endocytosis: Cells internalize substances.

  • Phagocytosis: Engulfing large particles.

  • Pinocytosis: Ingesting liquids and small molecules.

  • Receptor-Mediated Endocytosis: Specific uptake of macromolecules via clathrin-coated pits (e.g., LDL cholesterol uptake).

• Exocytosis: Cells release substances into the extracellular space.

  • Constitutive Exocytosis: Constant, unregulated in all cells.

  • Regulated Exocytosis: Occurs in specialized cells in response to specific signals (e.g., neurotransmitter release).

Role of Clathrin in Vesicle Formation

• Clathrin coat forms a polyhedral lattice around budding vesicles, invaginating the membrane for cellular intake, exemplified by LDL cholesterol uptake.

Lysosomes and Endosomes

• Endosomes: Sorting stations for internalized material and Golgi proteins/lipids. Early endosomes recycle material or progress to late endosomes.

• Lysosomes: Mature late endosomes, containing acid hydrolases and an acidic pH ($4.5-5.0$), for degrading macromolecules. Endosomes also recycle receptors.

Conclusion

Understanding vesicular transport, cytoskeletal dynamics, and cellular functions is crucial, with concepts like motor proteins and melanosome transport providing practical examples.