Bulk Transport Mechanisms in Eukaryotes

Exocytosis

• Definition: Process of releasing contents outside the cell

• Steps:

  • Movement of vesicles to cell surface

  • Fusion of vesicle membrane with plasma membrane

  • Discharge of vesicle contents

  • Vesicle membrane becomes part of cell membrane

• Importance of Fusion: Vesicles derived from ER and Golgi fuse with plasma membrane

• Role of Microtubules: Evidence suggests involvement in vesicle movement

• Immunostaining: Shows relationship between microtubule network and secretory vesicles

• Actin Coats: Provide mechanical force for vesicle content expulsion

Endocytosis

• Definition: Process of internalizing external materials

• Opposite Effect to Exocytosis: Removes lipids and proteins from plasma membrane

• Phagocytosis: Ingestion of large particles, important for defense in organisms

• Receptor-Mediated Endocytosis:

  • Limited to materials up to 100 nanometers

  • Cells use receptors to internalize macromolecules

  • Ligand-receptor binding triggers internalization

• Examples: Ingestion of hormones, growth factors, cholesterol, etc.

• LDL Internalization: Carries cholesterol into cells

• Steps:

  • Ligand-receptor binding on cell surface

  • Encounter of complexes with coated pits for internalization

Conclusion

• Balance of Exocytosis and Endocytosis: Determines plasma membrane composition

• Phagocytosis in Defense: Utilized by white blood cells in humans

• Future Discussion: Mechanism of phagocytosis and lysosome formation

This lecture highlights the essential processes of exocytosis and endocytosis in eukaryotic cells, emphasizing the roles of microtubules, actin coats, and receptor-mediated endocytosis in cellular transport mechanisms.

Chapter 2: More Coat Proteins

• Accumulation of proteins in coated pits

  • More receptors lead to recruitment of more coat proteins for accommodating ligands and forming endocytotic vesicles.

  • Coat proteins (adapter proteins, clathrin, dynamin) induce membrane bending and invagination of the pit.

• Formation of coated vesicles

  • Endocytotic vesicle forms and pinches off to become a coated vesicle.

  • Coated vesicle is coated by coat proteins and uncoats upon reaching its destination.

  • Uncoating process requires ATP hydrolysis.

• Recycling and fusion

  • Coat proteins and dynamin are recycled to the plasma membrane.

  • Uncoated vesicle fuses with early endosome or target rapidly due to the importance of endocytosis.

  • Membrane fluidity and protein mobility are crucial for the entire process.

Chapter 3: Endocytosis and Recycling

• Endosome maturation

  • Uncoated vesicles fuse with trans Golgi network vesicles to form early endosomes.

  • Early endosomes sort and recycle materials, acquiring lysosomal proteins to mature into late endosomes and then lysosomes.

• Acidification and recycling

  • ATP-dependent proton pump lowers pH in endosomes for receptor recycling.

  • Lower pH dissociates ligand-receptor complexes, favoring receptor return to the membrane.

• Importance of pH in recycling

  • Acidic environment protonates proteins, leading to ligand-receptor dissociation.

  • Electrostatic repulsion due to positive charges aids in efficient receptor recycling.

• Variations in endocytosis

  • Different variations exist, like epidermal growth factor endocytosis stimulating cell division.

  • Desensitization due to excess EGF internalization can lead to abnormal cell proliferation and tumor formation.

Chapter 3: Receptor Mediated Endocytosis

• Regulation of EGF receptor expression

  • Continuous expression of EGF receptor leads to uncontrolled cell growth and possible tumor formation.

  • Cooperative effect in receptor mediated endocytosis: ligand binding recruits more receptors to coated pits for internalization.

• Variations in endocytosis

  • Some receptors are concentrated in coated pits independent of ligand binding.

  • LDL receptors are constitutively concentrated and internalized regardless of ligand binding.

• Fate of ligand-receptor complexes

  • Some complexes are degraded in lysosomes.

  • Some complexes enter the trans Golgi network for various cellular processes.

  • Other complexes are transported to the plasma membrane for secretion in transcytosis.

• Importance of transcytosis

  • Transcytosis plays a crucial role in propagating chemical signals.

  • Facilitates the exchange of insulin signal between endothelial cells and muscle cells.

• Clathrin-dependent endocytosis

  • Receptor mediated endocytosis relies on clathrin coat proteins.

  • Clathrin-coated vesicles are formed by clathrin and adapter proteins inducing membrane bending.

  • Clathrin triskelions, composed of heavy and light chains, assemble into hexagons and pentagons for membrane curvature.

• Visualizing membrane bending

  • Clathrin triskelions' shape resembles a soccer ball with pentagons and hexagons.

  • Arrangement of clathrin triskelions induces membrane bending and curvature similar to how a flat soccer ball can form a sphere.

Clustering Code Proteins

• Coat proteins induce membrane bending and curvature

  • Triskelion lattices of clackrene forms (hexagon and pentagons) make membrane bending efficient.

  • Important for inducing endocytosis by bending the membrane.

• Coat proteins' role in receptor-mediated endocytosis

  • Initiation phase involves cargo/ligand binding to receptors, leading to lateral diffusion into coated pit.

  • Coated pits recruit more receptors and coat proteins cooperatively.

• Assembly of coat proteins in coated pits

  • Endocytotic vesicle shape varies by organism.

  • Coat proteins not directly bound to plasma membrane; adapter proteins involved.

  • Adapter proteins bound to plasma membrane via phosphatidyl inositol 45 bisphosphate.

• Adapter proteins and specificity for endocytosis

  • Adapter proteins recognize phosphatidyl inositol 45 bisphosphate for specificity.

  • Different variations of phosphatidyl inositol for different signaling pathways.

• Actin's role in membrane bending and curvature

  • Clactrin coat proteins serve as anchoring points for actin.

  • Actin remodeling helps induce membrane bending and curvature.

  • Coat proteins essential for actin to pull and induce bending.

• Vesicle cision mechanism

  • Dynamin plays a crucial role in vesicle cision.

  • Adapter proteins involved in membrane attachment of clactrin for bending and curvature induction.

Chapter 5: Membrane Because Vesicles

Dynamin and Vesicle Seizure

• Dynamin is a coat protein responsible for inducing vesicle scission.

• Two models for vesicle scission: disassembly model and constriction and ratchet model.

  • In the disassembly model, dynamin binds to the vesicle neck and constricts it upon GTP hydrolysis.

  • In the constriction and ratchet model, dynamin wraps around the vesicle neck and constricts it further.

• Dynamin's role in inducing vesicle scission is crucial for endocytotic vesicles to bud off from the plasma membrane.

Uncoating of Coated Vesicles

• Coated vesicles need to be uncoated for fusion with endosomes or the trans Golgi network.

• Adapter proteins, such as blacklin and dynamin, play essential roles in inducing membrane bending and curvature.

• Uncoating involves dephosphorylation of phosphatidylinositol 4,5-bisphosphate.

• ATP hydrolysis is required for complete uncoating, involving the auxilin-HSC70 protein complex.

Clathrin-Dependent and Clathrin-Independent Endocytosis

• Clathrin-dependent endocytosis involves concentration of materials to be internalized.

• Clathrin-independent endocytosis (pinocytosis) is a nonspecific internalization of extracellular fluid.

• Clathrin-independent endocytosis compensates for membrane segments added by exocytosis to maintain plasma membrane size and composition.

Role of Coat Proteins in Vesicle Targeting

• Coat proteins play a crucial role in directing vesicles to their correct destinations.

• Different coat proteins have distinct origins and destinations, ensuring vesicles reach their intended targets.

• COP 1 and COP 2 proteins facilitate vesicle formation from the Golgi and ER, respectively.

• Caveolin-coated vesicles in caveolae are a recent discovery, contributing to vesicle formation mechanisms.

Chapter 6: Conclusion

• Cellular logic similar to Glactylen

  • Different players but similar process

  • COP 1 starts with ARF protein bound to GDP

  • GEF exchanges GDP with GTP inducing conformational changes in ARF

  • ARF bound to GTP recruits CAP 1 protein complex for vesicle formation

• COC 2 Process

  • SAR 1 protein involved

  • SAR 1 exchanges GDP for GTP after encountering GEF

  • SAR 1 GTP binds to ER membrane, recruits protein complexes for vesicle formation

• Vesicle Routing Mechanisms

  • Protein tanks and coat proteins play roles in routing vesicles

  • Snap proteins ensure vesicles reach correct destination

  • V SNAREs on vesicles, T SNAREs on target membrane complementary

  • Rab GTPase facilitates v SNARE and t SNARE association for vesicle