HUBS2206 Lecture 31: Endocytosis Notes

Molecule Transport: Endocytosis

Key Concepts & Learning Goals

  • Endocytosis and Exocytosis: Understanding the definitions and when cells use these mechanisms.
  • Mechanisms of Endocytosis:
    • Phagocytosis
    • Macropinocytosis
    • Clathrin-mediated endocytosis
    • Caveolin-mediated endocytosis
  • Understanding How These Mechanisms Occur: Including coated pits and vesicles.
  • Understanding Why Cells Use These Processes: Examples include phagocytosis of pathogens and clathrin-mediated endocytosis of cholesterol (receptor-mediated).

Recap: Cell Communication

  • Extracellular Signaling Molecules:
    • Endocrine signaling (distant)
    • Paracrine signaling (local)
    • Autocrine signaling (themselves)
    • Neurotransmission
  • Binding to Receptors: Initiates a signaling cascade.
  • Focus: How cells release and internalize macromolecules through endocytosis and exocytosis.
  • Question: How are small molecules vs. large macromolecules transported?

Recap: Biological Membranes

  • Composition: Lipids (polar head, non-polar tail) + proteins.
  • Lipid Bilayer:
    • 'Self-sealing' (spontaneously close into vesicles).
    • Hydrophobic tails avoid contact with water.

Permeability of Cell Membranes

  • Selective Permeability: Lipid bilayer is permeable to some substances, impermeable to others.
  • Permeable Substances:
    • Hydrophobic molecules (e.g., dissolved gases, lipid hormones) pass through easily.
    • Small uncharged polar molecules are semi-permeable.
  • Less Permeable Substances:
    • Large uncharged polar molecules are even less permeable.
    • Ions are impermeable.
  • Key Factor: Permeability is primarily based on hydrophobicity, not just size.

Transport of Large Molecules, Particles, and Fluids

  • Exocytosis:
    • Cells release polar or charged molecules into the extracellular environment.
    • Maintains plasma membrane integrity (preservation of lipid/protein composition).
  • Endocytosis:
    • Cells uptake large polar or charged molecules and particles that cannot be taken up otherwise.
    • Cells take in fluids (small and large volumes).

Exocytosis and Endocytosis Linkage

  • Linked Events: Budding → Target → Fusion.
  • Recycling: Many exocytosed molecules are retrieved and recycled (restores components).
  • Maintenance: Keeps plasma membrane composition intact and returns proteins necessary for correct functioning.

Endocytosis: An Overview

  • Process: Involves plasma membrane, early endosome, recycling endosome, multivesicular body, lysosome, late endosome, and endolysosome.
  • Transport: Microtubule-mediated transport.
  • Fusion: Fusion between different compartments.

Mechanisms of Endocytosis

  • Different Types:
    • Phagocytosis
    • Pinocytosis
    • Receptor-mediated (Clathrin-mediated and Clathrin-independent)
      • Clathrin-mediated endocytosis
      • Clathrin-independent endocytosis
        • Caveolin-mediated endocytosis
        • Caveolin-independent endocytosis
  • Specificity: Each type internalizes different molecules.

Phagocytosis: "Cell Eating"

  • Function: Specialist cells (e.g., monocytes/macrophages, neutrophils) ingest large objects, bacteria, viruses coated with antibodies or complement proteins.
  • Mechanism:
    • Receptors on phagocyte recognize targets.
    • Cell engulfs object into a vacuole (phagosome).
    • Involves actin cytoskeleton.
Phases of Phagocytosis
  1. Chemotaxis and adherence of microbe to phagocyte.
  2. Ingestion of microbe by phagocyte.
  3. Formation of a phagosome (phagocytic vesicle).
  4. Fusion of the phagosome with a lysosome to form a phagolysosome.
  5. Digestion of ingested microbe by enzymes.
  6. Formation of residual body containing indigestible material.
  7. Discharge of waste materials.

Pathogen Evasion of Phagocytosis

  • Example: Listeria monocytogenes causes listeriosis.
  • Source: Found in raw milk, soft cheeses, ice cream, raw vegetables, fermented raw-meat sausages, poultry, raw meats, and can survive at low temperatures (<4 °C).
  • Survival Mechanism: L. monocytogenes can survive and replicate in phagosomes.

Macropinocytosis: "Cell Drinking"

  • Function: Absorption of liquids (i.e., extracellular fluids).
  • Occurrence: Almost all cells perform pinocytosis.
  • Regulation: Can be regulated (occurs in response to stimulus) or constitutive (always occurs).
  • Mechanism:
    • Cell engulfs molecules (e.g., water, carbohydrates, glycerol, growth factors, integrin ligands) into vacuole (macropinosome).
    • Involves actin.
  • Uses:
    • Ingestion of large volumes.
    • Cell motility.
    • Pathogen entry (Ebola, salmonella).

Receptor-Mediated Endocytosis: Clathrin-Mediated

  • Major Route: Best understood endocytosis mechanism.
  • Mechanism:
    • Molecules (e.g., cholesterol, iron) interact with membrane receptors at coated pit.
    • Recruit clathrin and adaptor proteins (between clathrin triskelion cage and the membrane).
    • Clathrin links to actin, leading to budding.
Clathrin-Coated Vesicle Pinching Off
  • Dynamin's Role:
    • Forces from clathrin coat assembly alone are insufficient to pinch off vesicles.
    • Dynamin assembles as a ring around the neck.
    • Bends membrane and changes lipid composition (recruitment of lipid-modifying enzymes).
  • Membrane Bending Proteins: Participate and contain BAR domains.
  • Uncoating: Vesicle loses clathrin coat; hsp70 chaperone protein acts as uncoating ATPase activated by auxilin.
Pathway of Clathrin-Mediated Endocytosis
  1. Ligand binds to membrane receptor.
  2. Receptor-ligand migrates to clathrin-coated pit.
  3. Endocytosis occurs.
  4. Vesicle loses clathrin coat.
  5. Receptors and ligands separate.
  6. Ligands go to lysosomes or Golgi for processing.
  7. Transport vesicle with receptors moves to the cell membrane.

Endosomes

  • Definition: Membrane-bound compartment where material is sorted before degradation in the lysosome.
  • Types:
    • Early endosomes (pH = 6; dynamic tubular vesicular network; cargo is sorted).
    • Late endosomes (more acidic; lack tubules, contain many close-packed lumenal vesicles; cargo is degraded).
    • Recycling endosomes (return to membrane).
  • Composition: Each type has a different protein composition.
  • Fate: Fuse with lysosomes to form endolysosomes.

Clathrin-Mediated Endocytosis: Cholesterol Uptake

  • LDL: Low-density lipoprotein, a lipid vesicle containing cholesterol, phospholipids, fatty acids, and proteins.

Receptor-Mediated Endocytosis: Caveolin-Mediated

  • Caveolae: "Little cavities" found in most cell types (especially endothelial cells).
  • Lipid Rafts: Formed at membrane microdomains rich in cholesterol, glycosphingolipids, proteins (integral membrane protein caveolin; dynamin).
  • Uptake: Takes up vitamins (e.g., folate), proteins (e.g., albumin, growth hormones, GPI-anchored proteins), lipids (e.g., sphingolipids), and pathogens (e.g., Poliovirus, Simian Virus 40).
Caveolin-Mediated Endocytosis Pathway
  • Mechanism:
    • Molecule binds to receptor, found in lipid raft domain (caveolae).
    • Binding activates signaling and causes phosphorylation of caveolar proteins and remodeling of actin.
    • 'Pinching off' by dynamin.
  • Caveosome: Endosomal compartment with a neutral pH; caveolin vesicles then fuse with caveosome and release contents.

Clathrin and Caveolin-Independent Endocytosis

  • Characteristics: Least studied; mechanisms based on different composition of plasma membrane (e.g., rich in certain lipids - cholesterol; contain proteins – IL2R β).

Summary

  • Endocytosis: Internalization.
  • Mechanisms:
    • Phagocytosis (cell eating)
    • Pinocytosis (cell drinking)
    • Receptor-mediated (clathrin and caveolin-mediated)
  • Clathrin-Mediated Endocytosis: Steps and role in cholesterol and iron uptake.
  • Caveolin-Mediated Endocytosis: Caveolae involvement.