T2206 - Endocytosis and Exocytosis

Endocytosis and Exocytosis

Introduction

  • Endocytosis and exocytosis are two linked processes that often occur in the same regions of cells.
  • Endocytosis: Bringing things into the cell.
  • Exocytosis: Things leaving or exiting the cell.
  • Focus on four different types of endocytosis:
    • Phagocytosis
    • Macropinocytosis (or pinocytosis)
    • Clathrin-mediated endocytosis (receptor-mediated)
    • Caveolin-mediated endocytosis (receptor-mediated)

Cell Communication and Transport

  • Extracellular signaling molecules:
    • Paracrine signaling (local)
    • Autocrine signaling (local)
    • Endocrine signaling (long-range)
    • Neurotransmission (as covered in previous lectures)
  • These signaling methods require the release of a signaling molecule from one cell to be received by a target cell.
  • Exocytosis releases molecules from cells that generate them, whereas endocytosis uptakes signaling molecules, pathogens, bacteria, macromolecules (glycerol), and water.

Plasma Membrane Structure

  • The plasma membrane is a phospholipid bilayer with polar (hydrophilic) heads and nonpolar (hydrophobic) tails.
  • The lipid bilayer automatically seals itself due to the hydrophobic tails wanting to be away from water; therefore the most energetically favorable confirmation is the vesicle form.
  • Plasma membranes are impermeable to some molecules:
    • Hydrophobic molecules (Oxygen, Carbon Dioxide, Nitrogen & steroid hormones) can easily cross the lipid bilayer.
    • Small, uncharged polar molecules (Water, Urea, Glycerol, and Amines) can cross, but not as easily.
    • Large, uncharged polar molecules (Sugars) require an active process to enter the cell.
    • Ions require ion channel receptors in order to pass.

Exocytosis

  • Exocytosis is the process of releasing molecules from the cell, specifically polar or charged molecules, into the extracellular environment.
  • Molecules are packaged into vesicles made of lipid bilayers, which then fuse with the plasma membrane to release their contents.
  • Maintains integrity of plasma membrane

Endocytosis

  • Endocytosis involves the generation of a vacuole or vesicle from the plasma membrane.
  • The plasma membrane buds into the inner side of the cell, engulfing molecules from the extracellular side.
  • The vesicle then goes through endosomes and lysosomes for processing.

Protein Production and Processing

  • Proteins destined for the membrane (integral membrane proteins) or secretion are processed through the endoplasmic reticulum (ER) and Golgi apparatus.
  • This process requires budding off of vesicles. Proteins within these vesicles then travel along the microtubule network to the cell surface, finally using exocytosis to incorporate into the phospholipid bilayer of the plasma membrane.

Key Processes

  • Budding, targeting, and fusion are key processes in both endocytosis and exocytosis.
  • Fusion is the fusion of two lipid bilayers together which keeps the membrane composition intact.
  • Endocytosis and exocytosis are linked in processes like neurotransmission, where rapid recycling occurs.

Molecular Mechanisms of Endocytosis

  • The plasma membrane bulges into the inner side of the cell, invaginates, and eventually buds off, forming a vesicle in the cytosol.
  • This vesicle travels to the early endosome where receptors on the surface of the vacuole can be recycled back to the plasma membrane, or the molecule can go through a recycling endosome to be processed further.
  • Molecules that are needed by the cell undergo processing through the endosomes and ultimately fuse with the lysosome to form an endolysosome.
  • Lysosomes contain enzymes activated at a low pH that digest the molecules into usable components.
  • Endosomes are more acidic than the cytosol (pH ≈ 6).

Types of Endocytosis

  • Phagocytosis: Engulfing large particles, including pathogens.
  • Pinocytosis: Uptaking extracellular fluid and molecules like glycerol and water.
  • Receptor-mediated endocytosis:
    • Clathrin-mediated endocytosis
    • Caveolin-mediated endocytosis (clathrin-independent)
  • Each mechanism has evolved to bring in different types of molecules into the cell.
Phagocytosis (Cell Eating)
  • Macrophages and neutrophils are specialist white blood cells that carry out phagocytosis to engulf pathogens and damaged cells.
  • Apoptotic cells have changes in their plasma membrane, signaling macrophages to engulf them.
  • Macrophages ingest large objects (bacteria, viruses, damaged cells).
  • Pathogens are coated with antibodies or complement proteins that are recognized by phagocytes, activating them.
  • Activation of phagocytes activates the PI3 kinase signaling pathway.
  • This pathway induces a change in the actin cytoskeleton at the plasma membrane, causing the membrane to extend out and engulf objects via pseudopods.
Mechanism:
  1. Microbe or particle is present.
  2. Pseudopods are extended out from the macrophage or neutrophil.
  3. Engulfment into a phagocytic vesicle (phagosome).
  4. The phagosome fuses with the lysosome.
  5. Digestive enzymes within the lysosome digest the microbe.
  6. Recycling of usable components takes place.
  7. Waste is excreted from the cell via exocytosis.
Listeria Example
  • Certain microbes, like Listeria, have evolved to overcome phagocytosis.
  • Listeria enters the cell via phagocytosis but survives the acidic conditions of the lysosome.
  • It induces breaks in the lipid bilayer of the lysosome, releasing digestive enzymes into the cytosol.
  • Listeria activates the actin cytoskeleton to induce release via exocytosis after replication.
Macropinocytosis (Cell Drinking)
  • Involves the extension of regions of the plasma membrane and is regulated by the actin cytoskeleton.
  • Occurs in virtually all cells.
  • Can be constitutive (always occurring) or regulated (in response to a stimulus).
  • Stimulus often involves activation of a receptor which then stimulates a change in the actin cytoskeleton causing protrusions or membrane ruffles emerging from the plasma membrane.
Mechanism:
  1. Activation of a receptor stimulates changes in the actin cytoskeleton.
  2. Protrusions or membrane ruffles come out of the plasma membrane, engulfing fluid.
  3. Sealing and breaking off of the vacuole (macropinosome).
  • This process brings in water, carbohydrates, glycerol, growth factors, and integrin ligands.
  • Ebola and Salmonella bacteria can also enter cells using this process.
Clathrin-Mediated Endocytosis
  • Clathrin molecules come together with an adaptor protein (adaptin) to form a cage, bending the plasma membrane.
  • It is the most well-understood form of receptor-mediated endocytosis.
  • Used to import molecules like cholesterol and iron.
  • Clathrin molecules are concentrated at the plasma membrane in regions called coated pits.
  • Clathrin is recruited by adaptor proteins in response to receptor activation.
  • Requires activation of the actin cytoskeleton for the movement of the plasma membrane.
  • Dynamin (protein) is the one that causes the pinching off of the plasma membrane.
Mechanism:
  1. Receptor is activated by its ligand.
  2. The cytoskeleton is activated leading to the rearrangement and accumulation of clathrin molecules, forming a triskellion structure to bend the membrane.
  3. Pinching off of the plasma membrane occurs by Dynamin, forming a clathrin-coated vesicle.
  4. Clathrin is removed by a protein called HSP90 or Cnumna.
  5. Naked transport vesicles are formed.
Clathrin Triskelion:
  • Composed of six subunits (three heavy chains and three light chains) that form a three-dimensional structure.
  • Adaptin molecules link the receptors to the clathrin molecule.
Dynamin:
  • Assembles as a ring around the neck of the budding vesicle.
  • Brings other proteins (bar domain proteins) or membrane-bending proteins.
  • Under GTP hydrolysis, it stretches and pinches off the vacuole.
HSP70:
  • A heat shock protein that acts as an ATPase to activate a protein called auxilin.
  • Auxilin cleaves and releases the clathrin molecules from the vacuole, leaving a naked vesicle.
Process Summary:
  1. Receptor activated by binding to its ligand.
  2. Clathrin-coated pit starts to bend the plasma membrane.
  3. Dynamin activates membrane-bending proteins, bending the membrane further.
  4. Dynamin completely pinches off the vesicle.
  5. HSP70 and auxilin remove the clathrin molecules, which return to the plasma membrane.
  6. The receptor and ligand remain within the vesicle.
  7. The vacuole fuses with the endosome.
  8. The lower pH induces dissociation of the ligand and receptor.
  9. The receptor is recycled back to the plasma membrane.
  10. The ligand stays within the endosome and fuses with the lysosome for processing.
Endosomes:
  • Membrane-bound compartments that process molecules coming in or going out of the cell.
  • Early endosomes have a pH of about six.
  • Later endosomes are more acidic.
  • Recycling endosomes process proteins back to the plasma membrane.
  • Fusion with the lysosome leads to digestion of contents.
  • Multivesicular bodies are where multiple vesicles are processed at once within large bodies.
Cholesterol Example:
  • Cholesterol is transported through the body in the form of low-density lipoprotein (LDL).
  • LDL particles bind to LDL receptors on target cells.
  • The cell uptakes the LDL via clathrin-mediated endocytosis.
Process:
  1. LDL receptor binds its ligand.
  2. Clathrin accumulates, bending the membrane.
  3. Dynamin pinches off the vesicle.
  4. Clathrin is removed.
  5. The vesicle fuses with the endosome.
  6. In the early endosome, the lower pH causes LDL to dissociate from the receptor.
  7. Receptor is recycled back to the plasma membrane.
  8. The endosome fuses with the lysosome.
  9. Lysosomal enzymes digest the LDL particles into individual components.
  10. Free cholesterol is released into the cell.