Comprehensive Notes on Endocytosis, Exocytosis, and Cell Signalling

Caveolin Mediated Endocytosis

  • Caveoli are small cavities formed by invagination of the plasma membrane, smaller than clathrin-coated pits.
  • Occur in lipid-rich microdomains (lipid rafts) high in cholesterol and glycosphingolipids.
  • Caveolin protein drives the process; dynamin protein also involved.
  • Used for uptake of vitamins (folate), plasma proteins (albumin), hormones bound to albumin, sphingolipids, and some pathogens.
  • Polio virus and SV40 virus (oncovirus) enter cells via this mechanism.
  • Caveolin has a "cabbage-like" texture on the inner surface.
  • Activated by receptors localized to lipid rafts; ligand binding leads to caveolin phosphorylation.
  • Phosphorylation triggers actin cytoskeleton rearrangement and membrane invagination.
  • Dynamin pinches off the vesicle; caveolin dephosphorylation reduces actin dynamics.
  • Vesicle fuses with an endosome.
  • Comparison:
    • Clathrin-dependent: Clathrin molecules coat the pit surface, removed after internalization.
    • Caveolin-dependent: Caveolin protein phosphorylated, vesicle fuses with caveosome after dephosphorylation.

Clathrin Mediated Endocytosis

  • After being pinched off by Dynamin, Clathrin is removed via HSP70 and auxilin protein to uncoat the vesicle, and the next thing would be then to fuse with the endosome.

Other Types of Endocytosis

  • Clathrin and caveolin-dependent endocytosis are not the only mechanisms.
  • Other receptor-mediated mechanisms exist in different cells.
  • Some use dynamins, others do not.
  • Macroproneocytosis is another type.

Summary of Endocytosis

  • Endocytosis = internalization.
  • Mechanisms:
    • Phagocytosis: Immune system (macrophages, neutrophils) engulfs cells/particles.
    • Pinocytosis: Cell drinking.
    • Receptor-mediated: Clathrin and caveolin-mediated processes.
  • Clathrin: Cholesterol, iron uptake.
  • Caveolin: (not specified in summary).

Exocytosis: Introduction

  • Molecules exit the cell.
  • Key steps: Docking and fusion of vesicle with plasma membrane, RAB proteins, effectors, and SNARE proteins are involved.
  • Constitutive (continuous) vs. regulated (stimulus-dependent) exocytosis.
  • Neurons communicate via neurotransmitters; synaptic vesicle cycle links endocytosis and exocytosis.
  • Exosomes and transcytosis are related processes.
  • Cells communicate by releasing signaling molecules that target other cells.
  • Exocytosis releases water-soluble molecules that cannot passively cross the plasma membrane.
  • Transcytosis: Uses both exocytosis and endocytosis to move molecules across cells (e.g., epithelial/endothelial).
  • Peptide hormones and neurotransmitters are synthesized in the ER, pass through the Golgi, and are packaged into secretory vesicles.
  • They exit cells via exocytosis.

Constitutive vs. Regulated Secretory Pathways

  • Constitutive secretion: Continuous release of molecules for plasma membrane incorporation or extracellular matrix secretion.
    • Molecules are packaged in the ER and Golgi.
    • Vesicles are transported to the plasma membrane via the microtubule network.
    • Fusion with the plasma membrane releases contents.
  • Regulated secretion: Molecules are packaged into vesicles near the plasma membrane but are released only upon a stimulus.
    • Examples:
      • Neurotransmission: Calcium spike.
      • Pancreatic beta cells: Increased blood glucose levels trigger insulin release.
      • Pancreatic acinar cells: Increasing blood glucose triggers secretion of digestive proteins.
      • Mast cells: IgE or complement activation triggers histamine secretion.

Mast Cells and Exocytosis

  • Mast cells secrete histamines in response to IgE or complement activation.
  • Histamines and cytokines are prepackaged in large vacuoles/granules.
  • Signal induction (immunoglobulin or complement protein) leads to a calcium spike.
  • Exocytosis occurs, releasing the contents; membrane remains intact without holes.
  • Mast cells serve as a model for studying exocytosis.

Molecular Mechanisms of Exocytosis

  • Vesicles from the Golgi need to reach and fuse with the plasma membrane.
  • Specific proteins on the vesicle surface ensure selectivity, targeting vesicles to the right membrane at the right time.
  • Receptors on vesicle and target membrane must match for fusion to occur.
  • Markers:
    • RAB proteins: Anchor vesicle to motor proteins for microtubule transport; tether to target membrane.
    • SNARE proteins: V-SNARE (vesicle) and T-SNARE (target membrane) interact for specificity and fusion.

Vesicle Fusion Process

  • RAB protein (GTPase) interacts with RAB effector protein (tethering protein) to dock the vesicle close to the plasma membrane.
  • SNARE proteins (V-SNARE on vesicle, T-SNARE on target membrane) twist around each other, pulling the vesicle close.
  • Water molecules are pushed out, enabling lipid bilayer fusion.
  • Receptor proteins are integrated into the plasma membrane; associated molecules are released.
  • RAB proteins are inactivated via GTP hydrolysis for reuse.
  • V-SNARE and T-SNARE interaction facilitates lipid bilayer fusion.

SNARE Complex Dissociation

  • NSFs (proteins cycling between cytoplasm and membrane) and SNAPs (associated proteins) use ATP-dependent manner to break apart V-SNARE and T-SNARE complexes.
  • This releases V-SNARE for recycling to the Golgi; T-SNARE remains in the plasma membrane.
  • This regulates snare activity and prevents non-specific vesicle binding.

Review Questions

  • What molecule targets vesicles to particular cellular membranes? Rab proteins
  • What's the role of SNARE proteins in exocytosis? To mediate membrane fusion.

Neurotransmission and Exocytosis

  • Nerve terminals constitutively release some vesicles for plasma membrane components.
  • Synaptic vesicles prepackaged with neurotransmitters undergo exocytosis upon action potential and calcium influx.
  • Target cells have receptors for neurotransmitters.
  • Small synaptic vesicles and dense core secretory vesicles are involved.
  • Action potential triggers neurotransmitter exocytosis.
  • Rapid Recycling: Exocytosis is followed by rapid endocytosis and repackaging of neurotransmitters at the nerve terminal, or recycling through the endosome.

Rapid Exocytosis and SNARE Proteins

  • Requires priming step - A protein blocks complete fusion until calcium rise occurs.
  • V-snare: Synaptic brevin protein.
  • T-snare: Complex between SNAP-25 and syntaxin protein.
  • Complex Protein: Blocks complete fusion
  • Calcium: Cause conformational change pushing out complexin protein.
  • Additional step ensures rapid nanosecond release.

Priming Step for Synaptic Vesicle Exocytosis

  • Additional step ensures rapid nanosecond release of neurotransmitters.
  • V-SNARE (synaptic brevin) interacts with T-SNARE (SNAP-25 and syntaxin).
  • Complexin protein blocks complete fusion until a calcium spike occurs.
  • Synaptotagmin binds calcium, causing a conformational change that pushes out complexin, enabling full vesicle fusion.

Toxins

  • Botox (Clostridium botulinum): Causes localized paralysis by preventing acetylcholine release; cleaves SNARE proteins.
  • Tetanus: Causes paralysis by preventing GABA release from inhibitory neurons; cleaves synaptobrevin protein (V-SNARE).
  • Red-backed spider toxin: Induces excessive exocytosis, hyperstimulation, and exhaustion of vesicles.

Neurotransmission Review Questions

  • What's the name of the chemical that neurons use to communicate? Neurotransmitters
  • What is the name of the protein that's involved in priming neurotransmitter-loaded synaptic vesicles in preparation for rapid exocytosis? D – Complexin

Exosomes

  • Vesicles exited or released from cells via exocytosis, completely pinching off into the extracellular space.
  • Contain proteins, DNA, RNA within vesicles.
  • Secreted by various cells, including cancer cells.
  • Used for:
    • Cancer detection (tumor load).
    • Detection of cancer mutations.
    • Cell communication.
    • Priming sites for metastases.
    • Immune system control.
    • Cancer vaccines and targeted drug delivery.

Transcytosis

  • Both endocytosis and exocytosis within the same cell.
  • Transports macromolecules from one side of a tissue to another.
  • Example: Antibodies from mother's milk across baby's intestine.
  • Antibodies bind to receptors on intestinal lumen surface, triggering endocytosis.
  • Vesicle fuses with the early endosome, and a transport vesicle is released on the other side of the endosome.
  • Antibody is released via exocytosis on the other side.
  • Example #2: Plasma proteins crossing the endothelium via caveolin-mediated endocytosis.

Review Question

  • What is transcytosis? The process of transporting macromolecules within a cell.

Summary of Exocytosis

  • Docking, fusion, and release are key steps.
  • Neurotransmission includes packaging and priming steps.
  • Target membrane must have proteins to interact with right vesicles: V snare and T snare Proteins.
  • SNAREs intertwine – induces membrane fusion for exocytosis to release.
  • NSF with SNAP proteins involved to separate them out for re use again.
  • In Neurotransmission, we have docking and release, but have a priming step which has connexin protein which blocks V and T snare induction before fusion. So packaging happens quickly in endosome.

Additional Notes from Catherine Skelding:

  • Multimeric proteins assemble in ER.
  • Post-translational modifications are reversible and change protein shape.
  • Proteasome degrades proteins tagged with ubiquitin; proteases are inside the 20S core.
  • Km measures enzyme affinity for substrate; Lineweaver-Burk plot can determine Vmax and Km.
  • Competitive inhibitors increase Km.
  • Different types of gene therapy: ex vivo (cells modified outside the body), direct gene therapy (virus injected into the patient).

Gap Junctions: Allows molecules to flow into the cell smaller than 1000 Da

Malginant Tumors: promote their own well being via autocrine communication process to produce more and more growth.

Water soluble hormones transport freely so it can bind to receptor on cell surface.

Enzymes the transfers from ATP: Protein kinase

What are the three stages of cell signaling: Reception, transduction, response.

Activated Map kinases return to its inactive state via protein phosphatases.

Ras is often activated in cancer so for is to be activated you must exchange GDP to GTP.

HER2 the woman with breast cancer: this drug works by inhibiting the dimerization of the receptor tyrosine kinase.