Endocytosis and Intracellular Vesicle Trafficking Course Notes

Cell Membrane Overview: The lipid bilayer (cell membrane) is approximately $4\,nm$ thick and acts as a barrier that is poorly permeable to ions and macromolecules.
Uptake Mechanisms by Size:
- Ions and Small Molecules: Particles up to $40\,A$ ( $4\,nm$ ) can enter the cell through protein pumps, carriers, or channels.
- Macromolecules: Larger molecules, such as proteins ranging from $2-10\,nm$ , require membrane-bound carriers known as endosomes, which are typically $50-100\,nm$ in size. This process is defined as endocytosis.
Definition of Endocytosis: The process of taking up extracellular material (fluids and macromolecules) through the invagination of the plasma membrane, followed by pinching off to form an intracellular vesicle.

micropinocytosis: enables cells to take up fluid and nutrients
Clathrin-coated vesicle: allows uptake of receptors
Noncoated vesicle:
Caveolae: enables cells to redistribute their plasma membrane and takes up very specific ions
phagocytosis: enables cells to engulf large particles such as bacteria

Primary Function: The ingestion or removal of bacteria, typically sized between $1-10\,\mu m$ .
Professional Phagocytic Cells: These cells possess specific receptors capable of binding to antibodies that tag bacteria (opsonization).
Molecular Mechanism of Phagocytosis:
  1. Receptor Activation: Receptors bind to the bacteria, triggering the activation of $F$ -actin polymerization.
  2. Zippering: The plasma membrane protrudes and "zippers" tightly around the particle.
  3. Sealing: The phagosome is sealed off from the external environment.
  4. Maturation: $F$ -actin disassembles, and the phagosome is transported into the cell.
  5. Degradation: The phagosome fuses with lysosomes to form a phagolysosome, where content is degraded.

Pathogenic Exploitation: Some bacteria induce their own phagocytosis to enter non-phagocytic mammalian cells:
  - Yersinia: Causes Bubonic Plague (characterized by swollen lymph nodes).
  - Listeria: Causes Listeriosis (fever, muscle aches, diarrhea).
  - Shigella: Causes Shigellosis (diarrhea, fever, stomach cramps).

Functions:
- Feeding: General uptake of extracellular fluid for feeding and removal of large numbers of growth factor receptors from plasma membrane.
- Cancer Metastasis: Cancer cells frequently utilize macropinocytosis for nutrient uptake to support rapid growth.
Mechanism: Growth factors stimulate actin-driven protrusions of the plasma membrane called "ruffles” that are taken up as large vesicles.
Bacterial Induction: Salmonella can trigger macropinocytosis to enter the cell, causing Salmonellosis (diarrhea, vomiting, abdominal cramps).

Function: receptor-mediated endocytosis allows the cell to take up specific macromolecules which are not abundant in the extracellular fluid
- more than 25 different types of receptors
- requires clathrin and adapter molecules
Major Types:
- Constitutive Receptor-Mediated: present all the time regardless of presence of macromolecule (like an escalator). Iron uptake - transferrin receptor, cholesterol uptake - LDL receptor
- Ligand-Induced Receptor-Mediated: can be stopped/turned on by growth signall]ing factors, e.g. EGF receptor
Structure of Clathrin: Clathrin forms a three-legged structure called a triskelion, consisting of heavy chains and light chains.
CME Molecular Mechanism:
  1. Selection (coat assembly and cargo selection): Adaptin molecules recruit specific receptors inside the cytosol (coat formation).
  2. Bud Formation: Adaptin recruits clathrin (membrane starts to bend).
  3. Vesicle formation: Recruitment of Dynamin, N-WASP and Arp2/3 leads to actin polymerization → pinches membrane → vesicle. plasma membrane cant produce its own dyamin as charged surfaces will repel eachother
  4. Uncoating: Once inside, the vesicle is uncoated by HSC70 and Auxillin.

Dynamin Specifics: A large ( $100\,kDa$ ) protein that oligomerizes into spirals at the neck of the budding vesicle. It contains a PH (Pleckstrin Homology) domain, a GED (GTPase Effector Domain), and a PRD (Proline-Rich Domain). It is essential for vesicle fission.
Role of $F$ -Actin:
  - Supports vesicle scission.
  - Transports the vesicle away from the plasma membrane.
  - Models of Invagination: The "Edge pushing model" and "Apical pulling model" describe actin's role in CCS (Clathrin-Coated Structure) invagination. Two alternative models for actin-dependent invagination of CCS

Structure: Features small ( $50\,nm$ ) invaginations known as Caveolae, which are highly enriched in cholesterol and the protein caveolin.
Formation: Caveolin self-associates to form a coat; this, combined with membrane cholesterol, induces membrane invagination.
Requirements: Dynamin and $F$ -actin are required for the internalization of caveolae.
Biological Function: Currently understood to function as a plasma membrane reservoir to balance membrane tension.

Organelle Progression:
  - Early Endosome: Initial sorting station; maintained at approximately $pH\,6.5$ .
  - MVB (Multi-Vesicular Body): Transition stage between early and late pathways.
  - Late Endosome: Maturing compartment; maintained at approximately $pH\,5$ .
  - Lysosome: Final degradation site.
  - Recycling Endosome: Returns membranes/receptors back to the surface.
  - TGN: Trans-Golgi Network.
Rab Proteins:
- regulate compartmental specificity
- recruit motor proteins that transport carriers on actin filaments or microtubules
- recruit effectors: targeting and docking components (e.g. SNARES)
- RAB’s and effector proteins are primary determinants of compartmental specificity
Rab protein classificiation:
- Rab4 - receptor will end up going to the plasma membrane acting transmembrane protein
- rab7 - sent to late endosome then degraded by lysosomes
- rab 11- sent to the recycling endosome then return back to the plasma membrane

SNARE Proteins (SNAP Receptors):
- protein complexes
- transmembrane proteins
- recruited by rabs and are specific for compartments
- v-SNAREs and t-SNAREs on opposing membrane form trans-SNARE complex

conversion of trans-SNARE to cis-SNARE complex provides energy for fusion of vesicle with target membrane
NSF (N-ethyl maleimide sensitive factor), an ATPase dissociate cis-SNARE complex and recycle SNAREs