cell lecture 7b (m2)

Cellular Trafficking II - Vesicular Trafficking

Transport Vesicles in the Cell

• Vesicles are small membrane-bound structures that transport materials within the cell.

• They originate from organelles like the endoplasmic reticulum (ER) and the Golgi apparatus.

• The diagram highlights pathways leading to:

  • Secretory vesicles (for exocytosis).

  • Endosomes (early and late), which are intermediates in endocytosis.

  • Lysosomes, which digest waste material.

Exocytosis

• Definition: The process by which cells release substances into the extracellular space.

• Steps:

  1. Vesicle Transport & Docking

     • A secretory vesicle (from the Golgi) moves towards the plasma membrane.

  2. Membrane Fusion

     • The vesicle’s membrane fuses with the plasma membrane.

  3. Contents Release & Membrane Recycling

     • The plasma membrane ruptures which allows for content the soluble contents inside the vesicle to be discharged outside.

     • The vesicle membrane becomes part of the plasma membrane.

  4. Protein Orientation

     • Proteins that were on the inner vesicle membrane now face the extracellular space.

Protein Orientation in the Plasma Membrane

• Proteins destined for the plasma membrane are synthesized in the ER.

• Their active domains face the ER lumen.

• This orientation is maintained through vesicle transport.

• Once embedded in the plasma membrane, their active domain faces the extracellular space.

Secretory Pathways

Constitutive vs. Regulated Secretion

Cells use two major secretion pathways to deliver proteins and other molecules to the plasma membrane.

Constitutive Secretion

• Definition: Continuous and unregulated movement of secretory vesicles from the trans-Golgi network (TGN) to the plasma membrane.

• Key Features:

  • Occurs automatically without requiring a cellular signal.

  • Default pathway for proteins that don’t require special regulation.

  • Used for continuous secretion of materials like:

    • Extracellular matrix proteins

    • Mucus secretion in epithelial cells.

Regulated Secretion

• Definition: Movement of secretory vesicles from the TGN to the plasma membrane, but only fuses and releases contents in response to a specific cellular signal.

• Key Features:

  • Secretory vesicles accumulate near the plasma membrane, waiting for a signal.

  • Requires coordinated fusion when a signal (e.g., hormone or neurotransmitter) is received.

  • Example: Neurons store neurotransmitters in vesicles and release them when a signal is triggered.

Polarized Secretion

• Definition: Exocytosis occurs at a specific surface of the cell rather than randomly.

• Key Features:

  • Plasma membrane has specific subdomains where vesicles fuse.

  • Ensures directional secretion in specialized cells.

  • Examples:

    • Synaptic vesicles release neurotransmitters at synapses.

    • Zymogen granules (inactive enzyme precursors) are secreted into the intestinal lumen.

    • Hormone release into the bloodstream from endocrine cells.

Importance of Golgi Sorting in Polarized Secretion

• The trans-Golgi network (TGN) directs vesicles to specific membrane regions.

• Tight junctions help maintain separate membrane domains (apical vs. basolateral).

• Ensures that proteins and vesicles go to the correct side of the cell.

Forms of Endocytosis

Endocytosis is the process by which cells internalize extracellular materials into vesicles. This occurs through membrane invagination, where the plasma membrane folds inward, engulfs the material, and pinches off to form a vesicle inside the cell.

General Steps of Endocytosis

1. Membrane Invagination

   • The plasma membrane forms a pocket around the extracellular material.

   • This pocket can contain macromolecules, nutrients, or signaling molecules.

2. Vesicle Formation

   • The pocket pinches off from the plasma membrane.

   • This requires GTP-dependent proteins like dynamin to assist in scission.

   • The newly formed endocytic vesicle contains the engulfed material.

3. Fusion with Endosomes

   • The vesicle delivers its cargo to an early endosome, which acts as a sorting station.

   • Depending on the cargo’s fate, it can be sent to:

     • Recycling endosome (returns molecules to the plasma membrane).

     • Late endosome → Lysosome (for degradation).

Forms of Endocytosis

Endocytosis occurs through three main pathways, each specialized for different types of cargo.

A. Receptor-Mediated Endocytosis (Clathrin-Mediated Endocytosis)

• Highly specific process that requires membrane-bound receptors.

• Steps:

  1. Ligands (e.g., LDL, iron, hormones) bind to receptors on the plasma membrane.

  2. The receptor-ligand complexes migrate to clathrin-coated pits.

  3. The membrane pinches off, forming a clathrin-coated vesicle.

  4. The vesicle loses its clathrin coat.

  5. Receptors and ligands separate

  6. Ligands go to lysosomes or Golgi for processing

• Example: LDL (low-density lipoprotein) uptake for cholesterol transport.

B. Phagocytosis (Cell Eating)

• Involves the engulfment of large particles (e.g., bacteria, dead cells).

• Single-celled eukaryotes = means of finding food

• Animal phagocytes = performed by specialized immune cells like neutrophils and macrophages

• Steps:

  1. Cell surface receptors recognize the target (e.g., bacteria).

  2. The cell extends pseudopods (cytoskeletal projections) to surround the particle.

  3. The particle is engulfed into a phagosome.

  4. The phagosome matures into a lysosome through transient contact with endosomes

• Example: Neutrophils engulf bacteria during an immune response.

C. Pinocytosis (Cell Drinking)

• Non-specific uptake of extracellular fluid.

• Used for bulk-phase transport of solutes.

• The plasma membrane forms small vesicles that pinch off into the cytoplasm.

• No receptor specificity—it simply engulfs fluid and dissolved solutes.

• Example: Intestinal cells absorbing nutrients from the gut.

Caveolae-Mediated Endocytosis

• Caveolae are flask-shaped invaginations of the plasma membrane.

• Lined by caveolin proteins and rich in cholesterol and sphingolipids.

• Used for nutrient uptake and cell signaling.

• In endothelial cells, caveolae allow transcytosis—transporting materials across the cell.

• Example: Albumin transport across blood vessel walls.

Balancing Endocytosis and Exocytosis

• Endocytosis and exocytosis are opposing processes that maintain plasma membrane balance.

• Exocytosis adds new membrane and proteins to the plasma membrane.

• Endocytosis removes membrane components and regulates cell surface receptor levels.

Coated Vesicles

Coated vesicles play a critical role in intracellular transport by helping to sort, package, and direct cargo between organelles. The coat proteins on vesicles assist in membrane curvature, vesicle formation, and cargo selection.

• Facilitate vesicle formation by bending the membrane.

• Ensure cargo selection (specific proteins or lipids).

• Direct vesicles to the correct destination within the cell.

• Enable efficient intracellular transport (ER → Golgi → Plasma Membrane, Endocytosis, etc.).

Types of Coated Vesicles & Their Roles:

• Clathrin-Coated Vesicles:

  • Coat Proteins: Clathrin, AP1, ARF (for Golgi transport); Clathrin, AP2 (for endocytosis)

  • Function: Facilitates vesicle transport between the trans-Golgi network (TGN) and endosomes, as well as receptor-mediated endocytosis from the plasma membrane to endosomes.

• COPI-Coated Vesicles:

  • Coat Proteins: COPI, ARF

  • Function: Involved in retrograde transport, moving vesicles from the Golgi complex back to the ER. This ensures that proteins that need to remain in the ER are properly returned.

• COPII-Coated Vesicles:

  • Coat Proteins: COPII (Sec13/31 and Sec23/24), Sar1

  • Function: Facilitates anterograde transport, moving vesicles from the endoplasmic reticulum (ER) to the Golgi complex. This is an essential step in protein processing and secretion.

• Caveolin-Coated Vesicles:

  • Coat Proteins: Caveolin

  • Function: Involved in endocytosis and transcytosis, particularly in cholesterol and lipid trafficking. These vesicles form flask-shaped invaginations in the plasma membrane and are found in endothelial cells.

Step-by-Step Mechanism of Coated Vesicle Formation

1. Cargo Selection & Sorting

• Cargo receptors in the donor membrane bind to specific proteins meant for transport.

• Adaptor proteins help recruit coat proteins (Clathrin, COPI, COPII).

• The coat assembles and begins curving the membrane.

2. Coat Assembly & Bud Formation

• Coat proteins organize into a lattice or cage-like structure.

• This bends the membrane into a bud ready for vesicle formation.

• Clathrin uses triskelion structures to form a basket-like shape.

3. Vesicle Budding & Scission

• The vesicle pinches off from the donor membrane.

• Dynamin (GTPase protein) helps in membrane scission, especially in clathrin-coated vesicles.

4. Vesicle Uncoating

• The coat proteins dissociate, leaving a naked vesicle ready for transport.

• The vesicle fuses with the target membrane (Golgi, plasma membrane, lysosome).

Clathrin-Coated Vesicles & Endocytosis

• Clathrin forms a triskelion structure that creates a curved lattice.

• Used in receptor-mediated endocytosis (e.g., LDL uptake).

• Dynamin helps vesicles pinch off from the plasma membrane.

COPI & COPII Vesicles in ER-Golgi Transport

• COPII transports proteins from ER → Golgi (Anterograde).

• COPI returns proteins from Golgi → ER (Retrograde).

• These vesicles are crucial for protein processing & trafficking.

LDL-Receptor Mediated Endocytosis

• Function: Cells take up LDL particles from the extracellular fluid to obtain cholesterol for membrane synthesis and other functions.

• Key Components:

  • LDL (Low-Density Lipoprotein): Contains cholesterol, phospholipids, and triacylglycerols (TG).

  • Apolipoprotein B-100 (ApoB): A protein on LDL that binds to the LDL receptor on the plasma membrane.

  • Clathrin-Coated Vesicles: Form around the LDL-receptor complex for internalization.

Cargo Sorting & Coat Assembly

• LDL particles bind to LDL receptors (LDLR) on the plasma membrane.

• The cytoplasmic tail of LDL receptors binds to adaptor protein 2 (AP2).

• AP2 recruits clathrin, forming the clathrin-coated pit.

• This coat induces membrane curvature, helping the vesicle form.

Vesicle Budding

• Amphiphysin recruits dynamin, a GTPase enzyme, to the neck of the clathrin-coated pit.

• Dynamin hydrolyzes GTP, causing a constriction and extension motion.

• This leads to scission, where the vesicle pinches off from the plasma membrane.

• Clathrin-coated vesicles are released into the cytoplasm.

Vesicle Uncoating

• Once inside the cytoplasm, the clathrin coat is disassembled.

• HSC70 (heat-shock cognate protein 70), a chaperone, dismantles the clathrin lattice.

• ATP is required for this uncoating process.

• The now uncoated vesicle is directed toward the early endosome.

Delivery to Early Endosome

• The uncoated vesicle fuses with the early endosome.

• V-ATPase pumps protons (H⁺ ions) into the endosome, lowering the pH.

• The acidic environment causes LDL to dissociate from its receptor.

• LDL receptors are recycled back to the plasma membrane for reuse.

Transport to Lysosomes & Cholesterol Release

• LDL-containing vesicles move from early endosomes → late endosomes → lysosomes.

• Lysosomal enzymes degrade LDL, releasing cholesterol, fatty acids, and amino acids.

• The released cholesterol is used for membrane synthesis or stored in lipid droplets.

ER to Golgi Transport (Anterograde Transport) via COPII-Coated Vesicles

• Proteins destined for the Golgi are packed into vesicles coated with COPII.

• Key players:

  • Sar1 (a small GTPase) regulates COPII coat formation.

  • Sec13/31 and Sec23/24 proteins help assemble the COPII coat.

• Steps:

  1. Sar1-GDP (inactive) is recruited to the ER membrane.

  2. A guanine nucleotide exchange factor (GEF) swaps GDP for GTP, activating Sar1-GTP.

  3. Sar1 undergoes a conformational change, inserting an amphipathic helix into the membrane.

  4. COPII coat proteins (Sec13/31 & Sec23/24) bind, forming the vesicle.

  5. The vesicle buds off and travels toward the cis-Golgi.

  6. Uncoating occurs so the vesicle can fuse with the Golgi membrane and deliver its cargo.

Golgi to ER Transport (Retrograde Transport) via COPI-Coated Vesicles

• Some proteins need to return from the Golgi to the ER, e.g., ER-resident proteins marked by the KDEL sequence.

• Key players:

  • ARF (ADP-ribosylation factor), another GTPase, regulates COPI coat formation.

  • COPI coat proteins assemble the vesicle.

• Steps:

  1. ARF-GDP (inactive) is recruited to the Golgi membrane.

  2. A GEF exchanges GDP for GTP, activating ARF-GTP.

  3. ARF exposes a fatty acid tail, inserting into the membrane.

  4. COPI coat proteins assemble, forming the vesicle.

  5. The vesicle buds off and transports retrieval cargo (e.g., KDEL proteins) back to the ER.

  6. Uncoating occurs, and the vesicle fuses with the ER membrane.

Regulation of Coat Assembly and Disassembly

• GEFs (Guanine nucleotide exchange factors): Activate Sar1 and ARF by exchanging GDP for GTP.

• GAPs (GTPase-activating proteins): Inactivate Sar1 and ARF by hydrolyzing GTP back to GDP.

• This GTP/GDP cycle controls vesicle formation and coat removal.

The key differences between COPI and COPII in text format:

• Direction:

  • COPI: Retrograde transport (Golgi → ER)

  • COPII: Anterograde transport (ER → Golgi)

• GTPase Involved:

  • COPI: ARF (ADP-ribosylation factor)

  • COPII: Sar1

• Coat Proteins:

  • COPI: COPI coat proteins

  • COPII: Sec13/31 and Sec23/24

• Membrane Binding Mechanism:

  • COPI: ARF exposes a fatty acid tail for membrane insertion

  • COPII: Sar1 exposes an amphipathic helix for membrane insertion

1. SNARE Proteins and Membrane Fusion

• SNARE proteins (Soluble NSF Attachment Protein Receptor) mediate vesicle fusion by bringing two membranes close together.

• The process involves overcoming an energy barrier caused by the repulsion between negatively charged membrane surfaces.

Stages of Membrane Fusion:

1. Contact: The vesicle and target membrane come into proximity.

2. Dimpling/Protrusion: The vesicle begins to deform as it interacts with the target membrane.

3. Stalk Formation: The outer leaflets of the two membranes start merging.

4. Hemifusion Diaphragm: The outer leaflets fully merge, but the inner leaflets remain separate.

5. Fusion Pore Formation: The inner leaflets also merge, creating a continuous passage between the vesicle and the target membrane.

• SNARE proteins play a role in bringing membranes close enough for fusion to occur.

• Fusogenic lipids (like phosphatidylethanolamine) assist in the deformation of the membrane.

2. SNARE Protein Families and Vesicle Docking

SNARE proteins are divided into:

• v-SNAREs (vesicle SNAREs) found on vesicles.

• t-SNAREs (target SNAREs) found on target membranes.

Steps of Vesicle Docking and Fusion:

1. Recognition and Tethering:

   • The vesicle is recognized by membrane-anchored tethering proteins.

   • Rab GTPases assist in the recognition process.

2. SNARE Complex Formation:

   • v-SNAREs and t-SNAREs bind together to bring membranes into close proximity.

   • This interaction forces membrane fusion.

3. Membrane Fusion:

   • The vesicle membrane merges with the target membrane, releasing contents into the target compartment.

• This mechanism is crucial for processes like neurotransmitter release at synapses.

3. Effects of Botulinum Toxin on SNAREs

One of your images shows how botulinum toxin disrupts SNARE function. This toxin cleaves SNARE proteins, preventing vesicle fusion and neurotransmitter release. This leads to paralysis, as seen in botulism.

4. Possible Fates of Endocytosed Receptors

The final images illustrate what happens to receptors and molecules after endocytosis:

1. Recycling: The receptor is returned to the plasma membrane (e.g., LDL receptor).

2. Degradation: The receptor is sent to lysosomes for breakdown.

3. Golgi Transport: Some receptors are sent to the Golgi for further processing.

Lysosomes and Autophagosomes

• Lysosomes are organelles that contain acid hydrolases, which degrade macromolecules such as proteins, lipids, and carbohydrates.

• Autophagosomes form around damaged organelles or misfolded proteins and then fuse with lysosomes for degradation.

• This process is essential for cellular homeostasis, preventing the accumulation of waste and damaged components.

Steps in Autophagy and Lysosomal Degradation:

1. Autophagosome Formation – A double-membrane vesicle forms around damaged organelles or cytoplasmic debris.

2. Fusion with the Lysosome – The autophagosome merges with the lysosome, delivering its contents.

3. Degradation and Recycling – Acid hydrolases inside the lysosome break down the contents into simpler molecules, which are then recycled.

Lysosomal Function and Acidic Environment

• Lysosomes maintain a low pH (4.0–5.0), which is necessary for their digestive enzymes to function.

• V-type ATPases in the lysosomal membrane pump H⁺ ions into the lysosome, keeping it acidic.

• The low pH helps degrade macromolecules efficiently and prevents enzyme activation in the cytoplasm.

Endosomal Pathway and Lysosome Maturation

• The endocytic pathway involves the transport and degradation of molecules via early and late endosomes.

• Endocytic Vesicle Fusion:

  1. Endocytic vesicles form at the plasma membrane.

  2. They fuse with early endosomes, which sort cargo for recycling or degradation.

  3. Multivesicular bodies (MVBs) form and mature into late endosomes.

  4. Late endosomes fuse with lysosomes to degrade their contents.

• Recycling endosomes return useful materials (e.g., LDL receptors) to the plasma membrane.

Lysosomal Storage Disorders

• Lysosomal storage disorders (LSDs) result from mutations in lysosomal enzymes, preventing proper degradation of substrates.

• These disorders lead to the accumulation of undigested macromolecules, causing cellular dysfunction.

• Examples include:

  • Fabry disease – Deficiency in α-galactosidase A, leading to kidney and heart issues.

  • Gaucher disease – Deficiency in β-glucosidase, affecting the spleen, liver, and bone marrow.

  • Niemann-Pick disease – Deficiency in sphingomyelinase, causing lipid accumulation.

  • Hurler syndrome (MPS I) – Deficiency in α-iduronidase, leading to skeletal and organ abnormalities.

What Are Autophagosomes?

• Autophagosomes are double-membrane vesicles that engulf damaged organelles, proteins, and other cellular components for degradation.

• Since lysosomes cannot degrade large structures directly, autophagosomes act as intermediaries by encapsulating these large components before fusing with lysosomes.

The Process of Autophagy

Autophagy is a cellular recycling system that degrades unnecessary or damaged components.

Steps of Autophagy:

1. Initiation:

   • Autophagy begins in response to cellular stress, such as nutrient starvation, oxidative stress, or damaged organelles.

   • The endoplasmic reticulum (ER) contributes membranes to form an isolation membrane (phagophore).

2. Formation of the Autophagosome:

   • The phagophore expands and engulfs damaged mitochondria, peroxisomes, or protein aggregates.

   • The enclosed material is tagged with ubiquitin to signal for degradation.

   • The double-membrane autophagosome is completed and separates from the ER.

3. Fusion with Lysosome:

   • The autophagosome fuses with a lysosome, forming an autolysosome.

   • The low pH of the lysosome and its acid hydrolases degrade the contents into basic building blocks (amino acids, fatty acids, sugars).

   • These molecules are recycled for energy production or used to build new cellular components.

Triggers of Autophagy

Autophagy can be triggered by:

• Starvation: The cell breaks down its own components for survival.

• Accumulation of Misfolded Proteins: Prevents neurodegenerative diseases.

• Damaged Organelles: Particularly mitochondria (mitophagy) and peroxisomes (pexophagy).

Role of Autophagy in Disease

• Dysregulated autophagy is linked to many diseases:

  • Neurodegenerative disorders (e.g., Parkinson’s, Alzheimer’s) due to protein aggregation.

  • Cancer (autophagy can either suppress or promote tumor growth depending on context).

  • Infections (autophagy can degrade invading pathogens in a process called xenophagy).