# Lecture 12: Endocytosis and Exocytosis

## Cell Biology

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## Learning Objectives

By the end of this lecture, students will be able to:

1. Describe the major forms of endocytosis and their mechanisms
2. Explain clathrin-mediated endocytosis in molecular detail
3. Describe the endosomal pathway and the role of pH in cargo sorting
4. Explain receptor-mediated endocytosis using LDL uptake as a model
5. Describe the mechanisms of constitutive and regulated exocytosis

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## Lecture Content

### I. Overview of Endocytosis

Endocytosis is the process by which cells internalize material from their surroundings through invagination of the plasma membrane. Several distinct forms exist, each suited to different purposes.

**Phagocytosis** ("cell eating") involves the uptake of large particles such as bacteria, dead cells, and debris. It is carried out primarily by professional phagocytes -- macrophages, neutrophils, and dendritic cells. The process is actin-dependent: pseudopods extend around the target particle, engulfing it into a large vesicle called a phagosome (greater than 250 nm in diameter). The phagosome then fuses with lysosomes to form a phagolysosome, where the contents are degraded. Phagocytosis is receptor-mediated, driven by Fc receptors (which bind antibody-coated particles), complement receptors, and scavenger receptors.

**Pinocytosis** ("cell drinking") is the non-specific uptake of fluid and small solutes. It is a continuous, constitutive process occurring in most cells, producing small vesicles of approximately 100 nm. **Receptor-mediated endocytosis** (clathrin-mediated) is highly specific and efficient, using receptors to concentrate ligands in clathrin-coated pits that bud to form coated vesicles of approximately 120 nm. **Caveolae-mediated endocytosis** uses flask-shaped invaginations of 50 to 80 nm, rich in the protein caveolin and cholesterol, and is implicated in transcytosis, signal transduction, and lipid homeostasis, particularly in endothelial cells, adipocytes, and muscle cells. **Macropinocytosis** is a non-specific, actin-dependent process in which large membrane ruffles engulf substantial volumes of extracellular fluid, forming macropinosomes of 0.2 to 5 micrometers, and is important for antigen sampling by dendritic cells.

### II. Clathrin-Mediated Endocytosis

Clathrin-mediated endocytosis is the principal pathway for the selective uptake of macromolecules. The molecular machinery centers on **clathrin**, a triskelion-shaped protein composed of three heavy chains and three light chains that self-assembles into a polyhedral lattice on the cytoplasmic face of the membrane. **Adaptor protein complexes**, particularly **AP-2** for plasma membrane endocytosis, link clathrin to the membrane and to cargo. AP-2 binds clathrin, PIP2 in the membrane, and sorting signals on receptor cytoplasmic tails, including YXXO motifs (where Y is tyrosine, X is any amino acid, and O is a bulky hydrophobic residue) and dileucine motifs. Accessory proteins such as epsin, Eps15, intersectin, and amphiphysin assist in cargo selection, membrane curvature, and coat assembly.

The process unfolds in a series of defined steps. **Nucleation** occurs when AP-2 binds PIP2 and cargo receptors at the membrane. During **cargo selection**, receptors bearing internalization signals are concentrated. **Coat assembly** follows as clathrin triskelia are recruited to form a lattice that drives membrane invagination. **Membrane curvature** is promoted by BAR domain proteins such as amphiphysin and by epsin. The critical **scission** step is performed by **dynamin**, a GTPase that assembles as a ring around the neck of the budding vesicle. GTP hydrolysis by dynamin constricts and severs the neck. The importance of dynamin was first recognized through the shibire mutant in Drosophila, which blocks vesicle scission at non-permissive temperatures. After scission, **uncoating** occurs when Hsc70 and auxilin remove the clathrin coat, while synaptojanin dephosphorylates PIP2 to destabilize AP-2 binding. The resulting uncoated vesicle is then delivered to the early endosome.

<image>Clathrin-mediated endocytosis step-by-step. Panel A: Molecular components — clathrin triskelion structure (three heavy chains and three light chains), AP-2 adaptor complex bridging clathrin to the membrane and cargo receptor. Panel B: Sequential stages — (1) Flat membrane with AP-2 and cargo receptors, (2) Clathrin recruitment and lattice assembly creating a coated pit, (3) Invagination deepens, (4) Dynamin ring assembles at the vesicle neck, (5) GTP hydrolysis by dynamin severs the vesicle, (6) Clathrin coat removed by Hsc70/auxilin, producing an uncoated vesicle ready to fuse with early endosome. Each stage shown as a cross-sectional diagram.</image>

### III. The Endosomal Pathway

The **early endosome (sorting endosome)** is the first compartment to receive endocytosed material. It has a mildly acidic pH of approximately 6.0 to 6.5, a tubular-vesicular morphology, localizes near the cell periphery, and is marked by the GTPase Rab5 and the tethering factor EEA1. It serves as a critical sorting station that determines the fate of internalized receptors and ligands.

Three major sorting decisions are made at the early endosome. **Recycling** returns receptors to the plasma membrane, either rapidly through Rab4-positive tubules directly from the early endosome or more slowly through the Rab11-positive perinuclear recycling endosome. **Degradation** directs cargo to late endosomes and lysosomes. Receptors destined for degradation are ubiquitinated and recognized by the **ESCRT** (Endosomal Sorting Complexes Required for Transport) machinery. ESCRT-0, -I, -II, and -III sequentially recognize ubiquitinated cargo and sort it into intraluminal vesicles (ILVs), creating multivesicular bodies (MVBs). **Transcytosis** transports cargo across polarized cells, such as the transfer of IgA across intestinal epithelium.

The **late endosome/multivesicular body** has a more acidic pH of approximately 5.0 to 5.5, contains numerous intraluminal vesicles bearing receptors destined for degradation, and is marked by Rab7 and LAMP proteins. The transition from early to late endosome involves a Rab switch, in which Rab5 is replaced by Rab7. Late endosomes ultimately fuse with lysosomes to form endolysosomes, where lysosomal enzymes degrade the contents.

Interestingly, when MVBs fuse with the plasma membrane instead of lysosomes, their ILVs are released extracellularly as **exosomes** (30 to 150 nm vesicles). Exosomes carry proteins, lipids, mRNAs, and miRNAs and serve as vehicles for cell-cell communication. They are also being explored as disease biomarkers.

### IV. Receptor-Mediated Endocytosis: The LDL Pathway

The LDL receptor pathway, elucidated by Michael Brown and Joseph Goldstein (Nobel Prize, 1985), is the classic example of receptor-mediated endocytosis. **LDL (Low-Density Lipoprotein)** carries cholesterol in the blood, with a core of cholesterol esters surrounded by a surface of phospholipids, free cholesterol, and apolipoprotein B-100.

The pathway proceeds in a well-characterized sequence. LDL receptors concentrated in coated pits bind LDL through its apoB-100 component. Clathrin-mediated endocytosis internalizes the LDL-receptor complex. In the mildly acidic environment of the early endosome, LDL dissociates from its receptor. The receptor is recycled to the plasma membrane, while LDL is delivered to lysosomes. There, lysosomal acid lipase hydrolyzes the cholesterol esters to release free cholesterol. This free cholesterol then regulates cellular cholesterol homeostasis at three levels: it suppresses HMG-CoA reductase (the rate-limiting enzyme for cholesterol synthesis), suppresses LDL receptor gene transcription through the SREBP pathway, and activates ACAT to esterify cholesterol for storage.

**Familial hypercholesterolemia (FH)** results from mutations in the LDL receptor gene. Heterozygotes (approximately 1 in 500 individuals) have LDL levels roughly twice normal and develop premature atherosclerosis. Homozygotes (approximately 1 in 1,000,000) have LDL levels 4 to 6 times normal and may suffer heart attacks in childhood. Five classes of mutations have been identified: no receptor synthesis (Class 1), failure of receptor transport from ER to Golgi due to a folding defect (Class 2), inability to bind LDL (Class 3), failure to localize to coated pits because of a defective internalization signal (Class 4), and failure to recycle from endosomes (Class 5).

The **transferrin receptor** provides another important model for receptor recycling. Transferrin binds two Fe3+ ions at neutral pH and is internalized by clathrin-mediated endocytosis. In the acidic endosome, Fe3+ is released from transferrin, but importantly, apo-transferrin remains bound to its receptor. The apo-transferrin-receptor complex recycles to the cell surface, where apo-transferrin is released at neutral pH.

<image>The LDL receptor pathway. Sequential diagram showing: (1) LDL binds LDL receptor in a clathrin-coated pit at the cell surface. (2) Coated pit invaginates and forms a coated vesicle. (3) Coat is removed. (4) Vesicle fuses with early endosome (pH 6.0); LDL dissociates from receptor. (5) LDL receptor recycles back to the plasma membrane via recycling vesicle. (6) LDL is delivered to lysosome. (7) Lysosomal enzymes degrade LDL; cholesterol esters hydrolyzed to free cholesterol. (8) Inset box showing regulatory effects of free cholesterol: inhibition of HMG-CoA reductase, inhibition of LDL receptor transcription, activation of ACAT. Side panel showing the five classes of LDL receptor mutations in familial hypercholesterolemia.</image>

### V. Exocytosis

Exocytosis is the fusion of intracellular vesicles with the plasma membrane, releasing their contents extracellularly. **Constitutive exocytosis** is a continuous, unregulated process that delivers newly synthesized membrane proteins and lipids to the plasma membrane and secretes extracellular matrix components such as collagen and proteoglycans. No specific signal is needed to trigger fusion.

**Regulated exocytosis** stores secretory vesicles or granules in the cytoplasm until a specific signal triggers their release. The trigger is typically a rise in intracellular Ca2+. Examples include neurotransmitter release at synapses, insulin secretion from pancreatic beta cells, histamine release from mast cells, and cortical granule exocytosis during fertilization. **Synaptic vesicle exocytosis** is particularly well-characterized. Vesicles are pre-docked at active zones, with the SNARE complex already partially assembled from synaptobrevin (v-SNARE) and syntaxin plus SNAP-25 (t-SNAREs). Synaptotagmin, the Ca2+ sensor on the synaptic vesicle, responds to Ca2+ influx through voltage-gated Cav channels by triggering rapid fusion in as little as 0.2 milliseconds. Fusion can occur in either "kiss-and-run" mode (transient pore opening) or full-collapse mode. **Compound exocytosis**, in which granules fuse sequentially with each other before merging with the plasma membrane, occurs in eosinophils and mast cells.

In **polarized epithelial cells**, exocytosis is directional, with different cargo sorted to apical versus basolateral membrane domains. Tight junctions prevent the mixing of apical and basolateral membrane proteins, maintaining this polarity.

### VI. Phagocytosis and Its Regulation

Phagocytosis is specialized for the engulfment of large particles exceeding 0.5 micrometers in diameter. Target particles display **"eat me" signals** such as phosphatidylserine on the outer leaflet of apoptotic cells or opsonins (antibodies and complement component C3b) that coat the target. Phagocytic receptors include Fc receptors (binding the Fc region of antibodies), complement receptors (binding complement fragments), scavenger receptors (binding modified LDL and bacterial components), and PS receptors (recognizing apoptotic cells).

The engulfment mechanism follows a "zipper model" in which sequential engagement of receptors along the particle surface drives progressive wrapping. Actin polymerization, regulated by Rho family GTPases Rac and Cdc42, powers pseudopod extension around the target. The engulfed particle is enclosed within a phagosome, which matures through acquisition of Rab5 and then Rab7 and progressive acidification. Fusion with lysosomes creates the phagolysosome, where degradation occurs through the combined action of lysosomal hydrolases and reactive oxygen species generated during the respiratory burst.

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