Endocytosis and Exocytosis: Transport Mechanisms in Cell Biology, Schemes and Mind Maps of Biology

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11. Endocytosis and Exocytosis

Lecturer: Zheng Fan, Ph.D.

Department of Physiology

Lecture Outline

i. Transport into the cell from the plasma membrane and

exterior: endocytosis

Types of endocytosis

Phagocytosis

Clathrin-mediated (receptor-mediated) endocytosis and

sorting of internalized molecules

Multivesicular body and sorting of endocytosed membrane

proteins

ii. Transport from the trans Golgi network (TGN) to the cell

exterior: exocytosis

Constitutive and regulated secretory pathways

Formation and release of secretory vesicle

Proteolytic processing of proproteins in secretory

pathways

Protein sorting in polarized cells

There are Different Types of Endocytosis

• Endocytosis is an energy-using process by which cells absorb large molecules (such as proteins) or substances by engulfing them with the cell membrane.

Endocytic Pathways

• Receptor-mediated endocytosis – Mediated by clathrin-coated vesicles (CCVs, approx. 100 nm in diameter). CCVs are found in most cells and form clathrin-coated pits of the plasma membrane. Coated pits concentrate large extracellular molecules that have different receptors responsible for the receptor-mediated endocytosis of ligands, e.g. low density lipoprotein.
• Pinocytosis – Usually occurs from highly ruffled regions of the plasma membrane. It is the invagination of the cell membrane to form a pocket, which then pinches off into the cell to form a vesicle (0.5 – 5 μm in diameter) filled with extracellular fluid and molecules in a non-specific manner. The vesicle then travels into the cytosol and fuses with other vesicles such as endosomes and lysosomes.
• Phagocytosis – A process by which cells bind and internalize particulate matter larger than around 0.75 μm in diameter, such as small-sized dust particles, cell debris, microorganisms and even apoptotic cells, which only occurs in specialized cells. These processes involve the uptake of larger membrane areas.

Phagocytosis by a Macrophage

• (a) A scanning EM of a macrophage phagocytosing two chemically altered red blood cells. (red arrows: pseudopods)
• (b) Phagocytosis takes place in three steps. Step 1 – Unbound phagocyte receptors do not trigger phagocytosis. Step 2 – Binding of receptors causes them to cluster. Step 3 – Phagocytosis is triggered and the particle is taken up by the phagocyte. Pseudopod and phagosome are formed following phosphoinositide accumulation and actin polymerization.

(a) (b)

Lecture Outline

i. Transport into the cell from the plasma membrane and

exterior: endocytosis

Types of endocytosis

Phagocytosis

Clathrin-mediated (receptor-mediated) endocytosis and

sorting of internalized molecules

Multivesicular body and sorting of endocytosed membrane

proteins

ii. Transport from the trans Golgi network (TGN) to the cell

exterior: exocytosis

Constitutive and regulated secretory pathways

Formation and release of secretory vesicle

Proteolytic processing of proproteins in secretory

pathways

Protein sorting in polarized cells

The Initial Stages of Receptor-Mediated Endocytosis of Low-

Density Lipoprotein (LDL) Particles by EM

• (a) A coated pit, showing the clathrin coat on the inner (cytosolic) surface of the pit.
• (b) A pit containing LDL apparently closing on itself to form a coated vesicle.
• (c) A coated vesicle containing ferritin-tagged LDL particles.
• (d) Ferritin-tagged LDL particles in a smooth-surfaced early endosome 6 minutes after internalization began.
• Sample preparation: Cultured human fibroblasts were incubated in a medium containing LDL particles that were linked to the iron-containing ferritin visible under the electron microscope. Internalization of LDL- ferritin particles was initiated by raising temperature from 4 to 37°C.

Structure and Model for pH-Dependent Binding of LDL

Particles by the LDL Receptor

• The human LDL receptor is a single- pass transmembrane glycoprotein of 840 residues, only 50 of which are on the cytoplasmic side of the membrane, and carries the Asn-Pro-X-Tyr (NPXY) sorting signal.
• Schematic depiction of LDL receptor at neutral pH found at the cell surface (below, left) and at acidic pH found in the interior of the late endosome (below, right). Ligand binding arm = 7 sequence repeats

Internalization of LDL – An Example of Receptor-Mediated

Endocytosis and the Sorting of Internalized Molecules

Recycling endosome

Steps Involved in Receptor-Mediated LDL Endocytosis

Step 1 – Cell-surface LDL receptors bind to an apoB protein embedded in the phospholipid outer layer of LDL particles. Interaction between an Asn- Pro-X-Tyr (NPXY) sorting signal in the cytosolic tail of the LDL receptor and the AP2 complex incorporates the receptor-ligand complex into forming endocytic vesicles. Step 2 – Clathrin-coated pits containing receptor-LDL complexes are pinched off by the dynamin mechanism. Step 3 – After the vesicle coat is shed, the uncoated endocytic vesicle (early endosome) fuses with the late endosome. The acidic pH in this compartment causes a conformational change in the LDL receptor that leads to release of the bound LDL particle. Step 4 – The late endosome fuses with the lysosome, and the proteins and lipids of the free LDL particle are broken down to their constituent parts by enzymes in the lysosome. Step 5 – The LDL receptor recycles to the cell surface where at the neutral pH of the exterior medium the receptor undergoes a conformational change so that it can bind another LDL particle.

Multivesicular Bodies Form on the Pathway to Late Endosome

• Why – Lysosomal hydrolases could not digest the cytosolic domains of endocytosed membrane proteins such as the epidermal growth factor receptor (EGFR), if the protein were not localized in internal vesicles.
• Endocytosed membrane proteins are sequestrated into internal membranes of multivesicular bodies (MVB); lysosomal proteases and lipases eventually digest the internal membranes with the proteins.

Model of Lysosomal Digestion of Endocytosed Membranes

• The endocytosed plasma membranes reach the lysosomes in multivesicular bodies when late endosomes are fused with primary lysosomes, and are digested by hydrolases there.
• The lysosomal membrane is protected from digestion by a glycocalix composed of glycoproteins highly N - glycosylated that lines the inner leaflet (marked in grey).
• GSL: glycosphingolipids (taken as an example of membrane lipids)
• EGFR: epidermal growth factor receptor (taken as an example of plasma membrane proteins)

• There are three major types of endocytic pathways.
• Specialized phagocytic cells can ingest large particles.
• Pinocytosis is used primarily for the absorption of extracellular fluids, and molecules non-specifically.
• Cells use receptor-mediated endocytosis to import selected extracellular macromolecules.
• Endocytosed materials that are not recycled from endosomes end up in lysosomes.
• Some proteins are recycled from early endosomes and returned to the plasma membrane.
• Membrane invagination into multivesicular bodies are essential to achieve complete digestion of endocytosed membrane protein in lysosomes.
• Epithelial cells have two distinct early endosomal compart-ments but a common late endosomal compartment. (see next section)

Summary of Endocytosis

Lecture Outline

i. Transport into the cell from the plasma membrane and

exterior: endocytosis

Types of endocytosis

Phagocytosis

Clathrin-mediated (receptor-mediated) endocytosis and

sorting of internalized molecules

Multivesicular body and sorting of endocytosed membrane

proteins

ii. Transport from the trans Golgi network (TGN) to the cell

exterior: exocytosis

Constitutive and regulated secretory pathways

Formation and release of secretory vesicle

Proteolytic processing of proproteins in secretory

pathways

Protein sorting in polarized cells