#endocytosis

Basics of Cell Biology

Which cellular process involves the intake of large molecules via vesicles?

Cells internalize macromolecules too large or polar to cross bilayer via endocytosis involving membrane invagination and vesicle formation consuming energy. Clathrin-mediated endocytosis concentrates receptors such as transferrin receptor, LDL receptor, and EGFR into clathrin-coated pits assembled by adaptor AP2 and accessory proteins EPS15, dynamin GTPase mediates scission producing 100 nm vesicles that shed clathrin via auxilin and Hsc70 and fuse with early endosomes marked by Rab5 and PI3P. Caveolin-dependent uptake forms 60 nm flask-shaped caveolae enriched in cholesterol, regulating lipid

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 13: Endocytosis Mechanisms.

View Details

Membrane Transportation

Which of the following is an example of vesicular transport?

Vesicular transport refers to bulk modality moving macromolecules particles and fluid that cannot cross via single carriers by formation of membrane vesicles requiring cytoskeletal remodeling. Endocytosis exemplifies inward trafficking where plasma membrane invaginates forming coated pits concentrated by adaptor AP2 epsin sorting cargo low-density lipoprotein receptor nutrients antigens pathogens. Clathrin triskelion lattice polymerizes imposing curvature dynamin large GTPase forms helical collar around neck hydrolyzing GTP to pinch vesicle releasing one hundred nanometer vesicle containing sa

Ref: Alberts et al., Molecular Biology of the Cell, 6th ed., Chapter 13: Vesicular Transport – Endocytosis as Example.

View Details

Cell Organelles

Endosomes are formed from:

Endosomal system is central node of vesicular traffic that integrates endocytic and biosynthetic routes to sort cargo after internalization or secretion. Classical early endosomes originate when clathrin-coated pits invaginate with assistance of AP2 adaptor, epsin and dynamin GTPase scission, or when caveolae containing caveolin internalize, delivering extracellular ligands, nutrient receptors such as transferrin receptor, signaling receptors like EGFR, and fluid-phase markers into cell. These primary vesicles rapidly lose coat through auxilin and Hsc70 and fuse homotypically via Rab5-GTP, EEA

Ref: Alberts et al., Molecular Biology of the Cell, 6th ed., Chapter 13: Endosome Formation from Plasma Membrane and TGN.

View Details

Cell Organelles

Early endosomes function in:

Endocytic trafficking relies on progressive maturation and sorting of internalized cargo to maintain membrane homeostasis and signaling fidelity. Early endosomes, also called sorting endosomes, receive clathrin-coated and non-clathrin vesicles from the plasma membrane containing receptors such as transferrin receptor, EGF receptor, ligands, low-density lipoprotein, and extracellular fluid. Their pleomorphic tubular-vesicular morphology and mildly acidic pH around 6.0 to 6.2, maintained by vacuolar ATPase delivering protons, facilitate dissociation of many ligands from receptors while preservin

Ref: Alberts et al., Molecular Biology of the Cell, 6th ed., Chapter 13: Intracellular Vesicular Traffic – Early Endosomes and Sorting.

View Details

Cytoskeleton

In receptor-mediated endocytosis, which protein activates the Arp2/3 complex to drive actin assembly?

Endocytosis of cargo such as EGFR transferrin requires actin assistance in mammalian cells against high membrane tension especially under high membrane stiffness. Clathrin coated pit maturation involves transient actin patch assembly lasting 10 to 20 seconds around neck. Key signaling axis Cdc42 GTP recruits N WASP, which in resting state autoinhibited by intramolecular interaction between basic region GBD and VCA. Activation occurs when Cdc42 GTP binds GBD, PIP2 binds basic region, and SH3 proteins TOCA1 syndapin bind proline rich stretch, releasing VCA. Free VCA binds one G actin via WH2 and

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 13: WASP Arp2/3 Activation in Endocytosis.

View Details

Protein Sorting and Trafficking

What is the primary function of amphisomes?

Interplay macroautophagy and endocytic system produces hybrid intermediate called amphisome fusion product before lysosomal degradation. Autophagosome double-membrane vesicle size up to 1 micron bearing lipidated LC3-II cargo sequestered including mitochondria protein aggregates bacteria forms upon closure isolation membrane phagophore. Separately endocytic pathway internalizes surface receptors extracellular material into early endosomes marked Rab5 PI3P then matures Rab7 positive late endosomes multivesicular bodies MVBs intraluminal vesicles tetraspanins CD63 LAMP1 LBPA. Amphisome formation

Ref: Nakamura & Yoshimori, Autophagy 2017: Amphisome formation from autophagosome-endosome fusion.

View Details

Protein Sorting and Trafficking

During receptor-mediated endocytosis of iron, Fe³⁺ is transported into cells via:

Vertebrates transport ferric iron safely via transferrin family to avoid oxidative Fenton radical damage and precipitation. Hepatocytes secrete apo-transferrin 80 kDa bilobed glycoprotein serum loads two Fe3+ each coordinated synergistically bidentate carbonate anion plus four protein ligands two tyrosines histidine aspartate each lobe producing salmon pink complex termed ferrotransferrin diferric transferrin. Doubly loaded species binds transferrin receptor TfR1 homodimer dissociation constant 1-10 nM neutral pH versus 50-fold lower affinity monoferric negligible apo providing selection iron

Ref: Lodish et al., MCB Figure 14-29: Ferrotransferrin carries Fe3+ for uptake via transferrin receptor.

View Details

Protein Sorting and Trafficking

Which adapter protein interacts with the NPXY motif of LDL receptors?

LDL receptor internalization requires highly specific recognition cytosolic tail by endocytic co-adaptors bridging to clathrin lattice enhancing efficiency. Tail 50 amino acids contains conserved FXNPXY motif positions 802-807 forming tight beta-turn favored aromatic phenylalanine tyrosine. PTB domains autosomal recessive hypercholesterolemia protein ARH LDLRAP1 and Dab2 Disabled-2 liver recognize NPXY tyrosine hydrophobic pocket interacting also basic residues upstream motif. Adaptors contain additional trafficking domains: clathrin box binding heavy chain terminal domain, AP2 beta2 appendage

Ref: He et al., Biochemistry: NPXY motif of LDLR binds ARH and AP2 adaptor.

View Details

Protein Sorting and Trafficking

Which protein is required for vesicle scission during receptor-mediated endocytosis?

Final separation stage clathrin-coated pit formation yielding free vesicle requires enzymatic fission beyond coat polymerization. Clathrin assembly with adaptor AP2 and BAR proteins epsin, amphiphysin, endophilin generates deeply invaginated bud connected plasma membrane narrow tubule neck diameter around 20 nm bilayer. Separation demands mechanical constriction cutting neck. Specialist protein dynamin large 96 kDa GTPase family founding member N-terminal GTPase domain, middle stalk, PH domain binding PI(4,5)P2 enriched site, GED and PRD binding SH3, polymerizes helical collar around neck. Rec

Ref: Kaksonen & Roux, Nat Rev Mol Cell Biol: Dynamin scission in clathrin-mediated endocytosis.

View Details

Protein Sorting and Trafficking

The acidic pH in the late endosome causes:

Endosomal maturation involves Rab conversion progressive luminal acidification orchestrated by vacuolar V-type ATPase multisubunit rotary pump hydrolyzing ATP pumping protons generating pH early endosome 6.0-6.5 late 5.5-6.0 lysosome 4.5-5.0. Gradient critical sorting function beyond hydrolysis activation many ligand-receptor complexes exhibit pH dependent affinity due histidine protonation altering charge complementarity. Examples: LDL separates from LDL receptor pH below 6 releasing particle for lysosomal degradation while receptor recycles via retromer, mannose-6-phosphate receptors release

Ref: Maxfield & McGraw, Nat Rev Mol Cell Biol: Endosomal acidification dissociates receptor-ligand complexes.

View Details

Protein Sorting and Trafficking

Which type of endocytosis involves the uptake of small extracellular fluid droplets?

Cells internalize extracellular material by mechanistically distinct endocytic routes distinguished by particle size, actin requirement, coat dependence. Phagocytosis mediates uptake large particulates above 0.5 micron such as bacteria, yeast, dead cells via extension actin-driven pseudopods triggered clustering Fc gamma receptors, complement receptors CR3, scavenger receptors signaling via Syk, PI3K, Rho GTPases prominent professional phagocytes macrophages neutrophils forming phagosome destined lysosome. Pinocytosis literally cell drinking from Greek pinein describes constitutive uptake smal

Ref: Alberts et al., MBC Chapter 13: Pinocytosis for fluid-phase uptake.

View Details

Protein Sorting and Trafficking

The process of vesicle scission during clathrin-mediated endocytosis requires:

Formation of endocytic vesicle demands energetically unfavorable fission of neck connecting bud to parent membrane against line tension and bending resistance. In clathrin-mediated pathway adaptor AP2 and accessory proteins FCHo, epsin together with clathrin lattice driving polymerization stabilize deeply invaginated bud with omega shape. Constriction of neck performed by dynamin large 96 kDa GTPase assembling into helical oligomer about two turns around neck 50 nm diameter PH domain binding PI(4,5)P2. Polymerization aligns G domains enhancing GTPase 100 fold. Upon concerted GTP binding and co

Ref: Schmid & Frolov, Annu Rev Cell Dev Biol: Dynamin GTP hydrolysis drives membrane scission.

View Details