Single vesicle analysis of endocytic fission on microtubules in vitro

John W. Murray, Souvik Sarkar, Allan W. Wolkoff

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Following endocytosis, internalized molecules are found within intracellular vesicles and tubules that move along the cytoskeleton and undergo fission, as demonstrated here using primary cultured rat hepatocytes. Although the use of depolymerizing drugs has shown that the cytoskeleton is not required to segregate endocytic protein, many studies suggest that the cytoskeleton is involved in the segregation of protein in normal cells. To investigate whether cytoskeletal-based movement results in the segregation of protein, we tracked the contents of vesicles during in vitro microscopy assays. These studies showed that the addition of ATP causes fission of endocytic contents along microtubules, resulting in the segregation of proteins that are targeted for different cellular compartments. The plasma membrane proteins, sodium (Na+) taurocholate cotransporting polypeptide (ntcp) and transferrin receptor, segregated from asialoorosomucoid (ASOR), an endocytic ligand that is targeted for degradation. Epidermal growth factor receptor, which is degraded, and the asialoglycoprotein receptor, which remains partially bound to ASOR, segregated less efficiently from ASOR. Vesicles containing ntcp and transferrin receptor had reduced fission in the absence of ASOR, suggesting that fission is regulated to allow proteins to segregate. A single round of fission resulted in 6.5-fold purification of ntcp from ASOR, and 25% of the resulting vesicles were completely depleted of the endocytic ligand.

Original languageEnglish (US)
Pages (from-to)833-847
Number of pages15
JournalTraffic
Volume9
Issue number5
DOIs
StatePublished - May 2008
Externally publishedYes

Fingerprint

Transport Vesicles
Microtubules
Taurocholic Acid
Cytoskeleton
Transferrin Receptors
Proteins
Peptides
Asialoglycoprotein Receptor
Ligands
Cell membranes
Endocytosis
Epidermal Growth Factor Receptor
Purification
Rats
Blood Proteins
Microscopy
Hepatocytes
Assays
Microscopic examination
Membrane Proteins

Keywords

  • Asialoglycoprotein receptor
  • Endosome
  • Fission
  • Microtubules
  • Sorting
  • Transferrin receptor

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Single vesicle analysis of endocytic fission on microtubules in vitro. / Murray, John W.; Sarkar, Souvik; Wolkoff, Allan W.

In: Traffic, Vol. 9, No. 5, 05.2008, p. 833-847.

Research output: Contribution to journalArticle

Murray, John W. ; Sarkar, Souvik ; Wolkoff, Allan W. / Single vesicle analysis of endocytic fission on microtubules in vitro. In: Traffic. 2008 ; Vol. 9, No. 5. pp. 833-847.
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