Single vesicle analysis of endocytic fission on microtubules in vitro.
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.
Murray
John W
JW
Marion Bessin Liver Research Center and Department of Medicine, and Division of Hepatology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.|Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Sarkar
Souvik
S
Marion Bessin Liver Research Center and Department of Medicine, and Division of Hepatology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.|Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
Wolkoff
Allan W
AW
Marion Bessin Liver Research Center and Department of Medicine, and Division of Hepatology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.|Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
D000818:Animals; D037263:Asialoglycoprotein Receptor / Q000378:metabolism; D001212:Asialoglycoproteins / Q000378:metabolism; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D015739:Nocodazole / Q000378:metabolism; D029363:Organic Anion Transporters, Sodium-Dependent / Q000378:metabolism; D009961:Orosomucoid / Q000031:analogs & derivatives / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D011990:Receptors, Transferrin / Q000378:metabolism; D027981:Symporters / Q000378:metabolism; D050257:Tubulin Modulators / Q000378:metabolism
D037263:Asialoglycoprotein Receptor; D001212:Asialoglycoproteins; D066246:ErbB Receptors; D015739:Nocodazole; D029363:Organic Anion Transporters, Sodium-Dependent; D009961:Orosomucoid; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011990:Receptors, Transferrin; D027981:Symporters; D050257:Tubulin Modulators; C017030:asialoorosomucoid; C071908:sodium-bile acid cotransporter