Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexity

Today it is generally accepted that there are several endocytic mechanisms, both the clathrin-dependent one and mechanisms which operate without clathrin and with different requirements when it comes to dynamin, small GTP-binding proteins of the Rho family and specific lipids. It should be noted that clathrin-independent endocytosis can occur even when the cholesterol level in the membrane has been reduced to so low levels that caveolae are gone and clathrin-coated membrane areas are flat. Although new investigators in the field take it for granted that there is a multitude of entry mechanisms, it has taken a long time for this to become accepted. However, more work needs to be done, because one can still ask the question: How many endocytic mechanisms does a cell have, what are their function, and how are they regulated? This article describes some of the history of endocytosis research and attempts to give an overview of the complexity of the mechanisms and their regulation

Analysis of GLUT4 Distribution in Whole Skeletal Muscle Fibers: Identification of Distinct Storage Compartments That Are Recruited by Insulin and Muscle Contractions

The effects of insulin stimulation and muscle contractions on the subcellular distribution of GLUT4 in skeletal muscle have been studied on a preparation of single whole fibers from the rat soleus. The fibers were labeled for GLUT4 by a preembedding technique and observed as whole mounts by immunofluorescence microscopy, or after sectioning, by immunogold electron microscopy. The advantage of this preparation for cells of the size of muscle fibers is that it provides global views of the staining from one end of a fiber to the other and from one side to the other through the core of the fiber. In addition, the labeling efficiency is much higher than can be obtained with ultracryosections. In nonstimulated fibers, GLUT4 is excluded from the plasma membrane and T tubules. It is distributed throughout the muscle fibers with ∼23% associated with large structures including multivesicular endosomes located in the TGN region, and 77% with small tubulovesicular structures. The two stimuli cause translocation of GLUT4 to both plasma membrane and T tubules. Quantitation of the immunogold electron microscopy shows that the effects of insulin and contraction are additive and that each stimulus recruits GLUT4 from both large and small depots. Immunofluorescence double labeling for GLUT4 and transferrin receptor (TfR) shows that the small depots can be further subdivided into TfR-positive and TfR-negative elements. Interestingly, we observe that colocalization of TfR and GLUT4 is increased by insulin and decreased by contractions. These results, supported by subcellular fractionation experiments, suggest that TfR-positive depots are only recruited by contractions. We do not find evidence for stimulation-induced unmasking of resident surface membrane GLUT4 transporters or for dilation of the T tubule system (Wang, W., P.A. Hansen, B.A. Marshall, J.O. Holloszy, and M. Mueckler. 1996. J. Cell Biol. 135:415–430)

Expression of Mutant Dynamin Inhibits Toxicity and Transport of Endocytosed Ricin to the Golgi Apparatus

Endocytosis and intracellular transport of ricin were studied in stable transfected HeLa cells where overexpression of wild-type (WT) or mutant dynamin is regulated by tetracycline. Overexpression of the temperature-sensitive mutant dynG273D at the nonpermissive temperature or the dynK44A mutant inhibits clathrin-dependent endocytosis (Damke, H., T. Baba, A.M. van der Blieck, and S.L. Schmid. 1995. J. Cell Biol. 131: 69–80; Damke, H., T. Baba, D.E. Warnock, and S.L. Schmid. 1994. J. Cell Biol. 127:915–934). Under these conditions, ricin was endocytosed at a normal level. Surprisingly, overexpression of both mutants made the cells less sensitive to ricin. Butyric acid and trichostatin A treatment enhanced dynamin overexpression and increased the difference in toxin sensitivity between cells with normal and mutant dynamin. Intoxication with ricin seems to require toxin transport to the Golgi apparatus (Sandirg, K., and B. van Deurs. 1996. Physiol. Rev. 76:949–966), and this process was monitored by measuring the incorporation of radioactive sulfate into a modified ricin molecule containing a tyrosine sulfation site. The sulfation of ricin was much greater in cells expressing dynWT than in cells expressing dynK44A. Ultrastructural analysis using a ricin-HRP conjugate confirmed that transport to the Golgi apparatus was severely inhibited in cells expressing dynK44A. In contrast, ricin transport to lysosomes as measured by degradation of 125I-ricin was essentially unchanged in cells expressing dynK44A. These data demonstrate that although ricin is internalized by clathrin-independent endocytosis in cells expressing mutant dynamin, there is a strong and apparently selective inhibition of ricin transport to the Golgi apparatus. Also, in cells with mutant dynamin, there is a redistribution of the mannose-6-phosphate receptor

Interplay between Toxin Transport and Flotillin Localization

The flotillin proteins are localized in lipid domains at the plasma membrane as well as in intracellular compartments. In the present study, we examined the importance of flotillin-1 and flotillin-2 for the uptake and transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin and we investigated whether toxin binding and uptake were associated with flotillin relocalization. We observed a toxin-induced redistribution of the flotillins, which seemed to be regulated in a p38-dependent manner. Our experiments provide no evidence for a changed endocytic uptake of Stx or ricin in cells silenced for flotillin-1 or -2. However, the Golgi-dependent sulfation of both toxins was significantly reduced in flotillin knockdown cells. Interestingly, when the transport of ricin to the ER was investigated, we obtained an increased mannosylation of ricin in flotillin-1 and flotillin-2 knockdown cells. The toxicity of both toxins was twofold increased in flotillin-depleted cells. Since BFA (Brefeldin A) inhibits the toxicity even in flotillin knockdown cells, the retrograde toxin transport is apparently still Golgi-dependent. Thus, flotillin proteins regulate and facilitate the retrograde transport of Stx and ricin

Endocytosis and intracellular transport of ricin: recent discoveries

AbstractThe plant toxin ricin has proven valuable as a membrane marker in studies of endocytosis as well as studies of different intracellular transport steps. The toxin, which consists of two polypeptide chains, binds by one chain (the B-chain) to both glycolipids and glycoproteins with terminal galactose at the cell surface. The other chain (the A-chain) enters the cytosol and inhibits protein synthesis enzymatically. After binding the toxin is endocytosed by different mechanisms, and it is transported via endosomes to the Golgi apparatus and the endoplasmic reticulum before translocation of the A-chain to the cytosol. The different transport steps have been analyzed by studying trafficking of ricin as well as modified ricin molecules