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Sphingolipids (SLs) play important roles in membrane structure and cell function. Here, we examine the SL requirements of various endocytic mechanisms using a mutant cell line and pharmacological inhibitors to disrupt SL biosynthesis. First, we demonstrated that in Chinese hamster ovary cells we could distinguish three distinct mechanisms of clathrin-independent endocytosis (caveolar, RhoA, and Cdc42 dependent) which differed in cargo, sensitivity to pharmacological agents, and dominant negative proteins. General depletion of SLs inhibited endocytosis by each clathrin-independent mechanism, whereas clathrin-dependent uptake was unaffected. Depletion of glycosphingolipids (GSLs; a subgroup of SLs) selectively blocked caveolar endocytosis and decreased caveolin-1 and caveolae at the plasma membrane. Caveolar endocytosis and PM caveolae could be restored in GSL-depleted cells by acute addition of exogenous GSLs. Disruption of RhoA- and Cdc42-regulated endocytosis by SL depletion was shown to be related to decreased targeting of these Rho proteins to the plasma membrane and could be partially restored by exogenous sphingomyelin but not GSLs. Both the in vivo membrane targeting and in vitro binding to artificial lipid vesicles of RhoA and Cdc42 were shown to be dependent upon sphingomyelin. These results provide the first evidence that SLs are differentially required for distinct mechanisms of clathrin-independent endocytosis.

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference-mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1-dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.

CD99 was recently reported to be under control of the osteoblast-specific transcription factor Cbfa1 (RUNX2) in osteoblasts, suggesting a role in the phato-physiology of these cells. No extensive information is available on the role(s) of this molecule in malignant phenotype, and osteosarcoma, in particular, has never been studied. We report that in 11 different cell lines and 17 clinical samples CD99 expression is either undetectable or very low. Being expressed in the normal counterpart, we tested the hypothesis that CD99 down-regulation may have a role in osteosarcoma development and progression. CD99-forced expression in two osteosarcoma cell lines significantly reduced resistance to anoikis, inhibited growth in anchorage independence as well as cell migration, and led to abrogation of tumorigenic and metastatic ability. Therefore, the molecule acts as a potent suppressor of malignancy in osteosarcoma. CD99 gene transfection induces caveolin-1 up-regulation and the two molecules were found to colocalize on the cell surface. Treatment with antisense oligonucleotides to caveolin-1 abrogates the effects of CD99 on migration. The findings point to an antioncogenic role for CD99 in osteosarcoma, likely through the regulation of caveolin-1 and inhibition of c-Src kinase activity.