Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent (“caveolar-like”) mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a “vital marker” for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.

Internalization of some plasma membrane constituents, bacterial toxins, and viruses occurs via caveolae; however, the factors that regulate caveolar internalization are still unclear. Here, we demonstrate that a brief treatment of cultured cells with natural or synthetic glycosphingolipids (GSLs) or elevation of cholesterol (either by acute treatment with m␤-cyclodextrin/cholesterol or by alteration of growth conditions) dramatically stimulates caveolar endocytosis with little or no effect on other endocytic mechanisms. These treatments also stimulated the movement of GFP-labeled vesicles in cells transfected with caveolin-1-GFP and reduced the number of surface-connected caveolae seen by electron microscopy. In contrast, overexpression of caveolin-1 decreased caveolar uptake, but treatment with GSLs reversed this effect and stimulated caveolar endocytosis. Stimulation of caveolar endocytosis did not occur using ceramide or phosphatidylcholine and was not due to GSL degradation because similar results were obtained using a nonhydrolyzable GSL analog. Stimulated caveolar endocytosis required src kinase and PKC-␣ activity as shown by i) use of pharmacological inhibitors, ii) expression of kinase inactive src or dominant negative PKC␣, and iii) stimulation of src kinase activity upon addition of GSLs or cholesterol. These results suggest that caveolar endocytosis is regulated by a balance of caveolin-1, cholesterol, and GSLs at the plasma membrane. INTRODUCTIONCaveolae are plasma membrane (PM) specializations that are rich in cholesterol, sphingolipids, and caveolin-1 (Cav1), a cholesterol-binding protein (Smart et al., 1999). By electron microscopy, they appear as flask-shaped structures at the PM and as smooth uncoated vesicles near the PM. Caveolae have been implicated in signaling, endocytosis, transcytosis, and potocytosis (for reviews see Smart et al., 1999;Fielding and Fielding, 2001;Mineo and Anderson, 2001;Carver and Schnitzer, 2003;Parton, 2003). Recently caveolae have gained attention because they have been documented to play a role in the cellular uptake and intracellular delivery of some bacterial toxins, viruses, and bacteria (Lencer et al., 1999;Shin et al., 2000;Norkin, 2001;Pelkmans et al., 2001;Richterova et al., 2001;Duncan et al., 2002;Marjomaki et al., 2002). One of the first markers to be used for caveolar endocytosis was the cholera toxin B subunit (CtxB), which binds to GM 1 ganglioside at the PM and is internalized through caveolae in some cell types (Orlandi and Fishman, 1998;Torgersen et al., 2001). SV40 virus has been shown to be internalized from the PM in small Cav1-containing vesicles (Pelkmans et al., 2001), and caveolar endocytosis of derivatized albumin has also been reported (Schnitzer et al., 1994;Shubert et al., 2001;Sharma et al., 2003;Singh et al., 2003). We previously showed that fluorescent glycosphingolipid (GSL) analogs (lactosylceramide [LacCer] and globoside) are selectively internalized via caveolae in human skin fibroblasts (HSFs) and other cell types using pathway-sp...

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With the overall goal being a better understanding of the sensing environment from the local perspective of a situated agent, we studied uniform flows and Kármán vortex streets in a frame of reference relevant to a fish or swimming robot. We visualized each flow regime with digital particle image velocimetry and then took local measurements using a rigid body with laterally distributed parallel pressure sensor arrays. Time and frequency domain methods were used to characterize hydrodynamically relevant scenarios in steady and unsteady flows for control applications. Here we report that a distributed pressure sensing mechanism has the capability to discriminate Kármán vortex streets from uniform flows, and determine the orientation and position of the platform with respect to the incoming flow and the centre axis of the Kármán vortex street. It also enables the computation of hydrodynamic features which may be relevant for a robot while interacting with the flow, such as vortex shedding frequency, vortex travelling speed and downstream distance between vortices. A Kármán vortex street was distinguished in this study from uniform flows by analysing the magnitude of fluctuations present in the sensor measurements and the number of sensors detecting the same dominant frequency. In the Kármán vortex street the turbulence intensity was 30% higher than that in the uniform flow and the sensors collectively sensed the vortex shedding frequency as the dominant frequency. The position and orientation of the sensor platform were determined via a comparative analysis between laterally distributed sensor arrays; the vortex travelling speed was estimated via a cross-correlation analysis among the sensors.

Glycosphingolipids are known to play roles in integrinmediated cell adhesion and migration; however, the mechanisms by which glycosphingolipids affect integrins are unknown. Here, we show that addition of the glycosphingolipid, C8-lactosylceramide (C8-LacCer), or free cholesterol to human fibroblasts at 10°C causes the formation of glycosphingolipidenriched plasma membrane domains as shown by visualizing a fluorescent glycosphingolipid probe, BODIPY-LacCer, incorporated into the plasma membrane of living cells. Addition of C8-LacCer or cholesterol to cells initiated the clustering of B 1 -integrins within these glycosphingolipid-enriched domains and the activation of the B 1 -integrins as assessed using a HUTS antibody that only binds activated integrin. On warming to 37°C, B 1 -integrins were rapidly internalized via caveolar endocytosis in cells treated with C8-LacCer or cholesterol, whereas little B 1 -integrin was endocytosed in untreated fibroblasts. Incubation of cells with C8-LacCer or cholesterol followed by warm-up caused src activation, a reorganization of the actin cytoskeleton, translocation of RhoA GTPase away from the plasma membrane as visualized using total internal reflection fluorescence microscopy, and transient cell detachment. These studies show that LacCer can regulate integrin function both by modulating integrin clustering in microdomains and by regulating integrin endocytosis via caveolae. Our findings suggest the possibility that aberrant levels of glycosphingolipids found in cancer cells may influence cell attachment events by direct effects on integrin clustering and internalization. (Cancer Res 2005; 65(18): 8233-41)