This article aims at providing an update on the complexity of clathrin-independent endocytosis. It is now almost 30 years since we first wrote a review about its existence; at that time many people believed that with the exception of macropinocytosis, which will only be briefly mentioned in this review, all uptake could be accounted for by clathrin-dependent endocytosis. Now it is generally accepted that there are different clathrin-independent mechanisms, some of them regulated by ligands and membrane lipid composition. They can be both dynamin-dependent and -independent, meaning that the uptake cannot be accounted for by caveolae and other dynamin-dependent processes such as tubular structures that can be induced by toxins, e.g. Shiga toxin, or the fast endophilin mediated endocytosis recently described. Caveolae seem to be mostly quite stable structures with other functions than endocytosis, but evidence suggests that they may have cell-type dependent functions. Although several groups have been working on endocytic mechanisms for years, and new advanced methods have improved our ability to study mechanistic details, there are still a number of important questions we need to address, such as: How many endocytic mechanisms does a cell have? How quantitatively important are they? What about the complexity in polarized cells where clathrin-independent endocytosis is differentially regulated on the apical and basolateral poles? These questions are not easy to answer since one and the same molecule may contribute to more than one process, and manipulating one mechanism can affect another. Also, several inhibitors of endocytic processes commonly used turn out to be less specific than originally thought. We will here describe the current view of clathrin-independent endocytic processes and the challenges in studying them.

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2-Deoxy-D-glucose (2DG) is a structural analogue of glucose with well-established applications as an inhibitor of glycolysis and N-glycosylation. Importantly, 2DG has been shown to improve the efficacy of several cancer chemotherapeutic agents in vivo and thus it is in clinical studies in combination with chemotherapy and radiotherapy. However, although 2DG has been demonstrated to modulate many cellular functions, including autophagy, apoptosis and cell cycle control, little is known about the effects of 2DG on intracellular transport, which is of great importance when predicting the effects of 2DG on therapeutic agents. In addition to proteins, lipids play important roles in cellular signalling and in controlling cellular trafficking. We have, in the present study, investigated the effects of 2DG on cellular lipid composition and by use of protein toxins we have studied 2DG-mediated changes in intracellular trafficking. By quantifying more than 200 individual lipid species from 17 different lipid classes, we have found that 2DG treatment changes the levels and/or species composition of several lipids, such as phosphatidylinositol (PI), diacylglycerol (DAG), cholesteryl ester (CE), ceramide (Cer) and lysophospho-lipids. Moreover, 2DG becomes incorporated into the carbohydrate moiety of glycosphingolipids (GSLs). In addition, we have discovered that 2DG protects cells against Shiga toxins (Stxs) and inhibits release of the cytotoxic StxA1 moiety in the endoplasmic reticulum (ER). The data indicate that the 2DG-induced protection against Stx is independent of inhibition of glycolysis or N-glycosylation, but rather mediated via the depletion of Ca(2+) from cellular reservoirs by 2DG. In conclusion, our results reveal novel actions of 2DG on cellular lipids and Stx toxicity.

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