Spoiled for Choice: Diverse Endocytic Pathways Function at the Cell Surface.

Endocytosis has long been identified as a key cellular process involved in bringing in nutrients, in clearing cellular debris in tissue, in the regulation of signaling, and in maintaining cell membrane compositional homeostasis. While clathrin-mediated endocytosis has been most extensively studied, a number of clathrin-independent endocytic pathways are continuing to be delineated. Here we provide a current survey of the different types of endocytic pathways available at the cell surface and discuss a new classification and plausible molecular mechanisms for some of the less characterized pathways. Along with an evolutionary perspective of the origins of some of these pathways, we provide an appreciation of the distinct roles that these pathways play in various aspects of cellular physiology, including the control of signaling and membrane tension.

N-BAR and F-BAR proteins-endophilin-A3 and PSTPIP1-control clathrin-independent endocytosis of L1CAM.

Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.

Regulation of Hook1-mediated endosomal sorting of clathrin-independent cargo by γ-taxilin.

In clathrin-independent endocytosis, Hook1, a microtubule- and cargo-tethering protein, participates in sorting of cargo proteins such as CD98 (encoded by SLC3A2) and CD147 (encoded by BSG) into recycling endosomes. However, the molecular mechanism that regulates Hook1-mediated endosomal sorting is not fully understood. In the present study, we found that γ-taxilin is a novel regulator of Hook1-mediated endosomal sorting. γ-Taxilin depletion promoted both CD98-positive tubular formation and CD98 recycling. Conversely, overexpression of γ-taxilin inhibited the CD98-positive tubular formation. Depletion of Hook1, or Rab10 or Rab22a (which are both involved in Hook1-mediated endosomal sorting), attenuated the effect of γ-taxilin depletion on the CD98-positive tubular formation. γ-Taxilin depletion promoted CD147-mediated spreading of HeLa cells, suggesting that γ-taxilin might be a pivotal player in various cellular functions in which Hook1-mediated cargo proteins are involved. γ-Taxilin bound to the C-terminal region of Hook1 and inhibited its interaction with CD98; the latter interaction is necessary for sorting CD98. We suggest that γ-taxilin negatively regulates the sorting of Hook1-mediated cargo proteins into recycling endosomes by interfering with the interactions between Hook1 and the cargo proteins.

Cytokine receptor cluster size impacts its endocytosis and signaling.

The interleukin-2 receptor (IL-2R) is a cytokine receptor essential for immunity that transduces proliferative signals regulated by its uptake and degradation. IL-2R is a well-known marker of clathrin-independent endocytosis (CIE), a process devoid of any coat protein, raising the question of how the CIE vesicle is generated. Here, we investigated the impact of IL-2Rγ clustering in its endocytosis. Combining total internal reflection fluorescence (TIRF) live imaging of a CRISPR-edited T cell line endogenously expressing IL-2Rγ tagged with green fluorescent protein (GFP), with multichannel imaging, single-molecule tracking, and quantitative analysis, we were able to decipher IL-2Rγ stoichiometry at the plasma membrane in real time. We identified three distinct IL-2Rγ cluster populations. IL-2Rγ is secreted to the cell surface as a preassembled small cluster of three molecules maximum, rapidly diffusing at the plasma membrane. A medium-sized cluster composed of four to six molecules is key for IL-2R internalization and is promoted by interleukin 2 (IL-2) binding, while larger clusters (more than six molecules) are static and inefficiently internalized. Moreover, we identified membrane cholesterol and the branched actin cytoskeleton as key regulators of IL-2Rγ clustering and IL-2-induced signaling. Both cholesterol depletion and Arp2/3 inhibition lead to the assembly of large IL-2Rγ clusters, arising from the stochastic interaction of receptor molecules in close correlation with their enhanced lateral diffusion at the membrane, thus resulting in a default in IL-2R endocytosis. Despite similar clustering outcomes, while cholesterol depletion leads to a sustained IL-2-dependent signaling, Arp2/3 inhibition prevents signal initiation. Taken together, our results reveal the importance of cytokine receptor clustering for CIE initiation and signal transduction.

Nutrient-responsive O-GlcNAcylation dynamically modulates the secretion of glycan-binding protein galectin 3.

Endomembrane glycosylation and cytoplasmic O-GlcNAcylation each play essential roles in nutrient sensing, and characteristic changes in glycan patterns have been described in disease states such as diabetes and cancer. These changes in glycosylation have important functional roles and can drive disease progression. However, little is known about the molecular mechanisms underlying how these signals are integrated and transduced into biological effects. Galectins are proteins that bind glycans and that are secreted by a poorly characterized nonclassical secretory mechanism. Once outside the cell, galectins bind to the terminal galactose residues of cell surface glycans and modulate numerous extracellular functions, such as clathrin-independent endocytosis (CIE). Originating in the cytoplasm, galectins are predicted substrates for O-GlcNAc addition and removal; and as we have shown, galectin 3 is a substrate for O-GlcNAc transferase. In this study, we also show that galectin 3 secretion is sensitive to changes in O-GlcNAc levels. We determined using immunoprecipitation and Western blotting that there is a significant difference in O-GlcNAcylation status between cytoplasmic and secreted galectin 3. We observed dramatic alterations in galectin 3 secretion in response to nutrient conditions, which were dependent on dynamic O-GlcNAcylation. Importantly, we showed that these O-GlcNAc-driven alterations in galectin 3 secretion also facilitated changes in CIE. These results indicate that dynamic O-GlcNAcylation of galectin 3 plays a role in modulating its secretion and can tune its function in transducing nutrient-sensing information coded in cell surface glycosylation into biological effects.

Structured clustering of the glycosphingolipid GM1 is required for membrane curvature induced by cholera toxin.

AB5 bacterial toxins and polyomaviruses induce membrane curvature as a mechanism to facilitate their entry into host cells. How membrane bending is accomplished is not yet fully understood but has been linked to the simultaneous binding of the pentameric B subunit to multiple copies of glycosphingolipid receptors. Here, we probe the toxin membrane binding and internalization mechanisms by using a combination of superresolution and polarized localization microscopy. We show that cholera toxin subunit B (CTxB) can induce membrane curvature only when bound to multiple copies of its glycosphingolipid receptor, GM1, and the ceramide structure of GM1 is likely not a determinant of this activity as assessed in model membranes. A mutant CTxB capable of binding only a single GM1 fails to generate curvature either in model membranes or in cells, and clustering the mutant CTxB-single-GM1 complexes by antibody cross-linking does not rescue the membrane curvature phenotype. We conclude that both the multiplicity and specific geometry of GM1 binding sites are necessary for the induction of membrane curvature. We expect this to be a general rule of membrane behavior for all AB5 toxins and polyomaviruses that bind glycosphingolipids to invade host cells.

ESCRT-dependent protein sorting is required for the viability of yeast clathrin-mediated endocytosis mutants.

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin-mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin-associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N-terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME-deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony-sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)-dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin-independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild-type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1-dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT-dependent trafficking on endocytic recycling and the PM.

Glycosylation and glycan interactions can serve as extracellular machinery facilitating clathrin-independent endocytosis.

In contrast to clathrin-mediated endocytosis (CME) which is well characterized and understood, little is known about the regulation and machinery underlying clathrin-independent endocytosis (CIE). There is also a wide variation in the requirements each individual CIE cargo has for its internalization. Recent studies have shown that CIE is affected by glycosylation and glycan interactions. We briefly review these studies and explore how these studies mesh with one another. We then discuss what this sensitivity to glycan interactions could indicate for the regulation of CIE. We address the spectrum of responses CIE has been shown to have with respect to changes in glycan interactions and attempt to reconcile disparate observations onto a shared conceptual landscape. We focus on the mechanisms by which cells can alter the glycan interactions at the plasma membrane and propose that glycosylation and glycan interactions could provide cells with a tool box with which cells can manipulate CIE. Altered glycosylation is often associated with a number of diseases and we discuss how under different disease settings, glycosylation-based modulation of CIE could play a role in disease progression.

APEX2-mediated RAB proximity labeling identifies a role for RAB21 in clathrin-independent cargo sorting.

RAB GTPases are central modulators of membrane trafficking. They are under the dynamic regulation of activating guanine exchange factors (GEFs) and inactivating GTPase-activating proteins (GAPs). Once activated, RABs recruit a large spectrum of effectors to control trafficking functions of eukaryotic cells. Multiple proteomic studies, using pull-down or yeast two-hybrid approaches, have identified a number of RAB interactors. However, due to the in vitro nature of these approaches and inherent limitations of each technique, a comprehensive definition of RAB interactors is still lacking. By comparing quantitative affinity purifications of GFP:RAB21 with APEX2-mediated proximity labeling of RAB4a, RAB5a, RAB7a, and RAB21, we find that APEX2 proximity labeling allows for the comprehensive identification of RAB regulators and interactors. Importantly, through biochemical and genetic approaches, we establish a novel link between RAB21 and the WASH and retromer complexes, with functional consequences on cargo sorting. Hence, APEX2-mediated proximity labeling of RAB neighboring proteins represents a new and efficient tool to define RAB functions.

Endocytosis in proliferating, quiescent and terminally differentiated cells.

Endocytosis mediates nutrient uptake, receptor internalization and the regulation of cell signaling. It is also hijacked by many bacteria, viruses and toxins to mediate their cellular entry. Several endocytic routes exist in parallel, fulfilling different functions. Most studies on endocytosis have used transformed cells in culture. However, as the majority of cells in an adult body have exited the cell cycle, our understanding is biased towards proliferating cells. Here, we review the evidence for the different pathways of endocytosis not only in dividing, but also in quiescent, senescent and terminally differentiated cells. During mitosis, residual endocytosis is dedicated to the internalization of caveolae and specific receptors. In non-dividing cells, clathrin-mediated endocytosis (CME) functions, but the activity of alternative processes, such as caveolae, macropinocytosis and clathrin-independent routes, vary widely depending on cell types and functions. Endocytosis supports the quiescent state by either upregulating cell cycle arrest pathways or downregulating mitogen-induced signaling, thereby inhibiting cell proliferation. Endocytosis in terminally differentiated cells, such as skeletal muscles, adipocytes, kidney podocytes and neurons, supports tissue-specific functions. Finally, uptake is downregulated in senescent cells, making them insensitive to proliferative stimuli by growth factors. Future studies should reveal the molecular basis for the differences in activities between the different cell states.

ALS2, the small GTPase Rab17-interacting protein, regulates maturation and sorting of Rab17-associated endosomes.

Small GTPase Rab17 has been shown to regulate a wide range of physiological processes including cell migration in tumor cells and dendrite morphogenesis in neurons. However, molecular mechanism underlying Rab17-mediated intracellular trafficking is still unclear. To address this issue, we focused on Rab17-interacting protein ALS2, which was also known as a guanine nucleotide exchange factor (GEF) for Rab5, and investigated how ALS2 contributed to Rab17-associated membrane trafficking in cells. Rab17 was primarily localized to endosomal compartments, particularly to recycling endosomes, which was dependent on Rab11 expression. Upon Rac1 activation, Rab17 along with ALS2 was recruited to membrane ruffles and early endosomes in a Rab5 activity-independent manner. While RABGEF1, another Rab17-interacting Rab5 GEF, functioned as a GEF for Rab17, ALS2 did not possess such catalytic activity but merely interacted with Rab17. Importantly, ALS2 acted downstream of RABGEF1, regulating the maturation of Rab17-residing nascent endosomes to early endosome antigen 1 (EEA1)-positive early endosomes. Further, these Rab17-residing nascent endosomes were arisen via clathrin-independent endocytosis (CIE). Collectively, ALS2 plays a crucial role in the regulation of Rab17-associated endosomal trafficking and maturation, probably through their physical interaction, in cells.

TBC1D24 regulates recycling of clathrin-independent cargo proteins mediated by tubular recycling endosomes.

Many plasma membrane proteins enter cells by clathrin-independent endocytosis (CIE). Rab family small GTPases play pivotal roles in CIE and following intracellular trafficking of cargo proteins. Here, we provide evidence that TBC1D24, which contains an atypical Rab GAP domain, facilitates formation of tubular recycling endosomes (TREs) that are a hallmark of the CIE cargo trafficking pathway in HeLa cells. Overexpression of TBC1D24 in HeLa cells dramatically increased TREs loaded with CIE cargo proteins, while deletion of TBC1D24 impaired TRE formation and delayed the recycling of CIE cargo proteins back to the plasma membrane. We also found that TBC1D24 binds to Rab22A, through which TBC1D24 regulates TRE-mediated CIE cargo recycling. These findings provide insight into regulatory mechanisms for CIE cargo trafficking.

Proteins involved in actin filament organization are key host factors for Japanese encephalitis virus life-cycle in human neuronal cells.

Multiple membrane trafficking networks operate in the eukaryotic cell and are hijacked by viruses to establish infection. Recent studied have highlighted that viruses can exploit distinct pathways depending on the cell type. Japanese encephalitis virus (JEV), a neurotropic flavivirus, can infect neuronal cells through a clathrin-independent endocytic mechanism. To further characterize the membrane trafficking requirements for JEV infection of neuronal cells, we have performed a RNA interference-based study targeting 136 proteins in the human cell line IMR-32. Through quantitative RT-PCR and plaque assays we have validated that JEV infection in neuronal cells was independent of clathrin, and identified host-factors that were crucial for establishment of infection. Several of these proteins were involved in regulation of actin filament organization such as RHOA, RAC1, proteins of the ARP2/3 complex and N-WASP family, LIMK1, PAK1 and ROCK2. The small molecule inhibitors of ARP2/3 complex, CK-548 and of the N-WASP, Wiskostatin inhibited virus replication highlighting the important roles of these proteins in the virus life-cycle. We also identified ATG12, BECN1, VAPA, VAPB and VCP proteins as crucial host-factors for JEV replication across epithelial and neuronal cell lineages.

Clathrin- and dynamin-dependent endocytosis limits canonical NF-κB signaling triggered by lymphotoxin ß receptor.

BACKGROUND: Lymphotoxin ß receptor (LTßR) is a member of tumor necrosis factor receptor (TNFR) superfamily which regulates the immune response. At the cellular level, upon ligand binding, the receptor activates the pro-inflammatory NF-κB and AP-1 pathways. Yet, the intracellular distribution of LTßR, the routes of its endocytosis and their connection to the signaling activation are not characterized. Here, we investigated the contribution of LTßR internalization to its signaling potential. METHODS: Intracellular localization of LTßR in unstimulated and stimulated cells was analyzed by confocal microscopy. Endocytosis impairment was achieved through siRNA- or CRISPR/Cas9-mediated depletion, or chemical inhibition of proteins regulating endocytic routes. The activation of LTßR-induced signaling was examined. The levels of effector proteins of the canonical and non-canonical branches of the NF-κB pathway, and the phosphorylation of JNK, Akt, ERK1/2, STAT1 and STAT3 involved in diverse signaling cascades, were measured by Western blotting. A transcriptional response to LTßR stimulation was assessed by qRT-PCR analysis. RESULTS: We demonstrated that LTßR was predominantly present on endocytic vesicles and the Golgi apparatus. The ligand-bound pool of the receptor localized to endosomes and was trafficked towards lysosomes for degradation. Depletion of regulators of different endocytic routes (clathrin-mediated, dynamin-dependent or clathrin-independent) resulted in the impairment of LTßR internalization, indicating that this receptor uses multiple entry pathways. Cells deprived of clathrin and dynamins exhibited enhanced activation of canonical NF-κB signaling represented by increased degradation of IκBα inhibitor and elevated expression of LTßR target genes. We also demonstrated that clathrin and dynamin deficiency reduced to some extent LTßR-triggered activation of the non-canonical branch of the NF-κB pathway. CONCLUSIONS: Our work shows that the impairment of clathrin- and dynamin-dependent internalization amplifies a cellular response to LTßR stimulation. We postulate that receptor internalization restricts responsiveness of the cell to subthreshold stimuli. Video Abstract.

Escherichia coli K1 utilizes host macropinocytic pathways for invasion of brain microvascular endothelial cells.

Eukaryotic cells utilize multiple endocytic pathways for specific uptake of ligands or molecules, and these pathways are commonly hijacked by pathogens to enable host cell invasion. Escherichia coli K1, a pathogenic bacterium that causes neonatal meningitis, invades the endothelium of the blood-brain barrier, but the entry route remains unclear. Here, we demonstrate that the bacteria trigger an actin-mediated uptake route, stimulating fluid phase uptake, membrane ruffling and macropinocytosis. The route of uptake requires intact lipid rafts as shown by cholesterol depletion. Using a variety of perturbants we demonstrate that small Rho GTPases and their downstream effectors have a significant effect on bacterial invasion. Furthermore, clathrin-mediated endocytosis appears to play an indirect role in E. coli K1 uptake. The data suggest that the bacteria effect a complex interplay between the Rho GTPases to increase their chances of uptake by macropinocytosis into human brain microvascular endothelial cells.

Distinct cargo-specific response landscapes underpin the complex and nuanced role of galectin-glycan interactions in clathrin-independent endocytosis.

Clathrin-independent endocytosis (CIE) is a form of endocytosis that lacks a defined cytoplasmic machinery. Here, we asked whether glycan interactions, acting from the outside, could be a part of that endocytic machinery. We show that the perturbation of global cellular patterns of protein glycosylation by modulation of metabolic flux affects CIE. Interestingly, these changes in glycosylation had cargo-specific effects. For example, in HeLa cells, GlcNAc treatment, which increases glycan branching, increased major histocompatibility complex class I (MHCI) internalization but inhibited CIE of the glycoprotein CD59 molecule (CD59). The effects of knocking down the expression of galectin 3, a carbohydrate-binding protein and an important player in galectin-glycan interactions, were also cargo-specific and stimulated CD59 uptake. By contrast, inhibition of all galectin-glycan interactions by lactose inhibited CIE of both MHCI and CD59. None of these treatments affected clathrin-mediated endocytosis, implying that glycosylation changes specifically affect CIE. We also found that the galectin lattice tailors membrane fluidity and cell spreading. Furthermore, changes in membrane dynamics mediated by the galectin lattice affected macropinocytosis, an altered form of CIE, in HT1080 cells. Our results suggest that glycans play an important and nuanced role in CIE, with each cargo being affected uniquely by alterations in galectin and glycan profiles and their interactions. We conclude that galectin-driven effects exist on a continuum from stimulatory to inhibitory, with distinct CIE cargo proteins having unique response landscapes and with different cell types starting at different positions on these conceptual landscapes.

Arf6 and Rab22 mediate T cell conjugate formation by regulating clathrin-independent endosomal membrane trafficking.

Endosomal trafficking can influence the composition of the plasma membrane and the ability of cells to polarize their membranes. Here, we examined whether trafficking through clathrin-independent endocytosis (CIE) affects the ability of T cells to form a cell-cell conjugate with antigen-presenting cells (APCs). We show that CIE occurs in both the Jurkat T cell line and primary human T cells. In Jurkat cells, the activities of two guanine nucleotide binding proteins, Arf6 and Rab22 (also known as Rab22a), influence CIE and conjugate formation. Expression of the constitutively active form of Arf6, Arf6Q67L, inhibits CIE and conjugate formation, and results in the accumulation of vacuoles containing lymphocyte function-associated antigen 1 (LFA-1) and CD4, molecules important for T cell interaction with the APC. Moreover, expression of the GTP-binding defective mutant of Rab22, Rab22S19N, inhibits CIE and conjugate formation, suggesting that Rab22 function is required for these activities. Furthermore, Jurkat cells expressing Rab22S19N were impaired in spreading onto coverslips coated with T cell receptor-activating antibodies. These observations support a role for CIE, Arf6 and Rab22 in conjugate formation between T cells and APCs.

Ikarugamycin: A Natural Product Inhibitor of Clathrin-Mediated Endocytosis.

Ikarugamycin (IKA) is a previously discovered antibiotic, which has been shown to inhibit the uptake of oxidized low-density lipoproteins in macrophages. Furthermore, several groups have previously used IKA to inhibit clathrin-mediated endocytosis (CME) in plant cell lines. However, detailed characterization of IKA has yet to be performed. Consequently, we performed biochemistry and microscopy experiments to further characterize the effects of IKA on CME. We show that IKA has an IC50 of 2.7 µm in H1299 cells and acutely inhibits CME, but not other endocytic pathways, in a panel of cell lines. Although long-term incubation with IKA has cytotoxic effects, the short-term inhibitory effects on CME are reversible. Thus, IKA can be a useful tool for probing routes of endocytic trafficking.

Local actin polymerization during endocytic carrier formation.

Extracellular macromolecules, pathogens and cell surface proteins rely on endocytosis to enter cells. Key steps of endocytic carrier formation are cargo molecule selection, plasma membrane folding and detachment from the cell surface. While dedicated proteins mediate each step, the actin cytoskeleton contributes to all. However, its role can be indirect to the actual molecular events driving endocytosis. Here, we review our understanding of the molecular steps mediating local actin polymerization during the formation of endocytic carriers. Clathrin-mediated endocytosis is the least reliant on local actin polymerization, as it is only engaged to counter forces induced by membrane tension or cytoplasmic pressure. Two opposite situations are coated pit formation in yeast and at the basolateral surface of polarized mammalian cells which are, respectively, dependent and independent on actin polymerization. Conversely, clathrin-independent endocytosis forming both nanometer [CLIC (clathrin-independent carriers)/GEEC (glycosylphosphatidylinositol (GPI)-anchored protein enriched endocytic compartments), caveolae, FEME (fast endophilin-mediated endocytosis) and IL-2ß (interleukin-2ß) uptake] and micrometer carriers (macropinocytosis) are dependent on actin polymerization to power local membrane deformation and carrier budding. A variety of endocytic adaptors can recruit and activate the Cdc42/N-WASP or Rac1/WAVE complexes, which, in turn, engage the Arp2/3 complex, thereby mediating local actin polymerization at the membrane. However, the molecular steps for RhoA and formin-mediated actin bundling during endocytic pit formation remain unclear.

Microtubule motors power plasma membrane tubulation in clathrin-independent endocytosis.

How the plasma membrane is bent to accommodate clathrin-independent endocytosis remains uncertain. Recent studies suggest Shiga and cholera toxin induce membrane curvature required for their uptake into clathrin-independent carriers by binding and cross-linking multiple copies of their glycosphingolipid receptors on the plasma membrane. But it remains unclear if toxin-induced sphingolipid crosslinking provides sufficient mechanical force for deforming the plasma membrane, or if host cell factors also contribute to this process. To test this, we imaged the uptake of cholera toxin B-subunit into surface-derived tubular invaginations. We found that cholera toxin mutants that bind to only one glycosphingolipid receptor accumulated in tubules, and that toxin binding was entirely dispensable for membrane tubulations to form. Unexpectedly, the driving force for tubule extension was supplied by the combination of microtubules, dynein and dynactin, thus defining a novel mechanism for generating membrane curvature during clathrin-independent endocytosis.