Clathrin-independent endocytosis contributes to uptake of glucose into BY-2 protoplasts

In eukaryotic cells, several pathways exist for the internalization of plasma membrane proteins and extracellular cargo molecules. These endocytic pathways can be divided into clathrin-dependent and clathrin-independent pathways. While clathrin-dependent pathways are known to be involved in a variety of cellular processes in plants, clathrin-independent pathways have so far only been identified in animal and yeast cells. Here we show that internalization of fluorescent glucose into BY-2 cells leads to accumulation of the sugar in compartments of the endocytic pathway. This endocytic uptake of glucose was not blocked by ikarugamycin, an inhibitor of clathrin-dependent endocytosis, suggesting a role for clathrin-independent endocytosis in glucose uptake. Investigations of fusion and fission of single vesicles by membrane capacitance measurements revealed stimulation of endocytic activity by extracellular glucose. Glucose-stimulated fission of vesicles was not affected by addition of ikarugamycin or blocking of clathrin coat formation by transient over-expression of HUB1 (the C-terminal part of the clathrin heavy chain). These data demonstrate that clathrin-independent endocytosis does occur in plant cells. This pathway may represent a common mechanism for the uptake of external nutrients.     

Double-stranded RNA is internalized by scavenger receptor-mediated endocytosis in Drosophila S2 cells

Double-stranded RNA (dsRNA) fragments are readily internalized and processed by Drosophila S2 cells, making these cells a widely used tool for the analysis of gene function by gene silencing through RNA interference (RNAi). The underlying mechanisms are insufficiently understood. To identify components of the RNAi pathway in S2 cells, we developed a screen based on rescue from RNAi-induced lethality. We identified Argonaute 2, a core component of the RNAi machinery, and three gene products previously unknown to be involved in RNAi in Drosophila: DEAD-box RNA helicase Belle, 26 S proteasome regulatory subunit 8 (Pros45), and clathrin heavy chain, a component of the endocytic machinery. Blocking endocytosis in S2 cells impaired RNAi, suggesting that dsRNA fragments are internalized by receptor-mediated endocytosis. Indeed, using a candidate gene approach, we identified two Drosophila scavenger receptors, SR-CI and Eater, which together accounted for more than 90% of the dsRNA uptake into S2 cells. When expressed in mammalian cells, SR-CI was sufficient to mediate internalization of dsRNA fragments. Our data provide insight into the mechanism of dsRNA internalization by Drosophila cells. These results have implications for dsRNA delivery into mammalian cells.     

The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing

Many metazoan cells can take up exogenous double-stranded (ds) RNA and use it to initiate an RNA silencing response, however, the mechanism for this uptake is ill-defined. Here, we identify the pathway for dsRNA uptake in Drosophila melanogaster S2 cells. Biochemical and cell biological analyses, and a genome-wide screen for components of the dsRNA-uptake machinery, indicated that dsRNA is taken up by an active process involving receptor-mediated endocytosis. Pharmacological inhibition of endocytic pathways disrupted exogenous dsRNA entry and the induction of gene silencing. This dsRNA uptake mechanism seems to be evolutionarily conserved, as knockdown of orthologues in Caenorhabditis elegans inactivated the RNA interference response in worms. Thus, this entry pathway is required for systemic RNA silencing in whole organisms. In Drosophila cells, pharmacological evidence suggests that dsRNA entry is mediated by pattern-recognition receptors. The possible role of these receptors in dsRNA entry may link RNA interference (RNAi) silencing to other innate immune responses.     

Endocytic pathways in pollen tube: Implications for in vivo growth regulation

By endocytosis eukaryotic cells can take up extracellular components and/or plasma membrane proteins for further delivering to endosomes. Although in animal cells different endocytic pathways were identified based on the requirement of a clathrin coating for vesicle internalization, endocytosis in plant cells still require to be fully characterized. The use of positively and negatively charged nanogold in combination with Ika, an inhibitor of the clathrindependent endocytosis, allowed to dissect the endocytic pathway and revealed the presence of clathrin-dependent and clathrin-independent degradative pathways.Key words: Nicotiana tabacum, pollen tube growth, endocytosis     

Clathrin Heavy Chain Is Important for Viability,Oviposition, Embryogenesis and,Possibly, Systemic RNAi Response in the Predatory Mite Metaseiulus occidentalis

Clathrin heavy chain has been shown to be important for viability, embryogenesis, and RNA interference (RNAi) in arthropods such as Drosophila melanogaster. However, the functional roles of clathrin heavy chain in chelicerate arthropods, such as the predatory mite Metaseiulus occidentalis, remain unknown. We previously showed that dsRNA ingestion, followed by feeding on spider mites, induced systemic and robust RNAi in M. occidentalis females. In the current study, we performed a loss-of-function analysis of the clathrin heavy chain gene in M. occidentalis using RNAi. We showed that ingestion of clathrin heavy chain dsRNA by M. occidentalis females resulted in gene knockdown and reduced longevity. In addition, clathrin heavy chain dsRNA treatment almost completely abolished oviposition by M. occidentalis females and the few eggs produced did not hatch. Finally, we demonstrated that clathrin heavy chain gene knockdown in M. occidentalis females significantly reduced a subsequent RNAi response induced by ingestion of cathepsin L dsRNA. The last finding suggests that clathrin heavy chain may be involved in systemic RNAi responses mediated by orally delivered dsRNAs in M. occidentalis.     

Regulation of clathrin-dependent endocytosis by diacylglycerol kinase delta: importance of kinase activity and binding to AP2alpha

DGKdelta (diacylglycerol kinase delta), which phosphorylates DAG (diacylglycerol) and converts it into PA (phosphatidic acid), has an important role in signal transduction. In the present study, we have demonstrated the molecular mechanism of DGKdelta-mediated regulation of clathrin-dependent endocytosis that controls the internalization, recycling and degradation of receptors. Involvement of DGKdelta in the regulation of clathrin-dependent endocytosis was previously proposed following genome-wide RNAi (RNA interference) screening. Clathrin-coated pits are mainly formed by clathrin and AP-2 (adaptor protein 2) complex. These proteins assemble a polyhedral lattice at the membrane and gather several endocytic accessory proteins. As the intracellular localization of DGKdelta2 overlapped with clathrin-coated pits, we predicted the possible regulation of clathrin-dependent endocytosis by DGKdelta2 and its interaction with some endocytosis-regulatory proteins. DGKdelta2 contained the DXF-type binding motifs, and DGKdelta2 bound to AP2alpha, a subunit of the AP-2 complex. DGKdelta2 interacted with the platform subdomain in the AP2alpha ear domain via F369DTFRIL and D746PF sequences in the catalytic domain of DGKdelta2. For further insight into the role for DGKdelta2 in clathrin-dependent endocytosis, we measured the transferrin and EGF (epidermal growth factor) uptake-expressing wild-type or mutant DGKdelta2 under knockdown of endogenous DGKdelta. Mutants lacking binding ability to AP2alpha as well as kinase-negative mutants could not compensate for the uptake of transferrin inhibited by siRNA (small interfering RNA) treatment, whereas overexpression of wild-type DGKdelta2 completely recovered the transferrin uptake. These results demonstrate that binding between DGKdelta2 and AP2alpha is involved in the transferrin internalization and that DGK activity is also necessary for the regulation of the endocytic process.     

The Tyro3 receptor kinase Axl enhances macropinocytosis of Zaire ebolavirus

Axl, a plasma membrane-associated Tyro3/Axl/Mer (TAM) family member, is necessary for optimal Zaire ebolavirus (ZEBOV) glycoprotein (GP)-dependent entry into some permissive cells but not others. To date, the role of Axl in virion entry is unknown. The focus of this study was to characterize entry pathways that are used for ZEBOV uptake in cells that require Axl for optimal transduction and to define the role of Axl in this process. Through the use of biochemical inhibitors, interfering RNA (RNAi), and dominant negative constructs, we demonstrate that ZEBOV-GP-dependent entry into these cells occurs through multiple uptake pathways, including both clathrin-dependent and caveola/lipid raft-mediated endocytosis. Other dynamin-dependent and -independent pathways such as macropinocytosis that mediate high-molecular-weight dextran uptake also stimulated ZEBOV-GP entry into these cells, and inhibitors that are known to block macropinocytosis inhibited both dextran uptake and ZEBOV infection. These findings provided strong evidence for the importance of this pathway in filovirus entry. Reduction of Axl expression by RNAi treatment resulted in decreased ZEBOV entry via macropinocytosis but had no effect on the clathrin-dependent or caveola/lipid raft-mediated endocytic mechanisms. Our findings demonstrate for the first time that Axl enhances macropinocytosis, thereby increasing productive ZEBOV entry.     

Gene Transfer by Means of Lipo- and Polyplexes: Role of Clathrin and Caveolae-Mediated Endocytosis

In this paper we address the contribution of different endocytic pathways to the intracellular uptake and processing of differently sized latex particles and of plasmid DNA complexes by means of fluorescence microscopy and FACS analysis. By using a number of specific inhibitors of either clathrin-dependent or caveolae-dependent endocytosis we were able to discriminate between these two pathways. Latex particles smaller than 200 nm were internalized exclusively by clathrin-mediated endocytosis, whereas larger particles entered the cells via a caveolae-dependent pathway.

The route of uptake of plasmid DNA complexes appears strongly dependent on the nature of the complexes. Thus, lipoplexes containing the cationic lipid DOTAP, were exclusively internalized by a clathrin-dependent mechanism, while polyplexes prepared from the cationic polymer polyethyleneimine (PEI) were internalized in roughly equal proportions by both pathways. Upon incubation of cells with lipoplexes containing the luciferase gene abundant luciferase expression was observed, which was effectively blocked by inhibitors of clathrin-dependent endocytosis but not by inhibitors of the caveolae-dependent uptake mechanism. By contrast, luciferase transfection of the cells with polyplexes was unaffected by inhibition of clathrin-mediated endocytosis, but was nearly completely blocked by inhibitors interfering with the caveolae pathway. The results are discussed with respect to possible differences in the mechanism by which plasmid DNA is released from lipoplexes and polyplexes into the cytosol and to the role of size in the uptake and processing of the complexes. Our data suggest that improvement of non-viral gene transfection could very much benefit from controlling particle size, which would allow targeting of particle internalization via a non-degradative pathway, involving caveolae-mediated endocytosis.     

Clathrin-independent endocytosis used by the IL-2 receptor is regulated by Rac1, Pak1 and Pak2

There are several endocytic pathways, which are either dependent on or independent of clathrin. This study focuses on a poorly characterized mechanism-clathrin- and caveolae-independent endocytosis-used by the interleukin-2 receptor beta (IL-2R beta). We address the question of its regulation in comparison with the clathrin-dependent pathway. First, we show that Ras-related C3 botulinum toxin substrate 1 (Rac1) is specifically required for IL-2R beta entry, and we identify p21-activated kinases (Paks) as downstream targets. By RNA interference, we show that Pak1 and Pak2 are both necessary for IL-2R beta uptake, in contrast to the clathrin-dependent route. We observe that cortactin, a partner of actin and dynamin-two essential endocytic factors-is required for IL-2R beta uptake. Furthermore, we find that cortactin acts downstream from Paks, suggesting control of its function by these kinases. Thus, we describe a cascade composed of Rac1, Paks and cortactin specifically regulating IL-2R beta internalization. This study indicates Paks as the first specific regulators of the clathrin-independent endocytosis pathway.     

Cortactin and dynamin are required for the clathrin-independent endocytosis of gammac cytokine receptor

Endocytosis is critical for many cellular functions. We show that endocytosis of the common gammac cytokine receptor is clathrin independent by using a dominant-negative mutant of Eps15 or RNA interference to knock down clathrin heavy chain. This pathway is synaptojanin independent and requires the GTPase dynamin. In addition, this process requires actin polymerization. To further characterize the function of dynamin in clathrin-independent endocytosis, in particular its connection with the actin cytoskeleton, we focused on dynamin-binding proteins that interact with F-actin. We compared the involvement of these proteins in the clathrin-dependent and -independent pathways. Thus, we observed that intersectin, syndapin, and mAbp1, which are necessary for the uptake of transferrin (Tf), a marker of the clathrin route, are not required for gammac receptor endocytosis. Strikingly, cortactin is needed for both gammac and Tf internalizations. These results reveal the ubiquitous action of cortactin in internalization processes and suggest its role as a linker between actin dynamics and clathrin-dependent and -independent endocytosis.     

Rift Valley Fever Virus Strain MP-12 Enters Mammalian Host Cells via Caveola-Mediated Endocytosis

Rift Valley fever virus (RVFV) is a zoonotic pathogen capable of causing serious morbidity and mortality in both humans and livestock. The lack of efficient countermeasure strategies, the potential for dispersion into new regions, and the pathogenesis in humans and livestock make RVFV a serious public health concern. The receptors, cellular factors, and entry pathways used by RVFV and other members of the family Bunyaviridae remain largely uncharacterized. Here we provide evidence that RVFV strain MP-12 uses dynamin-dependent caveola-mediated endocytosis for cell entry. Caveolae are lipid raft domains composed of caveolin (the main structural component), cholesterol, and sphingolipids. Caveola-mediated endocytosis is responsible for the uptake of a wide variety of host ligands, as well as bacteria, bacterial toxins, and a number of viruses. To determine the cellular entry mechanism of RVFV, we used small-molecule inhibitors, RNA interference (RNAi), and dominant negative (DN) protein expression to inhibit the major mammalian cell endocytic pathways. Inhibitors and RNAi specific for macropinocytosis and clathrin-mediated endocytosis had no effect on RVFV infection. In contrast, inhibitors of caveola-mediated endocytosis, and RNAi targeted to caveolin-1 and dynamin, drastically reduced RVFV infection in multiple cell lines. Expression of DN caveolin-1 also reduced RVFV infection significantly, while expression of DN EPS15, a protein required for the assembly of clathrin-coated pits, and DN PAK-1, an obligate mediator of macropinocytosis, had no significant impact on RVFV infection. These results together suggest that the primary mechanism of RVFV MP-12 uptake is dynamin-dependent, caveolin-1-mediated endocytosis.     

The endocytic adaptor protein Disabled-2 is required for cellular uptake of fibrinogen

Endocytosis is pivotal for uptake of fibrinogen from plasma into megakaryocytes and platelet α-granules. Due to the complex adaptor and cargo contents in endocytic vehicles, the underlying mechanism of fibrinogen uptake is not yet completely elucidated. In this study, we investigated whether the endocytic adaptor protein Disabled-2 (DAB2) mediates fibrinogen uptake in an adaptor-specific manner. By employing primary megakaryocytes and megakaryocytic differentiating human leukemic K562 cells as the study models, we found that fibrinogen uptake is associated with the expression of integrin αIIbβ3 and DAB2 and is mediated through clathrin-dependent manner. Accordingly, constitutive and inducible knockdown of DAB2 by small interfering RNA reduced fibrinogen uptake for 53.2 ± 9.8% and 59.0 ± 10.7%, respectively. Culturing the cells in hypertonic solution or in the presence of clathrin inhibitor chlorpromazine abrogated clathrin-dependent endocytosis and diminished the uptake of fibrinogen. Consistent with these findings, 72.2 ± 0.2% of cellular DAB2 was colocalized with clathrin, whereas 56.4±4.1% and 54.6 ± 2.0% of the internalized fibrinogen were colocalized with clathrin and DAB2, respectively. To delineate whether DAB2 mediates fibrinogen uptake in an adaptor-specific manner, K562 stable cell lines with knockdown of the adaptor protein-2 (AP-2) or double knockdown of AP-2/DAB2 were established. The AP-2 knockdown cells elicited normal fibrinogen uptake activity but the uptake of collagen was diminished. In addition, collagen uptake was further reduced in DAB2/AP-2 knockdown cells. These findings thereby define an adaptor-specific mechanism in the control of fibrinogen uptake and implicate that DAB2 is the key adaptor in the clathrin-associated endocytic complexes to mediate fibrinogen internalization.     

Notch down-regulation by endocytosis is essential for pigment cell determination and survival in the Drosophila retina

The clathrin heavy chain is a fundamental element in endocytosis and therefore, in the internalization of several cell-surface receptors through which cells interact with their environment. Here we show that the only non-lethal mutant allele of the clathrin heavy chain identified to date in metazoans, the Drosophila Chc⁴, involves the substitution of a residue at the knee region of the molecule that impairs clathrin-dependent endocytosis. We have investigated the consequences of this endocytic defect in Drosophila retinal development and found that it produces an inhibition of programmed cell death in the retinal lattice, followed by widespread death of interommatidial pigment cells once retinal development has been completed. Through genetic interactions and transgenic analyses, we show that Chc⁴ phenotypes are caused by a Notch receptor gain-of-function, providing a dramatic example of the importance of Notch down-regulation by endocytosis. An increase in Notch signaling is also observed in Drosophila wings in response to the mutant clathrin, suggesting that Notch levels are controlled by clathrin-dependent endocytosis. We discuss the implications of these findings for current models on eye-development and for the role of endocytosis in Notch signaling.     

Caenorhabditis elegans reveals a FxNPxY-independent low-density lipoprotein receptor internalization mechanism mediated by epsin1

Low-density lipoprotein receptor (LDLR) internalization clears cholesterol-laden LDL particles from circulation in humans. Defects in clathrin-dependent LDLR endocytosis promote elevated serum cholesterol levels and can lead to atherosclerosis. However, our understanding of the mechanisms that control LDLR uptake remains incomplete. To identify factors critical to LDLR uptake, we pursued a genome-wide RNA interference screen using Caenorhabditis elegans LRP-1/megalin as a model for LDLR transport. In doing so, we discovered an unanticipated requirement for the clathrin-binding endocytic adaptor epsin1 in LDLR endocytosis. Epsin1 depletion reduced LDLR internalization rates in mammalian cells, similar to the reduction observed following clathrin depletion. Genetic and biochemical analyses of epsin in C. elegans and mammalian cells uncovered a requirement for the ubiquitin-interaction motif (UIM) as critical for receptor transport. As the epsin UIM promotes the internalization of some ubiquitinated receptors, we predicted LDLR ubiquitination as necessary for endocytosis. However, engineered ubiquitination-impaired LDLR mutants showed modest internalization defects that were further enhanced with epsin1 depletion, demonstrating epsin1-mediated LDLR endocytosis is independent of receptor ubiquitination. Finally, we provide evidence that epsin1-mediated LDLR uptake occurs independently of either of the two documented internalization motifs (FxNPxY or HIC) encoded within the LDLR cytoplasmic tail, indicating an additional internalization mechanism for LDLR.     

Gene transfer by means of lipo- and polyplexes: role of clathrin and caveolae-mediated endocytosis

In this paper we address the contribution of different endocytic pathways to the intracellular uptake and processing of differently sized latex particles and of plasmid DNA complexes by means of fluorescence microscopy and FACS analysis. By using a number of specific inhibitors of either clathrin-dependent or caveolae-dependent endocytosis we were able to discriminate between these two pathways. Latex particles smaller than 200 nm were internalized exclusively by clathrin-mediated endocytosis, whereas larger particles entered the cells via a caveolae-dependent pathway.The route of uptake of plasmid DNA complexes appears strongly dependent on the nature of the complexes. Thus, lipoplexes containing the cationic lipid DOTAP, were exclusively internalized by a clathrin-dependent mechanism, while polyplexes prepared from the cationic polymer polyethyleneimine (PEI) were internalized in roughly equal proportions by both pathways. Upon incubation of cells with lipoplexes containing the luciferase gene abundant luciferase expression was observed, which was effectively blocked by inhibitors of clathrin-dependent endocytosis but not by inhibitors of the caveolae-dependent uptake mechanism. By contrast, luciferase transfection of the cells with polyplexes was unaffected by inhibition of clathrin-mediated endocytosis, but was nearly completely blocked by inhibitors interfering with the caveolae pathway. The results are discussed with respect to possible differences in the mechanism by which plasmid DNA is released from lipoplexes and polyplexes into the cytosol and to the role of size in the uptake and processing of the complexes. Our data suggest that improvement of non-viral gene transfection could very much benefit from controlling particle size, which would allow targeting of particle internalization via a non-degradative pathway, involving caveolae-mediated endocytosis.     

Endoplasmic reticulum stress enhances endocytosis in calreticulin deficient cells

Calreticulin an endoplasmic reticulum (ER) chaperone that is involved in the quality control process and plays an important role as a regulator of intracellular calcium homeostasis. Previously, we illustrated that loss of calreticulin (crt−/−) results in the activation of ubiquitin-proteasome pathway facilitating the increased resistance to apoptosis. Our preliminary data illustrated a significant increase in the endocytosis in the calreticulin knockout mouse embryonic fibroblast cells (crt−/−). Therefore, we hypothesized that the mechanism for this increased endocytosis in the crt−/− cells is due to onset of ER stress. To test this hypothesis, we measured endocytosis in the wild type (wt) and crt−/− cells using uptake of fluorescent dextran and showed a significant increase in the rate of its uptake in crt−/− cells as compared to wt cells.To determine the endocytic pathway involved we examined both clathrin and caveolin-1 dependent endocytosis. Our results illustrated no change in the expression of clathrin heavy chain while there was a significant increase in the expression of caveolin-1 in the crt−/− cells as compared to the wt cells. Furthermore, using shRNA we illustrated that knockdown of clathrin heavy chain had no effect on endocytosis in the crt−/− cells. While knock-down of caveolin-1 significantly reduced endocytosis in the crt−/− cells. Finally, we illustrated that a chemical chaperone, 4‑phenylbutyrate significantly reduced both the endoplasmic reticulum stress and endocytosis in the crt−/− cells. Our data shows for the first time, that ER stress led to enhanced caveolin-1 mediated endocytosis and reversal of ER stress reduces endocytosis.     

Clathrin-dependent targeting of receptors to the flagellar pocket of procyclic-form Trypanosoma brucei

In trypanosomatids, endocytosis and exocytosis occur exclusively at the flagellar pocket, which represents about 0.43% of the pellicle membrane and is a deep invagination of the plasma membrane where the flagellum extends from the cell. Receptor molecules are selectively retained at the flagellar pocket. We studied the function of clathrin heavy chain (TbCLH) in the trafficking of the flagellar pocket receptors in Trypanosoma brucei by using the double-stranded RNA interference approach. It appears that TbCLH is essential for the survival of both the procyclic form and the bloodstream form of T. brucei, even though structures resembling large coated endocytic vesicles are absent in procyclic-form trypanosomes. Down-regulation of TbCLH by RNA interference (RNAi) for 24 h rapidly and drastically reduced the uptake of macromolecules via receptor-mediated endocytosis in procyclic-form trypanosomes. This result suggested the importance of TbCLH in receptor-mediated endocytosis of the procyclic-form trypanosome, in which the formation of large coated endocytic vesicles may not be required. Surprisingly, induction of TbCLH RNAi in the procyclic T. brucei for a period of 48 h prohibited the export of the flagellar pocket-associated transmembrane receptor CRAM from the endoplasmic reticulum to the flagellar pocket, while trafficking of the glycosylphosphatidylinositol-anchored procyclin coat was not significantly affected. After 72 h of induction of TbCLH RNAi, procyclics exhibited morphological changes to an apolar round shape without a distinct structure of the flagellar pocket and flagellum. Although trypanosomes, like other eukaryotes, use similar organelles and machinery for protein sorting and transport, our studies reveal a novel role for clathrin in the secretory pathway of trypanosomes. We speculate that the clathrin-dependent trafficking of proteins to the flagellar pocket may be essential for the biogenesis and maintenance of the flagellar pocket in trypanosomes.     

Mouse hepatitis virus type 2 enters cells through a clathrin-mediated endocytic pathway independent of Eps15

It has recently been shown that cell entry of mouse hepatitis virus type 2 (MHV-2) is mediated through endocytosis (Z. Qiu et al., J. Virol. 80:5768-5776, 2006). However, the molecular mechanism underlying MHV-2 entry is not known. Here we employed multiple chemical and molecular approaches to determine the molecular pathways for MHV-2 entry. Our results showed that MHV-2 gene expression and infectivity were significantly inhibited when cells were treated with chemical and physiologic blockers of the clathrin-mediated pathway, such as chlorpromazine and hypertonic sucrose medium. Furthermore, viral gene expression was significantly inhibited when cells were transfected with a small interfering RNA specific to the clathrin heavy chain. However, these treatments did not affect the infectivity and gene expression of MHV-A59, demonstrating the specificity of the inhibitions. In addition, overexpression of a dominant-negative mutant of caveolin 1 did not have any effect on MHV-2 infection, while it significantly blocked the caveolin-dependent uptake of cholera toxin subunit B. These results demonstrate that MHV-2 utilizes the clathrin- but not caveolin-mediated endocytic pathway for entry. Interestingly, when the cells transiently overexpressed a dominant-negative form (DIII) of Eps15, which is thought to be an essential component of the clathrin pathway, viral gene expression and infectivity were unaffected, although DIII expression blocked transferrin uptake and vesicular stomatitis virus infection, which are dependent on clathrin-mediated endocytosis. Thus, MHV-2 entry is mediated through clathrin-dependent but Eps15-independent endocytosis.     

The length of vesicular stomatitis virus particles dictates a need for actin assembly during clathrin-dependent endocytosis

Microbial pathogens exploit the clathrin endocytic machinery to enter host cells. Vesicular stomatitis virus (VSV), an enveloped virus with bullet-shaped virions that measure 70 x 200 nm, enters cells by clathrin-dependent endocytosis. We showed previously that VSV particles exceed the capacity of typical clathrin-coated vesicles and instead enter through endocytic carriers that acquire a partial clathrin coat and require local actin filament assembly to complete vesicle budding and internalization. To understand why the actin system is required for VSV uptake, we compared the internalization mechanisms of VSV and its shorter (75 nm long) defective interfering particle, DI-T. By imaging the uptake of individual particles into live cells, we found that, as with parental virions, DI-T enters via the clathrin endocytic pathway. Unlike VSV, DI-T internalization occurs through complete clathrin-coated vesicles and does not require actin polymerization. Since VSV and DI-T particles display similar surface densities of the same attachment glycoprotein, we conclude that the physical properties of the particle dictate whether a virus-containing clathrin pit engages the actin system. We suggest that the elongated shape of a VSV particle prevents full enclosure by the clathrin coat and that stalling of coat assembly triggers recruitment of the actin machinery to finish the internalization process. Since some enveloped viruses have pleomorphic particle shapes and sizes, our work suggests that they may use altered modes of endocytic uptake. More generally, our findings show the importance of cargo geometry for specifying cellular entry modes, even when the receptor recognition properties of a ligand are maintained.     

Systemic RNA Interference Deficiency-1 (SID-1) Extracellular Domain Selectively Binds Long Double-stranded RNA and Is Required for RNA Transport by SID-1

During systemic RNA interference (RNAi) in Caenorhabditis elegans, RNA spreads across different cells and tissues in a process that requires the systemic RNA interference deficient-1 (sid-1) gene, which encodes an integral membrane protein. SID-1 acts cell-autonomously and is required for cellular import of interfering RNAs. Heterologous expression of SID-1 in Drosophila Schneider 2 cells enables passive uptake of dsRNA and subsequent soaking RNAi. Previous studies have suggested that SID-1 may serve as an RNA channel, but its precise molecular role remains unclear. To test the hypothesis that SID-1 mediates a direct biochemical recognition of RNA molecule and subsequent permeation, we expressed the extracellular domain (ECD) of SID-1 and purified it to near homogeneity. Recombinant purified SID-1 ECD selectively binds dsRNA but not dsDNA in a length-dependent and sequence-independent manner. Genetic missense mutations in SID-1 ECD causal for deficient systemic RNAi resulted in significant reduction in its affinity for dsRNA. Furthermore, full-length proteins with these mutations decrease SID-1-mediated RNA transport efficiency, providing evidence that dsRNA binding to SID-1 ECD is related to RNA transport. To examine the functional similarity of mammalian homologs of SID-1 (SIDT1 and SIDT2), we expressed and purified mouse SIDT1 and SIDT2 ECDs. We show that they bind long dsRNA in vitro, supportive of dsRNA recognition. In summary, our study illustrates the functional importance of SID-1 ECD as a dsRNA binding domain that contributes to RNA transport.