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.      

Endocytosis of Ubiquitylation‐Deficient EGFR Mutants via Clathrin‐Coated Pits is Mediated by Ubiquitylation

Signaling by epidermal growth factor receptor (EGFR) is controlled by endocytosis. However, mechanisms of EGFR endocytosis remain poorly understood. Here, we found that the EGFR mutant lacking known ubiquitylation, acetylation and clathrin adaptor AP‐2‐binding sites (21KRΔAP2) was internalized at relatively high rates via the clathrin‐dependent pathway in human duodenal adenocarcinoma HuTu‐80 cells. RNA interference analysis revealed that this residual internalization is strongly inhibited by depletion of Grb2 and the E2 ubiquitin‐conjugating enzyme UbcH5b/c, and partially affected by depletion of the E3 ubiquitin ligase Cbl and ubiquitin‐binding adaptors, indicating that an ubiquitylation process is involved. Several new ubiquitin conjugation sites were identified by mass spectrometry in the 21KRΔAP2 mutant, suggesting that cryptic ubiquitylation may mediate endocytosis of this mutant. Total internal reflection fluorescence microscopy imaging of HuTu‐80 cells transfected with labeled ubiquitin adaptor epsin1 demonstrated that the ubiquitylation‐deficient EGFR mutant was endocytosed through a limited population of epsin‐enriched clathrin‐coated pits (CCPs), although with a prolonged CCP lifetime. Native EGFR was recruited with the same efficiency into CCPs containing either AP‐2 or epsin1 that were tagged with fluorescent proteins by genome editing of MDA‐MD‐231 cells. We propose that two redundant mechanisms, ubiquitylation and interaction with AP‐2, contribute to EGFR endocytosis via CCPs in a stochastic fashion.      

Sorting of Clathrin‐Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin‐Mediated Endocytosis

Clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE) co‐exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6‐associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6‐GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6‐GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin‐coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6‐GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.      

Clathrin‐Mediated Endocytosis at the Synaptic Terminal: Bridging the Gap Between Physiology and Molecules

It has long been known that the maintenance of fast communication between neurons requires that presynaptic terminals recycle the small vesicles from which neurotransmitter is released. But the mechanisms that retrieve vesicles from the cell surface are still not understood. Although we have a wealth of information about the molecular details of endocytosis in non‐neuronal cells, it is clear that endocytosis at the synapse is faster and regulated in distinct ways. A satisfying understanding of these processes will require molecular events to be manipulated while observing endocytosis in living synapses. Here, we review recent work that seeks to bridge the gap between physiology and molecules to unravel the endocytic machinery operating at the synaptic terminal.     

AP180 Couples Protein Retrieval to Clathrin‐Mediated Endocytosis of Synaptic Vesicles

How clathrin‐mediated endocytosis (CME) retrieves vesicle proteins into newly formed synaptic vesicles (SVs) remains a major puzzle. Besides its roles in stimulating clathrin‐coated vesicle formation and regulating SV size, the clathrin assembly protein AP180 has been identified as a key player in retrieving SV proteins. The mechanisms by which AP180 recruits SV proteins are not fully understood. Here, we show that following acute inactivation of AP180 in Drosophila, SV recycling is severely impaired at the larval neuromuscular synapse based on analyses of FM 1‐43 uptake and synaptic ultrastructure. More dramatically, AP180 activity is important to maintain the integrity of SV protein complexes at the plasma membrane during endocytosis. These observations suggest that AP180 normally clusters SV proteins together during recycling. Consistent with this notion, SV protein composition and distribution are altered in AP180 mutant flies. Finally, AP180 co‐immunoprecipitates with SV proteins, including the vesicular glutamate transporter and neuronal synaptobrevin. These results reveal a new mode by which AP180 couples protein retrieval to CME of SVs. AP180 is also genetically linked to Alzheimer's disease. Hence, the findings of this study may provide new mechanistic insight into the role of AP180 dysfunction in Alzheimer's disease.      

Clathrin‐mediated endocytosis: What works for small,also works for big

Clathrin and the endocytosis machinery has recently been described as being required in mammalian cells for the internalization of large particles including pathogenic bacteria, fungi, and large viruses. These apparently unexpected observations, within the framework of the classical mechanisms for the formation of clathrin‐coated vesicles, are now considered as examples of a new non‐classical function of clathrin, which can promote the internalization of membrane domains associated to planar clathrin lattices. The role of actin downstream of clathrin seems to be critical for this still poorly characterized process. The historical frontier between endocytosis and phagocytosis is vanishing in the light of this new role for clathrin.     

Polarised Clathrin‐Mediated Endocytosis of EGFR During Chemotactic Invasion

Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin‐mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA‐MB‐231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin‐mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin‐mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin‐mediated endocytosis to directed cell motility.      

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.      

Disruption of Clathrin‐Mediated Trafficking Causes Centrosome Overduplication and Senescence

The Hsc70 cochaperone, G cyclin‐associated kinase (GAK), has been shown to be essential for the chaperoning of clathrin by Hsc70 in the cell. In this study, we used conditional GAK knockout mouse embryonic fibroblasts (MEFs) to determine the effect of completely inhibiting clathrin‐dependent trafficking on the cell cycle. After GAK was knocked out, the cells developed the unusual phenotype of having multiple centrosomes, but at the same time failed to divide and ultimately became senescent. To explain this phenotype, we examined the signaling profile and found that mitogenic stimulation of the GAK KO cells and the control cells were similar except for increased phosphorylation of Akt. In addition, the disruption of intracellular trafficking caused by knocking out GAK destabilized the lysosomal membranes, resulting in DNA damage due to iron leakage. Knocking down clathrin heavy chain or inhibiting dynamin largely reproduced the GAK KO phenotype, but inhibiting only clathrin‐mediated endocytosis by knocking down adaptor protein (AP2) caused growth arrest and centrosome overduplication, but no DNA damage or senescence. We conclude that disruption of clathrin‐dependent trafficking induces senescence accompanied by centrosome overduplication because of a combination of DNA damage and changes in mitogenic signaling that uncouples centrosomal duplication from DNA replication.     

Myosin VI binds to and localises with Dab2, potentially linking receptor-mediated endocytosis and the actin cytoskeleton

Myosin VI, an actin-based motor protein, and Disabled 2 (Dab2), a molecule involved in endocytosis and cell signalling, have been found to bind together using yeast and mammalian two-hybrid screens. In polarised epithelial cells, myosin VI is known to be associated with apical clathrin-coated vesicles and is believed to move them towards the minus end of actin filaments, away from the plasma membrane and into the cell. Dab2 belongs to a group of signal transduction proteins that bind in vitro to the FXNPXY sequence found in the cytosolic tails of members of the low-density lipoprotein receptor family. The central region of Dab2, containing two DPF motifs, binds to the clathrin adaptor protein AP-2, whereas a C-terminal region contains the binding site for myosin VI. This site is conserved in Dab1, the neuronal counterpart of Dab2. The interaction between Dab2 and myosin VI was confirmed by in vitro binding assays and coimmunoprecipitation and by their colocalisation in clathrin-coated pits/vesicles concentrated at the apical domain of polarised cells. These results suggest that the myosin VI–Dab2 interaction may be one link between the actin cytoskeleton and receptors undergoing endocytosis.     

Phenothiazine‐Derived Antipsychotic Drugs Inhibit Dynamin and Clathrin‐Mediated Endocytosis

Chlorpromazine is a phenothiazine‐derived antipsychotic drug (APD) that inhibits clathrin‐mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of dynamin. Eight of eight phenothiazine‐derived APDs inhibited dynamin I (dynI) in the 2–12 µm range, the most potent being trifluoperazine (IC₅₀ 2.6 ± 0.7 µm ). They also inhibited dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8‐ to 10‐fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine‐derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine‐derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All dynamin‐active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure–activity relationships (SAR) revealed dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP‐2 recruitment in the formation of clathrin‐coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP‐2 interaction with amphiphysin or clathrin. Super‐resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP‐2, nor AP‐2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent dynamin inhibition is a shared characteristic of phenothiazine‐derived APDs, but not other typical or atypical APDs, and the data indicate that dynamin is their likely in‐cell target in endocytosis.      

Cell‐surface Metalloprotease ADAM12 is Internalized by a Clathrin‐ and Grb2‐dependent Mechanism

ADAM12 (A Disintegrin And Metalloprotease 12), a member of the ADAMs family of transmembrane proteins, is involved in ectodomain shedding, cell‐adhesion and signaling, with important implications in cancer. Therefore, mechanisms that regulate the levels and activity of ADAM12 at the cell‐surface are possibly crucial in these contexts. We here investigated internalization and subsequent recycling or degradation of ADAM12 as a potentially important regulatory mechanism. Our results show that ADAM12 is constitutively internalized primarily via the clathrin‐dependent pathway and is subsequently detected in both early and recycling endosomes. The protease activity of ADAM12 does not influence this internalization mechanism. Analysis of essential elements for internalization established that proline‐rich regions in the cytoplasmic domain of ADAM12, previously shown to interact with Src‐homology 3 domains, were necessary for proper internalization. These sites in the ADAM12 cytoplasmic domain interacted with the adaptor protein growth factor receptor‐bound protein 2 (Grb2) and knockdown of Grb2 markedly reduced ADAM12 internalization. These studies establish that internalization is indeed a mechanism that regulates ADAM cell surface levels and show that ADAM12 internalization involves the clathrin‐dependent pathway and Grb2 .     

Nonmuscle Myosin Heavy Chain B Is Recognized by a Monoclonal Antibody that Inhibits GM1-Enhanced Neuritogenesis

Abstract: Addition of gangliosides to neuroblastoma cells maintained in vitro has been shown to enhance neuritogenesis. Although the mechanism by which they exert this effect is unknown, it has been postulated that they may act by adhering to cell surface proteins. In this article, we describe the isolation and identification of an S20Y murine neuroblastoma cell protein recognized by a monoclonal antibody that was prepared against putative GM1-binding proteins and shown to inhibit GM1-enhanced neuritogenesis. The protein identified was nonmuscle myosin heavy chain B, which appears to function in neurite formation but may not adhere to gangliosides.     

Myosin VI,an actin motor for membrane traffic and cell migration

The actin cytoskeleton and associated myosin motor proteins are essential for the transport and steady-state localization of vesicles and organelles and for the dynamic remodeling of the plasma membrane as well as for the maintenance of differentiated cell-surface structures. Myosin VI may be expected to have unique cellular functions, because it moves, unlike almost all other myosins, towards the minus end of actin filaments. Localization and functional studies indicate that myosin VI plays a role in a variety of different intracellular processes, such as endocytosis and secretion as well as cell migration. These diverse functions of myosin VI are mediated by interaction with a range of different binding partners .     

Clathrin‐dependent endocytosis predominantly mediates protein absorption by fat body from the hemolymph in Bombyx mori

During insect larval–pupal metamorphosis, proteins in the hemolymph are absorbed by the fat body for the maintenance of intracellular homeostasis; however, the type of proteins and how these proteins are internalized into the fat body are unclear. In Bombyx mori, the developmental profiles of total proteins in the hemolymph and fat body showed that hemolymph‐decreased protein bands (55–100 kDa) were in accordance with those protein bands that increased in the fat body. Inhibition of clathrin‐dependent endocytosis predominantly blocked the transportation of 55–100 kDa proteins from the hemolymph into the fat body, which was further verified by RNA interference treatment of Bmclathrin. Six hexamerins were shown to comprise ～90% of the total identified proteins in both the hemolymph and fat body by mass spectrum (MS) analysis. In addition, hemolymph‐specific proteins were mainly involved in material transportation, while fat body‐specific proteins particularly participated in metabolism. In this paper, four hexamerins were found for the first time, and potential proteins absorbed by the fat body from the hemolymph through clathrin‐dependent endocytosis were identified. This study sheds light on the protein absorption mechanism during insect metamorphosis.     

A Common Clathrin‐Mediated Machinery Co‐ordinates Cell–Cell Adhesion and Bacterial Internalization

Invasive bacterial pathogens often target cellular proteins involved in adhesion as a first event during infection. For example, Listeria monocytogenes uses the bacterial protein InlA to interact with E‐cadherin, hijack the host adherens junction (AJ) machinery and invade non‐phagocytic cells by a clathrin‐dependent mechanism. Here, we investigate a potential role for clathrin in cell–cell adhesion. We observed that the initial steps of AJ formation trigger the phosphorylation of clathrin, and its transient localization at forming cell–cell contacts. Furthermore, we show that clathrin serves as a hub for the recruitment of proteins that are necessary for the actin rearrangements that accompany the maturation of AJs. Using an InlA/E‐cadherin chimera, we show that adherent cells expressing the chimera form AJs with cells expressing E‐cadherin. We demonstrate that non‐adherent cells expressing the InlA chimera, as bacteria, can be internalized by E‐cadherin‐expressing adherent cells. Together these results reveal that a common clathrin‐mediated machinery may regulate internalization and cell adhesion and that the relative mobility of one of the interacting partners plays an important role in the commitment to either one of these processes.     

Days and knights discussing membrane dynamics in endocytosis: meeting report from the Euresco/EMBL Membrane Dynamics in Endocytosis, 6--11 October in Tomar, Portugal

The Euresco/EMBL sponsored meeting on 'Membrane Dynamics in Endocytosis' took place on 6–11 October in Tomar, Portugal. Here we report on the 5 full days of exciting talks and active poster sessions that covered topics ranging from the mechanisms of clathrin-mediated endocytosis, the regulation of phagocytosis, caveolae dynamics and function, the role of lipids in regulating endocytic transport, the formation of and sorting into and out of multivesicular bodies, new links between the actin cytoskeleton and vesicular transport, and emerging roles for endocytic trafficking in signal transduction and development.     

Pak1 Phosphorylation Enhances Cortactin–N‐WASP Interaction in Clathrin‐Caveolin‐Independent Endocytosis

Growing evidence indicates that kinases are central to the regulation of endocytic pathways. Previously, we identified p21‐activated kinase 1 (Pak1) as the first specific regulator of clathrin‐ and caveolae‐independent endocytosis used by the interleukin 2 receptor subunit (IL‐2R). Here, we address the mechanism by which Pak1 regulates IL‐2Rβ endocytosis. First, we show that Pak1 phosphorylates an activator of actin polymerization, cortactin, on its serine residues 405 and 418. Consistently, we observe a specific inhibition of IL‐2Rβ endocytosis when cells overexpress a cortactin, wherein these serine residues have been mutated. In addition, we show that the actin polymerization enhancer, neuronal Wiskott–Aldrich syndrome protein (N‐WASP), is involved in IL‐2Rβ endocytosis. Strikingly, we find that Pak1 phosphorylation of cortactin on serine residues 405 and 418 increases its association with N‐WASP. Thus, Pak1, by controlling the interaction between cortactin and N‐WASP, could regulate the polymerization of actin during clathrin‐independent endocytosis.     

A Second Las17 Monomeric Actin‐Binding Motif Functions in Arp2/3‐Dependent Actin Polymerization During Endocytosis

During clathrin‐mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G‐actin) and a central‐acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3‐dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G‐actin‐binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G‐actin‐binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two‐hybrid system, GST‐pulldown, fluorescence polarization and pyrene‐actin polymerization assays, we show that LGM binds G‐actin and is necessary for normal Arp2/3‐mediated actin polymerization in vitro. Live‐cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G‐actin‐binding motif, WH2. These results establish a second G‐actin‐binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME.