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Discovery of New Cargo Proteins that Enter Cells through Clathrin‐Independent Endocytosis
Abstract
Clathrin-independent endocytosis (CIE) allows internalization of plasma membrane proteins lacking clathrin-targeting sequences, such as the major histocompatibility complex class I protein (MHCI), into cells. After internalization, vesicles containing MHCI fuse with transferrin-containing endosomes generated from clathrin-dependent endocytosis. In HeLa cells, MHCI is subsequently routed to late endosomes or recycled back out to the plasma membrane (PM) in distinctive tubular carriers. Arf6 is associated with endosomal membranes carrying CIE cargo and expression of an active form of Arf6 leads to the generation of vacuolar structures that trap CIE cargo immediately after endocytosis, blocking the convergence with transferrin-containing endosomes. We isolated these trapped vacuolar structures and analyzed their protein composition by mass spectrometry. Here we identify and validate six new endogenous cargo proteins (CD44, CD55, CD98, CD147, Glut1, and ICAM1) that use CIE to enter cells. CD55 and Glut1 appear to closely parallel the trafficking of MHCI, merging with transferrin endosomes before entering the recycling tubules. In contrast, CD44, CD98, and CD147 appear to directly enter the recycling tubules and by-pass the merge with EEA1-positive, transferrin-containing endosomes. This divergent itinerary suggests that sorting may occur along this CIE pathway. Furthermore, the identification of new cargo proteins will assist others studying CIE in different cell types and tissues.
... Figure 3. Identification of cargo proteins that travel the endosomal pathways in virion and extracel lular nanoparticle preparations. Clathrin-independent endocytosis (CIE) [43][44][45][46] and clathrin-de pendent endocytosis (CDE) [47][48][49][50][51][52] cargo proteins, integrins (both CIE and CDE proteins) [53], G protein-coupled receptors (GPCRs) [54], and cargo proteins of the retrograde EE-to-TGN pathwa [48,55] were identified in five proteomes of HCMV virion preparations and one proteome of HSV 1 heavy particles as described in Figure 2. Proteome 7 [16] shows the abundance of a cargo protein in high-resolution density-gradient-purified non-vesicular (NV) samples of DKO-1 cells (left box and Gli36 cells (right box), lEV (L), and sEV (S) samples of DKO-1 cells. A color code of very low (less than 2 × log2 abundance relative to average signal) was set up as a threshold [16]. ...
... Tubular extensions of the EEs generate transport intermediates that carry cargo directly to the PM (recycling) and Golgi (retrograde pathway) or travel to the cluster o pericentriolar tubular compartments known as the endosomal recycling compartmen (ERC) for another round of sorting before returning to the PM or being diverted to th Golgi. This pleomorphic tubular traffic is apparently adapted to the type of cargo and fina [43][44][45][46] and clathrindependent endocytosis (CDE) [47][48][49][50][51][52] cargo proteins, integrins (both CIE and CDE proteins) [53], G protein-coupled receptors (GPCRs) [54], and cargo proteins of the retrograde EE-to-TGN pathway [48,55] were identified in five proteomes of HCMV virion preparations and one proteome of HSV-1 heavy particles as described in Figure 2. Proteome 7 [16] shows the abundance of a cargo protein in high-resolution density-gradient-purified non-vesicular (NV) samples of DKO-1 cells (left box) and Gli36 cells (right box), lEV (L), and sEV (S) samples of DKO-1 cells. A color code of very low (less than 2 × log 2 abundance relative to average signal) was set up as a threshold [16]. ...
... Rab and Arf family proteins are marked in blue, SNXs in black, and cargo molecule in green. Based on references [43,44,46,. . Schematic representation of known exit sites and recycling pathways from the early endosomal system. ...
Beta-herpesvirus infection completely reorganizes the membrane system of the cell. This system is maintained by the spatiotemporal arrangement of more than 3000 cellular proteins that continuously adapt the configuration of membrane organelles according to cellular needs. Beta-herpesvirus infection establishes a new configuration known as the assembly compartment (AC). The AC membranes are loaded with virus-encoded proteins during the long replication cycle and used for the final envelopment of the newly formed capsids to form infectious virions. The identity of the envelopment membranes is still largely unknown. Electron microscopy and immunofluorescence studies suggest that the envelopment occurs as a membrane wrapping around the capsids, similar to the growth of phagophores, in the area of the AC with the membrane identities of early/recycling endosomes and the trans-Golgi network. During wrapping, host cell proteins that define the identity and shape of these membranes are captured along with the capsids and incorporated into the virions as host cell signatures. In this report, we reviewed the existing information on host cell signatures in human cytomegalovirus (HCMV) virions. We analyzed the published proteomes of the HCMV virion preparations that identified a large number of host cell proteins. Virion purification methods are not yet advanced enough to separate all of the components of the rich extracellular material, including the large amounts of non-vesicular extracellular particles (NVEPs). Therefore, we used the proteomic data from large and small extracellular vesicles (lEVs and sEVs) and NVEPs to filter out the host cell proteins identified in the viral proteomes. Using these filters, we were able to narrow down the analysis of the host cell signatures within the virions and determine that envelopment likely occurs at the membranes derived from the tubular recycling endosomes. Many of these signatures were also found at the autophagosomes, suggesting that the CMV-infected cell forms membrane organelles with phagophore growth properties using early endosomal host cell machinery that coordinates endosomal recycling.
... Recent studies have demonstrated that the facilitation of endocytic recycling of CD147, mediated by the small GTPases Arf6, Rab5 and Rab22, increases surface CD147 in hepatocellular carcinoma and lung cancer, thereby enhancing their MMP production and malignant phenotypes (27)(28)(29). Notably, it has been reported that CD147 is internalized through CIE and recycled through Rab22A-dependent tubular endosomes that are a hallmark of the CIE cargo trafficking pathway (30,31), indicating that clathrin-independent endocytic recycling plays a crucial role in CD147-dependent tumor cell malignancies. ...
... This finding raises the possibility that TRE17 promotes cell invasion through regulating the recycling of an unknown CIE cargo protein(s), which activates the invasion signaling pathway. Several lines of evidence led us to speculate that CD147 is one of the potential candidate cargoes; first, elevated expression of CD147 as well as TRE17 increases production of MMPs, and CD147 plays a central role in tumor cell invasion and metastasis (15,22,23); second, the endocytic recycling determines the cell surface level of CD147, which in turn enhances malignant phenotypes of cancers (27)(28)(29); third, CD147 enters cells via CIE and recycled through tubular recycling endosomes specified for CIE cargoes (30,31). To test this hypothesis, we measured the cell surface level of CD147 by immunofluorescence staining using a monoclonal antibody directed to the extracellular portion of CD147 without cell permeabilization. ...
... From there the cargo is either targeted to lysosomal degradation or recycled back to the plasma membrane. Another group of CIE cargo proteins (CD44, CD98, and CD147) is predominantly recycled back to the plasma membrane in a process dependent on Rab22A, leading to the prolonged surface half-life of these cargo proteins (30). Eyster et al. (3) have shown that MARCH E3 ubiquitin ligases target most of these CIE cargoes from both groups except for the GPI-anchored proteins CD55 and CD59, which lack the cytoplasmic domain. ...
Disordered expression and distribution of plasma membrane proteins at the cell surface leads to diverse malignant phenotypes in tumors, including cell invasion. The ubiquitin-specific protease TRE17/USP6, an oncogene identified in Ewing sarcoma, is highly expressed in several cancers and locally aggressive tumor-like lesions. We have previously demonstrated that TRE17 regulates the trafficking of plasma membrane proteins that enter cells via clathrin-independent endocytosis (CIE); TRE17 prevents CIE cargo proteins from being targeted to lysosomes for degradation by deubiquitylating them. However, functional insights into the effects of TRE17-mediated CIE cargo trafficking on cell invasion remain unknown. Here, we show that increased expression of TRE17 enhances invasiveness of the human sarcoma cell line HT-1080 by elevating the cell surface levels of the membrane glycoprotein CD147, which plays a central role in tumor progression. We demonstrate overexpression of TRE17 decreases ubiquitylated CD147, which is accompanied by suppression of CD147 transport to lysosomes, resulting in the stabilization and increase of cell surface-localized CD147. On the other hand, we show knockdown of TRE17 decreases cell surface CD147, which is coupled with reduced production of matrix metalloproteinases (MMPs), the enzymes responsible for extracellular matrix degradation. Furthermore, we demonstrate that inhibition of CD147 by a specific inhibitor alleviated the TRE17-promoted tumor cell invasion. We therefore propose a model for the pathogenesis of TRE17-driven tumors in which TRE17 increases CD147 at the cell surface by preventing its lysosomal degradation, which in turn enhances MMP synthesis and matrix degradation, thereby promoting tumor cell invasion.
... We also investigated the effect of Vps9d1-KO on the trafficking of Rab22A-dependent CIE cargo proteins CD55 and CD147 in tubular endosomes by performing antibody uptake assays in HeLa cells (Eyster et al., 2009;Maldonado-Báez et al., 2013a;Higashi et al., 2022). In the control WT cells, antibodies against CD55 and CD147 were incorporated into the cells and could be observed as numerous dots, as described previously (Eyster et al., 2009;Higashi et al., 2022), and their signals were well colocalized with EGFP-Rab10-positive tubules (Fig. 5A, top rows), whereas in Vps9d1-KO cells, the CD55 and CD147 dots were rather dispersed, and no colocalization between them and EGFP-Rab10 was observed (Fig. 5A, middle rows). ...
... We also investigated the effect of Vps9d1-KO on the trafficking of Rab22A-dependent CIE cargo proteins CD55 and CD147 in tubular endosomes by performing antibody uptake assays in HeLa cells (Eyster et al., 2009;Maldonado-Báez et al., 2013a;Higashi et al., 2022). In the control WT cells, antibodies against CD55 and CD147 were incorporated into the cells and could be observed as numerous dots, as described previously (Eyster et al., 2009;Higashi et al., 2022), and their signals were well colocalized with EGFP-Rab10-positive tubules (Fig. 5A, top rows), whereas in Vps9d1-KO cells, the CD55 and CD147 dots were rather dispersed, and no colocalization between them and EGFP-Rab10 was observed (Fig. 5A, middle rows). Since some of the CD147 dots in the Vps9d1-KO cells were colocalized or closely associated with EEA1 (Fig. S4A), CD147 proteins that were not transported to tubular endosomes are likely to have been trapped in early endosomes. ...
The small GTPase Rab22A is an important regulator of the formation of tubular endosomes, which are one of the types of recycling endosome compartments of the clathrin-independent endocytosis pathway. In order to regulate tubular endosome formation, Rab22A must be activated by a specific guanine nucleotide exchange factor (GEF); however, all of the GEFs that have been reported to exhibit Rab22A-GEF activity in vitro also activate Rab5A, an essential regulator of the clathrin-mediated endocytosis pathway, and no Rab22A-specific GEF has ever been identified. Here, we identified Vps9d1, a previously uncharacterized VPS9 protein, as a novel Rab22A-GEF. Tubular endosome structures were found to be severely impaired in Vps9d1-depleted HeLa cells, but Rab5A localization was unaffected. Expression of a constitutively active Rab22A mutant in Vps9d1-depleted HeLa cells restored tubular endosomes, but expression of a GEF-activity-deficient Vps9d1 mutant did not. Moreover, Vps9d1 depletion altered the distribution of clathrin-independent endocytosed cargos and impaired their recycling. Our findings indicated that Vps9d1 promotes tubular endosome formation by specifically activating Rab22A.
... These different omics data together suggest a contribution of endocytosis to the depolarized V m -induced loss of cell surface nutrient transporters, which is consistent with the view that nutrient uptake is tightly controlled by the dynamic membrane trafficking of such transporters 5 . In addition, the membrane trafficking of nutrient transporters, including GLUT1 and 4F2hc, are usually co-regulated in response to different stresses, possibly because of their clustering in the same membrane domains 5,47,48 . We found that, after ML133 or elevated [K + ] e treatment, LPS-stimulated macrophages showed significantly enhanced internalization of both GLUT1 and 4F2hc (Fig. 6d). ...
... Consistent with this, the enrichment of H3K36me3 in the gene-body regions of Il1b, Il1a, Il18, and Cxcl10 loci in Kir2.1-depleted macrophages was also restored (Supplementary Fig. 5C). GLUT1 and 4F2hc are ARF6/GRP1 cargo proteins that can be recycled back to the plasma membrane via the tubular recycling endosome5,47,51,52 . ...
Immunometabolism contributes to inflammation, but how activated macrophages acquire extracellular nutrients to fuel inflammation is largely unknown. Here, we show that the plasma membrane potential (Vm) of macrophages mediated by Kir2.1, an inwardly-rectifying K+ channel, is an important determinant of nutrient acquisition and subsequent metabolic reprogramming promoting inflammation. In the absence of Kir2.1 activity, depolarized macrophage Vm lead to a caloric restriction state by limiting nutrient uptake and concomitant adaptations in nutrient conservation inducing autophagy, AMPK (Adenosine 5‘-monophosphate-activated protein kinase), and GCN2 (General control nonderepressible 2), which subsequently depletes epigenetic substrates feeding histone methylation at loci of a cluster of metabolism-responsive inflammatory genes, thereby suppressing their transcription. Kir2.1-mediated Vm supports nutrient uptake by facilitating cell-surface retention of nutrient transporters such as 4F2hc and GLUT1 by its modulation of plasma membrane phospholipid dynamics. Pharmacological targeting of Kir2.1 alleviated inflammation triggered by LPS or bacterial infection in a sepsis model and sterile inflammation in human samples. These findings identify an ionic control of macrophage activation and advance our understanding of the immunomodulatory properties of Vm that links nutrient inputs to inflammatory diseases. Potassium channels and membrane potential may influence macrophage function during inflammation. Here the authors show that the Kir2.1 potassium channel affects macrophage metabolism by altering cell surface retention of nutrient transporters and subsequently regulates inflammatory disease responses.
... It has been reported that the internalization of CD147 depends on clathrin-independent endocytosis, which is closely related to Arf6 [22,27]. Therefore, we investigated the possible role of Arf6 in CD147mediated SARS-CoV-2 infection. ...
... In our study, we also explored the specific endocytosis type for the CD147-mediated viral infection. A previous study showed that the endocytic recycling of CD147 is closely related to Arf6-mediated endocytosis [27,38]. Arf6-mediated internalization requires the involvement of cholesterol and cytoskeleton, and after internalization, cargo-containing endosomes fuse with Rab5-positive sorting endosomes [8,39,40]. ...
The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants is threatening the public health around the world. Endocytosis functions as an important way for viral infection, and SARS-CoV-2 bears no exception. However, the specific endocytic mechanism of SARS-CoV-2 remains unknown. In this study, we used endocytic inhibitors to evaluate the role of different endocytic routes in SARS-CoV-2 pseudovirus infection and found that the viral infection was associated with caveolar/lipid raft- and cytoskeleton-mediated endocytosis, but independent of the clathrin-mediated endocytosis and macropinocytosis. Meanwhile, the knockdown of CD147 and Rab5a in Vero E6 and Huh-7 cells inhibited SARS-CoV-2 pseudovirus infection, and the co-localization of spike protein, CD147, and Rab5 was observed in pseudovirus-infected Vero E6 cells, which was weakened by CD147 silencing, illustrating that SARS-CoV-2 pseudovirus entered host cells via CD147-mediated endocytosis. Additionally, Arf6 silencing markedly inhibited pseudovirus infection in Vero E6 and Huh-7 cells, while little change was observed in CD147 konckout-Vero E6 cells. This finding indicated Arf6-mediated CD147 trafficking plays a vital role in SARS-CoV-2 entry. Taken together, our findings provide new insights into CD147-Arf6 axis in mediating SARS-CoV-2 pseudovirus entry into the host cells and further suggest that blockade of this pathway seems to be a feasible approach to prevent SARS-CoV-2 infection clinically.
... Arfs are also important for maintaining the structure and function of the Golgi apparatus [11,27,68,71,78,80], whose stacks form the outer pre-AC but retain their basic functions, such as the loading and processing of the MCMV-encoded nonstructural protein m06 and the viral glycoproteins in the late phase of infection [6,7,11]. The recruitment of Arf effectors at membranes depends on the duration of the Arf GTP binding/hydrolysis cycle [14,81]. The accumulation of the Arfs on the membranes of intracellular compartments leads to their expansion and massive accumulation of cargo molecules within them [19,35,41,67]. ...
... Thus, the disruption of their endocytic trafficking would alter the virus entry. At the PM, Arf1 and Arf6 regulate both the clathrin-dependent and -independent endocytosis of different cargo molecules, including molecules identified to serve as CMV receptors, such as receptor tyrosine kinases such as PDGFR and EGFR [31,102], integrins [17,90], CD147 [43,81], MHC class I molecules [26,42,103], and GPI-anchored proteins such as CD90 and CD151 [31]. Thus, it is likely that the lack of Arf1 or Arf6 can interrupt the PM-associated processes of MCMV pathogenesis, especially because functions of Arf1 and Arf6 are not reported to be directly linked to the cell nucleus. ...
Shortly after entering the cells, cytomegaloviruses (CMVs) initiate massive reorganization of cellular endocytic and secretory pathways, which results in the forming of the cytoplasmic virion assembly compartment (AC). We have previously shown that the formation of AC in murine CMV- (MCMV) infected cells begins in the early phase of infection (at 4–6 hpi) with the pre-AC establishment. Pre-AC comprises membranes derived from the endosomal recycling compartment, early endosomes, and the trans-Golgi network, which is surrounded by fragmented Golgi cisterns. To explore the importance of Arf GTPases in the biogenesis of the pre-AC, we infected Balb 3T3 cells with MCMV and analyzed the expression and intracellular localization of Arf proteins in the early phases (up to 16 hpi) of infection and the development of pre-AC in cells with a knockdown of Arf protein expression by small interfering RNAs (siRNAs). Herein, we show that even in the early phase, MCMVs cause massive reorganization of the Arf system of the host cells and induce the over-recruitment of Arf proteins onto the membranes of pre-AC. Knockdown of Arf1, Arf3, Arf4, or Arf6 impaired the establishment of pre-AC. However, the knockdown of Arf1 and Arf6 also abolished the establishment of infection. Our study demonstrates that Arf GTPases are required for different steps of early cytomegalovirus infection, including the establishment of the pre-AC.
... There are two general types of endocytic pathways, the clathrindependent (CDE) and clathrin-independent endocytosis (CIE) [3]. As increasing PM proteins are found to enter cells by CIE pathways [4], CIE mechanisms are recognized to be fundamental for many physiological processes, such as immune surveillance, cell signaling, cell migration, and metastasis [5]. However, compared to the well-established CDE pathway, our understanding of the details of CIE and post-endocytic trafficking is particularly limited [6,7]. ...
... Usually the internalized CIE proteins such as the major histocompatibility complex class I (MHCI), the GPI-anchored protein CD59, and GLUT1 join the classical SEs containing the CDE cargo transferrin receptor (TfR) and the early endosomal antigen 1 (EEA1) soon after internalization, where they are sorted for recycling or degradation [7]. However, a subset of CIE proteins, like CD44, CD98, and CD147, enter cells with MHCI and directly join the recycling tubules, by-passing the merge with classical EEA1-positive SEs [4]. The existence of these divergent itineraries suggests that the CIE cargoes are sorted at different points along the endo-lysosomal pathway. ...
Early endosomes are the sorting hub on the endocytic pathway, wherein sorting nexins (SNXs) play important roles for formation of the distinct membranous microdomains with different sorting functions. Tubular endosomes mediate the recycling of clathrin-independent endocytic (CIE) cargoes back toward the plasma membrane. However, the molecular mechanism underlying the tubule formation is still poorly understood. Here we screened the effect on the ARF-6-associated CIE recycling endosomal tubules for all the SNX members in Caenorhabditis elegans ( C . elegans ). We identified SNX-3 as an essential factor for generation of the recycling tubules. The loss of SNX-3 abolishes the interconnected tubules in the intestine of C . elegans . Consequently, the surface and total protein levels of the recycling CIE protein hTAC are strongly decreased. Unexpectedly, depletion of the retromer components VPS-26/-29/-35 has no similar effect, implying that the retromer trimer is dispensable in this process. We determined that hTAC is captured by the ESCRT complex and transported into the lysosome for rapid degradation in snx-3 mutants. Interestingly, EEA-1 is increasingly recruited on early endosomes and localized to the hTAC-containing structures in snx-3 mutant intestines. We also showed that SNX3 and EEA1 compete with each other for binding to phosphatidylinositol-3-phosphate enriching early endosomes in Hela cells. Our data demonstrate for the first time that PX domain-only C . elegans SNX-3 organizes the tubular endosomes for efficient recycling and retrieves the CIE cargo away from the maturing sorting endosomes by competing with EEA-1 for binding to the early endosomes. However, our results call into question how SNX-3 couples the cargo capture and membrane remodeling in the absence of the retromer trimer complex.
... The interaction of antibody with the receptor would most likely affect the trafficking route across the BECs, but the intracellular sorting is almost unexplored. The internalization of TfR1 and basigin was observed to be clathrin-dependent and clathrinindependent, respectively, in epithelial cells [33], indicating that the sorting of basigin mAbs could be different from TfR1 mAbs. It is not clear how the surface expression is regulated in BECs, but the sorting of MCT1 to the plasma membrane has been suggested to be dependent on caveolin-1 in epithelial cells [34]. ...
... early endosomes followed by localization into TfR1-and VPS35-positive vesicles. This is in contrast to studies in HeLa cells, where basigin did not co-localize with EEA1positive endosomes or TfR1-containing vesicles [26,33]. However, in a human breast epithelial cell line, basigin was localized in EEA1 vesicles [39]. ...
The blood–brain barrier (BBB) poses challenges for delivering antibody-based therapeutics to the brain and is a main obstacle for the successful application of biotherapeutics for the treatment of brain disorders. As only a small fraction of monoclonal antibodies (mAbs) is penetrating the BBB, high doses of therapeutics are required to elicit a pharmacological effect. This limitation has evoked research to improve transport across the BBB through receptor-mediated transcytosis, and several receptors have been explored for mediating this process. A recently suggested candidate is the brain endothelial cells (BECs) expressed basigin. Here, we explore the transcytosis capacity of different basigin mAbs targeting distinct epitopes using the porcine in vitro BBB models and provide data showing the intracellular vesicle sorting of these basigin mAbs in porcine BECs. Our data suggest that basigin mAbs avoid the lysosomal degradation pathway and are internalized to vesicles used by recycling receptors. Engagement of basigin mAbs with basigin led to the translocation of the mAbs across the tight BECs into the astrocytes in our in vitro BBB co-culture model. Although mAbs with higher binding affinity to basigin showed a greater astrocyte internalization, based on our experiments, it is not clear whether the transcytosis is affinity- or epitope-dependent or a combination of both. Overall, this study provides information about the intra- and intercellular fate of basigin mAbs in BECs, which are valuable for the future design of basigin-mediated drug delivery platforms.
... Studies have shown that ARF6-dependent CIE participates in the endocytosis of the MHC class I (Radhakrishna and Donaldson 1997), the β-integrins (Powelka et al. 2004), the glucose transporter 1 (GLUT1), and other proteins that are involved in amino acid uptake and cell-extracellular matrix interactions (Eyster et al. 2009). In addition, RhoA and CDC42 endocytosis are dependent on the lipid rafts for vesicle formation. ...
... CD98 is a multifunctional protein that plays a role in nutrient uptake and cell-matrix interactions (Devés and Boyd 2000) while CD147 is a matrix metalloproteinase inducer that associates with monocarboxylate transporter proteins for lactate and pyruvate transportation across the plasma membrane (Iacono et al. 2007). It was previously shown that CD98 and CD147 receptors follow the CIE process for their internalization (Eyster et al. 2009). It was established that CD44 was a Gal3dependent cargo which got internalized via CIE. ...
Multiple endocytic processes operate in cells in tandem to uptake multiple cargoes involved in diverse cellular functions, including cell adhesion and migration. The best-studied clathrin-mediated endocytosis (CME) involves the formation of a well-defined cytoplasmic clathrin coat to facilitate cargo uptake. According to the glycolipid–lectin (GL–Lect) hypothesis, galectin-3 (Gal3) binds to glycosylated membrane receptors and glycosphingolipids (GSLs) to drive membrane bending and tubular membrane invaginations that undergo scission to form a morphologically distinct class of uptake structures, termed clathrin-independent carriers (CLICs). Which components from cytoskeletal machinery are involved in the scission of CLICs remains to be explored. In this study, we propose that dynein is recruited onto Gal3-induced tubular endocytic pits and provides the pulling force for friction-driven scission. The uptake of Gal3 and its cargoes (CD98/CD147) is significantly dependent on dynein activity, whereas only transferrin (CME marker) is slightly affected upon dynein inhibition. Our study reveals that Gal3 and Gal3-dependent (CD98 and CD147) clathrin-independent cargoes require dynein for the clathrin-independent endocytosis.
... Open conformer, fully load and suboptimal loaded MHC-I are internalized differently in the endosomallysosomal system. Inside non-specialist APCs, open and closed MHC-I are most likely to be dependent on Arf6 and clathrin-independent pathways with different dynamics to internalize and recycle [90].Study showed the open and closed MHC-I converges into Rab5-positive endosomes [89]. The CIE pathway, named after the GTPase Arf6, has been widely described as the endocytosis mechanism of MHC-I molecules. ...
Immunotherapy has changed the treatment landscape for multiple cancer types. In the recent decade, great progress has been made in immunotherapy, including immune checkpoint inhibitors, adoptive T-cell therapy, and cancer vaccines. ICIs work by reversing tumor-induced immunosuppression, resulting in robust activation of the immune system and lasting immune responses. Whereas, their clinical use faces several challenges, especially the low response rate in most patients. As an increasing number of studies have focused on membrane immune checkpoint protein trafficking and degradation, which interferes with response to immunotherapy, it is necessary to summarize the mechanism regulating those transmembrane domain proteins translocated into the cytoplasm and degraded via lysosome. In addition, other immune-related transmembrane domain proteins such as T-cell receptor and major histocompatibility are associated with neoantigen presentation. The endosomal-lysosomal system can also regulate TCR and neoantigen-MHC complexes on the membrane to affect the efficacy of adoptive T-cell therapy and cancer vaccines. In conclusion, we discuss the process of surface delivery, internalization, recycling, and degradation of immune checkpoint proteins, TCR, and neoantigen-MHC complexes on the endosomal-lysosomal system in biology for optimizing cancer immunotherapy.
... CD147 is reported to act as a molecular chaperone helping to recycle several membrane proteins, including CD44 and integrins [21,38,39]. To elucidate the efects of h4 # 147D on the molecular chaperone function of CD147, expressions of CD147 and its binding proteins on the cell surface were analyzed using fow cytometry for human cancer cells obtained from tumor samples of xenograft mice bearing MIA PaCa-2 after h4 # 147D treatment. ...
CD147 is an immunoglobulin-like receptor that is highly expressed in various cancers and involved in the growth, metastasis, and activation of inflammatory pathways via interactions with various functional molecules, such as integrins, CD44, and monocarboxylate transporters. Through screening of CD147-targeting antibodies with antitumor efficacy, we discovered a novel rat monoclonal antibody #147D. This humanized IgG4-formatted antibody, h4#147D, showed potent antitumor efficacy in xenograft mouse models harboring the human PDAC cell line MIA PaCa-2, HCC cell line Hep G2, and CML cell line KU812, which featured low sensitivity to the corresponding standard-of-care drugs (gemcitabine, sorafenib, and imatinib, respectively). An analysis of tumor cells derived from MIA PaCa-2 xenograft mice treated with h4#147D revealed that cell surface expression of CD147 and its binding partners, including CD44 and integrin α3β1/α6β1, was significantly reduced by h4#147D. Inhibition of focal adhesion kinase (FAK), activation of multiple stress responsible signal proteins such as c-JunN-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38MAPK), and expression of SMAD4, as well as activation of caspase-3 were obviously observed in the tumor cells, suggesting that h4#147D induced tumor shrinkage by inducing multiple stress responsible signals. These results suggest that the anti-CD147 antibody h4#147D offers promise as a new antibody drug candidate.
... Work on nonprofessional APCs has shown that upon arrival to the cell surface, MHC-I can divide into different membrane domains according to their peptide-loading status (Mahmutefendić et al, 2011), from where they are constantly internalized to endosomal compartments in a clathrin-independent manner (Eyster et al, 2009;Montealegre & van Endert, 2018). In such cell lines, MHC-I can recycle to the cell surface, in a process regulated by the small GTPases Arf6 (Radhakrishna & Donaldson, 1997;Jovanovic et al, 2006), Rab22 (Weigert et al, 2004) and the epsilon homology domain proteins 1 and 3 (EHD-1 and EHD-3). ...
Cross-presentation of internalized antigens by MHC class I molecules (MHC-I) plays a critical role in priming of cytotoxic T cells, recognizing pathogens and tumors. It is thought that peptides derived from cross-presented antigens can be loaded on MHC I in the endoplasmic reticulum as well as in endocytic or phagocytic compartments of murine dendritic cells (DCs). However, the origin of MHC I in the latter compartments is poorly understood. Recently MHC-I trafficking through a juxtanuclear Rab11+ recycling compartment has been suggested to be required for cross-presentation of phagocytosed antigens. We have critically examined the existence of MHC I recycling and the role of Arf6, described to regulate recycling in non-professional antigen presenting cells, in DCs. We confirm fully conformed MHC I accumulation in a juxtanuclear Rab11+ compartment and localize Arf6 to this compartment. DC MHC I molecules undergo fast recycling, however, both fully conformed and “open” internalized MHC I fail to enter a putative slow recycling pathway to the Rab11+Arf6+ compartment. Arf6 knockdown increases cell surface class I density and reduces degradation of internalized “open” MHC I but does not affect fast MHC-I recycling. Moreover, it compromises cross-presentation of antigen internalized via Fc receptors but not other antigens. In conclusion, we demonstrate fast recycling of MHC I in mouse DCs but find that recycling is not required for general cross-presentation. We propose that Rab11 and Rab22, previously reported to be required for cross-presentation, mediate delivery of MHC I from the juxtanuclear compartment to phagosomes/endosomes. However, the origin of the MHC I molecules in this compartment remains to be determined.
... Several nutrients and cellular components are internalized through endocytosis and affect cellular physiology. For example, Arf6mediated endocytosis is crucial for nutrient and ion transport, cell-cell interactions (Glut1, potassium channels, mucolipin-2, CD98, and CD147), and immunological functions (MHCI, Cd1a, and MHCII) [116]. Folate receptor endocytosis by Cdc42 is essential for nutrient balance [30]. ...
Endocytosis is a fundamental mechanism by which cells perform housekeeping functions. It occurs via a variety of mechanisms and involves many regulatory proteins. The GTPase dynamin acts as a “molecular scissor” to form endocytic vesicles and is a critical regulator among the proteins involved in endocytosis. Some GTPases (e.g., Cdc42, arf6, RhoA), membrane proteins (e.g., flotillins, tetraspanins), and secondary messengers (e.g., calcium) mediate dynamin-independent endocytosis. These pathways may be convergent, as multiple pathways exist in a single cell. However, what determines the specific path of endocytosis is complex and challenging to comprehend. This review summarizes the mechanisms of dynamin-independent endocytosis, the involvement of microRNAs, and factors that contribute to the cellular decision about the specific route of endocytosis.
... Accordingly, our data support the notion that CORO2A depletion impairs MHC-1 endosomal fission. Additionally, we compared the effects of CORO1C and CORO2A on the internalization and recycling of another clathrin-independent cargo, CD98 (Eyster et al., 2009). The internalization of CD98 was compared in cells subjected to scrambled siRNA, CORO2A siRNA, and CORO1C siRNA (reduced expression validated in Figure 8H). ...
Fission of transport vesicles from endosomes is a crucial step in the recycling of lipids and receptors to the plasma membrane, but this process remains poorly understood. Although key components of the fission machinery, including the actin cytoskeleton and the ATPase Eps15 Homology Domain Protein 1 (EHD1) have been implicated in endosomal fission, how this process is coordinately regulated is not known. We have identified the actin regulatory protein Coronin2A (CORO2A) as a novel EHD1 interaction partner. CORO2A localizes to stress fibers and actin microfilaments, but also can be observed in partial overlap with EHD1 on endosomal structures. siRNA knockdown of CORO2A led to enlarged lamellae-like actin-rich protrusions, consistent with a role of other Coronin-family proteins in attenuating actin-branching. Moreover, CORO2A depletion also caused a marked decrease in the internalization of clathrin-dependent cargo, but had little impact on the uptake of clathrin-independent cargo, highlighting key differences in the role of branched actin for different modes of endocytosis. However, CORO2A was required for recycling of clathrin-independent cargo, and its depletion led to enlarged endosomes, supporting a role for CORO2A in the fission of endosomal vesicles. Our data support a novel role for CORO2A in coordinating endosomal fission and recycling with EHD1. [Media: see text] [Media: see text].
... It has been reported that in both normal and cancerous cells the kinetics of GLUT1 internalization, endosomal sorting and recycling back to the plasma membrane play principal roles in maintaining glucose homeostasis [22][23][24]. To further investigate whether F806 regulated GLUT1's subcellular trafficking, we first generated a construct expressing HA-GLUT1-GFP fusion protein, in which the HA epitope was inserted in the first exofacial loop of GLUT1 N terminus and GFP was fused at GLUT1 C terminus (Fig. 3 A), thus allowing detection of GLUT1 on plasma membrane insertion by HA immunofluorescence (in non-permeabilized condition) and total GLUT1 content by direct GFP fluorescence. ...
Cancer cells are characterized by altered energetic metabolism with increasing glucose uptake. F806, a 16‐membered macrodiolide analogue, has anti‐tumour effects on oesophageal squamous cell carcinoma (ESCC) cells. However, its precise anti‐tumour mechanism remains unclear. Here, metascape analysis of our previous quantitative proteomics data showed that F806 induced glucose starvation response and inhibited energy production in ESCC cells. The reduced glucose uptake and ATP production were further validated by the fluorescent methods, using glucose‐conjugated bioprobe Glu‐1‐O‐DCSN, and the bioluminescent methods, respectively. Consistently, under F806 treatment the AMP‐activated protein kinase signalling was activated, and autophagy flux was promoted and more autophagosomes were formed. Moreover, live‐cell imaging and immunofluorescence analysis showed that F806 induced GLUT1 plasma membrane dissociation and promoted its internalization and autolysosome accumulation and lysosome degradation. Furthermore, molecular docking studies demonstrated that F806 bound to GLUT1 with a comparable binding energy to that of GLUT1’s direct interacting inhibitor cytochalasin B. Amino acid mutation was used to test which residues of GLUT1 may participate in F806 mediated‐GLUT1 internalization and degradation, and results showed that Thr137, Asn411 and Trp388 were required for GLUT1 internalization and degradation, respectively. Taken together, these findings shed light on a novel anti‐tumour mechanism of F806 by targeting and promoting GLUT1 internalization and further autolysosomal degradation.
... In contrast, in TBK1-knockdown cells, GLUT1 was trapped mainly in cytoplasm and intracellular vesicles. The inhibition of autophagy could decrease the membrane location of GLUT1 as indicated in previous studies and glucose uptake rate of CRC was also altered with different TBK1 expression [30,38,39]. In short, a deficiency in TBK1 could block the plasma membrane localization and stability of GLUT1 and TBK1 facilitates CRC development mediated by GLUT1. ...
Intestinal inflammation is a vital precipitating factor of colorectal cancer (CRC), but the underlying mechanisms are still elusive. TANK-binding kinase 1 (TBK1) is a core enzyme downstream of several inflammatory signals. Recent studies brought the impacts of TBK1 in malignant disease to the forefront, we found aberrant TBK1 expression in CRC is correlated with CRC progression. TBK1 inhibition impaired CRC cell proliferation, migration, drug resistance and tumor growth. Bioinformatic analysis and experiments in vitro showed overexpressed TBK1 inhibited mTORC1 signaling activation in CRC along with elevated GLUT1 expression without inducing GLUT1 translation. TBK1 mediated mTORC1 inhibition induces intracellular autophagy, which in turn decreasing GLUT1 degradation. As a rescue, blocking of autophagosome and retromer respectively via autophagy-related gene 7 (ATG7) or TBC1 Domain Family Member 5 (TBC1D5) silence diminished the regulation of TBK1 to GLUT1. GLUT1 staining presented that TBK1 facilitated GLUT1 membrane translocation which subsequently enhanced glucose consumption. Inhibitor of TBK1 also decreased GLUT1 expression which potentiated drug-sensitivity of CRC cell. Collectively, TBK1 facilitates glucose consumption for supporting CRC progression via initiating mTORC1 inhibition induced autophagy which decreases GLUT1 degradation and increases GLUT1 membrane location. The adaptive signaling cascade between TBK1 and GLUT1 proposes a new strategy for CRC therapy.
... However, it was shown using brefeldin A that a partially glycosylated form of this protein is able to reach the plasma membrane using an alternative mechanism that is independent of the classical secretory (ER/trans Golgi) pathway (Dalton et al. 2007). CD98hc can also be internalized by clathrin-independent endocytosis (CIE) for rapid recycling to the plasma membrane, together with CD147 (also known as basigin or EMMPRIN) (Eyster et al. 2009). Another study showed that CD98hc interacts with this transmembrane glycoprotein, suggesting that CD147 (basigin) may function as a chaperone for the trafficking of CD98hc from the ER to the surface, as well as for its recycling via CIE (Wu et al. 2015). ...
The eukaryotic protein CD98hc (also known as 4F2, FRP-1 or SLC3A2) is a membrane glycoprotein and one of the heavy chains of the family of heterodimeric amino acids transporters. It can associate with any of 6 different light chains to form distinct amino acid transporters. CD98hc is also involved in mediation of intracellular integrin signaling. Besides its physiological roles in the development of the placenta and the immune system, CD98hc is important during pathological processes such as tumorigenesis and host-pathogen interaction. Since its first identification as Fusion Regulatory Protein 1 regulating cell fusion in cells infected by the Newcastle disease virus, CD98hc has been reported to be mediating many viral, apicomplexan, and bacterial infectious processes. In this review we describe the role of CD98hc and its associated light chains in bacterial, apicomplexan, and viral pathogenesis. We also discuss the consequences of infection on the expression and localization of these proteins. The identification of the cellular processes in which CD98hc is involved during pathogenesis highlights the key role of this host protein in infectious diseases.
... Glucose transporter 1 (GLUT1) plays a major role in the regulation of glucose entry into the brain. It has been proposed that GLUT1 is internalized from the luminal membrane to the cytoplasm through endocytosis into brain capillary endothelial cells (BCECs) [1,2]. Among all transporters and receptors, GLUT1 is the most abundantly expressed in BCECs [3]. ...
Precise immunolocalization of molecules in relation to ultrastructural features is challenging, especially when the target is small and not frequent enough to be included in tiny ultrathin sections randomly selected for electron microscopy (EM). Glucose transporter 1 (GLUT1) is in charge of transporting glucose across brain capillary endothelial cells (BCECs). Paraformaldehyde-fixed floating sections (50 μm thick) of mouse brain were immunolabeled with anti-GLUT1 antibody and visualized with fluoronanogold. Fluorescent images encompassing the entire hemisphere were tiled to enable selection of GLUT1-positive BCECs suitable for subsequent EM and landmark placement with laser microdissection to guide trimming. Sections were then fixed with glutaraldehyde, gold enhanced to intensify the labeling and fixed with osmium tetroxide to facilitate ultrastructural recognition. Even though a region that contained target BCECs was successfully trimmed in the resin block, it was only after observation of serial ultrathin sections that GLUT1 signals in coated vesicles on the same cross section corresponding to the cross section preidentified by confocal laser microscope. This is the first ultrastructural demonstration of GLUT1 molecules in coated vesicles, which may well explain its functional relevance to transport glucose across BCECs. Successful ultrastructural localization of molecules in relation to well-preserved target structure in native tissue samples, as achieved in this study, will pave the way to understand the functional relevance of molecules and their relation to ultrastructural details.
... [27][28][29] CD44 is a known cargo of clathrin-independent endocytosis. 30 Acylation of CD44 is a critical driving force to CD44 association with lipid rafts, which is essential for the rates of hyaluronan endocytosis and CD44 turnover from cell surface. 31 Yu et al. show that receptor-meditated endocytosis by hyaluronic acid for targeting of CD44-overexpressing cancer cells has promising therapeutic prospects. ...
Aberrant expression of SNX5 can contribute to tumourigenesis, invasion, and metastasis of several human cancers. However, the clinicopathological and biological significance of SNX5 in clear cell renal cell carcinoma (ccRCC) remain unclear. In this study, we found that SNX5 expression was downregulated and negatively correlated with tumor size, AJCC stage, tumor thrombus of inferior vena cava (IVC) and poor prognosis in human ccRCC. Ectopic expression of SNX5 inhibited ccRCC cell proliferation and metastasis whereas knockdown of SNX5 increasedthese activities both in vitro and in vivo. Mechanistically, overexpression of SNX5 blocked internalization and intracellular trafficking of CD44 in ccRCC cells. Knockdown of SNX5 was associated with epithelial mesenchymal transition (EMT) in ccRCC cells.Overexpression of SNX5 inhibited TGF-β-induced migration, invasion and EMT in ccRCC cells. KLF9 directly bound to the SNX5 promoter and increased SNX5 transcription. Moreover, we found that the combination of SNX5 and CD44 or E-cadherin or KLF9 was a more powerful predictor of poor prognosis than either parameter alone.Collectively, our data reveal a mechanism that KLF9-mediated SNX5 expression was associated with poor prognosis via trafficking of CD44 and promoting EMT in ccRCC. SNX5 may be a potential prognostic biomarker and therapeutic target for patients with ccRCC.
... Endocytosis and membrane fusion are observed to be the two major entry pathways for viral infection (Harrison, 2015;Slonska et al., 2016). A sequential endocytosis of SARS-CoV-2 in Vero E6 cells is reported by an electron microscope, where CD147 is observed to invade the cells, via endocytosis, independent of clathrin (Maldonado-Baez et al., 2013;Eyster et al., 2009). Endocytosis is reported to be significantly regulated by Rab5, which is located at early endosome (Saitoh et al., 2017). ...
The combat against the Corona virus disease of 2019 (COVID-19), has created a chaos among the healthcare institutions and researchers, in turn accelerating the dire need to curtail the infection spread. The already established entry mechanism, via ACE2 has not yet successfully aided in the development of a suitable and reliable therapy. Taking in account the constant progression and deterioration of the cases worldwide, a different perspective and mechanistic approach is required, which has thrown light onto the cluster of differentiation 147 (CD147) transmembrane protein, as a novel route for SARS-CoV-2 entry. Despite lesser affinity towards COVID-19 virus, as compared to ACE2, this receptor provides a suitable justification behind elevated blood glucose levels in infected patients, retarded COVID-19 risk in women, enhanced susceptibility in geriatrics, greater infection susceptibility of T cells, infection prevalence in non-susceptible human cardiac pericytes and so on. The manuscript invokes the title role and distribution of CD147 in COVID-19 as an entry receptor and mediator of endocytosis-promoted entry of the virus, along with the “catch and clump” hypothesis, thereby presenting its Fundamental significance as a therapeutic target for potential candidates, such as Azithromycin, melatonin, statins, beta adrenergic blockers, ivermectin, Meplazumab etc. Thus, the authors provide a comprehensive review of a different perspective in COVID-19 infection, aiming to aid the researchers and virologists in considering all aspects of viral entry, in order to develop a sustainable and potential cure for the 2019 COVID-19 disease.
... Turning off ARFs is also crucial for their function as failure to inactivate ARFs can block downstream trafficking. For example, a GTP-locked ARF6 mutant blocked membrane recycling between the PM and endosomes (Eyster et al., 2009) and the GTP-locked form of ARF1 prevented cargo loading into COPI vesicles (Malsam et al., 1999), highlighting the importance of GTP hydrolysis beyond being a mere turning off switch. ...
Molecular switches of the ADP-ribosylation factor (ARF) GTPase family coordinate intracellular trafficking at all sorting stations along the secretory pathway, from the ER-Golgi-intermediate compartment (ERGIC) to the plasma membrane (PM). Their GDP-GTP switch is essential to trigger numerous processes, including membrane deformation, cargo sorting and recruitment of downstream coat proteins and effectors, such as lipid modifying enzymes. While ARFs (in particular ARF1) had mainly been studied in the context of coat protein recruitment at the Golgi, COPI/clathrin-independent roles have emerged in the last decade. Here we review the roles of human ARF1-5 GTPases in cellular trafficking with a particular emphasis on their roles in post-Golgi secretory trafficking and in sorting in the endo-lysosomal system.
... Anionic lipo 134 ± 26 -26 ± 1.9 PAR28-lipo 296 ± 70 -20 ± 3.0 PAR28-PEG-lipo 212 ± 56 -15 ± 0.9 endocytosis (CIE) cargo protein, and is translocated to tubular endosomes following internalization [20,21]. Thus, CD147 does not translocate to lysosomes, unlike the clathrin-dependent endocytosis cargo protein, transferrin receptor [22]. ...
Antibody-modified liposomes, immuno-liposomes, can selectively deliver encapsulated drug ‘cargos’ to cells via the interaction of cell surface proteins with antibodies. However, chemical modification of both the antibodies and phospholipids is required for the preparation of immuno-liposomes for each target protein using conventional methods, which is time-consuming. In the present study, we demonstrated that high-affinity protein A- (Protein A-R28: PAR28) displaying liposomes prepared by the post-insertion of PAR28-conjugated phospholipid through polyethylene glycol (PEG)-linkers (PAR28-PEG-lipo) can undergo rapid modification of antibodies on their surface, and the liposomes can be delivered to cells based on their modified antibodies. Anti-CD147 and anti-CD31 antibodies could be modified with PAR28-PEG-lipo within 1 h, and each liposome was specifically taken up by CD147- and CD31-positive cells, respectively. The cellular amounts of doxorubicin delivered by anti-CD147 antibody-modified PAR28-PEG-lipo were significantly higher than those of isotype control antibody-modified liposomes. PAR28-PEG-lipo can easily and rapidly undergo modification of various antibodies on their surface, which then makes them capable of selective drug delivery dependent on the antibodies.
... The ubiquitylation of CD98 by MARCH8 was reversed by Ub-specific protease 6 (USP6) and dependent on its deubiquitylating activity [68]. The DUb was also capable of counteracting the effect of MARCH ligases on the recycling of CD44, CD147 and, to a lesser degree, MHC-I [51,72]. Collectively, these findings support the possibility, raised above, that Tsg101 participates with E3 ligases and DUbs in regulating ubiquitylation and deubiquitylation events necessary for trafficking and sorting of cellular cargo. ...
Two decades ago, Tsg101, a component of the Endosomal Sorting Complexes Required for Transport (ESCRT) complex 1, was identified as a cellular factor recruited by the human immunodeficiency virus type 1 (HIV-1) to facilitate budding of viral particles assembled at the cell periphery. A highly conserved Pro-(Thr/Ser)-Ala-Pro [P(T/S)AP] motif in the HIV-1 structural polyprotein, Gag, engages a P(T/S)AP-binding pocket in the Tsg101 N-terminal domain. Since the same domain in Tsg101 that houses the pocket was found to bind mono-ubiquitin (Ub) non-covalently, Ub binding was speculated to enhance P(T/S)AP interaction. Within the past five years, we found that the Ub-binding site also accommodates di-Ub, with Lys63-linked di-Ub exhibiting the highest affinity. We also identified small molecules capable of disrupting Ub binding and inhibiting budding. The structural similarity of these molecules, prazoles, to nucleosides prompted testing for nucleic acid binding and led to identification of tRNA as a Tsg101 binding partner. Here, we discuss these recently identified interactions and their contribution to the viral assembly process. These new partners may provide additional insight into the control and function of Tsg101 as well as identify opportunities for anti-viral drug design.
... It is now well established that narrow endocytic pits can also form in endocytic processes that are not driven by the clathrin machinery [5][6][7][8]. A multitude of cellular proteins are involved in these clathrin-independent endocytosis events, such as small GT-Pases of the Rho/Rac/Cdc42 [9,10] and Arf families [11,12], actin [13,14], galectins [15,16], endophilins [17,18], and reticulon3 [19]. Whether these constitute different endocytic pathways or contribute at different steps to the same endocytic pathway has remained an open question (discussed in Reference [20]). ...
Lipid membranes are common to all forms of life. While being stable barriers that delimitate the cell as the fundamental organismal unit, biological membranes are highly dynamic by allowing for lateral diffusion, transbilayer passage via selective channels, and in eukaryotic cells for endocytic uptake through the formation of membrane bound vesicular or tubular carriers. Two of the most abundant fundamental fabrics of membranes—lipids and complex sugars—are produced through elaborate chains of biosynthetic enzymes, which makes it difficult to study them by conventional reverse genetics. This review illustrates how organic synthesis provides access to uncharted areas of membrane glycobiology research and its application to biomedicine. For this Special Issue on Chemical Biology Research in France, focus will be placed on synthetic approaches (i) to study endocytic functions of glycosylated proteins and lipids according to the GlycoLipid–Lectin (GL–Lect) hypothesis, notably that of Shiga toxin; (ii) to mechanistically dissect its endocytosis and intracellular trafficking with small molecule; and (iii) to devise intracellular delivery strategies for immunotherapy and tumor targeting. It will be pointed out how the chemical biologist’s view on lipids, sugars, and proteins synergizes with biophysics and modeling to “look” into the membrane for atomistic scale insights on molecular rearrangements that drive the biogenesis of endocytic carriers in processes of clathrin-independent endocytosis.
... CD44, a non-kinase transmembrane glycoprotein, is widely expressed on cell membrane implicated in many physiological and pathological processes, which include development, in ammation, immune responses, wound healing and cancer progression [26,27]. CD44 overexpression or alternative splicing was described for various types of cancers including ccRCC and associated with invasion, metastasis and resistance to chemotherapeutic drugs [28][29][30].CD44 is a known cargo of clathrin-independent endocytosis [31]. Acylation of CD44 is a critical driving force to CD44 association with lipid rafts, which is essential for the rates of hyaluronan endocytosis and CD44 turnover from cell surface [32].Yu,K.S et al ...
Background: Aberrant expression of SNX5 can contribute to tumourigenesis, invasion, and metastasis of several human cancers. However, the clinicopathological and biological significance of SNX5 in clear cell renal cell carcinoma (ccRCC) remain unclear. The aim of this study was to examine the role of SNX5 in the progression of ccRCC.
Methods: Immunohistochemical (IHC), Western blot, qRT-PCR, western blot, flow cytometry and immunofluorescence were used to detect the expression of indicated molecules. The biological role of SNX5 in ccRCC cells was evaluated by CCK8, colony formation, transwell assay, subcutaneous tumor formation as well as veil tail injection. ChIP assay and luciferase reporter assay were used to determine the direct binding of KLF9 to the promoter of the SNX5 gene.
Results: SNX5 expression was downregulated in human ccRCC tissues. SNX5 expression was negatively correlated with tumor size, AJCC stage, tumor thrombus of inferior vena cava (IVC) and poor prognosis of ccRCC. Ectopic expression of SNX5 inhibited ccRCC cell proliferation and metastasis whereas knockdown of SNX5 increase these activities both in vitro and in vivo. Mechanistically, overexpression of SNX5 blocked internalization and intracellular trafficking of CD44 in ccRCC cells. Exogenous expression of CD44 partially rescued the inhibitory effects of SNX5 on the proliferation and invasion activity of ccRCC cells. Knockdown of SNX5 in ccRCC cells was associated with epithelial mesenchymal transition (EMT), including the down-regulation of E-cadherin, ZO-1 and Claudin-1 and the concomitant up-regulation of Snail and N-cadherin. In addition, SNX5 inhibited TGF-β-induced migration, invasion and EMT in ccRCC cells. Moreover, we observed a significant correlation between SNX5 expression and E-cadherin levels in ccRCC patients. In addition, KLF9 directly bound to the SNX5 promoter and increased SNX5 transcription. SNX5 expression was closely correlated with KLF9 expression in ccRCC. Moreover, we found that the combination of SNX5 and CD44 or E-cadherin or KLF9 was a more powerful predictor of poor prognosis than either parameter alone.
Conclusion: Collectively, our data reveal a mechanism that KLF9-mediated SNX5 expression was associated with poor prognosis via trafficking of CD44 and promoting EMT in ccRCC. SNX5 may be a potential prognostic biomarker and therapeutic target for patients with ccRCC.
... As has been reported for other overexpressed endosomeassociated proteins (18,44,80,81), the average approximately fivefold increased expression of fluorescent SV needed for time-lapse microscopy (31) may affect endosome morphology by increasing tubulation. Signal overlaps with endogenous Rab5 in COS7 cells and with internalized integrin in primaquine-treated HeLa S3 cells appear mostly with vesicular SV structures (Figures 3 and 4), and not with the linear structures that are probably stress fibers or peripheral actomyosin filament bundles (30,31,36). ...
... CD44, CD98, CD147 and MHCI enter cells via a clathrin-independent pathway, and colocalize to the same endocytic vesicles after 5 min (ref. 64 ). However, CD44, CD98 and CD147 are rapidly sorted in a different way from MHCI into EEA1-negative endosomal compartments and avoid the degradative lysosomal pathway. ...
Endocytosis is a critical step in the process by which many therapeutic nanomedicines reach their intracellular targets. Our understanding of cellular uptake mechanisms has developed substantially in the past five years. However, these advances in cell biology have not fully translated to the nanoscience and therapeutics literature. Misconceptions surrounding the role of different endocytic pathways and how to study these pathways are hindering progress in developing improved nanoparticle therapies. Here, we summarize the latest insights into cellular uptake mechanisms and pathways. We highlight limitations of current systems to study endocytosis, particularly problems with non-specific inhibitors. We also summarize alternative genetic approaches to robustly probe these pathways and discuss the need to understand how cells endocytose particles in vivo. We hope that this critical assessment of the current methods used in studying nanoparticle uptake will guide future studies at the interface of cell biology and nanomedicine. Successful nanomedicine approaches rely on the efficient cellular uptake of nanoparticles, yet endocytic mechanisms remain challenging to probe. In this Review the authors describe the different cellular endocytic pathways and provide a critical discussion of the available tools and systems for their study.
... In hypoglycemic animals, the total GLUT1 of micro-vessel protein and luminal GLUT1 are increased [114]. GLUT1 on the cell membrane undergoes endocytosis and is pooled in intracellular vesicles [115,116]. The above reports suggest that the dynamic intracellular recycling of GLUT1 occurs in response to changes in blood glucose level. ...
The era of the aging society has arrived, and this is accompanied by an increase in the absolute numbers of patients with neurological disorders, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Such neurological disorders are serious costly diseases that have a significant impact on society, both globally and socially. Gene therapy has great promise for the treatment of neurological disorders, but only a few gene therapy drugs are currently available. Delivery to the brain is the biggest hurdle in developing new drugs for the central nervous system (CNS) diseases and this is especially true in the case of gene delivery. Nanotechnologies such as viral and non-viral vectors allow efficient brain-targeted gene delivery systems to be created. The purpose of this review is to provide a comprehensive review of the current status of the development of successful drug delivery to the CNS for the treatment of CNS-related disorders especially by gene therapy. We mainly address three aspects of this situation: (1) blood-brain barrier (BBB) functions; (2) adeno-associated viral (AAV) vectors, currently the most advanced gene delivery vector; (3) non-viral brain targeting by non-invasive methods.
... This further implies that significant uptake into AP-2 depleted cells by alternate clathrin-independent pathways is ongoing.The surface expression of cargo proteins that preferentially or exclusively utilize CIE should remain unaffected by loss of AP-2, or eventually even decrease if CIE is elevated as part of a compensatory response to the KD of AP-2. Inspection of the proteome data revealed that a number of markers for CIE including CD44 (hyaluronic acid receptor), 71 CD155 (poliovirus receptor),72 or IGF1R (IGF1 receptor),73 as well as various GPI-anchored proteins including the complement restricting factors CD55 and CD59,74,75 or the folate receptor FOLR1 remained almost unaffected by loss of AP-2 (seeFigure 2and ...
In eukaryotic cells, clathrin‐mediated endocytosis (CME) is a central pathway for the internalization of proteins from the cell surface, thereby contributing to the maintenance of the plasma membrane protein composition. A key component for the formation of endocytic clathrin‐coated vesicles (CCVs) is AP‐2, as it sequesters cargo membrane proteins, recruits a multitude of other endocytic factors and initiates clathrin polymerization. Here, we inhibited CME by depletion of AP‐2 and explored the consequences for the plasma membrane proteome. Quantitative analysis revealed accumulation of major constituents of the endosomal‐lysosomal system reflecting a block in retrieval by compensatory CME. The noticeable enrichment of integrins and blockage of their turnover resulted in severely impaired cell migration. Rare proteins such as the anti‐cancer drug target CA9 and tumor markers (CD73, CD164, CD302) were significantly enriched. The AP‐2 knockdown attenuated the global endocytic capacity, but clathrin‐independent entry pathways were still operating, as indicated by persistent internalization of specific membrane‐spanning and GPI‐anchored receptors (PVR, IGF1R, CD55, TNAP). We hypothesize that blocking AP‐2 function and thus inhibiting CME may be a novel approach to identify new druggable targets, or to increase their residence time at the plasma membrane, thereby increasing the probability for efficient therapeutic intervention. This article is protected by copyright. All rights reserved.
... CD147 is reported to enter the cells through clathrinindependent endocytosis. 24,25 Rab5 is a crucial regulator of endocytosis and locates at early endosome. 26 In our study, the co-localization of CD147, spike, and Rab5 was detected in BHK-21-CD147 cells and lung tissues from patient with COVID-19 (Fig. 6b), indicating that the receptor CD147 and virions were endocytosed and located at the early endosome. ...
In face of the everlasting battle toward COVID-19 and the rapid evolution of SARS-CoV-2, no specific and effective drugs for treating this disease have been reported until today. Angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, mediates the virus infection by binding to spike protein. Although ACE2 is expressed in the lung, kidney, and intestine, its expressing levels are rather low, especially in the lung. Considering the great infectivity of COVID-19, we speculate that SARS-CoV-2 may depend on other routes to facilitate its infection. Here, we first discover an interaction between host cell receptor CD147 and SARS-CoV-2 spike protein. The loss of CD147 or blocking CD147 in Vero E6 and BEAS-2B cell lines by anti-CD147 antibody, Meplazumab, inhibits SARS-CoV-2 amplification. Expression of human CD147 allows virus entry into non-susceptible BHK-21 cells, which can be neutralized by CD147 extracellular fragment. Viral loads are detectable in the lungs of human CD147 (hCD147) mice infected with SARS-CoV-2, but not in those of virus-infected wild type mice. Interestingly, virions are observed in lymphocytes of lung tissue from a COVID-19 patient. Human T cells with a property of ACE2 natural deficiency can be infected with SARS-CoV-2 pseudovirus in a dose-dependent manner, which is specifically inhibited by Meplazumab. Furthermore, CD147 mediates virus entering host cells by endocytosis. Together, our study reveals a novel virus entry route, CD147-spike protein, which provides an important target for developing specific and effective drug against COVID-19.
... In addition to biosynthetic regulation of CD98 expression, we previously reported that MARCH1-mediated ubiquitination of CD98 limits expression of endogenous CD98 and clonal expansion of T cells (16). The growing list of MARCH substrates indicates that MARCH proteins can influence a wide range of biological processes through ubiquitination of transmembrane receptors including CD98, CD44, MHC class I and II, CD86, CD25, ICAM, TRAILR1/2 and the Insulin receptor (14,(19)(20)(21)(22)(23)(24)(25). MARCH8 can also restrict HIV replication by directed ubiquitination of the ENV protein suggesting MARCH8 also plays a role in innate immunity (25). ...
CD98, which is required for the rapid proliferation of both normal and cancer cells, and MET, the hepatocyte growth factor receptor, are potential targets for therapeutic antitumor Abs. In this study, we report that the antiproliferative activity of a prototype anti-CD98 Ab, UM7F8, is due to Ab-induced membrane-associated ring CH (MARCH) E3 ubiquitin ligase-mediated ubiquitination and downregulation of cell surface CD98. MARCH1-mediated ubiquitination of CD98 is required for UM7F8's capacity to reduce CD98 surface expression and its capacity to inhibit the proliferation of murine T cells. Similarly, CD98 ubiquitination is required for UM7F8's capacity to block the colony-forming ability of murine leukemia-initiating cells. To test the potential generality of the paradigm that MARCH E3 ligases can mediate the antiproliferative response to antitumor Abs, we examined the potential effects of MARCH proteins on responses to emibetuzumab, an anti-MET Ab currently in clinical trials for various cancers. We report that MET surface expression is reduced by MARCH1, 4, or 8-mediated ubiquitination and that emibetuzumab-induced MET ubiquitination contributes to its capacity to downregulate MET and inhibit human tumor cell proliferation. Thus, MARCH E3 ligases can act as cofactors for antitumor Abs that target cell surface proteins, suggesting that the MARCH protein repertoire of cells is a determinant of their response to such Abs.
Human endogenous retroviruses (HERVs) are associated with the pathogenesis of amyotrophic lateral sclerosis (ALS); a disease characterized by motor neuron degeneration and cell death. The HERV-K subtype HML-2 envelope protein (HERV-K Env) is expressed in the brain, spinal cord, and cerebrospinal fluid of people living with ALS and through CD98 receptor-linked interactions causes neurodegeneration. HERV-K Env-induced increases in oxidative stress are implicated in the pathogenesis of ALS, and ferrous iron (Fe ²⁺ ) generates reactive oxygen species (ROS). Endolysosome stores of Fe ²⁺ are central to iron trafficking and endolysosome deacidification releases Fe ²⁺ into the cytoplasm. Because HERV-K Env is an arginine-rich protein that is likely endocytosed and arginine is a pH-elevating amino acid, it is important to determine HERV-K Env effects on endolysosome pH and whether HERV-K Env-induced neurotoxicity is downstream of Fe ²⁺ released from endolysosomes. Here, we showed using SH-SY5Y human neuroblastoma cells and primary cultures of human cortical neurons (HCNs, information on age and sex was not available) that HERV-K Env (1) is endocytosed via CD98 receptors, (2) concentration dependently deacidified endolysosomes, (3) decreased endolysosome Fe ²⁺ concentrations, (4) increased cytosolic and mitochondrial Fe ²⁺ and ROS levels, (5) depolarized mitochondrial membrane potential, and (6) induced cell death, effects blocked by an antibody against the CD98 receptor and by the endolysosome iron chelator deferoxamine. Thus, HERV-K Env-induced increases in cytosolic and mitochondrial Fe ²⁺ and ROS as well as cell death appear to be mechanistically caused by HERV-K Env endocytosis, endolysosome deacidification, and endolysosome Fe ²⁺ efflux into the cytoplasm.
Proteostasis requires oxidative metabolism (ATP) and mitigation of the associated damage by glutathione, in an increasingly dysfunctional relationship with aging. SLC3A2 (4F2hc, CD98) plays a role as a disulfide-linked adaptor to the SLC7A5 and SLC7A11 exchangers which import essential amino acids and cystine while exporting Gln and Glu, respectively. The positions of N-glycosylation sites on SLC3A2 have evolved with the emergence of primates, presumably in synchrony with metabolism. Herein, we report that each of the four sites in SLC3A2 has distinct profiles of Golgi-modified N-glycans. N-glycans at the primate-derived site N381 stabilized SLC3A2 in the galectin-3 lattice against coated-pit endocytosis, while N365, the site nearest the membrane promoted glycolipid-galectin-3 (GL-Lect)-driven endocytosis. Our results indicate that surface retention and endocytosis are precisely balanced by the number, position, and remodeling of N-glycans on SLC3A2. Furthermore, proteomics and functional assays revealed an N-glycan-dependent clustering of the SLC3A2∗SLC7A5 heterodimer with amino-acid/Na⁺ symporters (SLC1A4, SLC1A5) that balances branched-chain amino acids and Gln levels, at the expense of ATP to maintain the Na⁺/K⁺ gradient. In replete conditions, SLC3A2 interactions require Golgi-modified N-glycans at N365D and N381D, whereas reducing N-glycosylation in the endoplasmic reticulum by fluvastatin treatment promoted the recruitment of CD44 and transporters needed to mitigate stress. Thus, SLC3A2 N-glycosylation and Golgi remodeling of the N-glycans have distinct roles in amino acids import for growth, maintenance, and metabolic stresses.
Galectins, a family of evolutionarily conserved glycan-binding proteins, play key roles in diverse biological processes including tissue repair, adipogenesis, immune cell homeostasis, angiogenesis, and pathogen recognition. Dysregulation of galectins and their ligands has been observed in a wide range of pathologic conditions including cancer, autoimmune inflammation, infection, fibrosis, and metabolic disorders. Through protein–glycan or protein–protein interactions, these endogenous lectins can shape the initiation, perpetuation, and resolution of these processes, suggesting their potential roles in disease monitoring and treatment. However, despite considerable progress, a full understanding of the biology and therapeutic potential of galectins has not been reached due to their diversity, multiplicity of cell targets, and receptor promiscuity. In this article, we discuss the multiple galectin-binding partners present in different cell types, focusing on their contributions to selected physiologic and pathologic settings. Understanding the molecular bases of galectin–ligand interactions, particularly their glycan-dependency, the biochemical nature of selected receptors, and underlying signaling events, might contribute to designing rational therapeutic strategies to control a broad range of pathologic conditions.
Most cell surface proteins are decorated by glycans, and the plasma membrane is rich in glycosylated lipids. The mechanisms by which the enormous complexity of these glycan structures on proteins and lipids is exploited to control glycoprotein activity by setting their cell surface residence time and the ways by which they are taken up into cells are still under active investigation. Here, two mechanisms are presented, termed galectin lattices and glycolipid-lectin (GL-Lect)-driven endocytosis, which are among the most prominent to establish a link between glycan information and endocytosis. Types of glycans on glycoproteins and glycolipids are reviewed from the angle of their interaction with glycan-binding proteins that are at the heart of galectin lattices and GL-Lect-driven endocytosis. Examples are given to show how these mechanisms affect cellular functions ranging from cell migration and signaling to vascularization and immune modulation. Finally, outstanding challenges on the link between glycosylation and endocytosis are discussed.
Nano-structured systems have been attracting attention as carriers that can selectively transport bioactive substances such as nucleic acid medicine to a target location in the body. However, none of these systems have been able to cross the blood-brain barrier （BBB） via a systemic route to realize safe and efficient delivery of their cargo to the brain parenchyma. To tackle this problem, the authors focused on a transport protein, glucose transporter-1（GLUT1）, which is highly expressed in brain capillary endothelial cells （BCECs）, to facilitate the transport of nano-structured systems across the BBB. GLUT1 is known to migrate from the luminal side to the abluminal side in response to a rapid increase of blood glucose concentration from the hypoglycemic state. Utilizing such unique characteristic of GLUT1, the authors designed a self-assembled supramolecular nanocarrier with glucose integrated on the surface at an optimal configuration. Results obtained in vivo have shown significant accumulation of the nanocarrier within the brain parenchyma, at an efficiency that far surpasses that of conventional strategies. Precise control of glucose density on the surface of the nanocarrier allowed selection of the accumulation point in the brain, and a particularly high degree of accumulation in the neurocytes was achieved. The developed method can be used for efficiently delivering a variety of drug modalities to the brain, which offers new treatment strategies to cure neurological disorders such as the Alzheimer’s disease.
Sorting nexins (SNX) are a family of proteins containing the Phox homology domain, which shows a preferential endo-membrane association and regulates cargo sorting processes. Here, we established that SNX32, a SNX-BAR (Bin/Amphiphysin/Rvs) sub-family member associates with SNX4 via its BAR domain and the residues A226, Q259, E256, R366 of SNX32, and Y258, S448 of SNX4 that lie at the interface of these two SNX proteins mediates this association. SNX32, via its PX domain, interacts with the Transferrin receptor (TfR) and Cation Independent Mannose-6-Phosphate Receptor (CIMPR), and the conserved F131 in its PX domain is important in stabilizing these interactions. Silencing of SNX32 leads to a defect in intracellular trafficking of TfR and CIMPR. Further, using SILAC-based differential proteomics of the wild type and the mutant SNX32, impaired in cargo binding, we identified Basigin (BSG), an immunoglobulin superfamily member, as a potential interactor of SNX32 in SHSY5Y cells. We then demonstrated that SNX32 binds to BSG through its PX domain and facilitates its trafficking to the cell surface. In Neuro-Glial cell lines, silencing of SNX32 leads to defects in neuronal differentiation. Moreover, abrogation in lactate transport in the SNX32 depleted cells led us to propose that SNX32 may contribute to maintaining the neuro-glial coordination via its role in BSG trafficking and the associated Monocarboxylate transporter activity. Taken together, our study showed that SNX32 mediates the trafficking of specific cargo molecules along distinct pathways.
The human rhinovirus (HRV) A2 is endocytosed by clathrin-mediated endocytosis (CME) bound to the classical LDL receptor and releases its RNA during its transport to late endosomes. Here it is shown that - presumably due to an effect on virus recycling - a low concentration of the CME inhibitor chlorpromazine present during virus internalization (30 min) did not reduce HRV-A2 infection, but strongly inhibited short-time (5 min) endocytosis of HRV-A2. Chlorpromazine had no effect on the colocalization of the ICAM-1 ligand HRV-A89 with early endosomes, excluding CME as the main endocytosis pathway of this virus. As published for HRV-A2 and HRV-A14, HRV-A89 partially colocalized with lysosome-associated membrane protein 2 and the microtubule inhibitor nocodazole did not reduce virus infection when present only during virus internalization. Together with previous work these data suggest that there are no principal differences between endocytosis pathways of ICAM-1-binding rhinoviruses in different cell types.
Skin cancer is a global health issue and mainly composed of melanoma and nonmelanoma cancers. For the first clinical proof of concept on humans, we decided to study good prognosis skin cancers, i.e., carcinoma basal cell. In UE, the first-line treatment remains surgical resection, healing most of the tumors, but presents aesthetic disadvantages with a high reoccurrence rate on exposed areas. Moreover, the therapeutic indications could extend to melanoma and metastasis, which is a different medical strategy that could combine this treatment. Indeed, patients with late-stage melanoma are in a therapeutic impasse, despite recent targeted and immunological therapies. Photothermal therapy using gold nanoparticles is the subject of many investigations due to their strong potential to treat cancers by physical, thermal destruction. We developed gold nanoparticles synthesized by green chemistry (gGNPs), using endemic plant extract from Reunion Island, which have previously showed their efficiency at a preclinical stage. Here, we demonstrate that these gGNPs are less cytotoxic than gold nanoparticles synthesized by Turkevich's method. Furthermore, our work describes the optimization of gGNP coating and stabilization, also taking into consideration the gGNP path in cells (endocytosis, intracellular trafficking, and exocytosis), their specificity toward cancerous cells, their cytotoxicity, and their in vivo efficiency. Finally, based on the metabolic switch of cancerous cells overexpressing Glut transporters in skin cancers, we demonstrated that glucose-stabilized gGNP (gGNP@G) enables a quick internalization, fourfold higher in cancerous cells in contrast to healthy cells with no side cytotoxicity, which is particularly relevant to target and treat cancer.
A host of endocytic pathways exist at the surface of eukaryotic cells, which lead to the internalization of the bulk of membranes along with membrane proteins, signaling receptors, growth factors, and other cargoes (Smith et al. 2017). For decades, the clathrin-mediated pathway has been the major well characterized endocytic process where clathrin polymerizes along with the associated adaptor proteins to include ligand-bound receptors, leading to membrane bending, membrane scission, and endocytosis (Smith et al. 2017). Recently, multiple alternative mechanisms have been uncovered which facilitate the endocytic uptake of cargo molecules and membrane receptors even in the absence of clathrin machinery (Mayor et al.2014). A model of endocytosis that doesn’t require clathrin but rather sugar-binding galectins and glycolipids has been proposed by my host laboratory (Lakshminarayan et al. 2014). Galectins constitute a family of beta-galactoside–binding lectins, which to date consists of 15 members in mammals. Galectins are broadly distributed in a variety of cells and tissues (Leffler et al. 2004). They are translocated from the cytosol to the extracellular space by a process of non-classical secretion (Hughes 1999). Glycosphingolipids (GSLs) are ubiquitous membrane constituents that are subdivided in neutral or acidic fractions. The term GSLs applies to compounds that contain at least one monosaccharide and a ceramide. Of note, the enzyme UDP-glucose ceramide glucosyltransferase (Ugcg) catalyzes the initial step for the biosynthesis of glycosylceramide-based GSLs.Our current working model, which involves glycolipids and lectins, was termed the GL-Lect hypothesis (Johannes et al. 2016). It is backed up by experimental data as described in Ref. (Lakshminarayan et al. 2014) and can be described as follows:i) Monomeric Gal3 binds to glycoproteinsii) Gal3 then starts to oligomerizeiii) Oligomerized Gal3 has the capacity to bind to glycosphingolipids and this may induce clustering of GSLsiv) Gal3-GSL cluster are inducing the invagination of the plasma membrane to generate tubular endocytic pits from which clathrin-independent carriers (CLICs, which are pre-early endosomes) are generated.Oligomeric Gal3 is indeed able to bind to GSLs and to induce membrane deformation (Lakshminarayan et al. 2014) in a similar way the pathogenic lectin Shiga toxin-B subunit (STxB) does. Therefore, both processes could be summarized under the same hypothesis, the GL-Lect hypothesis, where GL stands for the glycosphingolipids (Gb3 for STxB and gangliosides for Gal3) and Lect summarizes the lectins (STxB, Gal3 and possibly others as well).Understanding if this clathrin-independent but Gal3-dependent internalization mechanism is conserved not only in vitro model systems but in vivo is a main challenge in the field of trafficking.We characterized for the first time that in the gut a new mechanism facilitates endocytic uptake of cargo. This mechanism is driven by Galectin3 and operates in intestinal enterocytes for transcytosis like process and is glycosphingolipid dependent. Indeed, we have found that the lactotransferrin (LTF), a Gal3 cargo that we have identified by Mass spec, strongly required Gal3 and GSLs for its efficient endocytosis and its transcytosis like distribution pattern, respectively. Based on these findings in mouse intestinal epithelium, we established a functional in vivo model system where the newly proposed endocytic mechanism termed in our lab as GL-Lect, was physiologically investigated.
Sorting nexins (SNX) are a family of proteins containing the Phox homology domain, which shows a preferential endo-membrane association and regulates cargo sorting processes. Even with the vast amount of information unveiled systematically, the underlying mechanism of sorting remains elusive. Here, we established that SNX32, a SNX-BAR (Bin/Amphiphysin/Rvs) sub-family member, is associated with SNX4 via its BAR domain. We identified A226, Q259, E256, R366 of SNX32, and Y258, S448 of SNX4 at the interface of these two SNX proteins that are important for maintaining the association. Via its PX domain, SNX32 interacts with the Transferrin receptor (TfR) and Cation Independent Mannose-6-Phosphate Receptor (CIMPR). We showed that the conserved F131 in its PX domain is important in stabilising the above interactions. Silencing of SNX32 led to a defect in intracellular trafficking of TfR and CIMPR, which could be rescued by overexpressing shRNA-resistant snx32. We also showed that both individual domains play an essential role in trafficking. Our results indicate that SNX4, SNX32 and Rab11 may participate in a common pathway regulating transferrin trafficking; however, the existence of an independent pathway for Rab11 and SNX32 could not be completely ruled out. Further, we established that the PX domain of SNX32 could bind to PI(3)P and PI(4)P, suggesting a possible explanation for its sub-cellular localization. Taken together, our study showed that SNX32 mediate the trafficking of specific cargo molecules along distinct pathway via its PX domain-directed binding to phosphoinositides and its BAR domain-mediated association with other SNX family members. Further, using SILAC-based differential proteomics of the wild type and the mutant SNX32, impaired in cargo binding, we identified Basigin (BSG), an immunoglobulin super family member, as a potential interactor of SNX32 in SH-SY-5Y cells. We then demonstrated that SNX32 binds to BSG through its PX domain and facilitates its trafficking to the cell surface. In Neuro-Glial cell lines, the silencing of SNX32 led to defects in neuronal differentiation. Moreover, abrogation in lactate transport in the SNX32 depleted cells led us to propose that the SNX may contribute to maintaining the neuro-glial coordination via its role in BSG trafficking and the associated Monocarboxylate transporter activity.
The properties of nanoparticle (NP) carriers, such as size, shape and surface state, have been proved to dramatically affect their uptake by tumor cells, thereby influencing and determining the effect of nanomedicine on tumor theranostics. However, the effect of the stiffness of NPs on their cellular internalization remains unclear, especially for circumstances involving the active or passive NP targeting. In this work, we constructed eutectic gallium indium liquid metal NPs with the same particle size, shape and surface charge properties but distinct stiffness via tailoring the surface oxidation and silica coating. It has been found that the softer NPs would be endocytosed much slower than the stiffer counterparts in the presence of specific ligand-receptor interaction. Interestingly, once the interaction is eliminated, softer NPs are internalized faster than the stiffer ones. Based on experimental observations and theoretical verification, we demonstrate that this phenomenon is mainly caused by varying degrees of the deformation of soft NPs induced by ligand-receptor interactions. Such finding of the stiffness effect of NPs implies great potential for fundamental biomedical applications, such as the rational design of nanomedicines.
Background:
The near impermeability of the blood-brain barrier (BBB) and the unique neuroimmune environment of the CNS prevents the effective use of antibodies in neurological diseases. Delivery of biotherapeutics to the brain can be enabled through receptor-mediated transcytosis via proteins such as the transferrin receptor, although limitations such as the ability to use Fc-mediated effector function to clear pathogenic targets can introduce safety liabilities. Hence, novel delivery approaches with alternative clearance mechanisms are warranted.
Methods:
Binders that optimized transport across the BBB, known as transcytosis-enabling modules (TEMs), were identified using a combination of antibody discovery techniques and pharmacokinetic analyses. Functional activity of TEMs were subsequently evaluated by imaging for the ability of myeloid cells to phagocytose target proteins and cells.
Findings:
We demonstrated significantly enhanced brain exposure of therapeutic antibodies using optimal transferrin receptor or CD98 TEMs. We found that these modules also mediated efficient clearance of tau aggregates and HER2+ tumor cells via a non-classical phagocytosis mechanism through direct engagement of myeloid cells. This mode of clearance potentially avoids the known drawbacks of FcγR-mediated antibody mechanisms in the brain such as the neurotoxic release of proinflammatory cytokines and immune cell exhaustion.
Conclusions:
Our study reports a new brain delivery platform that harnesses receptor-mediated transcytosis to maximize brain uptake and uses a non-classical phagocytosis mechanism to efficiently clear pathologic proteins and cells. We believe these findings will transform therapeutic approaches to treat CNS diseases.
Funding:
This research was funded by Janssen, Pharmaceutical Companies of Johnson & Johnson.
Brain delivery of therapeutic antibodies and biologics is restricted due to the presence of the blood-brain barrier (BBB). However, their delivery can be improved with the use of “carrier” antibodies that target receptors on the luminal surface of the BBB which initiate a process termed receptor-mediated transcytosis (RMT). This review describes key steps and transcellular pathways various BBB-crossing antibodies undertake to deliver therapeutic cargos into the brain via RMT. The pathway is initiated with the receptor-mediated endocytosis through clathrin- and/or caveolin-dependent or independent pathways. Once internalized the antibodies are routed to various endosomal compartments where decisions are made regarding their fate during endosomal protein sorting process. During this process antibodies with specific attributes will be either discarded and degraded in lysosomes or rerouted into compartments destined for release on the abluminal surface of the brain endothelial cells. Different RMT receptors may engage different shuttling pathways between the luminal and abluminal sides of the BBB. Based on this knowledge, antibodies can be engineered to add attributes that facilitate preferential routing through pathways that result in enhanced BBB crossing.
Glucose transporter 1 deficiency syndrome (GLUT1DS) is a neurometabolic disorder caused by haploinsufficiency of the GLUT1 glucose transporter (encoded by SLC2A1) leading to defective glucose transport across the blood–brain barrier. This work describes the genetic analysis of 56 patients with clinical or biochemical GLUT1DS hallmarks. 55.4% of these patients had a pathogenic variant of SLC2A1, and 23.2% had a variant in one of 13 different genes. No pathogenic variant was identified for the remaining patients. Expression analysis of SLC2A1 indicated a reduction in SLC2A1 mRNA in patients with pathogenic variants of this gene, as well as in one patient with a pathogenic variant in SLC9A6, and in three for whom no candidate variant was identified. Thus, the clinical and biochemical hallmarks generally associated with GLUT1DS may be caused by defects in genes other than SLC2A1.
The existence of endocytic pathways that do not rely on the coat protein clathrin has been known for some time. However, until recently the mechanisms involved in these pathways, as well as their functions, cargoes, and dynamics were largely unknown. Recent advances are leading to a new understanding of the magnitude of clathrin-independent pathways, their cellular functions, and the molecular mechanisms involved in the formation and post-endocytic trafficking of clathrin-independent carriers.
Recycling to the cell surface requires the scission of tubular membrane intermediates emanating from endosomes. Here we identify the monotopic membrane protein LITAF (LPS-Induced TNF Activating Factor) and the related CDIP1 (Cell Death Involved p53 target 1) as novel membrane curvature proteins that contribute to recycling tubule scission. Recombinant LITAF supports high membrane curvature, shown by its ability to reduce proteoliposome size. The membrane domains of LITAF and CDIP1 partition strongly into ∼50 nm diameter tubules labelled with the recycling markers Pacsin2, ARF6 and SNX1, and the recycling cargoes MHC Class I and CD59. Partitioning of LITAF into tubules is impaired by mutations linked to Charcot Marie Tooth disease type 1C. Meanwhile, depletion of LITAF and CDIP1 results in the expansion of tubular recycling compartments and stabilised Rab11 tubules, pointing to a function for LITAF/CDIP1 in membrane scission. Consistent with this, depletion of LITAF and CDIP1 impairs integrin recycling and cell migration.
The small GTPase Arf6 regulates many cellular processes, including cytoskeletal remodeling, receptor endocytosis, and pathogen phagocytosis. Arf6 silencing in neutrophil (PMN)-like cells is well-known to inhibit chemotactic peptide-mediated activation of phospholipase D, the oxidative burst, and β2 integrin-dependent adhesion. In conditional knockout (cKO) mice, the migration to inflammatory sites of Arf6-deficient PMNs was diminished and associated with reduced cell surface expression of β2 integrins. In this study we assessed the impact of Arf6 depletion on the functions and gene expression profile of PMNs isolated from the mouse air pouch. Numerous genes involved in response to oxygen levels, erythrocyte and myeloid differentiation, macrophage chemotaxis, response to chemicals, apoptosis, RNA destabilization, endosome organization, and vesicle transport were differentially expressed in PMNs cKO for Arf6. Lpar6 and Lacc-1 were the most up-regulated and down-regulated genes, respectively. The deletion of Arf6 also decreased Lacc-1 protein level in PMNs, and silencing of Arf6 in THP-1 monocytic cells delayed LPS-mediated Lacc-1 expression. We report that fMLP or zymosan-induced glycolysis and oxygen consumption rate were both decreased in air pouch PMNs but not in bone marrow PMNs of Arf6 cKO mice. Reduced oxygen consumption correlated with a decrease in superoxide and ROS production. Deletion of Arf6 in PMNs also reduced phagocytosis and interfered with apoptosis. The data suggest that Arf6 regulates energy metabolism, which may contribute to impaired phagocytosis, ROS production, and apoptosis in PMN-Arf6 cKO. This study provides new information on the functions and the inflammatory pathways influenced by Arf6 in PMNs.
Although agonist-dependent endocytosis of G protein-coupled receptors (GPCRs) as a means to modulate receptor signaling has been widely studied, the constitutive endocytosis of GPCRs has received little attention. Here we show that two prototypical class I GPCRs, the beta2 adrenergic and M3 muscarinic receptors, enter cells constitutively by clathrin-independent endocytosis and colocalize with markers of this endosomal pathway on recycling tubular endosomes, indicating that these receptors can subsequently recycle back to the plasma membrane (PM). This constitutive endocytosis of these receptors was not blocked by antagonists, indicating that receptor signaling was not required. Interestingly, the G proteins that these receptors couple to, Galpha(s) and Galpha(q), localized together with their receptors at the plasma membrane and on tubular recycling endosomes. Upon agonist stimulation, Galpha(s) and Galpha(q) remained associated with the PM and these endosomal membranes, whereas beta2 and M3 receptors now entered cells via clathrin-dependent endocytosis. Deletion of the third intracellular loop (i3 loop), which is thought to play a role in agonist-dependent endocytosis of the M3 receptor, had no effect on the constitutive internalization of the receptor. Surprisingly, with agonist, the mutated M3 receptor still internalized and accumulated in cells but through clathrin-independent and not clathrin-dependent endocytosis. These findings demonstrate that GPCRs are versatile PM proteins that can utilize different mechanisms of internalization depending upon ligand activation.
A new impermeant photoaffinity label has been used for identifying cell surface glucose transporters in isolated rat adipose cells. This compound is 2-N-4(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4- yloxy)-2- propylamine. We have used this reagent in combination with immunoprecipitation by specific antibodies against the GLUT4 and GLUT1 glucose transporter isoforms to estimate the relative abundance of these two transporters on the surface of the intact adipose cell following stimulation by insulin and phorbol 12-myristate 13-acetate (PMA). In the basal state, GLUT4 and GLUT1 are both present at the cell surface but GLUT4 is more abundant than GLUT1. In response to insulin, GLUT4 increases 15-20-fold and GLUT1 increases approximately 5-fold while 3-O-methyl-D-glucose transport is stimulated 20-30-fold. By contrast, PMA only induces a approximately 4-fold increase in GLUT4 while GLUT1 increases approximately 5-fold to the same level as seen with insulin. In addition, PMA stimulates 3-O-methyl-D-glucose transport approximately 3-fold to only 13% of the insulin-stimulated state. Thus GLUT4 is the major glucose transporter isoform under all conditions, and it is selectively and markedly enriched in response to insulin but not PMA which increases GLUT1 and GLUT4 equally. Furthermore, stimulation of glucose transport activity correlates closely with the appearance of GLUT4 on the cell surface in response to both insulin and PMA but does not correlate with the sum of GLUT1 and GLUT4 appearance. These results suggest that GLUT4 may be inherently more active than GLUT1 due to a higher TK (turnover/Km).
ADP-ribosylation factor (ARF) 6 localizes to the plasma membrane (PM) in its GTP state and to a tubulovesicular compartment in its GDP state in HeLa cells that express wild-type or mutant forms of this GTPase. Aluminum fluoride (AlF) treatment of ARF6-transfected cells redistributes ARF6 to the PM and stimulates the formation of actin-rich surface protrusions. Here we show that cytochalasin D (CD) treatment inhibited formation of the AlF-induced protrusions and shifted the distribution of ARF6 to a tubular membrane compartment emanating from the juxtanuclear region of cells, which resembled the compartment where the GTP-binding defective mutant of ARF6 localized. This membrane compartment was distinct from transferrin-positive endosomes, could be detected in the absence of ARF6 overexpression or CD treatment, and was accessible to loading by PM proteins lacking clathrin/AP-2 cytoplasmic targeting sequences, such as the IL-2 receptor alpha subunit Tac. ARF6 and surface Tac moved into this compartment and back out to the PM in the absence of pharmacologic treatment. Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively. Thus, the ARF6 GTP cycle regulates this membrane traffic pathway. The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton.
ADP-ribosylation factor (Arf) 6 regulates the movement of membrane between the plasma membrane (PM) and a nonclathrin-derived endosomal compartment and activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show that PIP2 visualized by expressing a fusion protein of the pleckstrin homology domain from PLCdelta and green fluorescent protein (PH-GFP), colocalized with Arf6 at the PM and on tubular endosomal structures. Activation of Arf6 by expression of its exchange factor EFA6 stimulated protrusion formation, the uptake of PM into macropinosomes enriched in PIP2, and recycling of this membrane back to the PM. By contrast, expression of Arf6 Q67L, a GTP hydrolysis-resistant mutant, induced the formation of PIP2-positive actin-coated vacuoles that were unable to recycle membrane back to the PM. PM proteins, such as beta1-integrin, plakoglobin, and major histocompatibility complex class I, that normally traffic through the Arf6 endosomal compartment became trapped in this vacuolar compartment. Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6 Q67L. These results demonstrate that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway.
The Eps15 homology (EH) domain-containing protein, EHD1, has recently been ascribed a role in the recycling of receptors internalized by clathrin-mediated endocytosis. A subset of plasma membrane proteins can undergo internalization by a clathrin-independent pathway regulated by the small GTP-binding protein ADP-ribosylation factor 6 (Arf6). Here, we report that endogenous EHD proteins, as well as transgenic tagged EHD1, are associated with long, membrane-bound tubules containing Arf6. EHD1 appears to induce tubule formation, which requires nucleotide cycling on Arf6 and intact microtubules. Mutations in the N-terminal P-loop domain or deletion of the C-terminal EH domain of EHD1 prevent association of EHD1 with tubules or induction of tubule formation. The EHD1 tubules contain internalized major histocompatibility complex class I (MHC-I) molecules that normally traffic through the Arf6 pathway. Recycling assays show that overexpression of EHD1 enhances MHC-I recycling. These observations suggest an additional function of EHD1 as a tubule-inducing factor in the Arf6 pathway for recycling of plasma membrane proteins internalized by clathrin-independent endocytosis.
Growth arrest specific 3 (Gas3)/peripheral myelin protein 22 (PMP22) is a component of the compact peripheral nerve myelin, and mutations affecting gas3/PMP22 gene are responsible for a group of peripheral neuropathies in humans. We have performed in vivo imaging in order to investigate in detail the phenotype induced by Gas3/PMP22 overexpression in cultured cells. Here we show that Gas3/PMP22 triggers the accumulation of vacuoles, before the induction of cell death or of changes in cell spreading. Overexpressed Gas3/PMP22 accumulates into two distinct types of intracellular membrane compartments. Gas3/PMP2 accumulates within late endosomes close to the juxtanuclear region, whereas in the proximity of the cell periphery, it induces the formation of actin/phosphatidylinositol (4,5)-bisphosphate (PIP(2))-positive large vacuoles. Gas3/PMP22-induced vacuoles do not contain transferrin receptor, but instead they trap membrane proteins that normally traffic through the ADP-ribosylation factor 6 (Arf6) endosomal compartment. Arf6 and Arf6-Q67L co-localize with Gas3/PMP22 in these vacuoles, and the dominant negative mutant of Arf6, T27N, blocks the appearance of vacuoles in response to Gas3/PMP22, but not its accumulation in the late endosomes. Finally a point mutant of Gas3/PMP22 responsible for the Charcot-Marie-Tooth 1A disease is unable to trigger the accumulation of PIP(2)-positive vacuoles. Altogether these results suggest that increased Gas3/PMP22 levels can alter membrane traffic of the Arf6 plasma-membrane-endosomal recycling pathway and show that, similarly to other tetraspan proteins, Gas3/PMP22 can accumulate in the late endosomes.
The trafficking of two plasma membrane (PM) proteins that lack clathrin internalization sequences, major histocompatibility complex class I (MHCI), and interleukin 2 receptor alpha subunit (Tac) was compared with that of PM proteins internalized via clathrin. MHCI and Tac were internalized into endosomes that were distinct from those containing clathrin cargo. At later times, a fraction of these internalized membranes were observed in Arf6-associated, tubular recycling endosomes whereas another fraction acquired early endosomal autoantigen 1 (EEA1) before fusion with the "classical" early endosomes containing the clathrin-dependent cargo, LDL. After convergence, cargo molecules from both pathways eventually arrived, in a Rab7-dependent manner, at late endosomes and were degraded. Expression of a constitutively active mutant of Arf6, Q67L, caused MHCI and Tac to accumulate in enlarged PIP(2)-enriched vacuoles, devoid of EEA1 and inhibited their fusion with clathrin cargo-containing endosomes and hence blocked degradation. By contrast, trafficking and degradation of clathrin-cargo was not affected. A similar block in transport of MHCI and Tac was reversibly induced by a PI3-kinase inhibitor, implying that inactivation of Arf6 and acquisition of PI3P are required for convergence of endosomes arising from these two pathways.
The plasma membrane is the interface between cells and their harsh environment. Uptake of nutrients and all communication among cells and between cells and their environment occurs through this interface. 'Endocytosis' encompasses several diverse mechanisms by which cells internalize macromolecules and particles into transport vesicles derived from the plasma membrane. It controls entry into the cell and has a crucial role in development, the immune response, neurotransmission, intercellular communication, signal transduction, and cellular and organismal homeostasis. As the complexity of molecular interactions governing endocytosis are revealed, it has become increasingly clear that it is tightly coordinated and coupled with overall cell physiology and thus, must be viewed in a broader context than simple vesicular trafficking.
The endocytosis of E-cadherin has recently emerged as an important determinant of cadherin function with the potential to participate in remodeling adhesive contacts. In this study we focused on the initial fate of E-cadherin when it predominantly exists free on the cell surface prior to adhesive binding or incorporation into junctions. Surface-labeling techniques were used to define the endocytic itinerary of E-cadherin in MCF-7 cells and in Chinese hamster ovary cells stably expressing human E-cadherin. We found that in this experimental system E-cadherin entered a transferrin-negative compartment before transport to the early endosomal compartment, where it merged with classical clathrin-mediated uptake pathways. E-cadherin endocytosis was inhibited by mutant dynamin, but not by an Eps15 mutant that effectively blocked transferrin internalization. Furthermore, sustained signaling by the ARF6 GTPase appeared to trap endocytosed E-cadherin in large peripheral structures. We conclude that in isolated cells unbound E-cadherin on the cell surface is predominantly endocytosed by a clathrin-independent pathway resembling macropinocytotic internalization, which then fuses with the early endosomal system. Taken with earlier reports, this suggests the possibility that multiple pathways exist for E-cadherin entry into cells that are likely to reflect cell context and regulation.
CD44 was once thought to simply be a transmembrane adhesion molecule that also played a role in the metabolism of its principal ligand hyaluronan. Investigations of CD44 over the past approximately 20 yr have established additional functions for CD44, including its capacity to mediate inflammatory cell function and tumor growth and metastasis. It has also become evident that intricate posttranslational modifications of CD44 regulate the affinity of the receptor for its ligands. In this review, we focus on emerging evidence that functional fragments of the cytoplasmic and ectodomain of CD44 can be liberated by enzymatic modification of cell surfaces as well as of cell-associated matrix. Based on the evidence discussed, we propose that CD44 exists in three phases, as a transmembrane receptor, as an integral component of the matrix, and as a soluble protein found in body fluids, each with biologically significant functions of which some are shared and some distinct. Thus, CD44 represents a model for understanding posttranslational processing and its emerging role as a general mechanism for regulating cell behavior.
Clathrin-independent endocytosis internalizes plasma membrane proteins that lack cytoplasmic sequences recognized by clathrin adaptor proteins. There is evidence for different clathrin-independent pathways but whether they share common features has not been systematically tested. Here, we examined whether CD59, an endogenous glycosylphosphatidyl inositol-anchored protein (GPI-AP), and major histocompatibility protein class I (MHCI), an endogenous, integral membrane protein, entered cells through a common mechanism and followed a similar itinerary. At early times of internalization, CD59 and MHCI were found in the same Arf6-associated endosomes before joining clathrin cargo proteins such as transferrin in common sorting endosomes. CD59 and MHCI, but not transferrin, also were observed in the Arf6-associated tubular recycling membranes. Endocytosis of CD59 and MHCI required free membrane cholesterol because it was inhibited by filipin binding to the cell surface. Expression of active Arf6 stimulated endocytosis of GPI-APs and MHCI to the same extent and led to their accumulation in Arf6 endosomes that labeled intensely with filipin. This blocked delivery of GPI-APs and MHCI to early sorting endosomes and to lysosomes for degradation. Endocytosis of transferrin was not affected by any of these treatments. These observations suggest common mechanisms for endocytosis without clathrin.
Plasma membrane proteins that are internalized independently of clathrin, such as major histocompatibility complex class I (MHCI), are internalized in vesicles that fuse with the early endosomes containing clathrin-derived cargo. From there, MHCI is either transported to the late endosome for degradation or is recycled back to the plasma membrane via tubular structures that lack clathrin-dependent recycling cargo, e.g., transferrin. Here, we show that the small GTPase Rab22a is associated with these tubular recycling intermediates containing MHCI. Expression of a dominant negative mutant of Rab22a or small interfering RNA-mediated depletion of Rab22a inhibited both formation of the recycling tubules and MHCI recycling. By contrast, cells expressing the constitutively active mutant of Rab22a exhibited prominent recycling tubules and accumulated vesicles at the periphery, but MHCI recycling was still blocked. These results suggest that Rab22a activation is required for tubule formation and Rab22a inactivation for final fusion of recycling membranes with the surface. The trafficking of transferrin was only modestly affected by these treatments. Dominant negative mutant of Rab11a also inhibited recycling of MHCI but not the formation of recycling tubules, suggesting that Rab22a and Rab11a might coordinate different steps of MHCI recycling.
Eps15 homology domain (EHD) 1 enables membrane recycling by controlling the exit of internalized molecules from the endocytic recycling compartment (ERC) en route to the plasma membrane, similar to the role described for Rab11. However, no physical or functional connection between Rab11 and EHD-family proteins has been demonstrated yet, and the mode by which they coordinate their regulatory activity remains unknown. Here, we demonstrate that EHD1 and EHD3 (the closest EHD1 paralog), bind to the Rab11-effector Rab11-FIP2 via EH-NPF interactions. The EHD/Rab11-FIP2 associations are affected by the ability of the EHD proteins to bind nucleotides, and Rab11-FIP2 is recruited to EHD-containing membranes. These results are consistent with a coordinated role for EHD1 and Rab11-FIP2 in regulating exit from the ERC. However, because no function has been attributed to EHD3, the significance of its interaction with Rab11-FIP2 remained unclear. Surprisingly, loss of EHD3 expression prevented the delivery of internalized transferrin and early endosomal proteins to the ERC, an effect differing from that described upon EHD1 knockdown. Moreover, the subcellular localization of Rab11-FIP2 and endogenous Rab11 were altered upon EHD3 knockdown, with both proteins absent from the ERC and retained in the cell periphery. The results presented herein promote a coordinated role for EHD proteins and Rab11-FIP2 in mediating endocytic recycling and provide evidence for the function of EHD3 in early endosome to ERC transport.
Extracellular signal-regulated kinase (Erk) is widely recognized for its central role in cell proliferation and motility. Although previous work has shown that Erk is localized at endosomal compartments, no role for Erk in regulating endosomal trafficking has been demonstrated. Here, we report that Erk signaling regulates trafficking through the clathrin-independent, ADP-ribosylation factor 6 (Arf6) GTPase-regulated endosomal pathway. Inactivation of Erk induced by a variety of methods leads to a dramatic expansion of the Arf6 endosomal recycling compartment, and intracellular accumulation of cargo, such as class I major histocompatibility complex, within the expanded endosome. Treatment of cells with the mitogen-activated protein kinase kinase (MEK) inhibitor U0126 reduces surface expression of MHCI without affecting its rate of endocytosis, suggesting that inactivation of Erk perturbs recycling. Furthermore, under conditions where Erk activity is inhibited, a large cohort of Erk, MEK, and the Erk scaffold kinase suppressor of Ras 1 accumulates at the Arf6 recycling compartment. The requirement for Erk was highly specific for this endocytic pathway, because its inhibition had no effect on trafficking of cargo of the classical clathrin-dependent pathway. These studies reveal a previously unappreciated link of Erk signaling to organelle dynamics and endosomal trafficking.
The ADP-ribosylation factor (ARF) small GTPases regulate vesicular traffic and organelle structure by recruiting coat proteins, regulating phospholipid metabolism and modulating the structure of actin at membrane surfaces. Recent advances in our understanding of the signalling pathways that are regulated by ARF1 and ARF6, two of the best characterized ARF proteins, provide a molecular context for ARF protein function in fundamental biological processes, such as secretion, endocytosis, phagocytosis, cytokinesis, cell adhesion and tumour-cell invasion.
Plasma membrane proteins such as receptors and ion channels allow a cell to communicate with its environment and regulate
many intracellular activities. Thus, the proper control of the surface number of these proteins is essential for maintaining
the structural and functional homeostasis of a cell. Internalization and recycling plays a key role in determining the surface
density of receptors and channels. Whereas the clathrin-mediated internalization and its associated recycling have been the
focus of research in this field, recent studies have revealed that an increasing number of receptors and channels enter a
cell via clathrin-independent pathways. However, little is known about the trafficking motifs involved in controlling clathrin-independent
internalization and various associated recycling pathways. By using a potassium channel as a model system, we identified a
class of trafficking motifs that function along a clathrin-independent pathway to increase the surface density of a membrane
protein by preventing its rapid internalization and/or facilitating its recycling via the ADP-ribosylation factor 6-dependent
recycling pathway. Moreover our data suggest that these motifs may enhance the association of membrane proteins with the EFA6
family of guanine nucleotide exchange factors for ADP-ribosylation factor 6.
Reduction in sample complexity enables more thorough proteomic analysis using mass spectrometry (MS). A solution-based two-dimensional (2D) protein fractionation system, ProteomeLab PF 2D, has recently become available for sample fractionation and complexity reduction. PF 2D resolves proteins by isoelectric point (pI) and hydrophobicity in the first and second dimensions, respectively. It offers distinctive advantages over 2D gel electrophoresis with respects to automation of the fractionation processes and characterization of proteins having extreme pIs. Besides fractionation, PF 2D is equipped with built-in UV detectors intended for relative quantification of proteins in contrasting samples using its software tools. In this study, we utilized PF 2D for the identification of basic and acidic proteins in mammalian cells, which are generally under-characterized. In addition, mass spectrometric methods (label-free and 18O-labeling) were employed to complement protein quantification based on UV absorbance. Our studies indicate that the selection of chromatographic fractions could impact protein identification and that the UV-based quantification for contrasting complex proteomes is constrained by coelution or partial coelution of proteins. In contrast, the quantification post PF 2D chromatography based on label-free or 18O-labeling mass spectrometry provides an alternative platform for basic/acidic protein identification and quantification. With the use of HCT116 colon carcinoma cells, a total of 305 basic and 183 acidic proteins was identified. Quantitative proteomics revealed that 17 of these proteins were differentially expressed in HCT116 p53-/- cells.
In a genome-wide RNA-mediated interference screen for genes required in membrane traffic - including endocytic uptake, recycling from endosomes to the plasma membrane, and secretion - we identified 168 candidate endocytosis regulators and 100 candidate secretion regulators. Many of these candidates are highly conserved among metazoans but have not been previously implicated in these processes. Among the positives from the screen, we identified PAR-3, PAR-6, PKC-3 and CDC-42, proteins that are well known for their importance in the generation of embryonic and epithelial-cell polarity. Further analysis showed that endocytic transport in Caenorhabditis elegans coelomocytes and human HeLa cells was also compromised after perturbation of CDC-42/Cdc42 or PAR-6/Par6 function, indicating a general requirement for these proteins in regulating endocytic traffic. Consistent with these results, we found that tagged CDC-42/Cdc42 is enriched on recycling endosomes in C. elegans and mammalian cells, suggesting a direct function in the regulation of transport.
Trafficking of H-Ras was examined to determine whether it can enter cells through clathrin-independent endocytosis (CIE). H-Ras colocalized with the CIE cargo protein, class I major histocompatibility complex, and it was sequestered in vacuoles that formed upon expression of an active mutant of Arf6, Q67L. Activation of Ras, either through epidermal growth factor stimulation or the expression of an active mutant of Ras, G12V, induced plasma membrane ruffling and macropinocytosis, a stimulated form of CIE. Live imaging of cells expressing H-RasG12V and fluorescent protein chimeras with pleckstrin homology domains that recognize specific phosphoinositides showed that incoming macropinosomes contained phosphatidylinositol 4,5-bisphosphate (PIP(2)) and phosphatiylinositol 3,4,5-trisphosphate (PIP(3)). PIP(2) loss from the macropinosome was followed by the recruitment of Rab5, a downstream target of Ras, and then PIP(3) loss. Our studies support a model whereby Ras can signal on macropinosomes that pass through three distinct stages: PIP(2)/PIP(3), PIP(3)/Rab5, and Rab5. Vacuoles that form in cells expressing Arf6Q67L trap Ras signaling in the first stage, recruiting the active form of the Ras effectors extracellular signal-regulated kinase and protein kinase B (Akt) but not Rab5. Arf6 stimulation of macropinocytosis also involves passage through the distinct lipid phases, but recruitment of Akt is not observed.
Today it is generally accepted that there are several endocytic mechanisms, both the clathrin-dependent one and mechanisms which operate without clathrin and with different requirements when it comes to dynamin, small GTP-binding proteins of the Rho family and specific lipids. It should be noted that clathrin-independent endocytosis can occur even when the cholesterol level in the membrane has been reduced to so low levels that caveolae are gone and clathrin-coated membrane areas are flat. Although new investigators in the field take it for granted that there is a multitude of entry mechanisms, it has taken a long time for this to become accepted. However, more work needs to be done, because one can still ask the question: How many endocytic mechanisms does a cell have, what are their function, and how are they regulated? This article describes some of the history of endocytosis research and attempts to give an overview of the complexity of the mechanisms and their regulation.
Major histocompatibility complex (MHC) class II molecules (MHC-II) function by binding antigenic peptides and displaying these
peptides on the surface of antigen presenting cells (APCs) for recognition by peptide-MHC-II (pMHC-II)-specific CD4 T cells.
It is known that cell surface MHC-II can internalize, exchange antigenic peptides in endosomes, and rapidly recycle back to
the plasma membrane; however, the molecular machinery and trafficking pathways utilized by internalizing/recycling MHC-II
have not been identified. We now demonstrate that unlike newly synthesized invariant chain-associated MHC-II, mature cell
surface pMHC-II complexes internalize following clathrin-, AP-2-, and dynamin-independent endocytosis pathways. Immunofluorescence
microscopy of MHC-II expressing HeLa-CIITA cells, human B cells, and human DCs revealed that pMHC enters Arf6+Rab35+EHD1+ tubular endosomes following endocytosis. These data contrast the internalization pathways followed by newly synthesized and
peptide-loaded MHC-II molecules and demonstrates that cell surface pMHC-II internalize and rapidly recycle from early endocytic
compartments in tubular endosomes.
In comparison to the internalization pathways of endocytosis, the recycling pathways are less understood. Even less defined is the process of regulated recycling, as few examples exist and their underlying mechanisms remain to be clarified. In this study, we examine the endocytic recycling of integrin β1, a process that has been suggested to play an important role during cell motility by mediating the redistribution of integrins to the migrating front. External stimulation regulates the endocytic itinerary of β1, mainly at an internal compartment that is likely to be a subset of the recycling endosomes. This stimulation-dependent recycling is regulated by ARF6 and Rab11, and also requires the actin cytoskeleton in an ARF6-dependent manner. Consistent with these observations being relevant for cell motility, mutant forms of ARF6 that affect either actin rearrangement or recycling inhibit the motility of a breast cancer cell line.
There is increasing interest in endocytosis that occurs independently of clathrin coats and the fates of membrane proteins internalized by this mechanism. The appearance of clathrin-independent endocytic and membrane recycling pathways seems to vary with different cell types and cargo molecules. In this review we focus on studies that have been performed using HeLa and COS cells as model systems for understanding this membrane trafficking system. These endosomal membranes contain signaling molecules including H-Ras, Rac1, Arf6 and Rab proteins, and a lipid environment rich in cholesterol and PIP(2) providing a unique platform for cell signaling. Furthermore, activation of some of these signaling molecules (H-Ras, Rac and Arf6) can switch the constitutive form of clathrin-independent endocytosis into a stimulated one, associated with PM ruffling and macropinocytosis.
Membrane-bound complement regulatory proteins (mCRPs) play an important role in the protection of cells from complement-mediated injury. It is now apparent that malignant tumor cells also express these proteins to escape complement attack. Here, Arko Gorter and Seppo Meri discuss the implications of complement resistance for the immunotherapeutic treatment of solid tumors with monoclonal antibodies.
In comparison to the internalization pathways of endocytosis, the recycling pathways are less understood. Even less defined is the process of regulated recycling, as few examples exist and their underlying mechanisms remain to be clarified. In this study, we examine the endocytic recycling of integrin beta1, a process that has been suggested to play an important role during cell motility by mediating the redistribution of integrins to the migrating front. External stimulation regulates the endocytic itinerary of beta1, mainly at an internal compartment that is likely to be a subset of the recycling endosomes. This stimulation-dependent recycling is regulated by ARF6 and Rab11, and also requires the actin cytoskeleton in an ARF6-dependent manner. Consistent with these observations being relevant for cell motility, mutant forms of ARF6 that affect either actin rearrangement or recycling inhibit the motility of a breast cancer cell line.
Syndecans are heparan sulfate proteoglycans that modulate the activity of several growth factors and cell adhesion molecules. PDZ domains in the adaptor protein syntenin interact with syndecans and with the phosphoinositide PIP(2), which is involved in the regulation of the actin cytoskeleton and membrane trafficking. Here, we show that the syntenin PDZ domain-PIP(2) interaction controls Arf6-mediated syndecan recycling through endosomal compartments. FGF receptor accompanies syndecan along the syntenin-mediated recycling pathway, in a heparan sulfate- and FGF-dependent manner. Syndecans that cannot recycle via this pathway become trapped intracellularly and inhibit cell spreading. This syntenin-mediated syndecan recycling pathway may regulate the surface availability of a number of cell adhesion and signaling molecules.
Coomassie blue staining of gels and blots is commonly employed for detection and quantitation of proteins by densitometry. We found that Coomassie blue or Fast Green FCF bound to protein fluoresces in the near infrared. We took advantage of this property to develop a rapid and sensitive method for detection and quantitation of proteins in gels and on blots. The fluorescence response is quantitative for protein content between 10 ng and 20 microg per band or spot. Staining and destaining require only 30 min, and the method is compatible with subsequent immunodetection.
A software program for label-free protein quantification using LC−MS/MS was developed. Complex proteomes were used to demonstrate the reproducibility and linearity of the label-free approach. The program was also applied for the identification of differentially expressed proteins in parental and p53-/- human colon cancer cell lines. The ability to process multiple data sets and the inclusion of algorithms for outlier-resistant mean estimation and probability threshold adjustment make the program a versatile tool for biomarker discovery.
Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that are widely expressed throughout the brain and are involved in synaptic development, transmission, and plasticity. The endocytosis of several members of the GPCR superfamily of receptors, such as beta-adrenergic receptors, has been studied extensively. In contrast, the mechanisms regulating mGluR endocytosis and intracellular trafficking remain poorly defined. We describe here for the first time a distinct endocytic and intracellular sorting pathway utilized by mGluR7. We show that mGluR7 constitutively internalizes via a non-clathrin mediated pathway in heterologous cells and in neurons. Unlike clathrin-mediated NMDAR endocytosis, mGluR7 traffics via an Arf6-positive endosomal pathway, similar to other well-characterized proteins such as major histocompatibility complex class I (MHC I) and the GPI-anchored protein CD59. Thus constitutive endocytosis of mGluR7 in neurons is not regulated by clathrin-dependent mechanisms, and this clathrin-independent pathway ultimately determines the amount of receptor present on the plasma membrane available to bind and respond to glutamate.
There are numerous ways that endocytic cargo molecules may be internalized from the surface of eukaryotic cells. In addition to the classical clathrin-dependent mechanism of endocytosis, several pathways that do not use a clathrin coat are emerging. These pathways transport a diverse array of cargoes and are sometimes hijacked by bacteria and viruses to gain access to the host cell. Here, we review our current understanding of various clathrin-independent mechanisms of endocytosis and propose a classification scheme to help organize the data in this complex and evolving field.
In mammals, the mucolipin family includes three members mucolipin-1, mucolipin-2, and mucolipin-3 (MCOLN1-3). While mutations in MCOLN1 and MCOLN3 have been associated with mucolipidosis type IV and the varitint-waddler mouse phenotype, respectively, little is known about the function and cellular distribution of MCOLN2. Here we show that MCOLN2 traffics via the Arf6-associated pathway and colocalizes with major histocompatibility protein class I (MHCI) and glycosylphosphatidylinositol-anchored proteins (GPI-APs), such as CD59 in both vesicles and long tubular structures. Expression of a constitutive active Arf6 mutant, or activation of endogenous Arf6 by transfection with EFA6 or treatment with aluminum fluoride, caused accumulation of MCOLN2 in enlarged vacuoles that also contain MHCI and CD59. In addition, overexpression of MCOLN2 promoted efficient activation of Arf6 in vivo, thus suggesting that MCOLN2 may have a role in the traffic of cargo through the Arf6-associated pathway. In support of this we found that overexpression of a MCOLN2 inactive mutant decreases recycling of CD59 to the plasma membrane. Therefore, our results indicate that MCOLN2 localizes to the Arf6-regulated pathway and regulates sorting of GPI-APs.
The immunoglobulin superfamily member CD147 plays an important role in fetal, neuronal, lymphocyte and extracellular matrix development. Here we review the current understanding of CD147 expression and protein interactions with regard to CD147 function and its role in pathologic conditions including heart disease, Alzheimer's disease, stroke and cancer. A model linking hypoxic conditions found within the tumor microenvironment to upregulation of CD147 expression and tumor progression is introduced.
Alix/Bro1p family proteins have recently been identified as important components of multivesicular endosomes (MVEs) and are involved in the sorting of endocytosed integral membrane proteins, interacting with components of the ESCRT complex, the unconventional phospholipid LBPA, and other known endocytosis regulators. During infection, Alix can be co-opted by enveloped retroviruses, including HIV, providing an important function during virus budding from the plasma membrane. In addition, Alix is associated with the actin cytoskeleton and might regulate cytoskeletal dynamics.
Here we demonstrate a novel physical interaction between the only apparent Alix/Bro1p family protein in C. elegans, ALX-1, and a key regulator of receptor recycling from endosomes to the plasma membrane, called RME-1. The analysis of alx-1 mutants indicates that ALX-1 is required for the endocytic recycling of specific basolateral cargo in the C. elegans intestine, a pathway previously defined by the analysis of rme-1 mutants. The expression of truncated human Alix in HeLa cells disrupts the recycling of major histocompatibility complex class I, a known Ehd1/RME-1-dependent transport step, suggesting the phylogenetic conservation of this function. We show that the interaction of ALX-1 with RME-1 in C. elegans, mediated by RME-1/YPSL and ALX-1/NPF motifs, is required for this recycling process. In the C. elegans intestine, ALX-1 localizes to both recycling endosomes and MVEs, but the ALX-1/RME-1 interaction appears to be dispensable for ALX-1 function in MVEs and/or late endosomes.
This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1.
CD1 proteins are a family of major histocompatibility complex (MHC) class I-like antigen-presenting molecules that present lipids to T cells. The cytoplasmic tails (CTs) of all human CD1 isoforms, with the exception of CD1a, contain tyrosine-based sorting motifs, responsible for the internalization of proteins by the clathrin-mediated pathway. The role of the CD1a CT, which does not possess any sorting motifs, as well as its mode of internalization are not known. We investigated the internalization and recycling pathways followed by CD1a and the role of its CT. We found that CD1a can be internalized by a clathrin- and dynamin-independent pathway and that it follows a Rab22a- and ADP ribosylation factor (ARF)6-dependent recycling pathway, similar to other cargo internalized independent of clathrin. We also found that the CD1a CT is S-acylated. However, this posttranslational modification does not determine the rate of internalization or recycling of the protein or its localization to detergent-resistant membrane microdomains (DRMs) where we found CD1a to be enriched. We also show that plasma membrane DRMs are essential for efficient CD1a-mediated antigen presentation. These findings place CD1a closer to MHC class I in its trafficking and potential antigen-loading compartments among CD1 isoforms. Furthermore, we identify CD1a as a new marker for the clathrin- and dynamin-independent and DRM-dependent pathway of internalization as well as the Rab22a- and ARF6-dependent recycling pathway.
Pathways of clathrin-independent endocytosis
- Mayor
Regulated portals of entry into the cell
- Conner
ARF proteins: roles in membrane traffic and beyond
- D'Souza-Schorey
CD147 immunoglobulin superfamily receptor function and role in pathology
- Kt Iacono
- Al Brown
- Mi Greene
- Sj Saouaf
CD147 immunoglobulin superfamily receptor function and role in pathology
- Iacono