Role In Receptor-mediated Endocytosis Research Articles

Opposite changes in the expression of clathrin and caveolin-1 in normal and cancerous human prostate tissue: putative clathrin-mediated recycling of EGFR

Endocytosis, an important macromolecule uptake process in cells, is known to be dysregulated in cancer. Clathrin and caveolin-1 proteins play a major role in receptor-mediated endocytosis. We have used a quantitative, unbiased and semi-automated method to measure in situ protein expression of clathrin and caveolin-1 in cancerous and paired normal (cancer adjacent, non-cancerous) human prostate tissue. There was a significant (p < 0.0001) increase in the expression of clathrin in prostate cancer samples (N = 29, n = 91) compared to normal tissue (N = 29, n = 67) (N = number of patients, n = number of cores in tissue arrays). Conversely, there was a significant (p < 0.0001) decrease in expression of caveolin-1 in prostate cancer tissue compared to normal prostate tissue. The opposite change in expression of the two proteins was highly correlated to increasing cancer aggressiveness. There was also a concurrent increase in the expression of epidermal growth factor receptor (EGFR), a key receptor in carcinogenesis, with clathrin in prostate cancer tissue, indicating recycling of EGFR through clathrin-mediated endocytosis (CME). These results indicate that in prostate cancer, caveolin-1-mediated endocytosis (CavME) may be acting as a brake and increase in CME may facilitate tumorigenicity and aggressiveness of prostate cancer through recycling of EGFR. Changes in the expression of these proteins can also potentially be used as a biomarker for prostate cancer to aid in diagnosis and prognosis and clinical decision-making.

Effects of receptor properties on particle internalization through receptor-mediated endocytosis.

Receptor-mediated endocytosis (RME) is a highly complex process carried out by bioparticles, such as viruses and drug carriers, to enter cells. The discovery of both clathrin-dependent and clathrin-free pathways makes the RME process even more intriguing. Numerical models have been developed to facilitate the exploration of the process. However, the impacts of the receptor properties on RME have been less studied partially due to the oversimplifications of the receptor models. In this paper, we implement a stochastic model to systematically investigate the effects of mechanical (receptor flexure), geometrical (receptor length) and biochemical (ligand-receptor cutoff) properties of receptors, on RME with and without the existence of clathrin. Our simulation results show that the receptor's flexural rigidity plays an important role in RME with clathrin. There is a threshold beyond which particle internalization will not occur. Without clathrin, it is very difficult to achieve complete endocytosis with ligand-receptor interactions alone. A shorter receptor length and longer ligand-receptor reaction cutoff promote the formation of ligand-receptor bonds and facilitate particle internalization. Complete internalization can only be obtained with an extremely short receptor length and long reaction cutoff. Therefore, there are most likely some additional mechanisms to drive the membrane deformation in clathrin-free RME. Our results yield important fundamental insights into RME and provide crucial guidance when correlating the simulation results with experimental observations.

WASP Restricts Active Rac to Maintain Cells' Front-Rear Polarization.

SummaryEfficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.

Specific Endocytosis Blockade of Trypanosoma cruzi Exposed to a Poly-LAcNAc Binding Lectin Suggests that Lectin-Sugar Interactions Participate to Receptor-Mediated Endocytosis.

Trypanosoma cruzi is a protozoan parasite transmitted by a triatomine insect, and causing human Chagas disease in South America. This parasite undergoes a complex life cycle alternating between non-proliferative and dividing forms. Owing to their high energy requirement, replicative epimastigotes of the insect midgut display high endocytic activity. This activity is mainly restricted to the cytostome, by which the cargo is taken up and sorted through the endosomal vesicular network to be delivered to reservosomes, the final lysosomal-like compartments. In African trypanosomes tomato lectin (TL) and ricin, respectively specific to poly-N-acetyllactosamine (poly-LacNAc) and β-D-galactose, allowed the identification of giant chains of poly-LacNAc in N-glycoproteins of the endocytic pathway. We show that in T. cruzi epimastigote forms also, glycoproteins of the endocytic pathway are characterized by the presence of N-linked glycans binding to both ricin and TL. Affinity chromatography using both TL and Griffonia simplicifolia lectin II (GSLII), specific to non-reducing terminal residue of N-acetylglucosamine (GlcNAc), led to an enrichment of glycoproteins of the trypanosomal endocytic pathway. Incubation of live parasites with TL, which selectively bound to the cytostome/cytopharynx, specifically inhibited endocytosis of transferrin (Tf) but not dextran, a marker of fluid endocytosis. Taken together, our data suggest that N-glycan modification of endocytic components plays a crucial role in receptor-mediated endocytosis of T. cruzi.

THE ROLE OF CD2AP IN APP PROCESSING

Genome-wide association studies (GWAS) have identified >20 risk loci for late-onset Alzheimer's disease (LOAD). Several of these risk genes are involved in endocytosis, a process believed to be disrupted during the early stages of LOAD. The CD2-associated protein (CD2AP) gene is strongly associated with LOAD risk (p=5.2x10-11) and plays a role in receptor-mediated endocytosis. We hypothesised that CD2AP contributes to amyloid precursor protein (APP) metabolism and subsequent amyloid –beta (Aβ) generation through its regulation of clathrin-mediated endocytosis. To test this, we initially characterised the effects of CD2AP knockdown on APP metabolism and Aβ production in a human neuronal cell line. A recent study found that another GWAS-identified risk gene PICALM, regulates trafficking of Aβ across the endothelial cells which form the blood brain barrier (BBB). We hypothesise that CD2AP acts in a similar way to PICALM and that knocking down CD2AP diminishes Aβ trafficking across the BBB, decreasing the amount of Aβ cleared from the brain. To investigate this, we are characterising the effects of CD2AP knockdown in an in vitro model of the BBB. Endogenous CD2AP expression was knocked down in H4 and hMEC/D3 cells using siRNA. Knockdown was quantified by western blot and Image J analysis. Levels of APP, Aβ, and APP metabolites were quantified using ELISAs. The rate of receptor-mediated endocytosis was measured by a transferrin uptake assay. Co-localisation of CD2AP with APP and endosomal markers was assessed using immunocytochemistry. Transcytosis in hMEC/D3 cells will be assessed using Transwell assays. We observed a significant 30.27±0.07 % increase in Aβ40 expression (p=0.0046). Expression levels of APP and APP processing metabolites was not significantly altered. Results from our experiments in progress will be made available at AAIC 2016. Knockdown of CD2AP expression resulted in a significant increase in extracellular amyloid-B (Aβ) levels, which did not appear to be directly related to changes in APP processing. This study is ongoing and novel data will be presented at AAIC 2016. Overall, our study provides valuable insights into how CD2AP contributes to LOAD susceptibility. Effect of CD2AP siRNA treatment of H4 cells on extracellular Aβ40 protein levels. Results are expressed as a percentage of oligofectamine only treated cells. Four biological replicates were analysed for each assay. Statistical significance of CD2AP siRNA vs. control siRNA treatment was determined by two-tailed student's t-test., **p = 0.0046; *= 0.0223 Error bars = SEM.

Phagocytic receptor signaling regulates clathrin and epsin-mediated cytoskeletal remodeling during apoptotic cell engulfment in C. elegans

The engulfment and subsequent degradation of apoptotic cells by phagocytes is an evolutionarily conserved process that efficiently removes dying cells from animal bodies during development. Here, we report that clathrin heavy chain (CHC-1), a membrane coat protein well known for its role in receptor-mediated endocytosis, and its adaptor epsin (EPN-1) play crucial roles in removing apoptotic cells in Caenorhabditis elegans. Inactivating epn-1 or chc-1 disrupts engulfment by impairing actin polymerization. This defect is partially suppressed by inactivating UNC-60, a cofilin ortholog and actin server/depolymerization protein, further indicating that EPN-1 and CHC-1 regulate actin assembly during pseudopod extension. CHC-1 is enriched on extending pseudopods together with EPN-1, in an EPN-1-dependent manner. Epistasis analysis places epn-1 and chc-1 in the same cell-corpse engulfment pathway as ced-1, ced-6 and dyn-1. CED-1 signaling is necessary for the pseudopod enrichment of EPN-1 and CHC-1. CED-1, CED-6 and DYN-1, like EPN-1 and CHC-1, are essential for the assembly and stability of F-actin underneath pseudopods. We propose that in response to CED-1 signaling, CHC-1 is recruited to the phagocytic cup through EPN-1 and acts as a scaffold protein to organize actin remodeling. Our work reveals novel roles of clathrin and epsin in apoptotic-cell internalization, suggests a Hip1/R-independent mechanism linking clathrin to actin assembly, and ties the CED-1 pathway to cytoskeleton remodeling.

Phagocytic receptor signaling regulates clathrin and epsin-mediated cytoskeletal remodeling during apoptotic cell engulfment inC. elegans

The engulfment and subsequent degradation of apoptotic cells by phagocytes is an evolutionarily conserved process that efficiently removes dying cells from animal bodies during development. Here, we report that clathrin heavy chain (CHC-1), a membrane coat protein well known for its role in receptor-mediated endocytosis, and its adaptor epsin (EPN-1) play crucial roles in removing apoptotic cells in Caenorhabditis elegans. Inactivating epn-1 or chc-1 disrupts engulfment by impairing actin polymerization. This defect is partially suppressed by inactivating UNC-60, a cofilin ortholog and actin server/depolymerization protein, further indicating that EPN-1 and CHC-1 regulate actin assembly during pseudopod extension. CHC-1 is enriched on extending pseudopods together with EPN-1, in an EPN-1-dependent manner. Epistasis analysis places epn-1 and chc-1 in the same cell-corpse engulfment pathway as ced-1, ced-6 and dyn-1. CED-1 signaling is necessary for the pseudopod enrichment of EPN-1 and CHC-1. CED-1, CED-6 and DYN-1, like EPN-1 and CHC-1, are essential for the assembly and stability of F-actin underneath pseudopods. We propose that in response to CED-1 signaling, CHC-1 is recruited to the phagocytic cup through EPN-1 and acts as a scaffold protein to organize actin remodeling. Our work reveals novel roles of clathrin and epsin in apoptotic-cell internalization, suggests a Hip1/R-independent mechanism linking clathrin to actin assembly, and ties the CED-1 pathway to cytoskeleton remodeling.

Epsin Family of Endocytic Adaptor Proteins as Oncogenic Regulators of Cancer Progression

Tumor angiogenesis, tumor cell proliferation, and tumor cell migration result from an accumulation of oncogenic mutations that alter protein expression and the regulation of various signaling cascades. Epsins, a small family of clathrin-mediated endocytic adaptor proteins, are reportedly upregulated in a variety of cancers. Importantly, loss of epsins protects against tumorigenesis, thus supporting an oncogenic role for epsins in cancer. Although a clear relationship between epsins and cancer has evolved, the importance of this relationship with regards to cancer progression and anti-cancer therapies remains unclear. In this review, we summarize epsins’ role as endocytic adaptors that modulate VEGF and Notch signaling through the regulated internalization of VEGFR2 and trans-endocytosis of Notch receptors. As both VEGF and Notch signaling have significant implications in angiogenesis, we focus on the newly identified role for epsins in tumor angiogenesis. In addition to epsins’ canonical role in receptor-mediated endocytosis, and the resulting downstream signaling regulation, we discuss the non-canonical role of epsins as regulators of small GTPases and the implications this has on tumor cell proliferation and invasion. Given epsins’ identified roles in tumor angiogenesis, tumor cell proliferation, and tumor cell invasion, we predict that the investigative links between epsins and cancer will provide new insights into the importance of endocytic adaptors and their potential use as future therapeutic targets.

The ArfGEF GBF-1 Is Required for ER Structure, Secretion and Endocytic Transport in C. elegans.

Small GTPases of the Sar/Arf family are essential to generate transport containers that mediate communication between organelles of the secretory pathway. Guanine nucleotide exchange factor (GEFs) activate the small GTPases and help their anchorage in the membrane. Thus, GEFs in a way temporally and spatially control Sar1/Arf1 GTPase activation. We investigated the role of the ArfGEF GBF-1 in C. elegans oocytes and intestinal epithelial cells. GBF-1 localizes to the cis-Golgi and is part of the t-ER-Golgi elements. GBF-1 is required for secretion and Golgi integrity. In addition, gbf-1(RNAi) causes the ER reticular structure to become dispersed, without destroying ER exit sites (ERES) because the ERES protein SEC-16 was still localized in distinct punctae at t-ER-Golgi units. Moreover, GBF-1 plays a role in receptor-mediated endocytosis in oocytes, without affecting recycling pathways. We find that both the yolk receptor RME-2 and the recycling endosome-associated RAB-11 localize similarly in control and gbf-1(RNAi) oocytes. While RAB5-positive early endosomes appear to be less prominent and the RAB-5 levels are reduced by gbf-1(RNAi) in the intestine, RAB-7-positive late endosomes were more abundant and formed aggregates and tubular structures. Our data suggest a role for GBF-1 in ER structure and endosomal traffic.

Diversity of Clathrin Function: New Tricks for an Old Protein

Clathrin is considered the prototype vesicle coat protein whose self-assembly mediates sorting of membrane cargo and recruitment of lipid modifiers. Detailed knowledge of clathrin biochemistry, structure, and interacting proteins has accumulated since the first observation, almost 50 years ago, of its role in receptor-mediated endocytosis of yolk protein. This review summarizes that knowledge, and focuses on properties of the clathrin heavy and light chain subunits and interaction of the latter with Hip proteins, to address the diversity of clathrin function beyond conventional receptor-mediated endocytosis. The distinct functions of the two human clathrin isoforms (CHC17 and CHC22) are discussed, highlighting CHC22's specialized involvement in traffic of the GLUT4 glucose transporter and consequent role in human glucose metabolism. Analysis of clathrin light chain function and interaction with the actin-binding Hip proteins during bacterial infection defines a novel actin-organizing function for CHC17 clathrin. By considering these diverse clathrin functions, along with intracellular sorting roles and influences on mitosis, further relevance of clathrin function to human health and disease is established.

Cytotoxicity and cellular uptake of tri-block copolymer nanoparticles with different size and surface characteristics

BackgroundPolymer nanoparticles (PNP) are becoming increasingly important in nanomedicine and food-based applications. Size and surface characteristics are often considered to be important factors in the cellular interactions of these PNP, although systematic investigations on the role of surface properties on cellular interactions and toxicity of PNP are scarce.ResultsFluorescent, monodisperse tri-block copolymer nanoparticles with different sizes (45 and 90 nm) and surface charges (positive and negative) were synthesized, characterized and studied for uptake and cytotoxicity in NR8383 and Caco-2 cells. All types of PNP were taken up by the cells. The positive smaller PNP45 (45 nm) showed a higher cytotoxicity compared to the positive bigger PNP90 (90 nm) particles including reduction in mitochondrial membrane potential (ΔΨm), induction of reactive oxygen species (ROS) production, ATP depletion and TNF-α release. The negative PNP did not show any cytotoxic effect. Reduction in mitochondrial membrane potential (ΔΨm), uncoupling of the electron transfer chain in mitochondria and the resulting ATP depletion, induction of ROS and oxidative stress may all play a role in the possible mode of action for the cytotoxicity of these PNP. The role of receptor-mediated endocytosis in the intracellular uptake of different PNP was studied by confocal laser scanning microscopy (CLSM). Involvement of size and charge in the cellular uptake of PNP by clathrin (for positive PNP), caveolin (for negative PNP) and mannose receptors (for hydroxylated PNP) were found with smaller PNP45 showing stronger interactions with the receptors than bigger PNP90.ConclusionsThe size and surface characteristics of polymer nanoparticles (PNP; 45 and 90 nm with different surface charges) play a crucial role in cellular uptake. Specific interactions with cell membrane-bound receptors (clathrin, caveolin and mannose) leading to cellular internalization were observed to depend on size and surface properties of the different PNP. These properties of the nanoparticles also dominate their cytotoxicity, which was analyzed for many factors. The effective reduction in the mitochondrial membrane potential (ΔΨm), uncoupling of the electron transfer chain in mitochondria and resulting ATP depletion, induction of ROS and oxidative stress likely all play a role in the mechanisms behind the cytotoxicity of these PNP.

Lipocalin-2 (24p3/Neutrophil Gelatinase-associated Lipocalin (NGAL)) Receptor Is Expressed in Distal Nephron and Mediates Protein Endocytosis

In the kidney, bulk reabsorption of filtered proteins occurs in the proximal tubule via receptor-mediated endocytosis (RME) through the multiligand receptor complex megalin-cubilin. Other mechanisms and nephron sites for RME of proteins are unclear. Recently, the secreted protein 24p3 (lipocalin-2, neutrophil gelatinase-associated lipocalin (NGAL)), which is expressed in the distal nephron, has been identified as a sensitive biomarker of kidney damage. A high-affinity receptor for 24p3 (24p3R) that is involved in endocytotic iron delivery has also been cloned. We investigated the localization of 24p3R in rodent kidney and its role in RME of protein-metal complexes and albumin. Immunostaining of kidney tissue showed expression of 24p3R in apical membranes of distal tubules and collecting ducts, but not of proximal tubule. The differential expression of 24p3R in these nephron segments was confirmed in the respective cell lines. CHO cells transiently transfected with 24p3R or distal tubule cells internalized submicromolar concentrations of fluorescence-coupled proteins transferrin, albumin, or metallothionein (MT) as well as the toxic cadmium-MT (Cd2+(7)-MT) complex, which caused cell death. Uptake of MT or transferrin and Cd2+(7)-MT toxicity were prevented by picomolar concentrations of 24p3. An EC50 of 123±50 nM was determined for binding of MT to 24p3R by microscale thermophoresis. Hence, 24p3R binds proteins filtered by the kidney with high affinity and may contribute to RME of proteins, including 24p3, and to Cd2+(7)-MT toxicity in distal nephron segments.

Pil1, an eisosome organizer, plays an important role in the recruitment of synaptojanins and amphiphysins to facilitate receptor-mediated endocytosis in yeast

The eisosome protein Pil1 is known to be implicated in the endocytosis of Ste3, but the precise biological function of it during endocytosis is poorly understood. Here, we present data to reveal Pil1's role in receptor-mediated endocytosis. Using live cell imaging, we show that endocytic patches carrying Abp1 and Las17 persisted much longer in PIL1-deficient cells. The loss of Pil1 also greatly affected both the scission efficiency and the frequency of formation of endocytic sites carrying Rvs161- and Rvs167-GFP. Furthermore, the mistargeting of the synaptojanins, Sjl1 and Sjl2, to the cytoplasm in pil1Δ cells suggests that Pil1 is required for the proper recruitment of the synaptojanins to endocytic sites. A severe motility defect of Abp1-GFP during its internalization in a codeletant of PIL1 and SJL2 indicates a functional interplay between them in endocytosis. Together, these results establish that Pil1 is involved in the recruitment of endocytic proteins to optimize endocytosis.

Endocytosis of PEGylated Agents Enhances Cancer Imaging and Anticancer Efficacy

PEGylated nanoparticles and macromolecules are increasingly used in cancer imaging and anticancer treatment. The role of receptor-mediated endocytosis in the efficacy of these agents, however, has not been clearly defined. Here, we developed a matched pair of endocytic and nonendocytic receptors to directly and unambiguously assess this issue. The ligand-binding domains of the low-density lipoprotein receptor (LDLR) or a truncated LDLR lacking the NPXY endocytosis motif (DeltaLDLR) were replaced with an anti-polyethylene glycol antibody (alphaPEG) to form endocytic alphaPEG-LDLR and nonendocytic alphaPEG-DeltaLDLR receptors. The receptors were stably expressed at similar levels on the surface of HCC36 cells. HCC36/alphaPEG-LDLR cells, but not HCC36/alphaPEG-DeltaLDLR cells, rapidly endocytosed PEG-quantum dots and PEG-liposomal doxorubicin (Lipo-Dox) in vitro and in vivo. Lipo-Dox was significantly more cytotoxic to HCC36/alphaPEG-LDLR cells than to HCC36/alphaPEG-DeltaLDLR cells. HCC36/alphaPEG-LDLR tumors also accumulated significantly more PEGylated near-IR probes (PEG-NIR797) and PEG-liposomal-(111)In than HCC36/alphaPEG-DeltaLDLR tumors in vivo. Furthermore, Lipo-Dox more significantly suppressed the growth of established HCC36/alphaPEG-LDLR tumors as compared with HCC36/alphaPEG-DeltaLDLR tumors. Our data show that endocytosis of PEGylated probes and drugs enhances both cancer imaging and anticancer efficacy, indicating that endocytic receptors are superior targets for the design of cancer imaging probes and immunoliposomal drugs.

Evolution and function of a novel clathrin isoform

Clathrin forms a lattice‐like intracellular membrane coat to organize adaptor molecules that interact with transmembrane protein cargo. This process facilitates protein sorting at the plasma membrane, on endosomes and in the trans‐Golgi network (TGN). The CHC17 isoform of the clathrin heavy chain is ubiquitously expressed in vertebrate tissues and plays a key role in receptor‐mediated endocytosis and lysosome biogenesis. A CHC17 ortholog is present in all eukaryotes where it performs related functions. During vertebrate evolution, a second isoform of clathrin heavy chain CHC22 arose by gene duplication. CHC22 is expressed highly in human skeletal muscle and adipocytes and at lower levels in other human cell types. CHC22 function is restricted primarily to the endosome‐TGN region of the cell, where it influences retrograde transport from endosomes to the TGN. In muscle cells and adipocytes, CHC22 regulates the formation of the storage compartment from which the GLUT4 glucose transporter is released upon insulin stimulation. The CHC22 gene was found to be a pseudogene in mice, revealing species‐restricted differences in membrane traffic components involved in glucose homeostasis. CHC22 appears to function by competition with CHC17 for clathrin adaptors. When introduced as a transgene into mice, it alters membrane traffic pathways involved in GLUT4 function and induces diabetic symptoms. Consistent with this observation, CHC22 distribution is altered in human patients with type 2 diabetes. This novel and species‐restricted function of CHC22 clathrin contributes to evolutionary diversity of metabolic pathways that have relevance to understanding human diabetes.

Regulation of clathrin‐mediated endocytosis by p53

The p53 gene encodes a multi-functional protein to prevent tumorigenesis. Although there have been many reports of the nuclear functions of p53, little is known about the cytosolic functions of p53. Here, we found that p53 is present in cytosol as well as nuclei under unstressed conditions and binds to clathrin heavy chain (CHC). CHC is known to play a role in receptor-mediated endocytosis. Based on our findings, we examined the effect of p53 on clathrin-mediated endocytosis of epidermal growth factor receptor (EGFR). Surprisingly, p53 co-localized with CHC at the plasma membrane in response to EGF stimulation. In cells with ablated p53 expression by RNAi, EGFR internalization was delayed and intracellular signaling from EGFR was altered. Thus, our findings provide evidence that cytosolic p53 may participate in the regulation of clathrin-mediated endocytosis to control the correct signaling from EGFR.

C. elegansDisabled is required for cell-type specific endocytosis and is essential in animals lacking the AP-3 adaptor complex

Disabled proteins are a conserved family of monomeric adaptor proteins that in mammals are implicated in the endocytosis of lipoprotein receptors. Previous studies have shown that the sole Caenorhabditis elegans Disabled homologue, DAB-1, is involved in the lipoprotein receptor-mediated secretion of a fibroblast growth factor. We show here that DAB-1 is essential for the uptake of yolk protein by developing oocytes, and for the localisation of the yolk receptor RME-2. The localisation of DAB-1 in oocytes is itself dependent upon clathrin and AP2, consistent with DAB-1 acting as a clathrin-associated sorting protein during yolk protein endocytosis. DAB-1 is also required for the endocytosis of molecules from the pseudocoelomic fluid by the macrophage-like coelomocytes, and is broadly expressed in epithelial tissues, consistent with a general role in receptor-mediated endocytosis. We also show that dab-1 mutations are synthetic lethal in combination with loss-of-function mutations affecting the AP-1 and AP-3 complexes, suggesting that the reduced fluid and membrane uptake exhibited by dab-1 mutants sensitises them to defects in other trafficking pathways.

Role of Receptor-Mediated Endocytosis in the Formation of Vaccinia Virus Extracellular Enveloped Particles

Infectious intracellular mature vaccinia virus particles are wrapped by cisternae, which may arise from trans-Golgi or early endosomal membranes, and are transported along microtubules to the plasma membrane where exocytosis occurs. We used EH21, a dominant-negative form of Eps15 that is an essential component of clathrin-coated pits, to investigate the extent and importance of endocytosis of viral envelope proteins from the cell surface. Several recombinant vaccinia viruses that inducibly or constitutively express an enhanced green fluorescent protein (GFP)-EH21 fusion protein were constructed. Expression of GFP-EH21 blocked uptake of transferrin, a marker for clathrin-mediated endocytosis, as well as association of adaptor protein-2 with clathrin-coated pits. When GFP-EH21 was expressed, there were increased amounts of viral envelope proteins, including A33, A36, B5, and F13, in the plasma membrane, and their internalization was inhibited. Wrapping of virions appeared to be qualitatively unaffected as judged by electron microscopy, a finding consistent with a primary trans-Golgi origin of the cisternae. However, GFP-EH21 expression caused a 50% reduction in released enveloped virions, decreased formation of satellite plaques, and delayed virus spread, indicating an important role for receptor-mediated endocytosis. Due to dynamic interconnection between endocytic and exocytic pathways, viral proteins recovered from the plasma membrane could be used by trans-Golgi or endosomal cisternae to form new viral envelopes. Adherence of enveloped virions to unrecycled viral proteins on the cell surface may also contribute to decreased virus release in the presence of GFP-EH21. In addition to a salvage function, the retrieval of viral proteins from the cell surface may reduce immune recognition.

Hypertonicity increases CLC-5 expression in mouse medullary thick ascending limb cells.

Genetic studies indicated that mutations of the chloride channel CLC-5 in the kidney are responsible for a group of clinical disorders, collectively called Dent's disease. In the kidney, CLC-5 was found to be expressed in the proximal tubule, medullary thick ascending limb (mTAL) of loop of Henle, and intercalated cells of the collecting tubule. In proximal tubular cells, CLC-5 was found to play an important role in receptor-mediated endocytosis. However, the functional roles of CLC-5 in mTAL and collecting tubules remain unclear. Because mTAL is normally exposed to a hypertonic environment, we aimed to examine the effect of hypertonicity on CLC-5 expression in this nephron segment. Our studies revealed that exposure to hypertonicity (up to 550 mosM) increased CLC-5 mRNA and protein levels in a murine mTAL cell line (MTAL) but not in an opossum kidney (OK) proximal tubular cell line. A similar effect was also found in mouse kidneys, where CLC-5 expression was enhanced in renal medulla, but not cortex, after 48 h of water deprivation. We also tested the effect of hypertonicity on endocytotic activity and found that exposure to hypertonicity caused a significant decrease in cellular uptake of FITC-labeled albumin in OK but not in MTAL cells. Our results suggest that CLC-5 expression is upregulated by hypertonicity in mTAL cells but not in proximal tubular cells. We speculate that the increased CLC-5 levels in mTAL may serve to maintain the endocytotic activity in a hypertonic environment.

Different types of glomerulopathic light chains interact with mesangial cells using a common receptor but exhibit different intracellular trafficking patterns

Patients with plasma cell dyscrasias may have circulating light chains (LCs), some of which are nephrotoxic. Nephrotoxic LCs can affect the various renal compartments. Some of these LCs may produce predominantly proximal tubular damage, while others are associated with distal nephron obstruction (the so-called ‘myeloma kidney'). Both these are considered tubulopathic (T) LCs. A receptor has been found in proximal tubular cells (cubilin/megalin complex), which mediates the absorption of LCs and is involved in the pathogenesis of tubulopathies that occurs in these patients. Another group of nephrotoxic LCs is associated with glomerular damage and these are considered as glomerulopathic (G). These patients with G-LCs may develop AL-amyloidosis (AL-Am) or LC deposition disease (LCDD). Recent evidence indicates that the physicochemical characteristics (amino-acid composition and conformation of the variable region) of a given nephrotoxic LC may be the most significant factor in determining the type and location of renal damage within the nephron. Other factors may also be involved, including yet uncharacterized host factors that may include genetic polymorphism, among others. Interestingly, the amount of LC production by the clone of plasma cells does not correlate directly with the severity of the renal alterations. Understanding the nature of the interactions between G-LCs and mesangial cells (MCs) is crucial to define key steps that may be targeted for therapeutic purposes. Experimental studies have delineated important aspects pertaining to interactions between G-LCs and MCs, indicating that these interactions are receptor mediated. The data presented in the current study support a single receptor present on MCs for both LCDD and AL-LCs, as clearly demonstrated with competition and colocalization immunofluorescence (IF) studies. This receptor resides in caveolae present on the plasma membrane of HMCs and is overexpressed when HMCs are incubated with G-LCs but not TLCs. Caveolae play a fundamental role in receptor-mediated endocytosis, a crucial process in the internalization of AL-LCs and amyloidogenesis. LC internalization is clathrin mediated. The data also indicate that intracellular trafficking in MCs is different for AL-LCs and LCDD-LCs. AL-LCs are delivered to the mature lysosomal compartment where amyloid formation occurs. LCDD-LCs alter mesangial function and phenotype by interacting with the MC surface membranes through similar receptors as the AL-LCs. The data also demonstrated that cubilin and megalin were absent on MCs, so the receptor involved is different from the one already characterized in the proximal tubules.