Nuclear Trafficking and Functions of Endocytic Proteins Implicated in Oncogenesis

A subset of proteins predominantly associated with early endosomes or implicated in clathrin‐mediated endocytosis can shuttle between the cytoplasm and the nucleus. Although the endocytic functions of these proteins have been extensively studied, much less effort has been expended in exploring their nuclear roles. Membrane trafficking proteins can affect signalling and proliferation and this can be achieved either at a nuclear or endocytic level. Furthermore, some proteins, such as Huntingtin interacting protein 1, are known as cancer biomarkers. This review will highlight the limits of our understanding of their nuclear functions and the relevance of this to signalling and oncogenesis.     

Role of Cell-Type-Specific Endoplasmic Reticulum-Associated Degradation in Polyomavirus Trafficking

BK polyomavirus (BKPyV) is a widespread human pathogen that establishes a lifelong persistent infection and can cause severe disease in immunosuppressed patients. BKPyV is a nonenveloped DNA virus that must traffic through the endoplasmic reticulum (ER) for productive infection to occur; however, it is unknown how BKPyV exits the ER before nuclear entry. In this study, we elucidated the role of the ER-associated degradation (ERAD) pathway during BKPyV intracellular trafficking in renal proximal tubule epithelial (RPTE) cells, a natural host cell. Using proteasome and ERAD inhibitors, we showed that ERAD is required for productive entry. Altered trafficking and accumulation of uncoated viral intermediates were detected by fluorescence in situ hybridization and indirect immunofluorescence in the presence of an inhibitor. Additionally, we detected a change in localization of partially uncoated virus within the ER during proteasome inhibition, from a BiP-rich area to a calnexin-rich subregion, indicating that BKPyV accumulated in an ER subcompartment. Furthermore, inhibiting ERAD did not prevent entry of capsid protein VP1 into the cytosol from the ER. By comparing the cytosolic entry of the related polyomavirus simian virus 40 (SV40), we found that dependence on the ERAD pathway for cytosolic entry varied between the polyomaviruses and between different cell types, namely, immortalized CV-1 cells and primary RPTE cells.     

Collapsin Response Mediator Protein-2 (Crmp2) Regulates Trafficking by Linking Endocytic Regulatory Proteins to Dynein Motors

Endocytosis is a conserved cellular process in which nutrients, lipids, and receptors are internalized and transported to early endosomes, where they are sorted and either channeled to degradative pathways or recycled to the plasma membrane. MICAL-L1 and EHD1 are important regulatory proteins that control key endocytic transport steps. However, the precise mechanisms by which they mediate transport, and particularly the mode by which they connect to motor proteins, have remained enigmatic. Here we have identified the collapsin response mediator protein-2 (Crmp2) as an interaction partner of MICAL-L1 in non-neuronal cells. Crmp2 interacts with tubulin dimers and kinesin and negatively regulates dynein-based transport in neuronal cells, but its expression and function in non-neuronal cells have remained poorly characterized. Upon Crmp2 depletion, we observed dramatic relocalization of internalized transferrin (Tf) from peripheral vesicles to the endocytic recycling compartment (ERC), similar to the effect of depleting either MICAL-L1 or EHD1. Moreover, Tf relocalization to the ERC could be inhibited by interfering with microtubule polymerization, consistent with a role for uncoupled motor protein-based transport upon depletion of Crmp2, MICAL-L1, or EHD1. Finally, transfection of dynamitin, a component of the dynactin complex whose overexpression inhibits dynein activity, prevented the relocalization of internalized Tf to the ERC upon depletion of Crmp2, MICAL-L1, or EHD1. These data provide the first trafficking regulatory role for Crmp2 in non-neuronal cells and support a model in which Crmp2 is an important endocytic regulatory protein that links MICAL-L1·EHD1-based vesicular transport to dynein motors.     

Rerouting of Plant Late Endocytic Trafficking Toward a Pathogen Interface

A number of plant pathogenic and symbiotic microbes produce specialized cellular structures that invade host cells where they remain enveloped by host‐derived membranes. The mechanisms underlying the biogenesis and functions of host–microbe interfaces are poorly understood. Here, we show that plant late endocytic trafficking is diverted toward the extrahaustorial membrane (EHM); a host–pathogen interface that develops in plant cells invaded by Irish potato famine pathogen Phytophthora infestans. A late endosome and tonoplast marker protein Rab7 GTPase RabG3c, but not a tonoplast‐localized sucrose transporter, is recruited to the EHM, suggesting specific rerouting of vacuole‐targeted late endosomes to a host–pathogen interface. We revealed the dynamic nature of this process by showing that, upon activation, a cell surface immune receptor traffics toward the haustorial interface. Our work provides insight into the biogenesis of the EHM and reveals dynamic processes that recruit membrane compartments and immune receptors to this host–pathogen interface.      

Calmodulin Localization and its Effects on Endocytic and Phagocytic Membrane Trafficking in Paramecium multimicronucleatum

ABSTRACT. In ciliates, calmodulin (CaM), as in other cells, has multiple functions, such as activation of regulatory enzymes and modulating calcium‐dependent cellular processes. By immunogold localization, CaM is concentrated at multiple sites in Paramecium. It is seen scattered over the cytosol, but bound to its matrix, and is concentrated at the pores of the contractile vacuole complexes and with at least three microtubular arrays. It was localized peripheral to the nine‐doublet microtubules of the ciliary axonemes. The most striking localization was on the akinetic side only of the cytopharyngeal microtubular ribbons opposite the side where the discoidal vesicles, acidosomes and the 100‐nm carrier vesicles bind and move. CaM was also present at the periphery of the postoral microtubular bundles along which the early vacuole moves and was associated with the cytoproct microtubules that guide the spent digestive vacuoles to the cytoproct. It was not found on the membranes of, or in the interior of nuclei, mitochondria, phagosomes, and trichocysts, and was only sparsely scattered over the cytosolic sides of discoidal vesicles, acidosomes, lysosomes, and digestive vacuoles. Together the associations with specific microtubular arrays and the effects of trifluoperazine and calmidazolium indicate that CaM is involved (i) in vesicle transport to the cytopharynx area for vacuole formation and subsequent vacuole acidification, (ii) in early vacuole transport along the postoral fiber, and (iii) in transporting the spent vacuole to the cytoproct. Higher CaM concentrations subjacent to the cell's pellicle and close to the decorated tubules of the contractile vacuole complex may support a role for CaM in ion traffic.     

Ganglioside and Non-ganglioside Mediated Host Responses to the Mouse Polyomavirus

Gangliosides serve as receptors for internalization and infection by members of the polyomavirus family. Specificity is determined by recognition of carbohydrate moieties on the ganglioside by the major viral capsid protein VP1. For the mouse polyomavirus (MuPyV), gangliosides with terminal sialic acids in specific linkages are essential. Although many biochemical and cell culture experiments have implicated gangliosides as MuPyV receptions, the role of gangliosides in the MuPyV-infected mouse has not been investigated. Here we report results of studies using ganglioside-deficient mice and derived cell lines. Knockout mice lacking complex gangliosides were completely resistant to the cytolytic and pathogenic effects of the virus. Embryo fibroblasts from these mice were likewise resistant to infection, and supplementation with specific gangliosides restored infectibility. Although lacking receptors for viral infection, cells from ganglioside-deficient mice retained the ability to respond to the virus. Ganglioside-deficient fibroblasts responded rapidly to virus exposure with a transient induction of c-fos as an early manifestation of a mitogenic response. Additionally, splenocytes from ganglioside-deficient mice responded to MuPyV by secretion of IL-12, previously recognized as a key mediator of the innate immune response. Thus, while gangliosides are essential for infection in the animal, gangliosides are not required for mitogenic responses and innate immune responses to the virus.     

Role of Ubiquitination in Endocytic Trafficking of G‐Protein‐Coupled Receptors

Lysyl ubiquitination has long been known to target cytoplasmic proteins for proteasomal degradation, and there is now extensive evidence that ubiquitination functions in vacuolar/lysosomal targeting of membrane proteins from both the biosynthetic and endocytic pathways. G‐protein‐coupled receptors (GPCRs) represent the largest and most diverse family of membrane proteins, whose function is of fundamental importance both physiologically and therapeutically. In this review, we discuss the role of ubiquitination in the vacuolar/lysosomal downregulation of GPCRs through the endocytic pathway, with a primary focus on lysosomal trafficking in mammalian cells. We will summarize evidence indicating that mammalian GPCRs are regulated by ubiquitin‐dependent mechanisms conserved in budding yeast, and then consider evidence for additional ubiquitin‐dependent and ‐independent regulation that may be specific to animal cells.     

Effects of Paclitaxel on EGFR Endocytic Trafficking Revealed Using Quantum Dot Tracking in Single Cells

Paclitaxel (PTX), a chemotherapeutic drug, affects microtubule dynamics and influences endocytic trafficking. However, the mechanism and the dynamics of altered endocytic trafficking by paclitaxel treatment in single living cells still remain elusive. By labeling quantum dots (QDs) to the epidermal growth factor (EGF), we continuously tracked the endocytosis and post-endocytic trafficking of EGF receptors (EGFRs) in A549 cells for a long time interval. A single-cell analysis method was introduced to quantitatively study the dynamics of endocytic trafficking. Compared with the control cells, the velocity of directed motion was reduced by 30% due to the suppression of high speed movements of EGF-QDs along the microtubules in PTX-treated cells. The endocytic trafficking in PTX-treated cells was mainly via super-diffusive mode of motion, whereas in control cells, it was mostly via sub-diffusive mode of motion. Moreover, PTX shortened endosomal trafficking and prevented EGF-QDs from moving to the perinuclear area via the rapid delivery of EGF-QDs into the peripheral lysosomes. The present study may shed light on the mechanism of the effect of PTX on the treatment of lung cancer.     

Endocytic Trafficking of Nanoparticles Delivered by Cell-penetrating Peptides Comprised of Nona-arginine and a Penetration Accelerating Sequence

Cell-penetrating peptides (CPPs) can traverse cellular membranes and deliver biologically active molecules into cells. In this study, we demonstrate that CPPs comprised of nona-arginine (R9) and a penetration accelerating peptide sequence (Pas) that facilitates escape from endocytic lysosomes, denoted as PR9, greatly enhance the delivery of noncovalently associated quantum dots (QDs) into human A549 cells. Mechanistic studies, intracellular trafficking analysis and a functional gene assay reveal that endocytosis is the main route for intracellular delivery of PR9/QD complexes. Endocytic trafficking of PR9/QD complexes was monitored using both confocal and transmission electron microscopy (TEM). Zeta-potential and size analyses indicate the importance of electrostatic forces in the interaction of PR9/QD complexes with plasma membranes. Circular dichroism (CD) spectroscopy reveals that the secondary structural elements of PR9 have similar conformations in aqueous buffer at pH 7 and 5. This study of nontoxic PR9 provides a basis for the design of optimized cargo delivery that allows escape from endocytic vesicles.     

Discrimination between Sialic Acid-Containing Receptors and Pseudoreceptors Regulates Polyomavirus Spread in the Mouse

Variations in the polyomavirus major capsid protein VP1 underlie important biological differences between highly pathogenic large-plaque and relatively nonpathogenic small-plaque strains. These polymorphisms constitute major determinants of virus spread in mice and also dictate previously recognized strain differences in sialyloligosaccharide binding. X-ray crystallographic studies have shown that these determinants affect binding to the sialic acids. Here we report results of further experiments designed to test the importance of specific contacts between VP1 and the carbohydrate moieties of the receptor. With minor exceptions, substitutions at positions predicted from crystallography to be important in binding the terminal alpha-2,3-linked sialic acid or the penultimate sugar (galactose) destroyed the ability of the virus to replicate in cell culture. Substitutions that prevented binding to a branched disialyloligosaccharide were found to result in viruses that were both viable in culture and tumorigenic in the mouse. Conversely, substitutions that allowed recognition and binding of the branched carbohydrate chain inhibited spread in the mouse, though the viruses remained viable in culture. Mice of five different inbred strains, all highly susceptible to large-plaque virus, showed resistance to the spread of polyomavirus strains bearing the VP1 type which binds the branched-chain receptor. We suggest that glycoproteins bearing the appropriate O-linked branched sialyloligosaccharide chains are effective pseudoreceptors in the host and that they block the spread of potentially tumorigenic or virulent virus strains.     

Imaging of Endocytic Trafficking and Extracellular Vesicles Released Under Neratinib Treatment in ERBB2+ Breast Cancer Cells

Breast cancers (BCa) with ERBB2 amplification show rapid tumor growth, increased disease progression, and lower survival rate. Deregulated intracellular trafficking and extracellular vesicle (EVs) release are mechanisms that support cancer progression and resistance to treatments. Neratinib (NE) is a Food and Drug Administration–approved pan-ERBB inhibitor employed for the treatment of ERBB2⁺ BCa that blocks signaling and causes survival inhibition. However, the effects of NE on ERBB2 internalization, its trafficking to multivesicular bodies (MVBs), and the release of EVs that originate from these organelles remain poorly studied. By confocal and electron microscopy, we observed that low nanomolar doses of NE induced a modest ERBB2 internalization along with an increase of clathrin-mediated endocytosis and of the CD63+ MVB compartment in SKBR-3 cells. Furthermore, we showed in the culture supernatant two distinct EV subsets, based on their size and ERBB2 positivity: small (30–100 nm) ERBB2⁻ EVs and large (>100 nm) ERBB2⁺ EVs. In particular, we found that NE increased the overall release of EVs, which displayed a reduced ERBB2 positivity compared with controls. Taken together, these results provide novel insight into the effects of NE on ERBB2⁺ BCa cells that may lead to a reduction of ERBB2 potentially transferred to distant target cells by EVs:     

Cytoskeletal Protein ABP-280 Directs the Intracellular Trafficking of Furin and Modulates Proprotein Processing in the Endocytic Pathway

Furin catalyzes the proteolytic maturation of many proproteins within the trans-Golgi network (TGN)/endosomal system. Furin's cytosolic domain (cd) directs both the compartmentalization to and transit between its manifold processing compartments (i.e., TGN/biosynthetic pathway, cell surface, and endosomes). Here we report the identification of the first furin cd sorting protein, ABP-280 (nonmuscle filamin), an actin gelation protein. The furin cd was used as bait in a yeast two-hybrid screen to identify ABP-280 as a furin-binding protein. Binding analyses in vitro and coimmunoprecipitation studies in vivo showed that furin and ABP-280 interact directly and that ABP-280 tethers furin molecules to the cell surface. Quantitative analysis of both ABP-280-deficient and genetically replete cells showed that ABP-280 modulates the rate of internalization of furin but not of the transferrin receptor, a cycling receptor. However, although ABP-280 directs the rate of furin internalization, the efficiency of sorting of the endoprotease from the cell surface to early endosomes is independent of expression of ABP-280. By contrast, efficient sorting of furin from early endosomes to the TGN requires expression of ABP-280. In addition, ABP-280 is also required for the correct localization of late endosomes (dextran bead uptake) and lysosomes (LAMP-1 staining), demonstrating a pleiotropic role for this actin binding protein in the organization of cellular compartments and directing protein traffic. Finally, and consistent with the trafficking studies on furin, we showed that ABP-280 modulates the processing of furin substrates in the endocytic but not the biosynthetic pathways. The novel roles of ABP-280 and the cytoskeleton in the sorting of furin in the TGN/ endosomal system and the formation of proprotein processing compartments are discussed.     

Caveolae Are Highly Immobile Plasma Membrane Microdomains, Which Are not Involved in Constitutive Endocytic Trafficking

To investigate whether caveolae are involved in constitutive endocytic trafficking, we expressed N- and C- terminally green fluorescent protein (GFP)-tagged caveolin- 1 fusion proteins in HeLa, A431, and Madin-Darby canine kidney cells. The fusion proteins were shown by immunogold labeling to be sorted correctly to caveolae. By using confocal microscopy and photobleaching techniques, it was found that although intracellular structures labeled with GFP-tagged caveolin were dynamic, GFP-labeled caveolae were very immobile. However, after incubation with methyl- beta-cyclodextrin, distinct caveolae disappeared and the mobility of GFP-tagged caveolin in the plasma membrane increased. Treatment of cells with cytochalasin D caused lateral movement and aggregation of GFP-labeled caveolae. Therefore, both cholesterol and an intact actin cytoskeleton are required for the integrity of GFP-labeled caveolae. Moreover, stimulation with okadaic acid caused increased mobility and internalization of the labeled caveolae. Although the calculated mobile fraction (for t = infinity) of intracellular, GFP-tagged caveolin- associated structures was 70-90%, GFP-labeled caveolae in unstimulated cells had a mobile fraction of <20%, a value comparable to that previously reported for E-cadherin in junctional complexes. We therefore conclude that caveolae are not involved in constitutive endocytosis but represent a highly stable plasma membrane compartment anchored by the actin cytoskeleton.     

Rabex-5 Protein Regulates the Endocytic Trafficking Pathway of Ubiquitinated Neural Cell Adhesion Molecule L1

Ubiquitination of integral membrane proteins is a common posttranslational modification used to mediate endocytosis and endocytic sorting of cell surface proteins in eukaryotic cells. Ubiquitin (Ub)-binding proteins (UBPs) regulate the stability, function, and localization of ubiquitinated cell surface proteins in the endocytic pathway. Here, I report that the immunoglobulin superfamily cell adhesion molecule L1 undergoes ubiquitination and dephosphorylation on the plasma membrane upon L1 antibody-induced clustering, which mimics L1-L1 homophilic binding, and that these modifications are critical for obtaining the maximal rate of internalization and trafficking to the lysosome, but not to the proteasome. Notably, L1 antibody-induced clustering leads to the association of ubiquitinated L1 with Rabex-5, a UBP and guanine nucleotide exchange factor for Rab5, via interaction with the motif interacting with Ub (MIU) domain, but not the A20-type zinc finger domain. This interaction specifically depends on the presence of an Ub moiety on lysine residues in L1. Rabex-5 expression accelerates the internalization rates of L1^WT and L1^Y1176A, a tyrosine-based motif mutant, but not L1^K11R, an ubiquitination-deficient mutant, leading to the accumulation of ubiquitinated L1 on endosomes. In contrast, RNA interference-mediated knockdown of Rabex-5 impairs the internalizations of L1^WT and L1^Y1176A, but not L1^K11R from the plasma membrane. Overall, these results provide a novel mechanistic insight into how Rabex-5 regulates internalization and postendocytic trafficking of ubiquitinated L1 destined for lysosomal degradation.     

HDAC6 Regulates Epidermal Growth Factor Receptor (EGFR) Endocytic Trafficking and Degradation in Renal Epithelial Cells

We present for the first time that histone deacetylase 6 (HDAC6) regulates EGFR degradation and trafficking along microtubules in Pkd1 mutant renal epithelial cells. HDAC6, the microtubule-associated α-tubulin deacetylase, demonstrates increased expression and activity in Pkd1 mutant mouse embryonic kidney cells. Targeting HDAC6 with a general HDAC inhibitor, trichostatin (TSA), or a specific HDAC6 inhibitor, tubacin, increased the acetylation of α-tubulin and downregulated the expression of EGFR in Pkd1 mutant renal epithelial cells. HDAC6 was co-localized with EGF induced endocytic EGFR and endosomes, respectively. Inhibition of the activity of HDAC6 accelerated the trafficking of EGFR from early endosomes to late endosomes along the microtubules. Without EGF stimulation EGFR was randomly distributed while after stimulation with EGF for 30 min, EGFR was accumulated around α-tubulin labeled microtubule bundles. These data suggested that the Pkd1 mutation induced upregulation of HDAC6 might act to slow the trafficking of EGFR from early endosomes to late endosomes along the microtubules for degradation through deacetylating α-tubulin. In addition, inhibition of HDAC activity decreased the phosphorylation of ERK1/2, the downstream target of EGFR axis, and normalized EGFR localization from apical to basolateral in Pkd1 knockout mouse kidneys. Thus, targeting HDAC6 to downregulate EGFR activity may provide a potential therapeutic approach to treat polycystic kidney disease.     

Slit-Dependent Endocytic Trafficking of the Robo Receptor Is Required for Son of Sevenless Recruitment and Midline Axon Repulsion

Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo) is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and in vitro Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. We identify two conserved motifs in Robo’s cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo. Our data support a model in which Robo’s ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment.