The role of clathrin in post-golgi trafficking in toxoplasma gondii

Apicomplexan parasites are single eukaryotic cells with a highly polarised secretory system that contains unique secretory organelles (micronemes and rhoptries) that are required for host cell invasion. In contrast, the role of the endosomal system is poorly understood in these parasites. With many typical endocytic factors missing, we speculated that endocytosis depends exclusively on a clathrin-mediated mechanism. Intriguingly, in Toxoplasma gondii we were only able to observe the endogenous clathrin heavy chain 1 (CHC1) at the Golgi, but not at the parasite surface. For the functional characterisation of Toxoplasma gondii CHC1 we generated parasite mutants conditionally expressing the dominant negative clathrin Hub fragment and demonstrate that CHC1 is essential for vesicle formation at the trans-Golgi network. Consequently, the functional ablation of CHC1 results in Golgi aberrations, a block in the biogenesis of the unique secretory microneme and rhoptry organelles, and of the pellicle. However, we found no morphological evidence for clathrin mediating endocytosis in these parasites and speculate that they remodelled their vesicular trafficking system to adapt to an intracellular lifestyle

Cortactin overexpression results in sustained epidermal growth factor receptor signaling by preventing ligand-induced receptor degradation in human carcinoma cells

The chromosome 11q13 region is frequently amplified in human carcinomas and results in an increased expression of various genes including cortactin, and is also associated with an increased invasive potential. Cortactin acts as an important regulator of the actin cytoskeleton. It is therefore very tempting to speculate that cortactin is the crucial gene within the 11q13 amplicon that mediates the invasive potential of these carcinomas. Cortactin also participates in receptor-mediated endocytosis, and recent findings have shown that, during receptor internalization, cortactin overexpression inhibits the ubiquitylation-mediated degradation of the epidermal growth factor receptor, resulting in a sustained ligand-induced epidermal growth factor receptor activity

Actin- and Dynamin-Dependent Maturation of Bulk Endocytosis Restores Neurotransmission following Synaptic Depletion

Bulk endocytosis contributes to the maintenance of neurotransmission at the amphibian neuromuscular junction by regenerating synaptic vesicles. How nerve terminals internalize adequate portions of the presynaptic membrane when bulk endocytosis is initiated before the end of a sustained stimulation is unknown. A maturation process, occurring at the end of the stimulation, is hypothesised to precisely restore the pools of synaptic vesicles. Using confocal time-lapse microscopy of FM1-43-labeled nerve terminals at the amphibian neuromuscular junction, we confirm that bulk endocytosis is initiated during a sustained tetanic stimulation and reveal that shortly after the end of the stimulation, nerve terminals undergo a maturation process. This includes a transient bulging of the plasma membrane, followed by the development of large intraterminal FM1-43-positive donut-like structures comprising large bulk membrane cisternae surrounded by recycling vesicles. The degree of bulging increased with stimulation frequency and the plasmalemma surface retrieved following the transient bulging correlated with the surface membrane internalized in bulk cisternae and recycling vesicles. Dyngo-4a, a potent dynamin inhibitor, did not block the initiation, but prevented the maturation of bulk endocytosis. In contrast, cytochalasin D, an inhibitor of actin polymerization, hindered both the initiation and maturation processes. Both inhibitors hampered the functional recovery of neurotransmission after synaptic depletion. Our data confirm that initiation of bulk endocytosis occurs during stimulation and demonstrates that a delayed maturation process controlled by actin and dynamin underpins the coupling between exocytosis and bulk endocytosis

Prognostic significance of cortactin levels in head and neck squamous cell carcinoma: comparison with epidermal growth factor receptor status

Cortactin is an actin-binding Src substrate involved in cell motility and invasion. In this study, we sought to examine the prognostic importance of cortactin protein expression in head and neck squamous cell carcinoma (HNSCC). To do so, cortactin and EGF receptor (EGFR) expression was retrospectively evaluated by immunohistochemistry in a tissue microarray composed of 176 HNSCCs with a mean follow-up time of 5 years. Cortactin immunoreactivity was weak to absent in normal epithelial tissue. Overexpression of the protein in 77 out of 176 tumours (44%) was associated with more advanced tumour-node-metastasis stage and higher histologic grade. Cortactin overexpression was associated with significantly increased local recurrence rates (49 vs 28% for high and low expressing carcinomas, respectively), decreased disease-free survival (17 vs 61%), and decreased the 5-year overall survival of (21 vs 58%), independently of the EGFR status. In multivariate analysis, cortactin expression status remained an independent prognostic factor for local recurrence, disease-free survival, and overall survival. Importantly, we identified a subset of patients with cortactin-overexpressing tumours that displayed low EGFR levels and a survival rate that equalled that of patients with tumoral overexpression of both EGFR and cortactin. These findings identify cortactin as a relevant prognostic marker and may have implications for targeted therapies in patients with HNSCC

The phocein homologue SmMOB3 is essential for vegetative cell fusion and sexual development in the filamentous ascomycete Sordaria macrospora

Members of the striatin family and their highly conserved interacting protein phocein/Mob3 are key components in the regulation of cell differentiation in multicellular eukaryotes. The striatin homologue PRO11 of the filamentous ascomycete Sordaria macrospora has a crucial role in fruiting body development. Here, we functionally characterized the phocein/Mob3 orthologue SmMOB3 of S. macrospora. We isolated the gene and showed that both, pro11 and Smmob3 are expressed during early and late developmental stages. Deletion of Smmob3 resulted in a sexually sterile strain, similar to the previously characterized pro11 mutant. Fusion assays revealed that ∆Smmob3 was unable to undergo self-fusion and fusion with the pro11 strain. The essential function of the SmMOB3 N-terminus containing the conserved mob domain was demonstrated by complementation analysis of the sterile S. macrospora ∆Smmob3 strain. Downregulation of either pro11 in ∆Smmob3, or Smmob3 in pro11 mutants by means of RNA interference (RNAi) resulted in synthetic sexual defects, demonstrating for the first time the importance of a putative PRO11/SmMOB3 complex in fruiting body development

A Novel Pseudopodial Component of the Dendritic Cell Anti-Fungal Response: The Fungipod

Fungal pathologies are seen in immunocompromised and healthy humans. C-type lectins expressed on immature dendritic cells (DC) recognize fungi. We report a novel dorsal pseudopodial protrusion, the “fungipod”, formed by DC after contact with yeast cell walls. These structures have a convoluted cell-proximal end and a smooth distal end. They persist for hours, exhibit noticeable growth and total 13.7±5.6 µm long and 1.8±0.67 µm wide at the contact. Fungipods contain clathrin and an actin core surrounded by a sheath of cortactin. The actin cytoskeleton, but not microtubules, is required for fungipod integrity and growth. An apparent rearward flow (225±55 nm/second) exists from the zymosan contact site into the distal fungipod. The phagocytic receptor Dectin-1 is not required for fungipod formation, but CD206 (Mannose Receptor) is the generative receptor for these protrusions. The human pathogen Candida parapsilosis induces DC fungipod formation strongly, but the response is species specific since the related fungal pathogens Candida tropicalis and Candida albicans induce very few and no fungipods, respectively. Our findings show that fungipods are dynamic actin-driven cellular structures involved in fungal recognition by DC. They may promote yeast particle phagocytosis by DC and are a specific response to large (i.e., 5 µm) particulate ligands. Our work also highlights the importance of this novel protrusive structure to innate immune recognition of medically significant Candida yeasts in a species specific fashion

P130Cas Attenuates Epidermal Growth Factor (EGF) Receptor Internalization by Modulating EGF-Triggered Dynamin Phosphorylation

BACKGROUND: Endocytosis controls localization-specific signal transduction via epidermal growth factor receptor (EGFR), as well as downregulation of that receptor. Extracellular matrix (ECM)-integrin coupling induces formation of macromolecular complexes that include EGFR, integrin, Src kinase and p130Cas, resulting in EGFR activation. In addition, cell adhesion to ECM increases EGFR localization at the cell surface and reduces EGFR internalization. The molecular mechanisms involved are not yet well understood. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the molecular mechanism by which p130Cas affects the endocytic regulation of EGFR. Biochemical quantification revealed that cell adhesion to fibronectin (FN) increases total EGFR levels and its phosphorylation, and that p130Cas is required for this process. Measurements of Texas Red-labeled EGF uptake and cell surface EGFR revealed that p130Cas overexpression reduces EGF-induced EGFR internalization, while p130Cas depletion enhances it. In addition, both FN-mediated cell adhesion and p130Cas overexpression reduce EGF-stimulated dynamin phosphorylation, which is necessary for EGF-induced EGFR internalization. Coimmunoprecipitation and GST pull-down assays confirmed the interaction between p130Cas and dynamin. Moreover, a SH3-domain-deleted form of p130Cas, which shows diminished binding to dynamin, inhibits dynamin phosphorylation and EGF uptake less effectively than wild-type p130Cas. CONCLUSIONS/SIGNIFICANCE: Our results show that p130Cas plays an inhibitory role in EGFR internalization via its interaction with dynamin. Given that the EGFR internalization process determines signaling density and specificity in the EGFR pathway, these findings suggest that the interaction between p130Cas and dynamin may regulate EGFR trafficking and signaling in the same manner as other endocytic regulatory proteins related to EGFR endocytosis

Murine Missing in Metastasis (MIM) Mediates Cell Polarity and Regulates the Motility Response to Growth Factors

Missing in metastasis (MIM) is a member of the inverse BAR-domain protein family, and in vitro studies have implied MIM plays a role in deforming membrane curvature into filopodia-like protrusions and cell dynamics. Yet, the physiological role of the endogenous MIM in mammalian cells remains undefined.We have examined mouse embryonic fibroblasts (MEFs) derived from mice in which the MIM locus was targeted by a gene trapping vector. MIM(-/-) MEFs showed a less polarized architecture characterized by smooth edges and fewer cell protrusions as compared to wild type cells, although the formation of filopodia-like microprotrusions appeared to be normal. Immunofluorescent staining further revealed that MIM(-/-) cells were partially impaired in the assembly of stress fibers and focal adhesions but were enriched with transverse actin filaments at the periphery. Poor assembly of stress fibers was apparently correlated with attenuation of the activity of Rho GTPases and partially relieved upon overexpressing of Myc-RhoA(Q63L), a constitutively activated RhoA mutant. MIM(-/-) cells were also spread less effectively than wild type cells during attachment to dishes and substratum. Upon treatment with PDGF MIM(-/-) cells developed more prominent dorsal ruffles along with increased Rac1 activity. Compared to wild type cells, MIM(-/-) cells had a slower motility in the presence of a low percentage of serum-containing medium but migrated normally upon adding growth factors such as 10% serum, PDGF or EGF. MIM(-/-) cells were also partially impaired in the internalization of transferrin, fluorescent dyes, foreign DNAs and PDGF receptor alpha. On the other hand, the level of tyrosine phosphorylation of PDGF receptors was more elevated in MIM depleted cells than wild type cells upon PDGF treatment.Our data suggests that endogenous MIM protein regulates globally the cell architecture and endocytosis that ultimately influence a variety of cellular behaviors, including cell polarity, motility, receptor signaling and membrane ruffling

Roles of Small GTPase Rac1 in the Regulation of Actin Cytoskeleton during Dengue Virus Infection

An important clinical characteristic of dengue hemorrhagic fever/dengue shock syndrome is increased vascular permeability. Actin cytoskeleton is a significant element of endothelial barrier function regulation. In vitro study showed that dengue virus infection could induce redistributions of actin cytoskeleton. It is not precisely clear the roles of actin and the mechanisms of its reorganization during the infection. Using immunochemical assays, drug inhibition assays and protein interaction profiling methods, we aimed to identify the ways in which dengue virus serotype 2 interacts with actin cytoskeleton. The study showed that dynamic treadmilling of actin is necessary for dengue virus entry, production and release, while small GTPase Rac1 also plays multiple roles during these processes. In addition, we demonstrated the association of viral E protein with actin, indicating a direct effect of viral protein on the structural modifications of actin cytoskeleton. Our results provide evidence for the participation of Rac1 signaling pathways in viral protein-induced actin reorganizations, which may be a mechanism involved in the etiology of dengue hemorrhagic fever