Cryptosporidium parvum induces host cell actin accumulation at the host-parasite interface.
ABSTRACT: Cryptosporidium parvum is an intracellular protozoan parasite that causes a severe diarrheal illness in humans and animals. Previous ultrastructural studies have shown that Cryptosporidium resides in a unique intracellular compartment in the apical region of the host cell. The mechanisms by which Cryptosporidium invades host intestinal epithelial cells and establishes this compartment are poorly understood. The parasite is separated from the host cell by a unique electron-dense structure of unknown composition. We have used indirect immunofluorescence microscopy and confocal laser scanning microscopy to characterize this structure. These studies indicate that host filamentous actin is assembled into a plaque-like structure at the host-parasite interface during parasite invasion and persists during parasite development. The actin-binding protein alpha-actinin is also present in this plaque early in parasite development but is lost as the parasite matures. Other actin-associated proteins, including vinculin, talin, and ezrin, are not present. We have found no evidence of tyrosine phosphorylation within this structure. Molecules known to link actin filaments to membrane were also examined, including alpha-catenin, beta-catenin, plakoglobin, and zyxin, but none was identified at the host-parasite junction. Thus, Cryptosporidium induces rearrangement of the host cell cytoskeleton and incorporates host cell actin and alpha-actinin into a host-parasite junctional complex.
Project description:Spatial and functional organization of cells in tissues is determined by cell-cell adhesion, thought to be initiated through trans-interactions between extracellular domains of the cadherin family of adhesion proteins, and strengthened by linkage to the actin cytoskeleton. Prevailing dogma is that cadherins are linked to the actin cytoskeleton through beta-catenin and alpha-catenin, although the quaternary complex has never been demonstrated. We test this hypothesis and find that alpha-catenin does not interact with actin filaments and the E-cadherin-beta-catenin complex simultaneously, even in the presence of the actin binding proteins vinculin and alpha-actinin, either in solution or on isolated cadherin-containing membranes. Direct analysis in polarized cells shows that mobilities of E-cadherin, beta-catenin, and alpha-catenin are similar, regardless of the dynamic state of actin assembly, whereas actin and several actin binding proteins have higher mobilities. These results suggest that the linkage between the cadherin-catenin complex and actin filaments is more dynamic than previously appreciated.
Project description:We examined gliding motility and cell invasion by an early-branching apicomplexan, Cryptosporidium parvum, which causes diarrheal disease in humans and animals. Real-time video microscopy demonstrated that C. parvum sporozoites undergo circular and helical gliding, two of the three stereotypical movements exhibited by Toxoplasma gondii tachyzoites. C. parvum sporozoites moved more rapidly than T. gondii sporozoites, which showed the same rates of motility as tachyzoites. Motility by C. parvum sporozoites was prevented by latrunculin B and cytochalasin D, drugs that depolymerize the parasite actin cytoskeleton, and by the myosin inhibitor 2,3-butanedione monoxime. Imaging of the initial events in cell entry by Cryptosporidium revealed that invasion occurs rapidly; however, the parasite does not enter deep into the cytosol but rather remains at the cell surface in a membrane-bound compartment. Invasion did not stimulate rearrangement of the host cell cytoskeleton and was inhibited by cytochalasin D, even in host cells that were resistant to the drug. Our studies demonstrate that C. parvum relies on a conserved actin-myosin motor for motility and active penetration of its host cell, thus establishing that this is a widely conserved feature of the Apicomplexa.
Project description:Germline and somatic inactivating mutations in the HRPT2 gene occur in the inherited hyperparathyroidism-jaw tumor syndrome, in some cases of parathyroid cancer and in some cases of familial hyperparathyroidism. HRPT2 encodes parafibromin. To identify parafibromin interacting proteins we used the yeast two-hybrid system for screening a heart cDNA library with parafibromin as the bait.Fourteen parafibromin interaction positive preys representing 10 independent clones encoding actinin-2 were isolated. Parafibromin interacted with muscle alpha-actinins (actinin-2 and actinin-3), but not with non-muscle alpha-actinins (actinin-1 and actinin-4). The parafibromin-actinin interaction was verified by yeast two-hybrid, GST pull-down, and co-immunoprecipitation. Yeast two-hybrid analysis revealed that the N-terminal region of parafibromin interacted with actinins. In actin sedimentation assays parafibromin did not dissociate skeletal muscle actinins from actin filaments, but interestingly, parafibromin could also bundle/cross-link actin filaments. Parafibromin was predominantly nuclear in undifferentiated proliferating myoblasts (C2C12 cells), but in differentiated C2C12 myotubes parafibromin co-localized with actinins in the cytoplasmic compartment.These data support a possible contribution of parafibromin outside the nucleus through its interaction with actinins and actin bundling/cross-linking. These data also suggest that actinins (and actin) participate in sequestering parafibromin in the cytoplasmic compartment.
Project description:The α-actinin proteins are a highly conserved family of actin crosslinkers that mediate interactions between several cytoskeletal and sarcomeric proteins. Nonsarcomeric α-actinin-1 and α-actinin-4 crosslink actin filaments in the cytoskeleton, while sarcomeric α-actinin-2 and α-actinin-3 serve a crucial role in anchoring actin filaments to the muscle Z-line. To assess the difference in turnover dynamics and structure/function properties between the α-actinin isoforms at the sarcomeric Z-line, we used Fluorescence Recovery After Photobleaching (FRAP) in primary myofiber cultures. We found that the recovery kinetics of these proteins followed three distinct patterns: α-actinin-2/α-actinin-3 had the slowest turn over, α-actinin-1 recovered to an intermediate degree, and α-actinin-4 had the fastest recovery. Interestingly, the isoforms' patterns of recovery were reversed at adhesion plaques in fibroblasts. This disparity suggests that the different α-actinin isoforms have unique association kinetics in myofibers and that nonmuscle isoform interactions are more dynamic at the sarcomeric Z-line. Protein domain-specific investigations using α-actinin-2/4 chimeric proteins showed that differential dynamics between sarcomeric and nonmuscle isoforms are regulated by cooperative interactions between the N-terminal actin-binding domain, the spectrin-like linker region and the C-terminal calmodulin-like EF hand domain. Together, these findings demonstrate that α-actinin isoforms are unique in binding dynamics at the Z-line and suggest differentially evolved interactive and Z-line association capabilities of each functional domain.
Project description:N-RAP is a striated muscle-specific scaffolding protein that organizes alpha-actinin and actin into symmetrical I-Z-I structures in developing myofibrils. Here we determined the order of events during myofibril assembly through time-lapse confocal microscopy of cultured embryonic chick cardiomyocytes coexpressing fluorescently tagged N-RAP and either alpha-actinin or actin. During de novo myofibril assembly, N-RAP assembled in fibrillar structures within the cell, with dots of alpha-actinin subsequently organizing along these structures. The initial fibrillar structures were reminiscent of actin fibrils, and coassembly of N-RAP and actin into newly formed fibrils supported this. The alpha-actinin dots subsequently broadened to Z-lines that were wider than the underlying N-RAP fibril, and N-RAP fluorescence intensity decreased. FRAP experiments showed that most of the alpha-actinin dynamically exchanged during all stages of myofibril assembly. In contrast, less than 20% of the N-RAP in premyofibrils was exchanged during 10-20 min after photobleaching, but this value increased to 70% during myofibril maturation. The results show that N-RAP assembles into an actin containing scaffold before alpha-actinin recruitment; that the N-RAP scaffold is much more stable than the assembling structural components; that N-RAP dynamics increase as assembly progresses; and that N-RAP leaves the structure after assembly is complete.
Project description:Host-parasite relationships are likely to be impacted by conservation management practices, potentially increasing the susceptibility of wildlife to emerging disease. Cryptosporidium, a parasitic protozoan genus comprising host-adapted and host-specific species, was used as an indicator of parasite movement between populations of a threatened marsupial, the brush-tailed rock-wallaby (Petrogale penicillata). PCR screening of faecal samples (n?=?324) from seven wallaby populations across New South Wales, identified Cryptosporidium in 7.1% of samples. The sampled populations were characterised as captive, supplemented and wild populations. No significant difference was found in Cryptosporidium detection between each of the three population categories. The positive samples, detected using 18S rRNA screening, were amplified using the actin and gp60 loci. Multi-locus sequence analysis revealed the presence of Cryptosporidium fayeri, a marsupial-specific species, and C.?meleagridis, which has a broad host range, in samples from the three population categories. Cryptosporidium meleagridis has not been previously reported in marsupials and hence the pathogenicity of this species to brush-tailed rock-wallabies is unknown. Based on these findings, we recommend further study into Cryptosporidium in animals undergoing conservation management, as well as surveying wild animals in release areas, to further understand the diversity and epidemiology of this parasite in threatened wildlife.
Project description:Localization of the actin crosslinking protein, alpha-actinin, to the cleavage furrow has been previously reported. However, its functions during cytokinesis remain poorly understood. We have analyzed the functions of alpha-actinin during cytokinesis by a combination of molecular manipulations and imaging-based techniques. alpha-actinin gradually dissipated from the cleavage furrow as cytokinesis progressed. Overexpression of alpha-actinin caused increased accumulation of actin filaments because of inhibition of actin turnover, leading to cytokinesis failure. Global depletion of alpha-actinin by siRNA caused a decrease in the density of actin filaments throughout the cell cortex, surprisingly inducing accelerated cytokinesis and ectopic furrows. Local ablation of alpha-actinin induced accelerated cytokinesis specifically at the site of irradiation. Neither overexpression nor depletion of alpha-actinin had an apparent effect on myosin II organization. We conclude that cytokinesis in mammalian cells requires tightly regulated remodeling of the cortical actin network mediated by alpha-actinin in coordination with actomyosin-based cortical contractions.
Project description:Alpha-actinin-4 is a widely expressed protein that employs an actin-binding site with two calponin homology domains to crosslink actin filaments (F-actin) in a Ca(2+)-sensitive manner in vitro. An inherited, late-onset form of kidney failure is caused by point mutations in the alpha-actinin-4 actin-binding domain. Here we show that alpha-actinin-4/F-actin aggregates, observed in vivo in podocytes of humans and mice with disease, likely form as a direct result of the increased actin-binding affinity of the protein. We document that exposure of a buried actin-binding site 1 in mutant alpha-actinin-4 causes an increase in its actin-binding affinity, abolishes its Ca(2+) regulation in vitro, and diverts its normal localization from actin stress fibers and focal adhesions in vivo. Inactivation of this buried actin-binding site returns the affinity of the mutant to that of the WT protein and abolishes aggregate formation in cells. In vitro, actin filaments crosslinked by the mutant alpha-actinin-4 exhibit profound changes of structural and biomechanical properties compared with WT alpha-actinin-4. On a molecular level, our findings elucidate the physiological importance of a dynamic interaction of alpha-actinin with F-actin in podocytes in vivo. We propose that a conformational change with full exposure of actin-binding site 1 could function as a switch mechanism to regulate the actin-binding affinity of alpha-actinin and possibly other calponin homology domain proteins under physiological conditions.
Project description:Regulated expression of cell adhesion molecules could be critical in the proliferation, sequestration, and maintenance of stem/progenitor cells. Therefore, we determined fetal and adult stage-specific roles of cell adhesion in liver cell compartments.We performed immunostaining for the adhesion molecules, E-cadherin and Ep-CAM, associated proteins, beta-catenin and alpha-actinin, hepatobiliary markers, albumin, alpha-fetoprotein, and cytokeratin-19, and the proliferation marker, Ki-67. Expression of albumin was verified by in situ mRNA hybridization.In the fetal liver, hepatoblasts showed extensive proliferation with wide expression of E-cadherin, beta-catenin, and alpha-actinin, although Ep-CAM was expressed in these cells less intensely and focally in the cell membrane to indicate weak cell adhesion. Hepatoblasts in ductal plate and bile ducts showed less proliferation and Ep-CAM was intensely expressed in these cells throughout the cell membrane, indicating strong adhesion. In some ductal plate cells, beta-catenin was additionally in the cytoplasm and nucleus, suggesting active cell signaling by adhesion molecules. In adult livers, cells were no longer proliferating and E-cadherin, beta-catenin, and alpha-actinin were expressed in hepatocytes throughout, whereas Ep-CAM was expressed in only bile duct cells. Some cells in ductal structures of the adult liver with Ep-CAM coexpressed albumin and cytokeratin-19, indicating persistence of fetal-like stem/progenitor cells.Regulated expression of Ep-CAM supported proliferation in fetal hepatoblasts through weak adhesion and helped in biliary morphogenesis by promoting stronger adhesion in hepatoblasts during this process. Restriction of Ep-CAM expression to bile ducts in the adult liver presumably facilitated sequestration of stem/progenitor cells. This stage-specific and cell compartment-related regulation of adhesion molecules should be relevant for defining how liver stem/progenitor cells enter, exit, and remain in hepatic niches during both health and disease.
Project description:alpha-Actinin is an actin bundling protein that regulates cell adhesion by directly linking actin filaments to integrin adhesion receptors. Phosphatidylinositol (4,5)-diphosphate (PtdIns (4,5)-P(2)) and phosphatidylinositol (3,4,5)-triphosphate (PtdIns (3,4,5)-P(3)) bind to the calponin homology 2 domain of alpha-actinin, regulating its interactions with actin filaments and integrin receptors. In this study, we examine the mechanism by which phosphoinositide binding regulates alpha-actinin function using mass spectrometry to monitor hydrogen-deuterium (H/D) exchange within the calponin homology 2 domain. The overall level of H/D exchange for the entire protein showed that PtdIns (3,4,5)-P(3) binding alters the structure of the calponin homology 2 domain increasing deuterium incorporation, whereas PtdIns (4,5)-P(2) induces changes in the structure decreasing deuterium incorporation. Analysis of peptic fragments from the calponin homology 2 domain showed decreased local H/D exchange within the loop region preceding helix F with both phosphoinositides. However, the binding of PtdIns (3,4,5)-P(3) also induced increased exchange within helix E. This suggests that the phosphate groups on the fourth and fifth position of the inositol head group of the phosphoinositides constrict the calponin homology 2 domain, thereby altering the orientation of actin binding sequence 3 and decreasing the affinity of alpha-actinin for filamentous actin. In contrast, the phosphate group on the third position of the inositol head group of PtdIns (3,4,5)-P(3) perturbs the calponin homology 2 domain, altering the interaction between the N and C terminus of the full-length alpha-actinin antiparallel homodimer, thereby disrupting bundling activity and interaction with integrin receptors.