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Articles by Scott Grieshaber in JoVE
Other articles by Scott Grieshaber on PubMed
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Determination of the Physical Environment Within the Chlamydia Trachomatis Inclusion Using Ion-selective Ratiometric Probes
Cellular Microbiology.
May, 2002 |
Pubmed ID: 12027956 Chlamydia trachomatis is an obligate intracellular bacterium with a biphasic life cycle that takes place entirely within a membrane-bound vacuole termed an inclusion. The chlamydial inclusion is non-fusogenic with endosomal or lysosomal compartments but intersects a pathway involved in transport of sphingomyelin from the Golgi apparatus to the plasma membrane. The physical conditions within the mature chlamydial inclusion are unknown. We used ratiometric imaging with membrane-permeant, ion-selective fluorescent dyes for microanalyis of the physical environment within the inclusion. Determination of H+, Na+, K+ and Ca(2+) concentrations using CFDA (carboxy fluorescein diacetate) or BCECF-AM (2',7'-bis (2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester, SBFI-AM, PBFI-AM and fura-PE3-acetomethoxyester (Fura-PE3-AM), respectively, indicated that all ions assayed within the lumenal space of the inclusion approximated the concentrations within the cytoplasm. Stimulation of purinergic receptors by addition of extracellular ATP triggered a dynamic Ca(2+) response that occurred simultaneously within the cytoplasm and interior of the inclusion. The chlamydial inclusion thus appears to be freely permeable to cytoplasmic ions. These results have implications for nutrient acquisition by chlamydiae and may contribute to the non-fusogenicity of the inclusion with endocytic compartments.
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Follicle-stimulating Hormone-responsive Cytoskeletal Genes in Rat Granulosa Cells: Class I Beta-tubulin, Tropomyosin-4, and Kinesin Heavy Chain
Endocrinology.
Jan, 2003 |
Pubmed ID: 12488327 FSH regulates gene expression for granulosa cell differentiation and follicular development. Therefore, FSH-responsive genes are crucial, but only a few genes have been identified for the early stage of follicular development. In particular, little is known about cytoskeletal genes, which likely play essential roles in the morphological changes such as the antrum formation, a major landmark. FSH is also known to induce the differentiation of an immature, undifferentiated rat ovary granulosa (ROG) cell line. Our data show that FSH induced massive yet distinct reorganization of microtubules and the actin cytoskeletons as well as morphological changes. To identify those genes responding to FSH during the differentiation, differential display was performed on ROG cells. Of the 80 FSH-responsive genes identified, there were three cytoskeleton-related genes (class I beta-tubulin, tropomyosin 4, and kinesin heavy chain), which are crucial for intracellular morphogenesis, transport, and differentiation. Northern blots show that the level of these gene transcripts reached a peak at 6 h after FSH treatment and subsided at 24 h. FSH induced the similar temporal expression not only in granulosa cells isolated from immature rats, but also in vivo. For instance, in situ hybridization showed that beta-tubulin mRNA was transiently expressed in the granulosa cells of large preantral and early antral follicles. Despite the same temporal expression, the regulatory mechanisms of the three genes were strikingly different. As an example, cycloheximide blocked the beta-tubulin mRNA expression, whereas it increased tropomyosin-4 (TM4) mRNA. Yet, it did not impact kinesin heavy chain (Khc) mRNA. In conclusion, FSH induces the massive reorganization of the cytoskeletons and morphological changes by the selective regulation of the gene expression, protein synthesis, and rearrangement of the cytoskeletal proteins in the ROG cells and probably, specific follicles and granulosa cells.
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Effects of Ectopically Expressed Neuronal Wiskott-Aldrich Syndrome Protein Domains on Rickettsia Rickettsii Actin-based Motility
Infection and Immunity.
Mar, 2003 |
Pubmed ID: 12595475 Neuronal Wiskott-Aldrich syndrome protein (N-WASP) and the actin-related protein 2/3 (Arp2/3) complex have emerged as critical host proteins that regulate pathogen actin-based motility. Actin tail formation and motility in Listeria monocytogenes require the Arp2/3 complex but bypasses N-WASP signaling. Motility of Shigella flexneri and vaccinia virus requires both N-WASP and the Arp2/3 complex. Functional roles for these cytoskeletal regulatory proteins in actin-based motility of Rickettsia rickettsii have not been established. In this study, functional domains of N-WASP tagged with green fluorescent protein that have characterized effects on Shigella and vaccinia virus actin-based motility were ectopically expressed in HeLa cells infected with R. rickettsii to assess their effects on rickettsial motility. S. flexneri-infected cells were used as a control. Expressed N-WASP domains did not localize to R. rickettsii or their actin tails. Expression of N-WASP missing the VCA domain (for "verprolin homology, cofilin homology, and acidic domains"), which acts as a dominant-negative form of N-WASP, completely inhibited actin-based motility of S. flexneri while only moderately inhibiting motility of R. rickettsii. Similarly, expression of the VCA domain, which acts as a dominant-negative with respect to Arp2/3 complex function, severely inhibited actin-based motility of S. flexneri (no motility observed in the majority of expressing cells) but only moderately inhibited R. rickettsii motility. These results, taken together with the differential effects on motility observed upon expression of other N-WASP domains, suggest that actin-based motility of R. rickettsii is independent of N-WASP and the Arp2/3 complex.
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Chlamydia Trachomatis Uses Host Cell Dynein to Traffic to the Microtubule-organizing Center in a P50 Dynamitin-independent Process
Journal of Cell Science.
Sep, 2003 |
Pubmed ID: 12902405 Chlamydiae are pathogenic obligate intracellular bacteria with a biphasic developmental cycle that involves cell types adapted for extracellular survival (elementary bodies, EBs) and intracellular multiplication (reticulate bodies, RBs). The intracellular development of chlamydiae occurs entirely within a membrane-bound vacuole termed an inclusion. Within 2 hours after entry into host cells, Chlamydia trachomatis EBs are trafficked to the perinuclear region of the host cell and remain in close proximity to the Golgi apparatus, where they begin to fuse with a subset of host vesicles containing sphingomyelin. Here, we provide evidence that chlamydial migration from the cell periphery to the peri-Golgi region resembles host cell vesicular trafficking. Chlamydiae move towards the minus end of microtubules and aggregate at the microtubule-organizing center (MTOC). In mammalian cells the most important minus-end-directed microtubule motor is cytoplasmic dynein. Microinjection of antibodies to a subunit of cytoplasmic dynein inhibited movement of chlamydiae to the MTOC, whereas microinjection of antibodies to the plus-directed microtubule motor, kinesin, had no effect. Surprisingly, overexpression of the protein p50 dynamitin, a subunit of the dynactin complex that links vesicular cargo to the dynein motor in minus directed vesicle trafficking, did not abrogate chlamydial migration even though host vesicle transport was inhibited. Nascent chlamydial inclusions did, however, colocalize with the p150(Glued) dynactin subunit, which suggests that p150(Glued) may be required for dynein activation or processivity but that the cargo-binding activity of dynactin, supplied by p50 dynamitin subunits and possibly other subunits, is not. Because chlamydial transcription and translation were required for this intracellular trafficking, chlamydial proteins modifying the cytoplasmic face of the inclusion membrane are probable candidates for proteins fulfilling this function.
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Requirement for the Rac GTPase in Chlamydia Trachomatis Invasion of Non-phagocytic Cells
Traffic (Copenhagen, Denmark).
Jun, 2004 |
Pubmed ID: 15117316 Chlamydiae are gram-negative obligate intracellular pathogens to which access to an intracellular environment is paramount to their survival and replication. To this end, chlamydiae have evolved extremely efficient means of invading nonphagocytic cells. To elucidate the host cell machinery utilized by Chlamydia trachomatis in invasion, we examined the roles of the Rho GTPase family members in the internalization of chlamydial elementary bodies. Upon binding of elementary bodies on the cell surface, actin is rapidly recruited to the sites of internalization. Members of the Rho GTPase family are frequently involved in localized recruitment of actin. Clostridial Toxin B, which is a known enzymatic inhibitor of Rac, Cdc42 and Rho GTPases, significantly reduced chlamydial invasion of HeLa cells. Expression of dominant negative constructs in HeLa cells revealed that chlamydial uptake was dependent on Rac, but not on Cdc42 or RhoA. Rac but not Cdc42 was found to be activated by chlamydial attachment. The effect of dominant negative Rac expression on chlamydial uptake is manifested through the inhibition of actin recruitment to the sites of chlamydial entry. Studies utilizing Green Fluorescent Protein fusion constructs of Rac, Cdc42 and RhoA, showed Rac to be the sole member of the Rho GTPase family recruited to the site of chlamydial entry.
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Tyrosine Phosphorylation of the Chlamydial Effector Protein Tarp is Species Specific and Not Required for Recruitment of Actin
Infection and Immunity.
Jul, 2005 |
Pubmed ID: 15972471 Chlamydiae are obligate intracellular pathogens that efficiently induce their endocytosis by susceptible eukaryotic host cells. Recently, a Chlamydia trachomatis type III secreted effector protein, Tarp, was found to be translocated and tyrosine phosphorylated at the site of entry and associated with the recruitment of actin that coincides with endocytosis. C. trachomatis Tarp possesses up to six direct repeats of approximately 50 amino acids each. The majority of the tyrosine residues are found within this repeat region. Here we have ectopically expressed distinct domains of Tarp in HeLa 229 cells and demonstrated that tyrosine phosphorylation occurs primarily within the repeat region, while recruitment of actin is mediated by the C-terminal domain of the protein. A comparison of other sequenced chlamydial genomes revealed that each contains an ortholog of Tarp, although Chlamydia muridarum, Chlamydophila caviae, and Chlamydophila pneumoniae Tarp lack the large repeat region. Immunofluorescence and immunoblotting using an antiphosphotyrosine antibody show no evidence of phosphotyrosine at the site of entry of C. muridarum, C. caviae, and C. pneumoniae, although each species similarly recruits actin. Ectopic expression of full-length C. trachomatis and C. caviae Tarp confirmed that both recruit actin but only C. trachomatis Tarp is tyrosine phosphorylated. The data indicate that the C-terminal domain of Tarp is essential for actin recruitment and that tyrosine phosphorylation may not be an absolute requirement for actin recruitment. The results further suggest the potential for additional, unknown signal transduction pathways associated specifically with C. trachomatis.
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Chlamydial Entry Involves TARP Binding of Guanine Nucleotide Exchange Factors
PLoS Pathogens.
Mar, 2008 |
Pubmed ID: 18383626 Chlamydia trachomatis attachment to cells induces the secretion of the elementary body-associated protein TARP (Translocated Actin Recruiting Protein). TARP crosses the plasma membrane where it is immediately phosphorylated at tyrosine residues by unknown host kinases. The Rac GTPase is also activated, resulting in WAVE2 and Arp2/3-dependent recruitment of actin to the sites of chlamydia attachment. We show that TARP participates directly in chlamydial invasion activating the Rac-dependent signaling cascade to recruit actin. TARP functions by binding two distinct Rac guanine nucleotide exchange factors (GEFs), Sos1 and Vav2, in a phosphotyrosine-dependent manner. The tyrosine phosphorylation profile of the sequence YEPISTENIYESI within TARP, as well as the transient activation of the phosphatidylinositol 3-kinase (PI3-K), appears to determine which GEF is utilized to activate Rac. The first and second tyrosine residues, when phosphorylated, are utilized by the Sos1/Abi1/Eps8 and Vav2, respectively, with the latter requiring the lipid phosphatidylinositol 3,4,5-triphosphate. Depletion of these critical signaling molecules by siRNA resulted in inhibition of chlamydial invasion to varying degrees, owing to a possible functional redundancy of the two pathways. Collectively, these data implicate TARP in signaling to the actin cytoskeleton remodeling machinery, demonstrating a mechanism by which C.trachomatis invades non-phagocytic cells.
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Tissue Distibution of Murine Muc19/Smgc Gene Products
The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society.
Oct, 2009 |
Pubmed ID: 19826070 The recently identified gene, Muc19/Smgc, encodes two diverse splice variants, Smgc (submandibular gland protein C) and Muc19 (mucin 19). Muc19 is a member of the large gel-forming mucin family and is an exocrine product of sublingual mucous salivary glands in mice. SMGC is a transiently expressed secretion product of developing rodent submandibular and sublingual glands. Little is known of the expression of Muc19/Smgc gene products in other murine salivary and non-salivary tissues containing the mucous cell phenotype. Muc19 expression was therefore initially assessed by RT-PCR and immunohistochemistry. As a complementary approach, we developed a knockin mouse model, Muc19-EGFP, in which mice express a fusion protein containing the first 69 residues of Muc19 followed by enhanced green fluorescent protein (EGFP) as a marker of Muc19 expression. Results from both approaches are consistent, with preferential Muc19 expression in salivary major and minor mucous glands as well as submucosal glands of the tracheolarynx and bulbourethral glands. Evidence also indicates individual mucous cells of minor salivary and bulbourethral glands produce another gel-forming mucin in addition to Muc19. We further find tissue expression of full-length Smgc transcripts, which encode for SMGC, are restricted to neonatal tracheolarynx and all salivary tissues.
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