RESUMEN
The engagement of a T cell with an antigen-presenting cell (APC) or activating surface results in the formation within the T cell of several distinct actin and actomyosin networks. These networks reside largely within a narrow zone immediately under the T cell's plasma membrane at its site of contact with the APC or activating surface, i.e., at the immunological synapse. Here we review the origin, organization, dynamics, and function of these synapse-associated actin and actomyosin networks. Importantly, recent insights into the nature of these actin-based cytoskeletal structures were made possible in several cases by advances in light microscopy.
Asunto(s)
Actinas/metabolismo , Actomiosina/metabolismo , Células Presentadoras de Antígenos/metabolismo , Citoesqueleto/metabolismo , Sinapsis Inmunológicas/metabolismo , Linfocitos T/metabolismo , Animales , Presentación de Antígeno , Humanos , Activación de LinfocitosRESUMEN
The human pathogen Chlamydia trachomatis targets epithelial cells lining the genital mucosa. We observed that infection of various cell types, including fibroblasts and epithelial cells resulted in the formation of unusually stable and mature focal adhesions that resisted disassembly induced by the myosin II inhibitor, blebbistatin. Superresolution microscopy revealed in infected cells the vertical displacement of paxillin and focal adhesion kinase from the signaling layer of focal adhesions, whereas vinculin remained in its normal position within the force transduction layer. The candidate type III effector TarP, which localized to focal adhesions during infection and when expressed ectopically, was sufficient to mimic both the reorganization and blebbistatin-resistant phenotypes. These effects of TarP, including its localization to focal adhesions, required a post-invasion interaction with the host protein vinculin through a specific domain at the C terminus of TarP. This interaction is repurposed from an actin-recruiting and -remodeling complex to one that mediates nanoarchitectural and dynamic changes of focal adhesions. The consequence of Chlamydia-stabilized focal adhesions was restricted cell motility and enhanced attachment to the extracellular matrix. Thus, via a novel mechanism, Chlamydia inserts TarP within focal adhesions to alter their organization and stability.
Asunto(s)
Infecciones por Chlamydia/metabolismo , Chlamydia trachomatis/fisiología , Adhesiones Focales/metabolismo , Animales , Células COS , Infecciones por Chlamydia/microbiología , Infecciones por Chlamydia/patología , Chlorocebus aethiops , Adhesiones Focales/microbiología , Adhesiones Focales/patología , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Mapas de Interacción de Proteínas , Vinculina/análisis , Vinculina/metabolismoRESUMEN
Chlamydiae are obligate intracellular pathogens. They undergo a biphasic developmental cycle differentiating between the infectious but metabolically quiescent elementary body and the vegetative, but non-infectious reticulate body. Chlamydia spends a significant portion of its development in the non-infectious stage, demanding an effective strategy of manipulating the host cells to ensure its intracellular survival and replication. A common target of all Chlamydia species studied so far is the host cell cytoskeleton, with past and recent findings revealing crucial roles in invasion, inclusion maintenance, nutrient acquisition, and egress. The molecular details of how Chlamydia co-opts the cytoskeleton is becoming clearer, with bacterial factors and their corresponding host cell targets identified.
Asunto(s)
Chlamydia/patogenicidad , Citoesqueleto/metabolismo , Citoesqueleto/microbiología , Interacciones Huésped-Patógeno , Animales , HumanosRESUMEN
Myosin 10 (Myo10) couples microtubules and integrin-based adhesions to movement along actin filaments via its microtubule-binding MyTH4 domain and integrin-binding FERM domain, respectively. Here we show that Myo10-depleted HeLa cells and mouse embryo fibroblasts (MEFs) both exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in Myo10-depleted MEFs and in Myo10-depleted HeLa cells lacking supernumerary centrosomes is pericentriolar material (PCM) fragmentation, which creates y-tubulin-positive acentriolar foci that serve as extra spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing the clustering of the extra spindle poles. Complementation experiments show that Myo10 must interact with both microtubules and integrins to promote PCM/pole integrity. Conversely, Myo10 only needs interact with integrins to promote supernumerary centrosome clustering. Importantly, images of metaphase Halo-Myo10 knockin cells show that the myosin localizes exclusively to the spindle and the tips of adhesive retraction fibers. We conclude that Myo10 promotes PCM/pole integrity in part by interacting with spindle microtubules, and that it promotes supernumerary centrosome clustering by supporting retraction fiber-based cell adhesion, which likely serves to anchor the microtubule-based forces driving pole focusing.
Asunto(s)
Centrosoma , Huso Acromático , Ratones , Humanos , Animales , Células HeLa , Huso Acromático/metabolismo , Centrosoma/metabolismo , Microtúbulos/metabolismo , Miosinas/metabolismo , Integrinas/metabolismo , MitosisRESUMEN
Myosin 10 (Myo10) has the ability to link actin filaments to integrin-based adhesions and to microtubules by virtue of its integrin-binding FERM domain and microtubule-binding MyTH4 domain, respectively. Here we used Myo10 knockout cells to define Myo10's contribution to the maintenance of spindle bipolarity, and complementation to quantitate the relative contributions of its MyTH4 and FERM domains. Myo10 knockout HeLa cells and mouse embryo fibroblasts (MEFs) both exhibit a pronounced increase in the frequency of multipolar spindles. Staining of unsynchronized metaphase cells showed that the primary driver of spindle multipolarity in knockout MEFs and knockout HeLa cells lacking supernumerary centrosomes is pericentriolar material (PCM) fragmentation, which creates γ-tubulin-positive acentriolar foci that serve as additional spindle poles. For HeLa cells possessing supernumerary centrosomes, Myo10 depletion further accentuates spindle multipolarity by impairing the clustering of the extra spindle poles. Complementation experiments show that Myo10 must interact with both integrins and microtubules to promote PCM/pole integrity. Conversely, Myo10's ability to promote the clustering of supernumerary centrosomes only requires that it interact with integrins. Importantly, images of Halo-Myo10 knock-in cells show that the myosin localizes exclusively within adhesive retraction fibers during mitosis. Based on these and other results, we conclude that Myo10 promotes PCM/pole integrity at a distance, and that it facilitates supernumerary centrosome clustering by promoting retraction fiber-based cell adhesion, which likely provides an anchor for the microtubule-based forces driving pole focusing.
RESUMEN
Bacterial type III secretion system (T3SS) chaperones pilot substrates to the export apparatus in a secretion-competent state, and are consequently central to the translocation of effectors into target cells. Chlamydia trachomatis is a genetically intractable obligate intracellular pathogen that utilizes T3SS effectors to trigger its entry into mammalian cells. The only well-characterized T3SS effector is TARP (translocated actin recruitment protein), but its chaperone is unknown. Here we exploited a known structural signature to screen for putative type III secretion chaperones encoded within the C. trachomatis genome. Using bacterial two-hybrid, co-precipitation, cross-linking and size exclusion chromatography we show that Slc1 (SycE-like chaperone 1; CT043) specifically interacts with a 200-amino-acid residue N-terminal region of TARP (TARP¹â»²°°). Slc1 formed homodimers in vitro, as shown in cross-linking and gel filtration experiments. Biochemical analysis of an isolated Slc1-TARP¹â»²°° complex was consistent with a characteristic 2:1 chaperone-effector stoichiometry. Furthermore, Slc1 was co-immunoprecipitated with TARP from C. trachomatis elementary bodies. Also, coexpression of Slc1 specifically enhanced host cell translocation of TARP by a heterologous Yersinia enterocolitica T3SS. Taken together, we propose Slc1 as a chaperone of the C. trachomatis T3SS effector TARP.
Asunto(s)
Proteínas Bacterianas/metabolismo , Infecciones por Chlamydia/microbiología , Chlamydia trachomatis/metabolismo , Chaperonas Moleculares/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Chlamydia trachomatis/química , Chlamydia trachomatis/genética , Células HeLa , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas , Alineación de SecuenciaRESUMEN
Background: Association of genetic polymorphisms in paired box 1 (PAX-1) gene can influence the development of adolescent idiopathic scoliosis (AIS). PAX-1 is mainly expressed in the region of the vertebral bodies and intervertebral discs, being important for the proper formation of spinal structures. Objectives: The objective of this study was to evaluate the association of polymorphisms in PAX-1 gene with the susceptibility of AIS. Settings and Design: This was an analytical observational case-control study. Materials and Methods: Samples of 59 AIS indicated for surgical treatment, and 119 controls, without spinal disease were genotyped for PAX-1 rs6137473 and rs169311 polymorphisms. Statistical Analysis: The association of the polymorphisms with AIS was evaluated by a multivariable logistic regression model, using odds ratios (OR) and 95% confidence intervals (CI). Results: According to Lenke's classification, 89.8% had Type I and 10.2% II curves. The mean value of the Cobb angle of the proximal thoracic curve was 30.8°, 58.7° thoracic, and 30.4° for the lumbar and on the bending films 14.6°, 40.7°, and 11°, respectively. Among the AIS group, there was a predominance of females (8.8:1). The PAX-1 rs169311 and rs6137473 polymorphisms were positively associated with developing the AIS (OR = 1.98; 95% CI = 1.2-3.3 and OR = 3.16; 95% CI = 1.4-7.3, respectively). The rs6137473 polymorphism was associated with the lumbar modifier B and C compared to A (OR = 2.52; 95% CI = 1.1-5.8). Conclusions: PAX-1 polymorphisms were associated with an increased risk of developing the AIS and with curve severity and can be used as a biomarker to map the risk of developing surgical-grade AIS, guiding the treatment of patients.
RESUMEN
The mammalian protein vinculin is often a target of bacterial pathogens to subvert locally host cell actin dynamics. In Chlamydia infection, vinculin has been implicated in RNA interference screens, but the molecular basis for vinculin requirement has not been characterized. In this report, we show that vinculin was involved in the actin recruitment and F-actin assembly at the plasma membrane to facilitate invasion. Vinculin was recruited to the plasma membrane via its interaction with a specific tripartite motif within TarP that resembles the vinculin-binding domain (VBD) found in the Shigella invasion factor IpaA. The TarP-mediated plasma membrane recruitment of vinculin resulted in the localized recruitment of actin. In vitro pulldown assays for protein-protein interaction and imaging-based evaluation of recruitment to the plasma membrane demonstrated the essential role of the vinculin-binding site 1 (VBS1), and the dispensability of VBS2 and VBS3. As further support for the functionality of VBD-vinculin interaction, VBD-mediated actin recruitment required vinculin. Interestingly, while both vinculin and the focal adhesion kinase (FAK) colocalized at the sites of adhesion, the recruitment of one was independent of the other; and the actin recruitment function of the VBD/vinculin signaling axis was independent of the LD/FAK pathway.
Asunto(s)
Actinas/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Chlamydia/fisiología , Multimerización de Proteína , Vinculina/metabolismo , Factores de Virulencia/metabolismo , Línea Celular , Endocitosis , Interacciones Huésped-Patógeno , Humanos , Unión Proteica , Mapeo de Interacción de ProteínasRESUMEN
PURPOSE: The purpose of this retrospective investigation was to evaluate the influence on the adjacent cervical bone of moving the implant-abutment microgap inward from the outer edge of an internal-hex implant platform (ie, a platform-switched configuration). MATERIALS AND METHODS: A convenience sample of 26 patients received 42 Frialit-2 (Dentsply-Friadent) implants for single tooth replacement and were divided into two groups. One group followed the original protocol (control group), and the other (study group) received narrower replaceable components. Follow-up time varied between 6 and 60 months (mean, 33.45 months). RESULTS: The control group showed noticeable bone remodeling (mean bone loss = 2.30 mm), and all the remodeled crests were apical to the implant platform. In contrast, all patients in the study (platform-switched) group showed stable levels (mean bone loss = 0.27) of the peri-implant crestal bone, coronal to the implant platform. CONCLUSION: The relocation of the implant-abutment microgap through platform switching with Frialit-2 System implants seems to be an effective means to minimize marginal bone loss in all circumstances employed.