Mechanosensitive mechanisms in transcriptional regulation

Advance Online Publication July 13, 2012 doi: 10.1242/?jcs.093005 Transcriptional regulation contributes to the maintenance of pluripotency, self-renewal and differentiation in embryonic cells and in stem cells. Therefore, control of gene expression at the level of transcription is crucial for embryonic development, as well as for organogenesis, functional adaptation, and regeneration in adult tissues and organs. In the past, most work has focused on how transcriptional regulation results from the complex interplay between chemical cues, adhesion signals, transcription factors and their co-regulators during development. However, chemical signaling alone is not sufficient to explain how three-dimensional (3D) tissues and organs are constructed and maintained through the spatiotemporal control of transcriptional activities. Accumulated evidence indicates that mechanical cues, which include physical forces (e.g. tension, compression or shear stress), alterations in extracellular matrix (ECM) mechanics and changes in cell shape, are transmitted to the nucleus directly or indirectly to orchestrate transcriptional activities that are crucial for embryogenesis and organogenesis. In this Commentary, we review how the mechanical control of gene transcription contributes to the maintenance of pluripotency, determination of cell fate, pattern formation and organogenesis, as well as how it is involved in the control of cell and tissue function throughout embryogenesis and adult life. A deeper understanding of these mechanosensitive transcriptional control mechanisms should lead to new approaches to tissue engineering and regenerative medicine.

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Mph1 kinetochore localization is crucial and upstream in the hierarchy of spindle assembly checkpoint protein recruitment to kinetochores

Advance Online Publication July 23, 2012 doi: 10.1242/?jcs.110387 The spindle assembly checkpoint (SAC) blocks entry into anaphase until all chromosomes have stably attached to the mitotic spindle through their kinetochores. The checkpoint signal originates from unattached kinetochores, where SAC proteins enrich. Whether the enrichment of all SAC proteins is crucial for SAC signalling is unclear. Here we provide evidence that in fission yeast, recruitment of the kinase Mph1 is of vital importance for a stable SAC arrest. An Mph1 mutant that eliminates kinetochore enrichment abolishes SAC signalling, whereas forced recruitment of this mutant to kinetochores restores SAC signalling. In bub3? cells, the SAC is functional with only Mph1 and the Aurora kinase Ark1, but no other SAC proteins, enriched at kinetochores. We analysed the network of dependencies for SAC protein localization to kinetochores and identify a three-layered hierarchy with Ark1 and Mph1 on top, Bub1 and Bub3 in the middle, and Mad3 as well as the Mad1-Mad2 complex at the lower end of the hierarchy. If Mph1 is artificially recruited to kinetochores, Ark1 becomes dispensable for SAC activity. Our results highlight the critical role of Mph1 at kinetochores and suggest that the Mad1-Mad2 complex does not necessarily need to enrich at kinetochores for functional SAC signalling.

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Visualisation of direct interaction of MDA5 and the dsRNA replicative intermediate form of positive strand RNA viruses

Advance Online Publication July 13, 2012 doi: 10.1242/?jcs.103887 The innate immune system is a vital part of the body's defences against viral pathogens. RIG-I and MDA5 function as cytoplasmic PRRs that are involved in the elimination of actively replicating RNA viruses. Their location and their differential responses to RNA viruses emphasises the complexity of the innate detection system. Despite the wealth of information on the types of RNA that trigger RIG-I, much less is known about the nature of the RNAs that act as agonists for MDA5. In order to identify which RNA species triggers MDA5 activation during infection, we isolated viral ssRNA and replicative intermediates of RNA from positive sense ssRNA viruses. We reveal that MDA5 recognises not the genomic ssRNA but the dsRNA generated by the replication of these viruses. Furthermore, using fluorescent imaging we present the first report of the visualization of dsRNA and MDA5, which provides unique evidence between the relationship of viral dsRNA and MDA5 and proves without a doubt that MDA5 is the key sensor for the dsRNA replicative intermediate form of positive sense ssRNA viruses.

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Quantitative proteomics and dynamic imaging reveal that G3BP-mediated stress granule assembly is poly(ADP-ribose)-dependent following exposure to MNNG-induced DNA alkylation

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United we stand - integrating the actin cytoskeleton and cell-matrix adhesions in cellular mechanotransduction

Advance Online Publication July 13, 2012 doi: 10.1242/?jcs.093716 Many essential cellular functions in health and disease are closely linked to the ability of cells to respond to mechanical forces. In the context of cell adhesion to the extracellular matrix, the forces that are generated within the actin cytoskeleton and transmitted through integrin-based focal adhesions are essential for the cellular response to environmental clues, such as the spatial distribution of adhesive ligands or matrix stiffness. Whereas substantial progress has been made in identifying mechanosensitive molecules that can transduce mechanical force into biochemical signals, much less is known about the nature of cytoskeletal force generation and transmission that regulates the magnitude, duration and spatial distribution of forces imposed on these mechanosensitive complexes. By focusing on cell-matrix adhesion to flat elastic substrates, on which traction forces can be measured with high temporal and spatial resolution, we discuss our current understanding of the physical mechanisms that integrate a large range of molecular mechanotransduction events on cellular scales. Physical limits of stability emerge as one important element of the cellular response that complements the structural changes affected by regulatory systems in response to mechanical processes.

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Wnt5a signaling controls cytokinesis by positioning ESCRT-III to the proper site at the midbody

Advance Online Publication July 23, 2012 doi: 10.1242/?jcs.108142 Wnts activate at least two signaling pathways, the ß-catenin-dependent and -independent pathways. Although the ß-catenin-dependent pathway is known to contribute to G1/S transition, involvement of the ß-catenin-independent pathway in cell cycle regulation remains unclear. Here, we show that Wnt5a signaling, which activates the ß-catenin-independent pathway, is required for cytokinesis. Dishevelled 2 (Dvl2), a mediator of Wnt signaling pathways, was localized to the midbody during cytokinesis. Beside the localization of Dvl2, Fz2, a Wnt receptor, was detected in the midbody with an endosomal sorting complex required for transport III (ESCRT-III) subunit, CHMP4B. Depletion of Wnt5a, its receptors, and Dvl increased multinucleated cells. The phenotype observed in Wnt5a-depleted cells was rescued by the addition of purified Wnt5a but not that of Wnt3a, which is a ligand for the ß-catenin-dependent pathway. Moreover, depletion of Wnt5a signaling caused loss of stabilized microtubules and mislocalization of CHMP4B in the midbody, which affected abscission. Inhibition of the stabilization of microtubules at the midbody lead to the mislocalization of CHMP4B, while depletion of CHMP4B did not affect the stabilization of microtubules, suggesting that the correct localization of CHMP4B depends on microtubules. Fz2 was localized to the midbody in a Rab11-dependent manner probably along stabilized microtubules. Fz2 formed a complex with CHMP4B upon Wnt5a stimulation and was required for proper localization of CHMP4B at the midbody, while CHMP4B was not necessary for the localization of Fz2. These results suggest that Wnt5a signaling positions ESCRT-III in the midbody properly for abscission by stabilizing midbody microtubules.

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