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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Finding the weakest link - exploring integrin-mediated mechanical molecular pathways

Advance Online Publication July 13, 2012 doi: 10.1242/?jcs.095794 From the extracellular matrix to the cytoskeleton, a network of molecular links connects cells to their environment. Molecules in this network transmit and detect mechanical forces, which subsequently determine cell behavior and fate. Here, we reconstruct the mechanical pathway followed by these forces. From matrix proteins to actin through integrins and adaptor proteins, we review how forces affect the lifetime of bonds and stretch or alter the conformation of proteins, and how these mechanical changes are converted into biochemical signals in mechanotransduction events. We evaluate which of the proteins in the network can participate in mechanotransduction and which are simply responsible for transmitting forces in a dynamic network. Besides their individual properties, we also analyze how the mechanical responses of a protein are determined by their serial connections from the matrix to actin, their parallel connections in integrin clusters and by the rate at which force is applied to them. All these define mechanical molecular pathways in cells, which are emerging as key regulators of cell function alongside better studied biochemical pathways.

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Human mesenchymal stem cells shift CD8+ T cells towards a suppressive phenotype by inducing tolerogenic monocytes

Advance Online Publication July 5, 2012 doi: 10.1242/?jcs.108860 Irit Hof-Nahor, Lucy Leshansky, Shoham Shivtiel, Liron Eldor, Daniel Aberdam, Joseph Itskovitz-Eldor and Sonia Berrih-Aknin?Corresponding author: Sonia Berrih-Aknin, UMRS 974 - UPMC Univ. Paris 6/U974 - Inserm/UMR7215 - CNRS, Hôpital La Pitié Salpêtrière, 105 Bd de l'hôpital, 75013 Paris, sonia.berrih-aknin{at}upmc.fr, Tel: +33 1 40 77 81 28; Fax: +33 1 40 77 81 29 The mechanisms underlying the immunomodulatory effects of mesenchymal stem cells (MSCs) have been essentially studied in conditions of strong T cell activation that represents extreme situation and induces rapid death of activated lymphocytes. The objective of this study was to investigate these mechanisms in absence of additional polyclonal activation. In cocultures of peripheral mononuclear blood cells with hMSC, we observed a striking decreased expression of CD8 level on CD8+ cells, together with decreased CD28 and CD44 expression and impaired IFN-gamma and Granzyme B production. This effect was specific to hMSCs, since it was not observed with several other cell lines. Down-regulation of CD8 expression required CD14+ monocytes in direct contact with the CD8+ cells, while the effects of hMSCs on the CD14+ cells were essentially mediated by soluble factors. The CD14+ monocytes exhibited a tolerogenic pattern when co-cultured with hMSCs, with a clear decrease in CD80 and CD86 co-stimulatory molecules, and an increase in the inhibitory receptors ILT-3 and ILT-4. MSC-preconditioned CD8+ cells had similar effects on monocytes and were able to inhibit lymphocyte proliferation. Injection of human MSCs in humanized NSG mice showed similar trends, in particular decreased CD44 and CD28 on human immune cells. Altogether, our study demonstrates a new immunomodulation mechanism of action of hMSCs through the modulation of CD8+ cells towards a non-cytotoxic/suppressive phenotype. This mechanism of action has to be taken into account in clinical trials, where it should be beneficial in grafts and autoimmune diseases, but potentially detrimental in malignant diseases.

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