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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Rab11 regulates exocytosis of recycling vesicles at the plasma membrane

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.102913 Senye Takahashi, Keiji Kubo, Satoshi Waguri, Atsuko Yabashi, Hye-Won Shin, Yohei Katoh and Kazuhisa Nakayama*?*Corresponding author: Kazuhisa Nakayama, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan; Phone: +81-75-753-4527; Fax: +81-75-753-4557; E-mail: kazunaka{at}pharm.kyoto-u.ac.jp. Rab11 is known to associate primarily with perinuclear recycling endosomes and regulate recycling of endocytosed proteins. However, the recycling step in which Rab11 participates remains unknown. We here show that, in addition to causing tubulation of recycling endosomes, Rab11 depletion gives rise to accumulation of recycling carriers containing endocytosed transferrin and transferrin receptor beneath the plasma membrane. We also show that the carriers are transported from perinuclear recycling endosomes to the cell periphery along microtubules. Total internal reflection fluorescence microscopy of cells expressing EGFP-tagged transferrin receptor revealed that Rab11 depletion inhibits tethering and fusion of recycling carriers to the plasma membrane. Depletion of a component of the exocyst tethering complex, Sec15 or Exo70, the former which interacts with Rab11, leads to essentially the same phenotypes as those of Rab11 depletion. Thus, in addition to its role in recycling processes at perinuclear recycling endosomes, Rab11 is transported along microtubules to the cell periphery through association with recycling carriers, and directly regulates vesicle exocytosis at the plasma membrane in concert with the exocyst.

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APPL1 regulates basal NF-{kappa}B activity by stabilizing NIK

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.105171 APPL1 is a multifunctional adaptor protein that binds membrane receptors, signaling proteins and nuclear factors, thereby acting in endosomal trafficking and in different signaling pathways. Here we uncover a novel role of APPL1 as a positive regulator of transcriptional activity of NF-?B under basal but not TNFa-stimulated conditions. APPL1 was found to directly interact with TRAF2, an adaptor protein known to activate the canonical NF-?B signaling. APPL1 synergized with TRAF2 to induce NF-?B activation and both proteins were necessary for this process by functioning upstream of the IKK complex. Although TRAF2 was not detectable on APPL endosomes, endosomal recruitment of APPL1 was required for its function in the NF-?B pathway. Importantly, in the canonical pathway APPL1 appeared to regulate the proper spatial distribution of p65 in the absence of cytokine stimulation, since its overexpression enhanced and its depletion reduced the nuclear accumulation of p65. Analyzing the patterns of gene transcription upon APPL1 overproduction or depletion we found altered expression of NF-?B target genes encoding cytokines. At the molecular level, overexpressed APPL1 markedly increased the level of NIK, the key component of the noncanonical NF-?B pathway, by reducing its association with the degradative complex containing TRAF2, TRAF3 and cIAP1. In turn, high levels of NIK triggered nuclear translocation of p65. Collectively, we propose that APPL1 regulates basal NF-?B activity by modulating the stability of NIK, which affects the activation of p65. This places APPL1 as a novel link between the canonical and noncanonical machineries of NF-?B activation.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted non-commercial use, distribution and reproduction in any medium provided that the original work is properly cited and all further distributions of the work or adaptation are subject to the same Creative Commons License terms.

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Toxofilin upregulates the host cortical actin cytoskeleton dynamics facilitating Toxoplasma invasion

Advance Online Publication May 28, 2012 doi: 10.1242/?jcs.103648 Violaine Delorme-Walker, Marie Abrivard, Vanessa Lagal, Karen Anderson, Audrey Perazzi, Virginie Gonzalez, Christopher Page, Juliette Chauvet, Wendy Ochoa, Niels Volkmann, Dorit Hanein and Isabelle Tardieux*?5 Correspondence should be addressed to I. T. (email : isabelle.tardieux{at}inserm.fr) Toxoplasma, a human pathogen and a model apicomplexan parasite, actively and rapidly invades host cells. To initiate invasion, the parasite induces the formation of a parasite-cell junction, progressively propels itself through the junction inside a newly formed vacuole that encloses the entering parasite. Litle is known how a few micron-large diameter parasite overcome the host cell cortical actin barrier to support these remarkably rapid process of internalization (< few seconds). Correlative light and electron microscopy in conjunction with electron tomography and three-dimensional image analysis indicate that toxofilin an actin-binding protein, secreted by invading parasites correlates with localized sites of disassembly of the host cell actin meshwork. Moreover, quantitative fluorescence speckle microscopy in cells expressing toxofilin indicates that toxofilin regulates actin filament disassembly and turnover. Furthermore, Toxoplasma tachyzoites lacking toxofilin, are impaired in cortical actin disassembly and exhibit delayed invasion kinetics. We propose that toxofilin locally upregulates actin turnover thus increasing depolymerization events at the site of entry that, in turn loosens the local host cell actin meshwork, facilitating parasite internalization and vacuole folding.

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Autophagy and cancer - issues we need to digest

Advance Online Publication May 28, 2012 doi: 10.1242/?jcs.093708 May 15, 2012 J Cell Sci 125, 2349-2358. Emma Y. Liu and Kevin M. Ryan*

Tumour Cell Death Laboratory, Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Glasgow G61 1BD, UK ?* Author for correspondence (k.ryan{at}beatson.gla.ac.uk) Autophagy is an evolutionarily conserved catabolic pathway that has multiple roles in carcinogenesis and cancer therapy. It can inhibit the initiation of tumorigenesis through limiting cytoplasmic damage, genomic instability and inflammation, and the loss of certain autophagy genes can lead to cancer. Conversely, autophagy can also assist cells in dealing with stressful metabolic environments, thereby promoting cancer cell survival. In fact, some cancers rely on autophagy to survive and progress. Furthermore, tumour cells can exploit autophagy to cope with the cytotoxicity of certain anticancer drugs. By contrast, it appears that certain therapeutics require autophagy for the effective killing of cancer cells. Despite these dichotomies, it is clear that autophagy has an important, if complex, role in cancer. This is further exemplified by the fact that autophagy is connected with major cancer networks, including those driven by p53, mammalian target of rapamycin (mTOR), RAS and glutamine metabolism. In this Commentary, we highlight recent advances in our understanding of the role that autophagy has in cancer and discuss current strategies for targeting autophagy for therapeutic gain.

Key words This article is part of a Minifocus on Autophagy. For further reading, please see related articles: ‘Ubiquitin-like proteins and autophagy at a glance’ by Tomer Shpilka et al. (J. Cell Sci. 125, 2343-2348) and ‘Autophagy and cell growth – the yin and yang of nutrient responses’ by Thomas Neufeld (J. Cell Sci. 125, 2359-2368).

Funding

Work in the Tumour Cell Death Laboratory is supported by Cancer Research UK and the Association for International Cancer Research.

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SNX9, SNX18 and SNX33 are required for progression through and completion of mitosis

Advance Online Publication June 20, 2012 doi: 10.1242/?jcs.105981 Maggie P.C. Ma and Megan Chircop?Address correspondence to:
Dr Megan Chircop, Children's Medical Research Institute, The University of Sydney, Locked Bag 23, Wentworthville, NSW, 2145, Australia, Phone: +61-2-9687 2800, Fax: +61-2-9687 2120, Email: mchircop{at}cmri.org.auMitosis involves considerable membrane remodelling and vesicular trafficking to generate two independent cells. Consequently, endocytosis and endocytic proteins are required for efficient mitotic progression and completion. Several endocytic proteins also participate in mitosis in an endocytosis-independent manner. Here, we report that the sorting nexin (SNX) 9 subfamily members – SNX9, SNX18 and SNX33 – are required for progression and completion of mitosis. Depletion of any one of these proteins using siRNA induces multinucleation, an indicator of cytokinesis failure, as well as an accumulation of cytokinetic cells. Time-lapse microscopy on siRNA-treated cells reveals a role for SNX9 subfamily members in progression through the ingression and abscission stages of cytokinesis. Depletion of these three proteins disrupted MRLCS19 localization during ingression and recruitment of Rab11-positive recycling endosomes to the intracellular bridge between nascent daughter cells. SNX9 depletion also disrupted the localization of Golgi during cytokinesis. Endocytosis of transferrin (Tfn) was blocked during cytokinesis by depletion of the SNX9 subfamily members, suggesting that these proteins participate in cytokinesis in an endocytosis-dependent manner. In contrast, depletion of SNX9 did not block Tfn uptake during metaphase but did delay chromosome alignment and segregation, suggesting that SNX9 plays an additional non-endocytic role at early mitotic stages. We conclude that SNX9 subfamily members are required for mitosis through both endocytosis-dependent and -independent processes.

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