dictyNews Electronic Edition Volume 33, number 15 December 4, 2009 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= Tug-of-war between Dissimilar Teams of Microtubule Motors Regulates Transport and Fission of Endosomes Soppina V, Rai AK, Ramaiya AJ, Barak P, Mallik R. Proceedings of the National Academy of Sciences,106, 19381 (2009) Intracellular transport is interspersed with frequent reversals in direction due to the presence of opposing kinesin and dynein motors on organelles that are carried as cargo. The cause and the mechanism of reversals are unknown, but are a key to understanding how cargos are delivered in a regulated manner to specific cellular locations. Unlike established single-motor biophysical assays,this problem requires understanding of the cooperative behavior of multiple interacting motors. Here we present measurements inside live Dictyostelium cells, in a cell extract and with purified motors to quantify such an ensemble function of motors. We show through precise motion analysis that reversals during endosome motion are caused by a tug-of-war between kinesin and dynein. Further, we use a combination of optical trap-based force measurements and Monte Carlo simulations to make the surprising discovery that endosome transport uses many (approximately four to eight) weak and detachment-prone dyneins in a tug-of-war against a single strong and tenacious kinesin. We elucidate how this clever choice of dissimilar motors and motor teams achieves net transport together with endosome fission, both of which are important in controlling the balance of endocytic sorting. To the best of our knowledge, this is a unique demonstration that dynein and kinesin function differently at the molecular level inside cells and of how this difference is used in a specific cellular process, namely endosome biogenesis. Our work may provide a platform to understand intracellular transport of a variety of organelles in terms of measurable quantities. Submitted by Roop Mallik [roop@tifr.res.in] -------------------------------------------------------------------------------- Myosin II Is Essential for the Spatiotemporal Organization of Traction Forces during Cell Motility Ruedi Meili(1),* Baldomero Alonso-Latorre (1)+, Juan C. del Álamo+, Richard A. Firtel(2),* and Juan C. Lasheras(2)+ *Section of Cell and Developmental Biology, Division of Biological Sciences, +Department of Mechanical and Aerospace Engineering, and ||Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093 1- co-first authors 2- co-senior authors Mol Bio Cell, in press Amoeboid motility requires spatiotemporal coordination of biochemical pathways regulating force generation and consists of the quasi-periodic repetition of a motility cycle driven by actin polymerization and actomyosin contraction. Using new analytical tools and statistical methods, we provide, for the first time, a statistically-significant quantification of the spatial distribution of the traction forces generated at each phase of the cycle (protrusion-contraction-retraction-relaxation). We show that cells are constantly under tensional stress and that wild-type cells develop two opposing "pole" forces pulling the front and back toward the center whose strength is modulated up and down periodically in each cycle. We demonstrate that MyoII cross-linking and motor functions have different roles in controlling the spatiotemporal distribution of traction forces, the changes in cell shape, and the duration of all the phases. We show that the time required to complete each phase is dramatically increased in cells with altered MyoII-motor-function, demonstrating that it is required not only for contraction but also for protrusion. Concomitant loss of MyoII-actin-cross-linking leads to a force redistribution throughout the cell perimeter pulling inwards toward the center. However, it does not reduce significantly the magnitude of the traction forces, uncovering a nonMyoII-mediated mechanism for the contractility of the cell. Submitted by Rick Firtel [rafirtel@ucsd.edu] -------------------------------------------------------------------------------- The WD-Repeat Domain of Dictyostelium Myosin Heavy Chain Kinase C Functions in Both Substrate Targeting and Cellular Localization Atiya Franklin, Linzi Hyatt, Alyssa Chowdhury, Paul A. Steimle J Eukaryotic Cell, in press In nonmuscle cells, contraction-dependent processes such as cytokinesis and cell migration rely on the proper assembly and localization of myosin II bipolar filaments; however, the signalling pathways regulating these processes are poorly understood.  Studies in Dictyostelium discoideum, and recently in mammalian cells, have demonstrated that myosin II filament disassembly is driven by phosphorylation of the tail¡region of the myosin II heavy chain (MHC), preventing myosin-mediated contraction.   In Dictyostelium, MHC phosphorylation is catalyzed by three MHC kinases (MHCK-A, -B, and -C) that share homologous alpha-kinase and WD-repeat domains. As a means of understanding how each of the MHCKs functions in the cell, we have focused our studies on the structure-function relationships defining the activity of MHCK-C.  Our cellular and biochemical studies revealed that the WD-repeat domain of MHCK-C is required for targeting of the kinase to phosphorylate MHC.  Localization studies demonstrated that MHCK-C translocation to the cell cortex occurs in the presence of chemoattractant, with a timing that lags behind myosin II cortical enrichment.  We also show that the MHCK-C WD-repeat domain is unique among its MHCK-A and MHCK-B counterparts since it is both necessary and sufficient for proper localization of MHCK-C in the cell.   The posterior localization of this domain in chemotaxing cells occurs with a periodicity similar to that exhibited by myosin II.  The unique functions that we have identified for the MHCK-C WD-repeat domain allow for the possibility that the seemingly redundant MHCKs in Dictyostelium could be regulated by a single signal but to perform distinct roles in regulating myosin II activity in the cell. Submitted by Paul Steimle [p_steiml@uncg.edu] ============================================================== [End dictyNews, volume 33, number 15]