dictyNews Electronic Edition Volume 40, number 5 February 14, 2014 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 ========= NNucleocytoplasmic Protein Translocation during Mitosis in the Social Amoebozoan Dictyostelium discoideum Danton H. O'Day a,b* and Aldona Budniaka a a: Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road N., Mississauga, Ontario, Canada, L5L 1C6 b: Department of Cell and Systems Biology, University of Toronto, 25 Harbord St., Toronto, Ontario, Canada, M5S 3G5 Biological Reviews, in press Mitosis is a fundamental and essential life process. It underlies the duplication and survival of all cells and, as a result, all eukaryotic organisms. Since uncontrolled mitosis is a dreaded component of many cancers, a full understanding of the process is critical. Evolution has led to the existence of three types of mitosis: closed, open, and semi-open. The significance of these different mitotic species, how they can lead to a full understanding of the critical events that underlie the asexual duplication of all cells, and how they may generate new insights into controlling unregulated cell division remains to be determined. The eukaryotic microbe Dictyostelium discoideum has proven to be a valuable biomedical model organism. While it appears to utilize closed mitosis, a review of the literature suggests that it possesses a form of mitosis that lies in the middle between truly open and fully closed mitosisÑit utilizes a form of semi-open mitosis. Here, the nucleocytoplasmic translocation patterns of the proteins that have been studied during mitosis in the social amoebozoan Dictyostelium are detailed followed by a discussion of how some of them provide support for the hypothesis of semi-open mitosis. Submitted by Danton H. O'Day [danton.oday@utoronto.ca] --------------------------------------------------------------------------- Identification of the protein kinases Pyk3 and Phg2 as regulators of the STATc-mediated response to hyperosmolarity Linh Hai Vu, Tsuyoshi Araki, Jianbo Na, Christoph S. Clemen, Jeffrey G.Williams and Ludwig Eichinger PLoS ONE, accepted Cellular adaptation to changes in environmental osmolarity is crucial for cell survival. In Dictyostelium, STATc is a key regulator of the transcriptional response to hyperosmotic stress. Its phosphorylation and consequent activation is controlled by two signaling branches, one cGMP- and the other Ca2+-dependent, of which many signaling components have yet to be identified. The STATc stress signalling pathway feeds back on itself by upregulating the expression of STATc and STATc-regulated genes. Based on microarray studies we chose two tyrosine-kinase like proteins, Pyk3 and Phg2, as possible modulators of STATc phosphorylation and generated single and double knock-out mutants to them. Transcriptional regulation of STATc and STATc dependent genes was disturbed in pyk3-, phg2-, and pyk3-/phg2- cells. The absence of Pyk3 and/or Phg2 resulted in diminished or completely abolished increased transcription of STATc dependent genes in response to sorbitol, 8-Br-cGMP and the Ca2+ liberator BHQ. Also, phospho-STATc levels were significantly reduced in pyk3- and phg2- cells and even further decreased in pyk3-/phg2- cells. The reduced phosphorylation was mirrored by a significant delay in nuclear translocation of GFP-STATc. The protein tyrosine phosphatase 3 (PTP3), which dephosphorylates and inhibits STATc, is inhibited by stress-induced phosphorylation on S448 and S747. Use of phosphoserine specific antibodies showed that Phg2 but not Pyk3 is involved in the phosphorylation of PTP3 on S747. In pull-down assays Phg2 and PTP3 interact directly, suggesting that Phg2 phosphorylates PTP3 on S747 in vivo. Phosphorylation of S448 was unchanged in phg2- cells. We show that Phg2 and an, as yet unknown, S448 protein kinase are responsible for PTP3 phosphorylation and hence its inhibition, and that Pyk3 is involved in the regulation of STATc by either directly or indirectly activating it. Our results add further complexities to the regulation of STATc, which presumably ensure its optimal activation in response to different environmental cues. Submitted by Ludwig Eichinger [ludwig.eichinger@uni-koeln.de] --------------------------------------------------------------------------- Excitable Signal Transduction Induces Both Spontaneous and Directional Cell Asymmetries in the Phosphatidylinositol Lipid Signaling System for Eukaryotic Chemotaxis Masatoshi Nishikawa(1), Marcel Hšrning(1), Masahiro Ueda(2), and Tatsuo Shibata(1) (1)Laboratory for Physical Biology, RIKEN Center for Developmental Biology, (2) Laboratory of Single Molecule Biology, Graduate School of Science, Osaka University Biophysical Journal, in press 106: 723Ð734, http://dx.doi.org/10.1016/j.bpj.2013.12.023 Intracellular asymmetry in the signaling network works as a compass to navigate eukaryotic chemotaxis in response to guidance cues. Although the compass variable can be derived from a self-organization dynamics, such as excit- ability, the responsible mechanism remains to be clarified. Here, we analyzed the spatiotemporal dynamics of the phosphatidy- linositol 3,4,5-trisphosphate (PtdInsP3) pathway, which is crucial for chemotaxis. We show that spontaneous activation of PtdInsP3-enriched domains is generated by an intrinsic excitable system. Formation of the same signal domain could be trig- gered by various perturbations, such as short impulse perturbations that triggered the activation of intrinsic dynamics to form signal domains. We also observed the refractory behavior exhibited in typical excitable systems. We show that the chemotactic response of PtdInsP3 involves biasing the spontaneous excitation to orient the activation site toward the chemoattractant. Thus, this biased excitability embodies the compass variable that is responsible for both random cell migration and biased random walk. Our finding may explain how cells achieve high sensitivity to and robust coordination of the downstream activation that allows chemotactic behavior in the noisy environment outside and inside the cells. Submitted by Tatsuo Shibata [tatsuoshibata@cdb.riken.jp]] --------------------------------------------------------------------------- Reversible Membrane Pearling in Live Cells Upon Destruction of the Actin Cortex Doris Heinrich, Mary Ecke, Marion Jasnin, Ulrike Engel and GŸnther Gerisch Biophysical Journal, in press http://dx.doi.org/10.1016/j.bpj.2013.12.054 Membrane pearling in live cells is observed when the plasma membrane is depleted of its support, the cortical actin network. Upon efficient depolymerization of actin, pearls of variable size are formed, which are connected by nanotubes of about 40 nm diameter. We show that formation of the membrane tubes and their transition into chains of pearls do not require external tension, and that they neither depend on microtubule-based molecular motors nor pressure generated by myosin-II. Pearling thus differs from blebbing. The pearling state is stable as long as actin is prevented from polymerizing. When polymerization is restored, the pearls are retracted into the cell, indicating continuity of the membrane. Our data suggest that the alternation of pearls and strings is an energetically favored state of the unsupported plasma membrane, and that one of the functions of the actin cortex is to prevent the membrane from spontaneously assuming this configuration. Submitted by GŸnther Gerisch [gerisch@biochem.mpg.de] --------------------------------------------------------------------------- Bleb driven chemotaxis of Dictyostelium cells Evgeny Zatulovskiy1, Richard Tyson2, Till Bretschneider2 and Robert R. Kay1 J Cell Biol., in press Blebs and F-actin-driven pseudopods are alternative ways of extending the leading edge of migrating cells. We show that Dictyostelium cells switch from using predominantly pseudopods to blebs when migrating under agarose overlays of increasing stiffness. Blebs expand faster than pseudopods leaving behind F-actin scars, but are less persistent. Blebbing cells are strongly chemotactic to cyclic-AMP, producing nearly all of their blebs up- gradient. When cells re-orientate to a needle releasing cyclic-AMP, they stereotypically produce first microspikes, then blebs and pseudopods only later. Genetically, blebbing requires myosin-II and increases when actin polymerization or cortical function are impaired. Cyclic-AMP induces transient blebbing independently of much of the known chemotactic signal transduction machinery, but requiring PI3-kinase and downstream PH-domain proteins, CRAC and PhdA. Impairment of this PI3-kinase pathway results in slow movement under agarose and cells that produce few blebs, though actin polymerization appears unaffected. We propose that mechanical resistance induces bleb-driven movement in Dictyostelium, which is chemotactic and controlled through PI3-kinase. Submitted by Rob Kay [rrk@mrc-lmb.cam.ac.uk] ============================================================== [End dictyNews, volume 40, number 5]