dictyNews Electronic Edition Volume 40, number 4 February 7, 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 ========= Both contractile axial and lateral traction force dynamics drive amoeboid cell motility Effie Bastounis, Ruedi Meili, Begoña Alvarez-González, Joshua Francois, Juan C. del Álamo, Richard A. Firtel*, and Juan C. Lasheras* (*co-senior authors). (2013) J. Cell Biol., in press Chemotaxing Dictyostelium cells adapt their morphology and migration speed in response to intrinsic and extrinsic cues. Using Fourier Traction Force Microscopy, we measured the spatiotemporal evolution of shape and traction stresses and constructed traction tension kymographs to analyze cell motility as a function of the dynamics of the cells’ mechanically active traction adhesions. We show that wild-type cells migrate in a step-wise fashion mainly forming stationary traction adhesions along their anterior-posterior axes and exerting strong contractile axial forces. We demonstrate that lateral forces are also important for motility, especially for migration on highly adhesive substrates. Analysis of two mutant strains lacking distinct actin crosslinkers (mhcA- and abp120- cells) on normal and highly adhesive substrates supports a key role for lateral contractions in amoeboid cell motility, while the differences in their traction adhesion dynamics suggest these two strains use distinct mechanisms to achieve migration. Finally, we provide evidence that the above patterns of migration may be conserved in mammalian amoeboid cells. Rick Firtel [rafirtel@ucsd.edu] --------------------------------------------------------------------------- Visualization of the Actin Cytoskeleton: Different F-Actin-Binding Probes Tell Different Stories Michael G. Lemieux, Dani Janzen, Rander Hwang, Jeannette Roldan, Irene Jarchum, and David A. Knecht* *Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut 06269 Cytoskeleton, In Press 2013 Dec 17. doi: 10.1002/cm.21160. [Epub ahead of print] PMID: 24347465 The actin cytoskeleton is necessary for cell viability and plays crucial roles in cell motility, endocytosis, growth, and cytokinesis. Hence visualization of dynamic changes in F-actin distribution in vivo is of central importance in cell biology. This has been accomplished by the development of fluorescent protein fusions to actin itself or to various actin-binding proteins, actin cross-linking proteins, and their respective actin-binding domains (ABD’s). Although these protein fusions have been shown to bind to F-actin in vivo, we show that the fluorescent protein used for visualization changes the subset of F-actin labeled by an F-actin ABD probe. Further, different amino acid linkers between the fluorescent protein and ABD induced a similar change in localization. Although different linkers and fluorescent proteins can alter the subset of actin bound by a particular ABD, in most cases, the fusion protein did not label all of a cell’s F-actin all of the time. Even LimEÄcoil and GFP-actin, which have been used extensively for cytoskeletal visualization, were highly variable in the subsets of actin that they labeled. Lifeact, conversely, clearly labeled cortical F-actin as well as F-actin in the anterior pseudopods of motile cells and in macropinocytotic cups. We conclude that Lifeact most accurately labels F-actin and is the best currently available probe for visualization of dynamic changes in F-actin networks. Michael Lemieux [michael.lemieux@uconn.edu] -------------------------------------------------------------------------- Linh Hai Vu, Tsuyoshi Araki, Jianbo Na, Christoph S. Clemen, Jeffrey G. Williams and Ludwig Eichinger PLoS ONE, accepted Abstract 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] ============================================================== [End dictyNews, volume 40, number 4]