dictyNews Electronic Edition Volume 42, number 22 September 23, 2016 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 ========= Autophagy in Dictyostelium: mechanisms, regulation and disease in a simple biomedical model Ana Mesquita,1,7 Elena Cardenal-Muñoz,2 Eunice Dominguez,1,5 Sandra Muñoz-Braceras,1 Beatriz Nuñez-Corcuera,1 Ben A. Phillips,3 Luis C. Tábara,1 Qiuhong Xiong,4 Roberto Coria,5 Ludwig Eichinger,4 Pierre Golstein,6 Jason S. King,3 Thierry Soldati,2 Olivier Vincent1 and Ricardo Escalante1 Autophagy, in press Autophagy is a fast-moving field with an enormous impact on human health and disease. Understanding the complexity of the mechanism and regulation of this process often benefits from the use of simple experimental models such as the social amoeba Dictyostelium discoideum. Since the publication of the first review describing the potential of D. discoideum in autophagy, significant advances have been made that demonstrate both the experimental advantages and interest in using this model. Since our previous review, research in D. discoideum has shed light on the mechanisms that regulate autophagosome formation and contributed significantly to the study of autophagy-related pathologies. Here, we review these advances, as well as the current techniques to monitor autophagy in D. discoideum. The comprehensive bioinformatics search of autophagic proteins that was a substantial part of the previous review has not been revisited here except for those aspects that challenged previous predictions such as the composition of the Atg1 complex. In recent years our understanding of, and ability to investigate autophagy in D. discoideum has evolved significantly and will surely enable and accelerate future research using this model. submitted by: Ricardo Escalante [rescalante@iib.uam.es] ——————————————————————————————————————— Analysis of Relevant Parameters for Autophagic Flux Using HeLa Cells Expressing EGFP-LC3. Sandra Muñoz-Braceras and Ricardo Escalante Methods Mol Biol. 2016;1449:313-29 Macroautophagy (called just autophagy hereafter) is an intracellular degradation machinery essential for cell survival under stress conditions and for the maintenance of cellular homeostasis. The hallmark of autophagy is the formation of double membrane vesicles that engulf cytoplasmic material. These vesicles, called autophagosomes, mature by fusion with endosomes and lysosomes that allows the degradation of the cargo. Autophagy is a dynamic process regulated at multiple steps. Assessment of autophagy is not trivial because the number autophagosomes might not necessarily reflect the real level of autophagic degradation, the so-called autophagic flux. Here, we describe an optimised protocol for the analysis of relevant parameters of autophagic flux using HeLa cells stably expressing EGFP-LC3. These cells are a convenient tool to determine the influence of the downregulation or overexpression of specific proteins in the autophagic flux as well as the analysis of autophagy-modulating compounds. Western blot analysis of relevant parameters, such as the levels of EGFP-LC3, free EGFP generated by autophagic degradation and endogenous LC3·I-II are analyzed in the presence and absence of the autophagic inhibitor chloroquine. submitted by: Ricardo Escalante [rescalante@iib.uam.es] ——————————————————————————————————————— Glutathione S-transferase 4 is a putative DIF-binding protein that regulates the size of fruiting bodies in Dictyostelium discoideum Hidekazu Kuwayama*, Haruhisa Kikuchi**, Yoshiteru Oshima**, and Yuzuru Kubohara*** *Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba 305-8572, Japan **Laboratory of Natural Product Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan ***Department of Molecular and Cellular Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan ***Laboratory of Health Life Science, Graduate School of Health and S ports Science, Juntendo University, Inzai, Chiba 270-1695, Japan Biochem. Biophys. Rep., in press In the development of the cellular slime mold Dictyostelium discoideum, two chlorinated compounds, the differentiation-inducing factors DIF-1 and DIF-2, play important roles in the regulation of both cell differentiation and chemotactic cell movement. However, the receptors of DIFs and the components of DIF signaling systems have not previously been elucidated. To identify the receptors for DIF-1 and DIF-2, we here performed DIF-conjugated affinity gel chromatography and liquid chromatography–tandem mass spectrometry and identified the glutathione S-transferase GST4 as a major DIF-binding protein. Knockout and overexpression mutants of gst4 (gst4– and gst4OE, respectively) formed fruiting bodies, but the fruiting bodies of gst4– cells were smaller than those of wild-type Ax2 cells, and those of gst4OE cells were larger than those of Ax2 cells. Both chemotaxis regulation and in vitro stalk cell formation by DIFs in the gst4 mutants were similar to those of Ax2 cells. These results suggest that GST4 is a DIF-binding protein that regulates the sizes of cell aggregates and fruiting bodies in D. discoideum. submitted by: Yuzuru Kubohara [ykuboha@juntendo.ac.jp] ——————————————————————————————————————— Acanthamoeba and Dictyostelium use different foraging strategies Nick A. Kuburich#, Nirakar Adhikari#, and Jeffrey A. Hadwiger* Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK 74078-3020 # These authors contributed equally to this work Protist, in press Amoeba often use cell movement as a mechanism to find food, such as bacteria, in their environment. The chemotactic movement of the soil amoeba Dictyostelium to folate or other pterin compounds released by bacteria is a well-documented foraging mechanism. Acanthamoeba can also feed on bacteria but relatively little is known about the mechanism(s) by which this amoeba locates bacteria. Acanthamoeba movement in the presence of folate or bacteria was analyzed in above agar assays and compared to that observed for Dictyostelium. The overall mobility of Acanthamoeba was robust like that of Dictyostelium but Acanthamoeba did not display a chemotactic response to folate. In the presence of bacteria, Acanthamoeba only showed a marginal bias in directed movement whereas Dictyostelium displayed a strong chemotactic response. A comparison of genomes revealed that Acanthamoeba and Dictyostelium share some similarities in G protein signaling components but that specific G proteins used in Dictyostelium chemotactic responses were not present in current Acanthamoeba genome sequence data. The results of this study suggest that Acanthamoeba does not use chemotaxis as the primary mechanism to find bacterial food sources and that the chemotactic responses of Dictyostelium to bacteria may have co-evolved with chemotactic responses that facilitate multicellular development. submitted by: Jeff Hadwiger [jeff.hadwiger@okstate.edu] ——————————————————————————————————————— WASH drives early recycling from macropinosomes and phagosomes to maintain surface phagocytic receptors Catherine M. Buckley1,2*, Navin Gopaldass3,4*, Cristina Bosmani3, Simon A. Johnston2,5, Thierry Soldati3 and Robert H. Insall6# and Jason S. King1,2# Proc. Nat. Acad. Sciences USA, in press http://www.pnas.org/content/early/2016/09/15/1524532113.full Macropinocytosis is an ancient mechanism that allows cells to harvest nutrients from extracellular media, which also allows immune cells to sample antigens from their surroundings. During macropinosome formation, bulk plasma membrane is internalized with all its integral proteins. It is vital for cells to salvage these proteins before degradation, but the mechanisms for sorting them are not known. Here we describe the evolutionarily conserved recruitment of the WASH (WASP and SCAR homolog) complex to both macropinosomes and phagosomes within a minute of internalization. Using Dictyostelium, we demonstrate that WASH drives protein sorting and recycling from macropinosomes and is thus essential to maintain surface receptor levels and sustain phagocytosis. WASH functionally interacts with the retorter complex at both early and late phases of macropinosome maturation, but mediates recycling via retromer-dependent and -independent pathways. WASH mutants consequently have decreased membrane levels of integrins and other surface proteins. This study reveals an important pathway enabling cells to sustain macropinocytosis without bulk degradation of plasma membrane components. submitted by: Jason King [jason.king@sheffield.ac.uk] ============================================================== [End dictyNews, volume 42, number 22]