dictyNews Electronic Edition Volume 38, number 3 January 27, 2012 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 ========= Functional Characterization of a Novel Aquaporin from Dictyostelium discoideum Amoebae Implies a Unique Gating Mechanism Julia von Buelow, Annika Mueller-Lucks, Lei Kai, Frank Bernhard, Eric Beitz J. Biol. Chem., in press The social amoeba Dictyostelium discoideum is a widely used model organism for studying basic functions of protozoan and metazoan cells, such as osmoregulation and cell motility. There is evidence from other species that cellular water channels, aquaporins (AQP), are central to both processes. Yet, data on D. discoideum AQPs is almost absent. Despite cloning of two putative D. discoideum AQPs, WacA and AqpA, water permeability has not been shown. Further, WacA and AqpA are expressed at the late multicellular stage and in spores but not in amoebae. We cloned a novel AQP, AqpB, from amoeboidal D. discoideum cells. Wildtype AqpB was impermeable to water, glycerol, and urea when expressed in Xenopus laevis oocytes. Neither stepwise truncation of the N-terminus nor selected point mutations activated the water channel. However, mutational truncation by 12 amino acids of an extraordinary long intracellular loop induced water permeability of AqpB hinting at a novel gating mechanism. This AqpB mutant was inhibited by mercuric chloride confirming the presence of a cysteine residue in the selectivity filter as predicted by our structure model. We detected AqpB by Western blot in a glycosylated and a non-glycosylated form throughout all developmental stages. When expressed in D. discoideum amoebae, AqpB-GFP fusion constructs localized to vacuolar structures, to the plasma membrane, and to lamellipodia-like membrane protrusions. We conclude, that the localization pattern in conjunction with channel gating may be indicative of AqpB functions in osmoregulation as well as cell motility of D. discoideum. Submitted by Eric Beitz [ebeitz@pharmazie.uni-kiel.de] -------------------------------------------------------------------------------------- Evidence of an evolutionarily conserved LMBR1 domain-containing protein that associates with endocytic cups and plays a role in cell migration in Dictyostelium. Jessica S Kelsey, Nathan M Fastman and Daphne D Blumberg Eukaryotic Cell, in press The ampA gene plays a role in Dictyostelium discoideum cell migration. Loss of ampA function results in reduced ability of growing cells to migrate to folic acid and results in small plaques on bacterial lawns, while overexpression of AmpA results in a rapid migration phenotype and correspondingly larger plaques than seen with wild type cells. To help understand how the ampA gene functions, second site suppressors were created by REMI mutageneis. These mutants were selected for their ability to reduce the large plaque size of the AmpA overexpresser strain. The lmbd2B gene was identified as a suppressor of an AmpA overexpressing strain. The lmbd2B gene belongs to the evolutionarily conserved LMBR1 protein family, some of whose known members are endocytic receptors associated with human diseases such as anemia. In order to understand lmbd2B function, mRFP fusion proteins were created and lmbd2B knockout cell lines were established. Our findings indicate that the LMBD2B protein is found associated with endocytic cups. It colocalizes with proteins that play key roles in endocytic events and is localized to ruffles on the dorsal surface of growing cells. Vegetative lmbd2B null cells display defects in cell migration. These cells have difficulty sensing the chemoattractant folic acid as indicated by a decrease in their chemotactic index. Lmbd2B null cells also appear to have difficulty establishing a front/back orientation to facilitate migration. A role for lmbd2B in development is also suggested. Our research gives insight into the function of a previously uncharacterized branch of the LMBR1 family of proteins. We provide evidence of an LMBR1 family plasma membrane protein that associates with endocytic cups and plays a role in chemotaxis. Submitted by Daphne D.Blumberg [blumberg@umbc.edu] -------------------------------------------------------------------------------------- eIF2alpha Kinases Regulate Development Through the BzpR Transcription Factor in Dictyostelium discoideum. Charles K. Singleton, Yanhua Xiong, Janet H. Kirsten, and Kelsey P. Pendleton. Department of Biological Sciences, Vanderbilt University, VU Station B 351634, Nashville TN 37235 PloS ONE, in press Background. A major mechanism of translational regulation in response to a variety of stresses is mediated by phosphorylation of eIF2alpha to reduce delivery of initiator tRNAs to scanning ribosomes. For some mRNAs, often encoding a bZIP transcription factor, eIF2alpha phosphorylation leads to enhanced translation due to delayed reinitiation at upstream open reading frames. Dictyostelium cells possess at least three eIF2alpha kinases that regulate various portions of the starvation induced developmental program. Cells possessing an eIF2alpha that cannot be phosphorylated (BS167) show abnormalities in growth and development. We sought to identify a bZIP protein in Dictyostelium whose production is controlled by the eIF2alpha regulatory system. Principle Findings. Cells disrupted in the bzpR gene had similar developmental defects as BS167 cells, including small entities, stalk defects, and reduced spore viability. beta-galactosidase production was used to examine translation from mRNA containing the bzpR 5Õ UTR. While protein production was readily apparent and regulated temporally and spatially in wild type cells, essentially no beta-galactosidase was produced in developing BS167 cells even though the lacZ mRNA levels were the same as those in wild type cells. Also, no protein production was observed in strains lacking IfkA or IfkB eIF2alpha kinases. GFP fusions, with appropriate internal controls, were used to directly demonstrate that the bzpR 5Õ UTR, possessing 7 uORFs, suppressed translation by 12 fold. Suppression occurred even when all but one uORF was deleted, and translational suppression was removed when the ATG of the single uORF was mutated. Conclusions. The findings indicate that BzpR regulates aspects of the development program in Dictyostelium, serving as a downstream effector of eIF2alpha phosphorylation. Its production is temporally and spatially regulated by eIF2alpha phosphorylation by IfkA and IfkB and through the use of uORFs within the bzpR 5Õ UTR. Submitted by Charles Singleton [charles.k.singleton@Vanderbilt.Edu] ============================================================== [End dictyNews, volume 38, number 3]