dictyNews
Electronic Edition
Volume 38, number 3
January 27, 2012

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=========
Abstracts
=========


Functional Characterization of a Novel Aquaporin from Dictyostelium 
discoideum Amoebae Implies a Unique Gating Mechanism

Julia von Bülow, Annika Müller-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 [[log in to unmask]]
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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 [[log in to unmask]]
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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 
5’ 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 5’ 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 5’ UTR.


Submitted by Charles Singleton [[log in to unmask]]
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[End dictyNews, volume 38, number 3]