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dictyNews Volume 38 Number 03
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
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Abstracts
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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]
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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 [blumberg@umbc.edu]
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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
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]
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[End dictyNews, volume 38, number 3]
