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dictyNews Volume 28 Number 08
dictyNews
Electronic Edition
Volume 28, number 8
April 6, 2007
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.
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Abstracts
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The role of SP65 in assembly of the Dictyostelium spore coat
Talibah Metcalf1, Hanke van der Wel1, Ricardo Escalante2, Leandro Sastre2
and Christopher M. West1
1Dept. of Biochemistry & Molecular Biology, University of Oklahoma Health
Sciences Center, Oklahoma City, OK 73104 USA;
2Instituto de Investigaciones Biomedicas Alberto Sols. C.S.I.C./U.A.M.,
Arturo Duperier 4, 28029 Madrid, Spain
Eukaryotic Cell, in press
Like the cyst walls of other protists, the spore coat of Dictyostelium is
formed de novo to protect the enclosed dormant cell from stress. Spore coat
assembly is initiated by exocytosis of protein and polysaccharide precursors
at the cell surface, followed by the infusion of nascent cellulose fibrils
resulting in an asymmetrical trilaminar sandwich with cellulose filling the
middle layer. A molecular complex consisting of cellulose and two proteins,
SP85 and SP65, is associated with the inner and middle layers and is required
for proper organization of distinct proteins in the outer layer. Here we show
that, unlike SP85 and other protein precursors, which are stored in prespore
vesicles, SP65 is, like cellulose, synthesized just in time. By tagging the
SP65 locus with GFP, we find that SP65 is delivered to the cell surface via
largely distinct vesicles, suggesting that separate delivery of components of
the cellulose-SP85-SP65 complex regulates its formation at the cell surface.
In support of previous in vivo studies, recombinant SP65 and SP85 are shown
to interact directly. In addition, truncation of SP65 causes a defect of the
outer layer permeability barrier as seen previously for SP85 mutants. These
observations suggest that assembly of the cellulose-SP85-SP65 triad at the
cell surface is biosynthetically regulated both temporally and spatially, and
that the complex contributes an essential function to outer layer architecture
and function.
Submitted by: Chris West [Cwest2@ouhsc.edu]
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Chemotaxis in the absence of PIP3 gradients
Oliver Hoeller and Robert R. Kay
MRC Laboratory of Molecular Biology,
Hills Rd,
Cambridge, CB2 2QH, UK.
Current Biology (in press)
Chemotaxing neutrophils and Dictyostelium amoebae produce gradients of the
signalling lipid PI(3,4,5)P3 (PIP3) in their plasma membranes [1-3], which
are orientated with the external chemotactic gradient and have been proposed
to act as an internal compass, guiding movement of the cell [4, 5]. Evidence
for and against this idea exists, but in all cases it depends on the use of
inhibitors or gene knockouts, which may only incompletely abolish the PIP3
gradient. We have created a multiple gene knockout strain in Dictyostelium
lacking all five type-1 phosphoinositide 3-kinases encoded in the genome and
the PTEN phosphatase and have thus removed all known ways for chemoattractant
to produce PIP3 gradients in the plasma membrane. The resulting sextuple
mutant is able to chemotax to cyclic-AMP with near wild-type efficiency and
to trigger actin polymerization without apparent defect. There is however,
a consistent defect in movement speed in chemotaxis and especially random
movement. This work shows that polarization of membrane PIP3 is not necessary
for accurate chemotaxis, but can affect cell speed. A signalling pathway from
receptor to cytoskeleton must exist which is able to guide cells independently
of polarized PIP3 and type-1 phosphoinositide 3-kinases.
Submitted by: Rob Kay [rrk@mrc-lmb.cam.ac.uk]
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Development of soil amoeba Dictyostelium discoideum as an expression system
for recombinant human erythropoietin
Bhavesh Vats, Harish Padh*
*Corresponding author
B. V. Patel Pharmaceutical Education and Research Development Centre,
Thaltej- Gandhinagar Highway, Thaltej, Ahmedabad 380054, India.
Telephone No.: +91-79-2743 9375
Fax No.: +91-79- 2745 0449
World Journal of Microbiology and Biotechnology, in press
EPO is the block buster biopharmaceutical product presently being produced by
recombinant DNA technology from mammalian cell lines. Other available
expression systems have not been useful in producing this protein due to the
requirement of N- glycosylation for in-vivo activity. In order to develop an
alternative expression system, the human epo gene was expressed in the cellular
slime mold Dictyostelium discoideum. The 2.43 kbp epo gene from the mammalian
expression vector was cloned in the Dictyostelium expression cassette and used
to transform cells. Positive clones were selected on the basis of antibiotic
resistance. The clones were screened for the presence of the transgene. The
copies of the gene inserted in the genome were identified and the transcript
too was ascertained. The protein was identified by immuno-blotting and appears
to be glycosylated though differently from that of humans or CHO cell lines.
Submitted by: Harish Padh [hpadh@yahoo.com]
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Evidence that non-coding RNA dutA is a multicopy suppressor of Dictyostelium
STAT protein, Dd-STATa
Nao Shimada and Takefumi Kawata*
Department of Biology, Faculty of Science, Toho University, Funabashi, Japan
*Corresponding author. Mailing address: Department of Biology, Faculty of
Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan.
Phone: 81-47-472-5156
FAX: 81-47-472-5156
E-mail: tkawata@bio.sci.toho-u.ac.jp
Eukaryotic Cell, in press.
Dd-STATa, a Dictyostelium homologue of metazoan STAT transcription factors,
is necessary for culmination. We created a mutant strain with partial Dd-STATa
activity, and used it to screen for unlinked suppressor genes. We screened
approximately 450,000 clones from a slug-stage cDNA library for their ability
to rescue the culmination defect when overexpressed. There were 12 multicopy
suppressors of Dd-STATa, of which 4 encode segments of a known non-coding
RNA, dutA. Expression of dutA is specific to the pstA zone, the region where
Dd-STATais activated. In suppressed strains the expression patterns of several
putative Dd-STATa target genes become similar to the wild-type strain. In addition,
the amount of the tyrosine phosphorylated form of Dd-STATa is significantly
increased in the suppressed strain. These results indicate that partial copies of
dutA may act upstream of Dd-STATa to regulate tyrosine phosphorylation by an
unknownmechanism.
Submitted by: Takefumi Kawata [tkawata@bio.sci.toho-u.ac.jp]
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[End dictyNews, volume 28, number 8]
