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dictyNews Volume 22 Number 12
Dicty News
Electronic Edition
Volume 22, number 12
May 14, 2004
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 Dicty-News, the Dicty Reference database and other
useful information is available at dictyBase - http://dictybase.org.
=============
Abstracts
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Specificity of a Soluble UDP-Galactose:Fucoside a1,3Galactosyltransferase
that Modifies the Cytoplasmic Glycoprotein Skp1 in Dictyostelium*
Catherine Ketcham¤, Fei Wang¤, S. Z. Fisher¤, Altan Ercan||, Hanke van der
Wel¤||, R. D. Locke¦, S. ud-Doulah.k¦, Khushi L. Matta¦ and Christopher M.
West¤||
From the ¤Department of Anatomy & Cell Biology, University of Florida College
of Medicine, Gainesville, FL 32610-0235 USA, the ¦Department of Molecular &
Cellular Biophysics, Roswell Park Cancer Institute, Elm & Carlton Streets,
Buffalo, NY 14263 USA, and the ||Department of Biochemistry & Molecular
Biology, Oklahoma Center for Medical Glycobiology, University of Oklahoma
Health Sciences Center, Oklahoma City, OK 73104 USA
J. Biol. Chem., in press
Skp1 is an adaptor-like protein in E3SCF-ubiquitin ligases and other
multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1
is modified by an unusual pentasaccharide containing a Gala1-Fuc linkage,
whose formation is examined here. A cytosolic extract from Dictyostelium was
found to yield, after 2400-fold purification, an activity that could transfer
Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic
Fuc-conjugates in the presence of Mn2+ and dithiothreitol. The microsomal
fraction was devoid of activity. The linkage formed was Gala1,3Fuc based on
co-chromatography with only this synthetic isomer conjugate, and sensitivity
to a1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower Km and
35-fold higher Vmax compared to a simple a-fucoside, but this advantage was
abolished by denaturation or alkylation of Cys-residues. A comparison of a
complete series of synthetic glycosides representing the non-reducing
terminal mono-, di- and tri-saccharides of Skp1 revealed, surprisingly, that
the disaccharide is most active owing primarily to a Vmax advantage, but
still much less active than Skp1 itself owing to a Km difference. These
findings indicate that aGalT1 is a cytoplasmic enzyme whose modification of
Skp1 requires proper presentation of the terminal acceptor disaccharide by
a folded Skp1 polypeptide, which correlates with previous evidence that the
Gala1,3Fuc-linkage is deficient in expressed mutant Skp1 proteins.
Submitted by: Chris West [Christopher-West@ouhsc.edu]
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Chemoattractant-Induced PI(3,4,5)P3 Accumulation is Spatially Amplified and
Adapts, Independent of the Actin Cytoskeleton
1Chris Janetopoulos, 2Lan Ma, 1Peter N. Devreotes, and 2,3Pablo A. Iglesias
1Dept Cell Biology
Johns Hopkins University School of Medicine
725 N. Wolfe St
Baltimore, MD 21205
410-955-3225
410-614-9461 (Fax)
2Dept Electrical & Computer Engineering
Johns Hopkins University
3400 N. Charles St.
Baltimore, MD, 21218
410-516-6026
410-516-5566 (Fax)
3Corresponding Author
Proceedings of the National Academy of Sciences USA, in press
Experiments in amoebae and neutrophils have shown that local accumulations
of PI(3,4,5)P3 mediate the ability of cells to migrate during gradient
sensing. To define the nature of this response, we subjected D. discoideum
cells to measurable temporal and spatial chemotactic inputs and analyzed the
accumulation of PI(3,4,5)P3 on the membrane, as well as the recruitment of
the enzymes PI3K and PTEN. In latrunculin-treated cells, spatial gradients
elicited a PI(3,4,5)P3 response only on the front portion of the cell where
the response increased more steeply than the gradient and did not depend on
its absolute concentration. PI3K bound to the membrane only at the front,
although it was less sharply localized than PI(3,4,5)P3. Membrane-bound
PTEN was highest at the rear and varied inversely with receptor occupancy.
The localization of PI(3,4,5)P3 was enhanced further in untreated, polarized
cells, containing an intact cytoskeleton. Interestingly, the treated cells
could respond to two independent gradients simultaneously, demonstrating that
a response at the front does not necessarily inhibit the back. Combinations
of temporal and spatial stimuli provided evidence of an inhibitory process
and showed that a gradient generates a persistent steady-state response
independent of previous history of exposure to chemoattractant. These results
support a local excitation/global inhibition model and argue against other
schemes proposed to explain directional sensing.
Submitted by: Chris Janetopoulos [cjanetop@jhmi.edu]
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A gene encoding prespore-cell-inducing factor in Dictyostelium discoideum
Takefumi Kawata1,4,*, Manabu Nakagawa3, Nao Shimada1, Shigeru Fujii3 and
Akiko A. Oohata2,4
1Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama,
Funabashi, Chiba 274-8510, JAPAN
2Biological Laboratory and 3Chemical Laboratory, Kansai Medical University,
Hirakata, Osaka 573-1136, JAPAN
4Correspondence authors
Dev. Growth Diff., in press
Two factors that exist in conditioned medium (CM) of Dictyostelium discoideum
induce amoebae to differentiate into prespore cells when they are incubated at
a very low cell density in submerged monolayer culture. Previously, we
purified one of them, a glycoprotein factor with an apparent molecular mass
of 106 kDa, and we named it y factor (psi, prespore-inducing factor). Based
on partial amino acid sequence of the purified y factor, we have isolated the
corresponding cDNA clone, which is expressed maximally at the loose mound
stage. The cDNA encodes a novel protein and the predicted molecular mass of
the mature secreted protein is 60 kDa. Knockout mutant strains of the y
factor gene, psiA-, were created by targeted integration. Although these
mutant strains appear to develop normally, CM from these mutants showed
reduced prespore-cell-inducing activity. Rescuing the mutant strains by
expression of y factor under control of a constitutive promoter causes
overproduction of y factor protein and CM from such cells showed a 20-fold
higher level of prespore-cell-inducing activity than that from wild-type
cells. Further, CM from parental cells induced prespore cell division,
while that from psiA null strains showed no cell division inducing activity.
Our results indicate that y factor protein is a novel type of growth factor
that does not belong to any of the families of growth factor so far
identified in animals.
Submitted by: Takefumi Kawata [tkawata@bio.sci.toho-u.ac.jp]
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Cell movements and mechanical force distribution during the migration of
Dictyostelium slugs
Jean-Paul Rieu 1,*, Catherine Barentin 1, Satoshi Sawai 2, Yasuo Maeda 3 and
Yasuji Sawada 4
1 Laboratoire de Physique de la Matiere Condensee et des
Nanostructures, Universite
J. Biol. Phys., in press
Migration of Dictyostelium discoideum slugs results from coordinated
movement of its constituent cells. It is generally assumed that each cell
contributes to the total motive force of the slug. However, the basic
mechanisms by which mechanical forces (traction and resistive forces) are
transmitted to the substrate, their magnitude and their location, are
largely unknown. In this work, we performed detailed observations of cell
movements by fluorescence microscopy using two-dimensional (2D) slugs. We
show that 2D slugs share most of the properties of 3D ones. In particular,
waves of movement propagate in long 2D slugs, and slug speed correlates with
slug length as found in 3D slugs. We also present the first measurements of
the distribution of forces exerted by 2D and 3D slugs using the elastic
substrate method. Traction forces are mainly exerted in the central region
of the slug. The large perpendicular forces around slug boundary and the
existence of parallel resistive forces in the tip and/or the tail suggest an
important role of the sheath in the transmission of forces to the substrate.
Submitted by: Yasuo Maeda [ymaeda@mail.tains.tohoku.ac.jp]
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[End Dicty News, volume 22, number 12]
