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dictyNews Volume 18 Number 10
Dicty News
Electronic Edition
Volume 18, number 10
June 1, 2002
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu.
Back issues of Dicty-News, the Dicty Reference database and other useful
information is available at DictyBase--http://dictybase.org.
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DictyBase and Stock Center Funded By NIH
===========================================
We are pleased to announce that the National Institutes of Health has
notified us that they will be funding both DictyBase and The Dictyostelium
stock center. The support of the Dictyostelium community has been
instrumental in obtaining the support for these resources. We look
forward to working with you to establish these resources.
Rex Chisholm, DictyBase
Jakob Franke and Rich Kessin, Stock Center
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Abstracts
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The BEACH Family of Proteins: Phylogenetic & Functional Analysis of Six
Dictyostelium BEACH Proteins.
Ning Wang, Wei-I Wu, and Arturo De Lozanne
Section of Molecular Cell & Developmental Biology
and Institute for Cellular and Molecular Biology,
University of Texas at Austin, Austin, TX 78712.
a.delozanne@mail.utexas.edu
Journal of Cellular Biochemistry, in press.
ABSTRACT
The BEACH-domain containing proteins constitute a new family of proteins
found in all eukaryotes. The function of these proteins, which include the
Chediak-Higashi syndrome protein, Neurobeachin, LvsA and FAN, is still poorly
understood. To understand the diversity of this novel protein family, we
analyzed a large array of BEACH-family protein sequences from several
organisms. Comparison of all these sequences suggests that they can be
classified into five distinct groups that may represent five distinct
functional classes. In Dictyostelium we identified six proteins in this
family, named LvsA-F, that belong to four of those classes. To test the
function of these proteins in Dictyostelium we created disruption mutants
in each of the lvs genes. Phenotypic analyses of these mutants indicate
that LvsA is required for cytokinesis and osmoregulation and LvsB functions
in lysosomal traffic. The LvsC-F proteins are not required for these or
other processes such as growth and development. These results strongly
support the concept that BEACH proteins from different classes have distinct
cellular functions. Having six distinct BEACH proteins, Dictyostelium
should be an excellent model system to dissect the molecular function of
this interesting family of proteins.
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Disruption of the gene encoding the cell adhesion molecule DdCAD-1 leads to
aberrant cell sorting and cell-type proportioning during Dictyostelium
development
Estella Wong*, Chunzhong Yang*, Jun Wang*, Danny Fuller, William F. Loomis,
and Chi-Hung Siu*
1 *Banting and Best Department of Medical Research and Department of
Biochemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada; and
Center for Molecular Genetics, Department of Biology, University of California
at San Diego, La Jolla, California 92037
Development, in press.
SUMMARY
The cadA gene in Dictyostelium encodes the Ca2+-dependent cell adhesion
molecule DdCAD-1, which is expressed soon after the initiation of development.
To investigate the biological role of DdCAD-1, the cadA gene was disrupted by
homologous recombination. The cadA-null cells showed a 50% reduction in
EDTA-sensitive cell adhesion. The remaining EDTA-sensitive adhesion sites
were resistant to dissociation by anti-DdCAD-1 antibody, suggesting that they
were distinct adhesion sites. Cells lacking DdCAD-1 were able to complete
development and form fruiting bodies. However, they displayed abnormal slug
morphology and culmination was delayed by ~6 hours. The yield of spores was
reduced by ~50%. The proportion of prestalk cells in cadA- slugs showed a
2.5-fold increase over the parental strain. When cadA- cells were
transfected with pcotB::GFP to label prespore cells, aberrant cell sorting
patterns in slugs became apparent. When mutant prestalk cells were mixed
with wild-type prespore cells, mutant prestalk cells were unable to return
to the anterior position of chimeric slugs, suggesting defects in the
sorting mechanism. The wild-type phenotype was restored when cadA- cells
were transfected with a cadA-expression vector. These results indicate
that, in addition to cell-cell adhesion, DdCAD-1 plays a role in cell type
proportioning and pattern formation.
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Spatial and temporal regulation of 3-phosphoinositides by PI3 kinase and
PTEN mediates chemotaxis
Satoru Funamoto, Ruedi Meili1, Susan Lee, Lisa Parry, and Richard A. Firtel
Cell, in press.
Abstract
We have investigated the mechanisms of leading edge formation in
chemotaxing Dictyostelium cells. We demonstrate that while phosphatidyl
inositol-3 kinase (PI3K) transiently translocates to the plasma membrane
in response to chemoattractant stimulation and to the leading edge in
chemotaxing cells, while PTEN, a negative regulator of PI3K pathways, exhibits
a reciprocal pattern of localization. By uniformly localizing PI3K along the
plasma membrane, we show that chemotaxis pathways are activated along the
lateral sides of cells and PI3K can initiate pseudopod formation, providing
evidence for a direct instructional role of PI3K in leading edge formation.
These findings provide evidence that differential subcellular localization
and activation of PI3K and PTEN is required for proper chemotaxis.
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Evidence for a novel, strongly bound acto-S1 complex carrying ADP and
phosphate stabilized in the G680V mutant of Dictyostelium myosin II.
Taro Q. P. Uyeda0, Kiyotaka Tokuraku and Bruce Patterson0
0 Gene Discovery Research Center, National Institute of Advanced Industrial
Science and Technology (AIST), Tsukuba, Ibaraki 305-8562, Japan, National
Institute for Medical Research, Mill Hill, London NW7 1AA, UK. Department of
Molecular and Cellular Biology, University of Arizona, Tucson, AZ 85721, USA
Biochemistry, in press.
Abstract
Gly 680 of Dictyostelium myosin II sits at a critical position within
the reactive thiol helices. We have previously shown that G680V mutant
subfragment 1 largely remains in strongly actin-bound states in the presence
of ATP. We speculated that acto-G680V subfragment 1 complexes accumulate in
the A0M0ADP0Pi state, based on the biochemical phenotypes conferred by mutations
which suppress the G680V mutation in vivo [Wu, Y. et al. (1999) Genetics, 153,
107-116]. Here, we report further characterization of the interaction between
actin and G680V subfragment 1. Light scattering data demonstrates that the
majority of G680V subfragment 1 is bound to actin in the presence of ATP. These
acto-G680V subfragment 1 complexes in the presence of ATP do not efficiently
quench the fluorescence of pyrene-actin, unlike those in rigor complexes or in
the presence of ADP alone. Kinetic analyses demonstrated that phosphate
release, but not ATP hydrolysis or ADP release, is very slow and rate limiting
in the acto-G680V subfragment 1 ATPase cycle. Single turnover kinetic
analysis demonstrates that, during ATP hydrolysis by the acto-G680V
subfragment 1 complex, quenching of pyrene fluorescence significantly lags
the increase of light scattering. This is unlike the situation with
wild-type subfragment 1, where the two signals have similar rate constants.
These data support the hypothesis that the main intermediate during ATP
hydrolysis by acto-G680V subfragment 1 is an acto- subfragment 1 complex
carrying ADP and Pi, which scatters light but does not quench the pyrene
fluorescence and so has a different conformation from the rigor complex.
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Pseudopodium Dynamics and Rapid Cell Movement in Dictyostelium Ras
Pathway Mutants
Jonathan R. Chubb*, Andrew Wilkins*, Deborah J. Wessels+, David R.
Soll+ and Robert Insall*
Cell Motil. Cytoskel., in press.
Abstract
Loss of either of the Ras pathway members RasS or GefB causes growing
Dictyostelium cells to move aberrantly rapidly. In this study, we
describe the changes in motility which underlie these phenotypes
using computer-assisted 3D dynamic image analysis. Unexpectedly, the
two mutants use different mechanisms to achieve rapid migration. The
rasS- cells' motility is characterised by highly dynamic cell
morphology, with rapidly extending and retracting pseudopodia. The
gefB- cells do not have an unusually dynamic morphology, and achieve
their efficient translocation by the continual remodelling of an
existing dominant anterior pseudopodium. In spite of these dramatic
changes in pseudopodium behaviour, the underlying motility cycle of
both mutants remains normal. The levels of F-actin in both mutant
cell lines are significantly elevated with respect to the wild-type
parental cells, suggesting a possible biochemical basis for these
emphatic phenotypes.
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Myosin II dynamics in Dictyostelium: Determinants for filament assembly and
translocation to the cell cortex during chemoattractant responses
Stephanie Levi, Mark V. Polyakov, and Thomas T. Egelhoff
Department of Physiology and Biophysics, Case Western Reserve School of
Medicine, Cleveland, OH
Cell Motility and the Cytoskeleton, IN PRESS
ABSTRACT
In the simple amoeba Dictyostelium discoideum, myosin II filament assembly
is regulated primarily by the action of a set of myosin heavy chain (MHC)
kinases and by MHC phosphatase activity. Chemoattractant signals acting
via G-protein coupled receptors lead to rapid recruitment of myosin II to
the cell cortex, but the structural determinants on myosin necessary for
translocation, and the second messengers upstream of MHC kinases and
phosphatases are not well understood. We report here the use of GFP-myosin
II fusions to characterize the domains necessary for myosin II filament
assembly and cytoskeletal recruitment during responses to global
stimulation with the developmental chemoattractant cAMP. Analysis
performed with GFP-myosin fusions, and with latrunculin A-treated cells
demonstrated that F-actin binding via the myosin motor domain together with
concomitant filament assembly mediates the rapid cortical translocation
observed in response to chemoattractant stimulation. A "headless"
GFP-myosin construct lacking the motor domain was unable to translocate to
the cell cortex in response to chemoattractant stimulation, suggesting that
myosin motor-based motility may drive translocation. This lack of
localization contrasts with earlier work demonstrating accumulation of the
same construct in the cleavage furrow of dividing cells, suggesting that
recruitment signals and interactions during cytokinesis differ from those
during chemoattractant responses. Evaluating upstream signaling, we find
that iplA null mutants, devoid of regulated calcium fluxes during
chemoattractant stimulation, display full normal chemoattractant-stimulated
myosin assembly and translocation. These results indicate that calcium
transients are not necessary for chemoattractant-regulated myosin II
filament assembly and translocation.
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A novel PCR-mediated method for one-tube generation of a gene disruption
construct
Hidekazu Kuwayama1, Shinji Obara1, Takahiro Morio1, Mariko Katoh1, Satoru
Kuhara2 and Yoshimasa Tanaka1.
1. Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki
305-8572, Japan.
2. Graduate School of Genetic Resources Technology, Kyushu University,
Fukuoka, 812-8581, Japan.
Biotechnology letters, in press
ABSTRACT
We report a novel PCR-based method for generating a gene disruption
construct, which requires no purification of PCR fragments and enables the
whole procedure to be completed in one tube very rapidly. The procedure
starts with PCR amplification of both the 5' and 3' regions of a particular
gene in one tube. Then, exonuclease I is added to the tube to remove the
residual primers. After heat inactivation of the enzyme, a marker cassette
DNA fragment is added and fusion PCR is performed to build up a gene
disruption construct. The gene disruption construct is subsequently
amplified with the outermost primers in the amount necessary for
transformation. In order to distinguish the gene disruption construct from
the remaining intact gene allele, the outermost primers are designed to have
GC-rich tag sequences that anneal at a higher temperature, ensuring the
specific amplification of the gene disruption construct.
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[End Dicty News, volume 18, number 10]
