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dictyNews Volume 20 Number 07
Dicty News
Electronic Edition
Volume 20, number 7
April 11, 2003
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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Abstracts
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Crawling into a new era - the Dictyostelium genome project
Ludwig Eichinger and Angelika A. Noegel
Center for Biochemistry, Medical Faculty, University of Cologne,
Joseph-Stelzmann-Str. 52, 50931 Kln, FRG
EMBO J., in press
The social amoeba Dictyostelium discoideum is a well-established model
organism for the study of basic aspects of differentiation, signal
transduction, phagocytosis, cytokinesis and cell motility. Its genome is
currently being sequenced by an international consortium using a whole
chromosome shotgun (WCS) approach. The pacemaker of the D. discoideum
genome project has been chromosome 2, the largest chromosome, which at 8 Mb
represents approximately 25% of the genome and whose sequence and analysis
has recently been published. Chromosomes 1 and 6 are close to being
finished. To accelerate completion of the genome sequence the next step in
the project will be a whole genome assembly followed by the analysis of the
complete gene content. The completed genome sequence and its analysis
provide the basis for genome-wide functional studies. It will position
Dictyostelium at the same level as other model organisms and further
enhance its experimental attractiveness.
Submitted by :Ludwig Eichinger [ludwig.eichinger@uni-koeln.de]
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TagA, a putative serine protease/ABC transporter of Dictyostelium that is required for cell
fate determination at the onset of development
J. Randall Good1,3,4,5, Matthew Cabral2,4, Sujata Sharma2, Jun Yang1, Nancy Van Driessche2,3,
Chad A. Shaw2, Gad Shaulsky2,3 and Adam Kuspa1,2,3,6 1Department of Biochemistry and Molecular
Biology, 2Department of Molecular and Human Genetics and the 3Program in Developmental Biology,
Baylor College of Medicine, Houston, Texas, USA, 77030, USA
Development, in press
Summary
The tag genes of Dictyostelium are predicted to encode multi-domain proteins consisting of
serine protease and ATP-binding cassette transporter domains. We have identified a novel tag
gene, tagA, which is involved in cell type differentiation. TagA mutant aggregates elaborate
multiple prestalk cell regions during development and produce spores asynchronously and with
low viability. The tagA mRNA reaches its highest level by two hours of development and
persists at lower levels, whereas TagA protein accumulates between two and ten hours of
development and decreases thereafter. Wild type cells express tagA in prespore cells and
mature spores, defining tagA expression as prespore-specific. However, tagA mutant cells
that activate the tagA promoter do not sporulate, but instead form part of the outer basal
disc and lower cup of the fruiting body. These results suggest that TagA is required for the
specification of an initial population of prespore cells in which tagA is expressed. TagA
mutants produce about twice as many prestalk cells as the wild type as judged by the expression
of a prestalk reporter construct. Furthermore, when mixed with wild-type cells tagA mutant
cells become overrepresented in the prestalk cell population, suggesting that this phenotype is
cell-autonomous. Expression profiling uncovered a delay in the transcriptional program between
2 and 6 hours, coincident with initial TagA expression, revealing a function for TagA prior to
the detectable overproduction of prestalk cells. TagA also appears to play a general role in
cell fate determination since tagA mutants express the spore coat protein gene, cotB, within
vacuolated cells that form part of the stalk and they express the prestalk/stalk-specific gene
ecmB within cells that become spores. The expression of TagA at two hours of development, the
observed coincident delay in the transcriptional program and the subsequent mis-expression of
cell-type specific genes provide evidence for cell fate determination beginning in some cells
much earlier than previously believed.
Submitted by: Adam Kuspa [akuspa@bcm.tmc.edu]
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IfkA, a presumptive eIF2alpha kinase of Dictyostelium, is required for proper timing of
aggregation and regulation of mound size.
Rui Fang, Yanhua Xiong, and Charles K. Singleton
Department of Biological Sciences, Vanderbilt University, VU Station B 351634, Nashville
TN 37235-1634
Accepted: BMC-Developmental Biology
ABSTRACT
Background. The transition from growth to development in Dictyostelium is initiated by amino
acid starvation of growing amobae. In other eukaryotes, a key sensor of amino acid starvation
and mediator of the resulting physiological responses is the GCN2 protein, an eIF2alpha kinase.
GCN2 downregulates the initiation of translation of bulk mRNA and enhances translation of
specific mRNAs by phosphorylating the translation initiation factor eIF2alpha. Two eIF2alpha
kinases were identified in Dictyostelium and studied herein.
Results. Neither of the eIF2alpha kinases appeared to be involved in sensing amino acid
starvation to initiate development. However, one of the kinases, IfkA, was shown to
phosphorylate eIF2alpha from 1 to 7 hours after the onset of development, resulting in a shift
from polysomes to free ribosomes for bulk mRNA. In the absence of the eIF2alpha phosphorylation,
ifkA null cells aggregated earlier than normal and formed mounds and ultimately fruiting bodies
that were larger than normal. The early aggregation phenotype in ifkA null cells reflected an
apparent, earlier than normal establishment of the cAMP pulsing system. The large mound
phenotype resulted from a reduced extracellular level of Countin, a component of the counting
factor that regulates mound size. In wild type cells, phosphorylation of eIF2alpha by IfkA
resulted in a specific stabilization and enhanced translational efficiency of countin mRNA even
though reduced translation resulted for bulk mRNA.
Conclusions. IfkA is an eIF2alpha kinase of Dictyostelium that normally phosphorylates
eIF2alpha from 1 to 7 hours after the onset of development, or during the preaggregation phase.
This results in an overall reduction in the initiation of protein synthesis during this time
frame and a concomitant reduction in the number of ribosomes associated with most mRNAs. For
some mRNAs, however, initiation of protein synthesis is enhanced or stabilized under the
conditions of increased eIF2alpha phosphorylation. This includes countin mRNA.
Submitted by: charles singleton [charles.k.singleton@vanderbilt.edu]
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Receptor mediated regulation of PI3Ks confines PI(3,4,5)P3 to the leading edge of chemotaxing
cells.
Huang, Y.E., Iijima, M., Parent, C.A., Funamoto, S., Firtel, R.A., and Devreotes, P.N.
Mol. Bio. Cell, in press.
Recent studies have demonstrated that PH domains specific for PI(3,4,5)P3 accumulate at the
leading edge of a number of migrating cells and that PI3Ks and PTEN associate with the
membrane at the front and back, respectively, of chemotaxing D. discoideum cells. However,
the dependence of chemoattractant induced changes in PI(3,4,5)P3 on PI3K and PTEN activities
have not been defined. We find that bulk PI(3,4,5)P3 levels increase transiently upon
chemoattractant stimulation, and the changes are greater and more prolonged in pten- cells.
PI3K activation increases within 5 seconds of chemoattractant addition and then declines to
a low level of activity identically in wild type and pten- cells. Reconstitution of the PI3K
activation profile can be achieved by mixing membranes from stimulated pi3k1-/pi3k2- cells
with cytosolic PI3Ks from unstimulated cells. These studies show that significant control of
chemotaxis occurs upstream of the PI3Ks and that regulation of the PI3Ks and PTEN cooperate
to shape the temporal and spatial localization of PI(3,4,5)P3.
Submitted by Pam Antol [pjantol@jhmi.edu]
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On the origin of differentiation
J. T. Bonner*
Department of Ecology and Evolutionary Biology, Princeton University,
Princeton, Princeton, NJ 08544, USA <jtbonner@princeton.edu>
J. Biosciences, in press
ABSTRACT
Following the origin of multicellularity in many groups of primitive
organisms there evolved more than one cell type. It has been
assumed that this early differentiation is related to size-the larger
the organism the more cell types. Here two very different kinds of
organisms are considered: the volvocine algae that become
multicellular by growth, and the cellular slime molds that become
multicellular by aggregation. In both cases there are species that
have only one cell type and others that have two. It has been
possible to show that there is a perfect correlation with size: the
forms with two cell types are significantly larger than those with
one. Also in both groups there are forms of intermediate size that
will vary from one to two cell types depending on the size of the
individuals, suggesting a form of quorum sensing. These observations
reinforce the view that size plays a critical role in influencing the
degree of differentiation.
Submitted by : John Bonner [jtbonner@Princeton.EDU]
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MFE1, a member of the peroxisomal hydroxyacyl-CoA dehydrogenase family, affects fatty acid
metabolism necessary for morphogenesis in Dictyostelium
Satomi Matsuoka1) #, Tamao Saito2) #, Hidekazu Kuwayama1)+, Naoki. Morita3), Hiroshi Ochiai2)
and Mineko Maeda1)*
1Department of Biology, Graduate school of Science, Osaka University, Machikaneyama-cho 1-16,
Toyonaka, Osaka 560-0043, Japan
2Division of Biological Science, Graduate school of Science, Hokkaido University, Sapporo,
Hokkaido 060-0810, Japan
3Research Institute of Biological Resources, National Institute of Advanced Industrial Science
and technology (AIST), Toyohira-ku, Sapporo 062-8517, Japan
Eukaryotic Cell (in press)
Oxidation of long chain fatty acids and branched-chain fatty acids is carried out in mammalian
peroxisomes by multifunctional enzyme MFE or DBP, with separate domains for hydroxyacyl-CoA
dehydrogenase, enoyl-CoA hydratase and steroid carrier protein, SCP2. We have found that
Dictyostelium has a gene, mfeA, encoding MFE1 with homology to the hydroxyacyl-CoA
dehydrogenase and SCP2 domains. A separate gene, mfeB, encodes MFE2 with homology to the
enoyl-CoA hydratase domain. When grown on a diet of bacteria, Dictyostelium cells in which
mfeA is disrupted accumulate excess cyclopropane fatty acids and are unable to develop beyond
early aggregation. Axenically grown mutant cells, however, developed into normal fruiting bodies
composed of spores and stalk cells. Comparative analysis of whole-cell lipid compositions
revealed that bacterially grown mutant cells accumulated cyclopropane fatty acids which remained
throughout the developmental stages. Such a persistent accumulation was not detected in wild type
cells or axenically grown mutant cells. Bacterial phosphatidylethanolamine which contains abundant
cyclopropane fatty acids inhibited the development of even axenically grown mutant cells, while
dipalmitoyl phosphatidylethanolamine did not. These results suggest that MFE1 protects the cells
from the increase of the harmful xenobiotic fatty acids incorporated from their diets and optimizes
cellular lipid composition for proper development. Hence we propose that this enzyme plays an
irreplaceable role in the survival strategy of Dictyostelium cells to form spores for their
efficient dispersal in nature.
Submitted by: Mineko Maeda [mmaeda@bio.sci.osaka-u.ac.jp]
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Phagocytosis of Dictyostelium discoideum Studied by Particle Tracking Method
J. Ishikawa1, J. Okano1, K. Ohki1, A. Amagai2, Y. Maeda2 and H. Miyata1*
1Physics Department, Graduate School of Science, Tohoku University, Aoba-ku, Sendai, Miyagi
980-8578, Japan
and 2Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences,
Tohoku University, Aoba-ku, Sendai, Miyagi 980-8578, Japan
*Corresponding author. E-mail: miyata@bio.phys.tohoku.ac.jp.
Exp. Cell Res. (in press)
Abstract
Single phagocytic events of cellular slime mold Dictyostelium discoideum were studied by the
method of particle tracking. A 2 um polystyrene bead, which had been covalently coated with
folate, was attached to the advancing edge of a Dictyostelium ameba with the aid of an optical
trap. The bead was transported backward on the cell surface. Forty-five percent of the
transported beads were internalized. The bead motion was analyzed by determining every 33 msec
the x-y coordinate of the centroid of the phase contrast image of the bead. The x(t) and y(t)
traces were smoothed over 1 sec and the difference between the smoothed (x'(t) and y'(t)) and
the original traces, delta x = x(t)-x'(t) and delta y = y(t)-y'(t), were calculated, which
represented relatively rapid components of the bead motion . The plot of delta2 = [(delta x)2
+ (delta y)2] against time could be divided into three phases on the basis of the variance of
delta2. Comparison of the plot with the video sequence indicated that the first phase
corresponded to the transport, the second phase the internalization and the third phase the
post-internalization process (intracellular movement). Cytochalasin A at 5 uM completely
inhibited phagocytosis without affecting the binding of bead to the cell surface, indicating
importance of actin cytoskeleton in all the phases. At 1 uM cytochalasin A the ( value of the
post-internalization process decreased, and the duration of the transport phase increased. At
0.25 uM cytochalasin A the duration of the internalization phase exhibited significant increase,
but other parameters did not appreciably change. The complex and differential effects of
cytochalasin A on the parameters characterizing the three phases in phagocytic process indicate
that various aspects of actin dynamics are involved in the individual process of phagocytosis.
Submitted by: Hidetake Miyata [miyata@brain.phys.tohoku.ac.jp]
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[End Dicty News, volume 20, number 7]
