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dictyNews Volume 23 Number 14
Dicty News
Electronic Edition
Volume 23, number 14
October 22, 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.
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Abstracts
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Dictyostelium discoideum strains lacking the RtoA protein are defective
for maturation of the Legionella pneumophila replication vacuole
Zhiru Li2, Jonathan M. Solomon2,º, and Ralph R. Isberg1,2*
1 Howard Hughes Medical Institute and
2 Department of Molecular Biology and Microbiology,
Tufts University School of Medicine
150 Harrison Ave., Boston, MA 02111
Cellular Microbiology, in press
To identify host proteins involved in Legionella pneumophila intracellular
replication, the soil amoeba Dictyostelium discoideum was analyzed. The
absence of the amoebal RtoA protein is demonstrated here to depress
L. pneumophila intracellular growth. Uptake of L. pneumophila into a
D. discoideum rtoA- strain was marginally defective, but this effect was
not sufficient to account for the defective intracellular growth of
L. pneumophila. The rtoA mutant was also more resistant to
high-multiplicity killing by the bacterium. A targeting assay testing
the co-localization of L. pneumophila-containing vacuole with an
endoplasmic reticulum/pre-Golgi intermediate compartment marker protein,
GFP-HDEL, was used to analyze these defects. In parental D. discoideum,
the L. pneumophila vacuole showed recruitment of GFP-HDEL within 40 min
after introduction of bacteria to the amoebae. By 6 hours after infection
it was clear that the rtoA mutant acquired and retained the GFP-HDEL less
efficiently than the parental strain, and that the mutant was defective for
promoting the physical expansion of the membranous compartment surrounding
the bacteria. Depressed intracellular growth of L. pneumophila in a
D. discoideum rtoA- mutant, therefore, appeared to result from a lowered
efficiency of vesicle trafficking events that are essential for the
modification and expansion of the L. pneumophila-containing compartment.
Submitted by: Zhiru Li [li.zhiru@tufts.edu]
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Dictyostelium PAKc is required for proper chemotaxis
Susan Lee1, Francisco Rivero2, Kyung Chan Park1, Emerald Huang1,
Satoru Funamoto1, and Richard A. Firtel1
1Section of Cell and Developmental Biology
Division of Biological Sciences
and Center for Molecular Genetics
University of California, San Diego
9500 Gilman Drive
La Jolla CA 92093-0634
USA
2Zentrum fr Biochemie I der Medizinischen Fakultt
Universitt zu Kln
Joseph-Stelzmann-Strasse 52
50931 Kln
Germany
Mol. Biol. Cell, in press.
We have identified a new Dictyostelium p21-activated protein kinase, PAKc,
that we demonstrate to be required for proper chemotaxis. PAKc contains a
Rac-GTPase binding (CRIB) and autoinhibitory domain, a PAK-related kinase
domain, an N-terminal phosphatidylinositol binding domain, and a C-terminal
extension related to the G?? binding domain of S. cerevisiae Ste20, the
latter two domains being required for PAKc transient localization to the
plasma membrane. In response to chemoattractant stimulation, PAKc kinase
activity is rapidly and transiently activated, with activity levels peaking
at ~10 sec. pakc null cells exhibit a loss of polarity and produce multiple
lateral pseudopodia when placed in a chemoattractant gradient. PAKc
preferentially binds the Dictyostelium Rac protein RacB, and point mutations
in the conserved CRIB that abrogate this binding result in mis-regulated
kinase activation and chemotaxis defects. We also demonstrate that a null
mutation lacking the PAK family member myosin I heavy chain kinase (MIHCK)
shows mild chemotaxis defects, including the formation of lateral
pseudopodia. A null strain lacking both PAKc and the PAK family member
MIHCK exhibits severe loss of cell movement, suggesting that PAKc and MIHCK
may cooperate to regulate a common chemotaxis pathway.
Submitted by: Rick Firtel [rafirtel@ucsd.edu]
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Involvement of the TRAP-1 homologue, Dd-TRAP1, in spore differentiation
during Dictyostelium development
Tsuyoshi Morita1, Hitomi Yamaguchi2, Aiko Amagai, and Yasuo Maeda*
Department of Developmental Biology and Neurosciences, Graduate School of
Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan
Present address: 1 Department of Neuroscience (D13), Osaka University
Graduate School of Medicine, Yamadaoka 2-2, Suita City, Osaka 565-0871,
Japan; 2 Department of Cell Genetics, National Institute of Genetics,
Mishima, Shizuoka-ken 411-8540, Japan.
Exp. Cell Res., in press
TRAP1 (tumor necrosis factor receptor-associated protein 1) is a member
of the molecular chaperone HSP90 (90-kDa heat shock protein) family. We have
previously demonstrated that Dd-TRAP1 (Dictyostelium discoideum TRAP1)
synthesized at the vegetative growth phase is retained during the whole
course of D. discoideum development, and that at the multicellular slug
stage it is located in prespore-specific vacuoles (PSVs) of prespore cells
as well as in the cell membrane and mitochondria. Thereupon, we examined the
function of Dd-TRAP1 in prepore and spore differentiation, using
Dd-TRAP1-knockdown cells (TRAP1-RNAi cells) produced by the RNA interference
method. As was expected, Dd-TRAP1 contained in the PSV was found to be
exocytosed during sporulation to constitute the outer-most layer of the
spore cell wall. In the TRAP1-RNAi cells, PSV formation and therefore
prespore differentiation were significantly impaired, particularly under a
heat stress condition. Although the TRAP1-RNAi cells formed apparently
normal-shaped spores with a cellulosic wall, the spores were less resistant
to heat and detergent treatments, as compared with those of parental MB35
cells derived from Ax-2 cells. These findings strongly suggest that Dd-TRAP1
may be closely involved in late development including spore differentiation,
as well as in early development as realized by its induction of
prestarvation response.
Submitted by: Yasuo Maeda [ymaeda@mail.tains.tohoku.ac.jp]
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Sphingosine kinase regulates the sensitivity of Dictyostelium discoideum
cells to the anticancer drug cisplatin
Junxia Min1, David Traynor2, Andrew L. Stegner1, Lei Zhang3,
Marie H. Hanigan3, Hannah Alexander1 and Stephen Alexander1#
1Division of Biological Sciences, University of Missouri,
Columbia, MO 65211-7400;
2 MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK;
3Department of Cell Biology, University of Oklahoma Health Sciences Center,
Oklahoma City, OK 73190.
Eukaryotic Cell, In press.
The drug cisplatin is widely used to treat a number of tumor types. However,
resistance to the drug, which remains poorly understood, limits its
usefulness. Previous work using D. discoideum as a model for studying drug
resistance, showed that mutants lacking sphingosine-1-phosphate (S-1-P)
lyase, the enzyme which degrades S-1-P, had increased resistance to cisplatin,
while mutants overexpressing the enzyme were more sensitive to the drug.
S-1-P is synthesized from sphingosine and ATP by the enzyme sphingosine
kinase. We have identified two sphingosine kinase genes in
D. discoideum - sgkA and sgkB - which are homologous to those of other
species. The biochemical properties of the SgkA and SgkB enzymes suggest
that they are the equivalent of the human Sphk1 and Sphk2 enzymes,
respectively. Disruption of the kinases by homologous recombination (both
single and double mutants) or overexpression of the sgkA gene resulted in
altered growth rates, and altered response to cisplatin. The null mutants
showed increased sensitivity to cisplatin, while mutants overexpressing the
sphingosine kinase resulted in increased resistance compared to the parental
cells. The results indicate that both the SgkA and SgkB enzymes function in
regulating cisplatin sensitivity. The increase in sensitivity of the
sphingosine kinase null mutants was reversed by addition of S-1-P, and the
increased resistance of the sphingosine kinase overexpressor mutant was
reversed by the inhibitor N, N-dimethylsphingosine. Parallel changes in
sensitivity of the null mutants are seen with the platinum based drug
carboplatin, but not with doxorubicin, 5-flurouracil and etoposide. This
pattern of specificity is similar to that observed with the S-1-P lyase
mutants, and should be useful in designing therapeutic schemes involving more
than one drug. This work identifies the sphingosine kinases as new drug
targets for modulating the sensitivity to platinum based drugs.
Submitted by: Hannah Alexander [AlexanderH@missouri.edu]
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Temperature adaptation in Dictyostelium: role of delta 5 fatty acid desaturase
Tamao SAITO1,3, Atsushi KATO1, Hiroshi OCHIAI1 and Naoki MORITA2
1 Division of Biological Sciences, Graduate School of Science, Hokkaido
University, Sapporo, 060-0810 Japan, 2 Research Institute of Genome-based
Biofactory, National Institute of Advanced Industrial Science and Technology
(AIST), Toyohira-ku, Sapporo 062-8517 Japan.
Microbiology, in press
Membrane fluidity is critical for proper membrane function and is regulated
in part by the proportion of unsaturated fatty acids present in the membrane
lipids. The proportion of these lipids in turn varies with temperature and
may contribute to temperature adaptation in poikilothermic organisms. The
fundamental question in this study is whether the unsaturation of fatty acids
contributes to the ability of adaptation to the temperature stress in
Dictyostelium. We firstly analyzed fatty acid composition and detected that
the relative proportions of dienoic acids changed with temperature. In
order to investigate the role of dienoic fatty acids in temperature
adaptation, we have created null mutants in the two known delta 5 fatty acid
desaturases (FadA and FadB) that are responsible for the production of the
dienoic fatty acids. The fadB null mutant showed no significant alteration
in fatty acid composition or in phenotype. However, the disruption of fadA
resulted in a large drop in dienoic fatty acid content from 51.2% to 4.1 %
and a possibly compensatory increase in monoenoic fatty acids
(40.9% to 92.4%). We detected no difference for the temperature adaptation
with that of wild type cells in the growth phase. However, surprisingly
mutant cells develop more efficiently than the wild-type at elevated
temperatures. Our results show that fatty acid composition of Dictyostelium
changes with temperature and suggest that the regulation of dienoic fatty
acid synthesis is involved in the development of Dictyostelium at elevated
temperature but not in the growth.
Submitted by: Tamao Saito [tasaito@sci.hokudai.ac.jp]
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[End Dicty News, volume 23, number 14] 
