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dictyNews Volume 16 Number 11
Dicty News
Electronic Edition
Volume 16, number 11
May 26, 2001
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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Ammonia Differentially Suppresses the cAMP Chemotaxis of Anterior-Like
Cells and Prestalk Cells in Dictyostelium discoideum
Ira N. Feit, Erika J. Medynski, and Michael J. Rothrock
Department of Biology, Franklin and Marshall College, Lancaster, PA 17603
Journal of Biosciences, In Press
A drop assay for chemotaxis to cAMP confirms that both anterior-like cells
(ALC) and prestalk cells (pst cells) respond to cAMP gradients. We present
evidence that the chemotactic response of both ALC and pst cells is
suppressed by ammonia, but a higher concentration of ammonia is required
to suppress the response in pst cells.
We also confirm that ALC show a chemotactic response to cAMP when
moving on a substratum of prespore cells in isolated slug posteriors
incubated under oxygen. ALC chemotaxis on a prespore cell substratum is
suppressed by the same concentration of ammonia that suppresses ALC
chemotaxis on the agar substratum of the drop assays. Chemotaxis
suppression is mediated by the unprotonated (NH3) species of ammonia.
The observed suppression, by ammonia, of ALC chemotaxis to cAMP
supports our earlier hypothesis that ammonia is the tip-produced
suppressor of such chemotaxis. We discuss implications of ammonia
sensitivity of pst cells and ALC with regard to the movement and
localization of ALC and pst cells in the slug and to the roles played
by ALC in fruiting body formation. In addition, we suggest that a
progressive decrease in sensitivity to ammonia is an important part
of the maturation of ALC into pst cells.
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Sequential Activities of PI 3-Kinase, PKB/Akt and Rab7 during Macropinosome
Formation in Dictyostelium
Adam Rupper1, Kyung Lee3, David Knecht3 and James Cardelli1,2
1Department of Microbiology and Immunology
2Feist/Weiller Cancer Center
LSU Health Sciences Center
Shreveport, LA 71130
318-675-5756
jcarde@lsuhsc.edu
3Department of Molecular and Cellular Biology
University of Connecticut
Storrs, CT 06269-3125
Molecular Biology of the Cell, in press
Abstract
Macropinocytosis plays an important role in the internalization of
antigens by dendritic cells, and is the route of entry for many bacterial
pathogens; however, little is known about the molecular mechanisms that
regulate the formation or maturation of macropinosomes. Like dendritic cells,
Dictyostelium amoebae are active in macropinocytosis, and a variety of proteins
have been identified that contribute to this process. As described here,
microscopic analysis of null mutants have revealed that the Class I PI 3-kinases,
PIK1 and PIK2, and the downstream effector PKB/AKT are important in regulating
completion of macropinocytosis. Although actin-rich membrane protrusions form
in these cell-lines, they recede without forming macropinosomes. Imaging of
cells expressing GFP fused to the pleckstrin homology domain (PH) of PKB
indicates that D3 phosphoinositides are enriched in the forming macropinocytic
cup, and remain associated with newly formed macropinosomes for less than
one minute. A fusion protein, consisting of GFP fused to an F-actin binding
domain, overlaps with GFP-PHPKB in the timing of association with forming
macropinosomes. Although macropinocytosis is reduced in cells expressing
dominant negative Rab7, microscopic imaging studies reveal that GFP-Rab7
associates only with formed macropinosomes at approximately the time that
F-actin and D3 phosphoinositide levels decrease. These results support a
model in which F-actin modulating proteins and vesicle trafficking proteins
coordinately regulate the formation and maturation of macropinosomes.
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A cell number-counting factor regulates group size in Dictyostelium by
differentially modulating cAMP-induced cAMP and cGMP pulse sizes
Lei Tang, Robin Amman, Tong Gao, and Richard H. Gomer
Howard Hughes Medical Institute and Department of Biochemistry and Cell
Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX
77005-1892
Journal of Biological Chemistry, in press; ms available at
http://www.jbc.org/cgi/reprint/M102205200v1
Abstract
A secreted counting factor (CF), regulates the size of Dictyostelium
discoideum fruiting bodies in part by regulating cell-cell adhesion.
Aggregation and the expression of adhesion molecules is mediated by relayed
pulses of cAMP. Cells also respond to cAMP with a short cGMP pulse. We
find that CF slowly down-regulates the cAMP-induced cGMP pulse by
inhibiting guanylyl cyclase activity. A one-minute exposure of cells to
purified CF increases the cAMP-induced cAMP pulse. CF does not affect the
cAMP receptor or its interaction with its associated G proteins, or the
translocation of the cytosolic regulator of adenylyl cyclase (CRAC) to the
membrane in response to cAMP. Pulsing streaming wild-type cells with a
high concentration of cAMP results in the formation of small groups, while
reducing cAMP pulse size with exogenous cAMP phosphodiesterase during
stream formation causes cells to form large groups. Altering the
extracellular cAMP pulse size does not phenocopy the effects of CF on the
cAMP-induced cGMP pulse size or cell-cell adhesion, indicating that CF does
not regulate cGMP pulses and adhesion via CF's effects on cAMP pulses. The
results suggest that regulating either cell-cell adhesion, the cGMP, or the
cAMP pulse size can control group size, and that CF regulates all three of
these independently.
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On the problem of establishing the subcellular localization of
Dictyostelium retrotransposon TRE5-A proteins by biochemical analysis of
nuclear extracts
Ulrich Hentschel, Ilse Zuendorf, Theodor Dingermann and Thomas Winckler
Institut fuer Pharmazeutische Biologie, Universitaet Frankfurt/M.
(Biozentrum), Frankfurt am Main, Germany
Analytical Biochemistry, in press
Abstract:
At first sight a protein that is enriched in extracts prepared from
nuclei by means of biochemical methods can be considered to be a nuclear
protein in vivo. Although this assumption will hold true for most of the
analysed proteins, it could also lead to false interpretations. We
analysed the subcellular distribution of endogenous and plasmid-borne
proteins derived from the retrotransposon TRE5-A of Dictyostelium
discoideum. In biochemical fractionation experiments the proteins
encoded by TRE5-A open reading frame 1 (ORF1p) and the putative
endonuclease encoded in ORF2 (ENp) were found in the detergent-insoluble
material containing the nuclei. However, salt extraction of isolated
nuclei did not considerably release the TRE5-A proteins. Instead, the
TRE5-A proteins were strongly enriched in a fraction that contained the
chromosomal DNA after removal of most cytoskeletal and histone proteins.
These observations implied that ORF1p and ENp were both attached to
chromatin in vivo, but this conclusion was disproved by the expression
of genetic fusions of green fluorescent protein with either ORF1p or
ENp. We show conclusive evidence that both fusion proteins were located
as large aggregates of native protein in the cytoplasm of D. discoideum
cells.
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Supriya Srinivasan*, Mathew TrainiÝ, Ben HerbertÝ, Diane SextonÝ, Jenny
HarryÝ, Hannah Alexander*, Keith L. WilliamsÝ and Stephen Alexander*
* Division of Biological Sciences, 303 Tucker Hall, University of Missouri,
Columbia, MO 65211, USA
Ý Proteome Systems Limited, 1/35-41 Waterloo Road, North Ryde, Sydney,
NSW 2113, Australia
Proteomics, In Press
SUMMARY
Secretion of spore coat proteins from the prespore secretory vesicles (PSVs)
in Dictyostelium discoideum is a signal mediated event that underlies
terminal cell differentiation, and represents an important case of
developmentally regulated secretion. In order to study the biochemical
mechanisms that govern the regulated fusion of the PSVs with the plasma
membrane and the subsequent secretion of their cargo, we purified this
organelle from prespore cells. Analysis of protein extracts of highly
purified PSVs indicated that, in addition to the cargo of structural spore
coat proteins, many more proteins are associated with the PSVs. Their
identification is paramount to the understanding of the mechanism of
regulated secretion in this system. In this study we have taken the
first comprehensive proteomic approach to the analysis of an entire -
previously uncharacterized - organelle, with the goal of identifying
the major proteins associated with the PSVs. We show that in addition
to the structural spore coat proteins, the PSVs contain the enzymes
needed for proper spore coat assembly (thioredoxin 2 and 3), regulatory
proteins which we predict receive and transduce the developmental signal
for secretion (rab7 GTPase, PI-3 kinase, NDP kinase and the calcium binding
proteins calfumirin-1 and calreticulin) as well as proteins that interact
with the cytoskeleton to mediate movement of the PSVs to the plasma membrane
(actin binding proteins coactosin and profilin 1). In addition, the results
suggest that proteins can play multiple roles in the cell, and that protein
function can be dictated in part by subcellular localization. The
identification of the PSV proteins is allowing us to develop testable
hypotheses about the roles of these proteins within the functional context
of developmentally regulated secretion.
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A Rab11-like GTPase Associates with and Regulates the Structure and Function
of the Contractile Vacuole System in Dictyostelium.
Edward Harris*, Kunito Yoshida, James Cardelli* and John Bush+
+Department of Biology
University of Arkansas at Little Rock
Little Rock, AR 72204
* Department of Microbiology and Immunology
LSU Health Sciences Center
Shreveport, LA 71130.
+ Corresponding author:
JOURNAL OF CELL SCIENCE, in press
ABSTRACT
Screening of a cDNA library revealed the existence of a Dictyostelium cDNA
encoding a protein 80% identical at the amino acid level to mammalian Rab11.
Subcellular fractionation and immunofluorescence studies revealed that
DdRab11 was exclusively associated with the ATPase proton pump-rich
contractile vacuole membrane system, consisting of a reticular network
and bladder-like vacuoles. Video microscopy of cells expressing GFP-DdRab11
revealed that this Rab was associated with contractile vacuolar bladders
undergoing formation, fusion, and expulsion of water. The association of
DdRab11 with contractile vacuole membranes was disrupted when cells were
exposed to either hypo-osmotic conditions or an inhibitor of the ATPase
proton pump. Cells over-expressing a dominant negative form of DdRab11
were analyzed biochemically and microscopically to measure changes in the
structure and function of the contractile vacuole system. Compared to
wild-type cells, the dominant negative DdRab11 expressing cells contained
a more extensive contractile vacuole network and abnormally enlarged
contractile vacuole bladders, most likely the result of defects in membrane
trafficking. Also, the mutant cells enlarged, detached from surfaces, and
contained large vacuoles when exposed to water, suggesting a functional
defect in osmotic regulation. No changes were observed in mutant cells in
the rate of fluid phase internalization or release, suggesting the
DdRab11-mediated membrane trafficking defects were not general in nature.
Surprisingly, the rate of phagocytosis was increased in the dominant
negative DdRab11 expressing cells as compared to control cells. Our results
are consistent with a role for DdRab11 in regulating membrane traffic to
maintain the normal morphology and function of the contractile vacuole.
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[End Dicty News, volume 16, number 11]
