Dicty News
Electronic Edition
Volume 19, number 10
November 15, 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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Abstracts
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Dynamic organization of the actin system in the motile cells of
Dictyostelium
Till Bretschneider +1, James Jonkman +2, Jana Khler +1, Ohad Medalia +1,
Karmela Barisic +1,4, Igor Weber +3, Ernst H.K. Stelzer +2, Wolfgang
Baumeister +1 and G. Gerisch +1
+1 Max-Planck-Institut fr Biochemie, D-82152 Martinsried, Germany;
+2 European Molecular Biology Laboratory,
Meyerhofstrasse 1, D-69117 Heidelberg, Germany;
+3 Rudjer Boskovic Institut, Bijenicka cesta 54, 10000 Zagreb, Croatia;
+4 Permanent address: University of Zagreb, Faculty of Pharmacy and
Biochemistry, Department of Medical Biochemistry and Hematology,
Domagojeva 2, 10000 Zagreb, Croatia.
Journal of Muscle Research and Cell Motility, Special Issue: Dictyostelium,
Ed. Dietmar J. Manstein, in press
Abstract
The actin system forms a supramolecular, membrane-associated network that
serves multiple functions in Dictyostelium cells, including cell motility
controlled by chemoattractant, phagocytosis, macropinocytosis, and
cytokinesis. In executing these functions the monomeric G-actin polymerizes
reversibly, and the actin filaments are assembled into membrane-anchored
networks together with other proteins involved in shaping the networks and
controlling their dynamics. Most impressive is the speed at which actin-
based structures are built, reorganized, or disassembled. We used GFP-
tagged coronin and Arp3, an intrinsic constituent of the Arp2/3 complex,
as examples of proteins that are recruited to highly dynamic actin-
filament networks. By fluorescence recovery after photobleaching (FRAP),
average exchange rates of cell-cortex bound coronin were estimated. A
nominal value of 5 seconds for half-maximal incorporation of coronin into
the cortex, and a value of 7 seconds for half-maximal dissociation from
cortical binding sites has been obtained. Actin dynamics implies also flow
of F-actin from sites of polymerization to sites of depolymerization, i.e.
to the tail of a migrating cell, the base of a phagocytic cup, and the
cleavage furrow in a mitotic cell. To monitor this flow, we expressed in
Dictyostelium cells a GFP-tagged actin-binding fragment of talin. This
fragment (GFP-TalC63) translocates from the front to the tail during cell
migration and from the polar regions to the cleavage furrow during mitotic
cell division. The intrinsic dynamics of the actin system can be manipulated
in vivo by drugs or other probes that act either as inhibitors of actin
polymerization or as stabilizers of filamentous actin. In order to
investigate structure-function relationships in the actin system, a
technique of reliably arresting transient network structures is in demand.
We discuss the potential of electron tomography of vitrified cells to
visualize actin networks in their native association with membranes.
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Multiple activities of a novel substance, dictyopyrone C isolated from
Dictyostelium discoideum, in cellular growth and differentiation
Y. Maeda1*, H. Kikuchi2*, K. Sasaki1, A. Amagai1, J. Sekiya2, Y. Takaya3 and
Y. Oshima2
1Department of Developmental Biology and Neurosciences, Graduate School of
Life Sciences , Tohoku University, Aoba, Sendai 980-8578, 2Graduate School
of Pharmaceutical Sciences, Tohoku University, Aoba, Sendai 980-8578, and
3Faculty of Pharmacy, Meijo University, Yagotoyama, Tempaku, Nagoya 468-8503
(Japan)
*equal contribution
Protoplasma (in press)
Summary.
We report here that a novel substance named dictyopyrone C(DPC) has
remarkable effects on growth and differentiation of D. discoideum Ax-2
cells, in a dose-dependent manner. In the presence of 3-15M DPC,
differentiation of starving Ax-2 (clone MS) cells was greatly enhanced
under submerged conditions, when vegetative MS-cells were harvested at
the mid-late exponential growth phase (3 X 106 cells/ml) and starved.
In contrast, DPC above 30M markedly impaired the progression of
differentiation including cell aggregation, most of starved cells being
rounded in shape 3-4 h after DPC-application and then lysed during further
incubation. In the presence of 30M DPC however, MS-cells that had been
harvested at the early exponential growth phase (5 X 105 cells/ml) and
starved were neither rounded in shape nor lysed, and exhibited rather
enhanced differentiation. Essentially the same results were obtained in
cultures of starved cells on nonnutrient agar. With respect to DPC-effect
on MS-cells growing in axenic medium, cell lysis and growth inhibition by
more than 15M DPC were realized in the mid-late exponential growth phase
cells (3 X 106 cells/ml), but not in the early exponential growth phase
cells (5 X 105 cells/ml). Moreover, analysis using synchronized MS-cells
has demonstrated that the DPC-effect changes in a cell-cycle dependent
manner. In contrast to such unique DPC-actions, the pyrone ring of DPC
had no effects on growth and differentiation within a range of 3-120M
tested. These findings have strongly suggested the importance of a combined
structure of the pyrone ring (PDP) and the linear carbon chain in revelation
of the DPC activities.
submitted by: Yasuo MAEDA [ymaeda@mail.cc.tohoku.ac.jp]
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Cytoskeleton Interactions Involved in the Assembly and Function of
Glycoprotein-80 Adhesion Complexes in Dictyostelium
Tony J. C. Harris*, Amir Ravandi*, Donald E. Awrey and Chi-Hung Siu*
*Banting and Best Department of Medical Research and Department of
Biochemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada,
Affinium Pharmaceuticals, 100 University Avenue, Toronto, Ontario M5J 1V6,
Canada.
J. Biol. Chem. (in press.)
SUMMARY
Adhesion complexes typically assemble from clustered receptors that link to
the cytoskeleton via cytoplasmic adapter proteins. However, it is unclear how
phospholipid-anchored adhesion molecules, such as the Dictyostelium receptor
gp80, interact with the cytoskeleton. gp80 has been found to form adhesion
complexes from raft-like membrane domains, which can be isolated as a Triton
X-100-insoluble floating fraction (TIFF). We report here that the actin-
binding protein ponticulin mediates TIFF-cytoskeleton interactions. Analysis
of gp80-null cells revealed that these interactions were minimal in the
absence of gp80. During development, gp80 was required to enhance these
interactions as its adhesion complexes assembled. While ponticulin and
gp80 could partition independently into TIFF, gp80 was shown to recruit
ponticulin to cell-cell contacts and to increase its partitioning into
TIFF. However, these proteins did not co-immunoprecipitate. Furthermore,
sterol sequestration abrogated the association of ponticulin with TIFF
without affecting gp80, suggesting that sterols may mediate the interactions
between ponticulin and gp80. In ponticulin-null cells, large gp80 adhesion
complexes assembled in the absence of ponticulin despite the lack of
cytoskeleton association. We propose that such nascent gp80 adhesion
complexes produce expanded raft-like domains that recruit ponticulin and
thereby establish stable cytoskeleton interactions to complete the assembly
process.
submited by: chi.hung.siu@utoronto.ca
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Reciprocal Raft-Receptor Interactions and the Assembly of Adhesion Complexes
Tony J. C. Harris and Chi-Hung Siu*
Banting and Best Department of Medical Research and Department of
Biochemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
BioEssays (in press)
Summary
Cell adhesion complexes are critical for the physical coordination of cell-
cell interactions and the morphogenesis of tissues and organs. Many adhesion
receptors are anchored to the plasma membrane by a
glycosylphosphatidylinositol (GPI) moiety and are thereby partitioned into
membrane rafts. In this review, we focus on reciprocal interactions between
rafts and adhesion molecule, leading to receptor clustering and raft
expansion and stability. A model for a 3-stage adhesion complex assembly
process is also proposed. First, GPI-anchored adhesion molecules are
recruited into rafts, which in turn promote receptor cis-oligomerization
and thereby produce precursory complexes primed for avid trans-interactions.
Second, trans-interactions of the receptors cross-link and stabilize large
amalgams of rafts at sites of adhesion complex assembly. Finally, the
enlarged and stabilized rafts acquire enhanced abilities to recruit the
cytoskeleton and induce signaling. This process exemplifies how the domain
structure of the plasma membrane can impact the function of its receptors.
submitted by: chi.hung.siu@utoronto.ca
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Mutations in the relay loop region result in dominant-negative inhibition of
myosin II function in Dictyostelium
Georgios Tsiavaliaris, Setsuko Fujita-Becker, Renu Batra, Dmitrii I.
Levitsky 1, F. Jon Kull 2, Michael A. Geeves 3 & Dietmar J. Manstein +
Max-Planck-Institut fr medizinische Forschung, Jahnstrasse 29, D-69120
Heidelberg, Germany, 1 A.N.Bach Institute of Biochemistry, Russian Academy
of Science, Moscow 119071, Russia, 2 Dartmouth College, 6128 Burke
Laboratory, Hanover, NH 03755, USA, 3 Department of Biosciences, University
of Kent, Canterbury, Kent CT2 7NJ, UK
EMBO Reports, in press
Dominant-negative inhibition is a powerful genetic tool for the
characterization of gene function in vivo, based on the specific impairment
of a gene product by the coexpression of a mutant version of the same gene
product. Here we describe the detailed characterization of two myosin
constructs containing either point mutations F487A or F506G in the relay
region. Dictyostelium cells transformed with F487A or F506G myosin are
unable to undergo processes that require myosin II function including
fruiting-body formation, normal cytokinesis and growth in suspension. Our
results show that the dominant-negative inhibition of myosin function is
caused by disruption of the communication between active site and lever
arm, which blocks motor activity completely, and perturbation of the
communication between active site and actin-binding site, leading to an
approximately 100-fold increase in the mutants affinity for actin in the
presence of ATP.
submitted by: Manstein.Dietmar@mh-hannover.de
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[End Dicty News, volume 19, number 10]