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dictyNews Volume 31 Number 06
dictyNews
Electronic Edition
Volume 31, number 6
August 8, 2008
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.
Upon publication of your paper, please send strains and plamids to
the Dicty Stock Center. For more information see
http://dictybase.org/StockCenter/Deposit.html.
Back issues of dictyNews, the Dicty Reference database and other
useful information is available at dictyBase - http://dictybase.org.
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Abstracts
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Correlated waves of actin filaments and PIP3 in Dictyostelium cells
Yukako Asano, Akira Nagasaki, and Taro Q. P. Uyeda
National Institute of Advanced Industrial Science and Technology (AIST)
Cell Motility and the Cytoskeleton, in press
Chemotaxis-deficient amiB-null mutant Dictyostelium cells show two distinct
movements: 1) they extend protrusions randomly without net displacements;
2) they migrate persistently and unidirectionally in a keratocyte-like manner.
Here, we monitored the intracellular distribution of phosphatidylinositol
(3,4,5)-trisphosphate (PIP3) to gain insight into roles PIP3 plays in those
spontaneous motilities. In keratocyte-like cells, PIP3 showed convex
distribution over the basal membrane, with no anterior enrichment.
In stalled cells, as well as in wild type cells, PIP3 repeated wave-like
changes, including emergence, expansion and disappearance, on the basal
membrane. The waves induced lamellipodia when they approached the cell
edge, and the advancing speed of the waves was comparable to the migration
speed of the keratocyte-like cells. LY294002, an inhibitor of PI3 kinase,
abolished PIP3 waves in stalled cells and stopped keratocyte-like cells.
These results together suggested that keratocyte-like cells are 'surfing'
on the PIP3 waves by coupling steady lamellipodial protrusions to the PIP3
waves. Simultaneous live observation of actin filaments and PIP3 in wild
type or stalled amiB- cells indicated that the PIP3 waves were correlated
with wave-like distributions of actin filaments. Most notably, PIP3 waves
often followed actin waves, suggesting that PIP3 induces local
depolymerization of actin filaments. Consistent with this idea, cortical
accumulation of PIP3 was often correlated with local retraction of the
periphery. We propose that the waves of PIP3 and actin filaments are
loosely coupled with each other and play important roles in generating
spontaneous cell polarity.
Submitted by: Taro Uyeda [t-uyeda@aist.go.jp]
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Chemotaxis-mediated scission contributes to efficient cytokinesis in
Dictyostelium.
Akira Nagasaki, and Taro Q. P. Uyeda
National Institute of Advanced Industrial Science and Technology (AIST)
Cell Motility and the Cytoskeleton, in press
Interphase amoeba of Entamoeba invadens are attracted to the furrowing
region of a neighboring dividing cell to assist with the division. A
seemingly similar behavior has been observed in Dictyostelium discoideum,
but in this case, it has not been shown whether the movements were truly
directed toward the furrowing region or whether they have any relevance.
We thus used myosin II-null cells, which spend more time than wild type
cells in cytokinesis, and successfully demonstrated that nearly half of the
division events involve the attraction of a neighbor cell to the furrowing
region. Cells lacking the beta subunit of the trimeric G protein (Gbeta),
which are incapable of chemotaxis, did not show such midwifery. Culturing
wild type cells flattened under agarose sheets also slowed the cytokinesis
process, and this allowed us to demonstrate that phosphatidylinositol
trisphosphate was enriched in the anterior region of midwifing cells,
consistent with the view that midwifery in D. discoideum is also chemotaxis.
On substrates, while only 3.6% of wild type cells were multinucleate, 9% of
Gbeta-null cells were multinucleate, and this was reduced to 3.4% when they
were surrounded by wild type cells. Conversely, multinucleated wild type
cells increased to 6.8% when they were surrounded by Gbeta-null cells.
Thus, Gbeta-null cells frequently fail to divide because they cannot assist
each other’s division and midwifery ensures successful cytokinesis in
Dictyostelium discoideum.
Submitted by: Taro Uyeda [t-uyeda@aist.go.jp]
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Cell adhesion molecules regulate contractile ring-independent cytokinesis in
Dictyostelium discoideum.
Akira Nagasaki, Masamitsu Kanada, and Taro Q. P. Uyeda
National Institute of Advanced Industrial Science and Technology (AIST)
Cell Research, in press
To investigate the roles of substrate adhesion in cytokinesis, we established
cell lines lacking paxillin (PAXB) or vinculin (VINA), and those expressing
the respective GFP fusion proteins in Dictyostelium discoideum. As in
mammalian cells, GFP-PAXB and GFP-VINA formed focal adhesion-like
complexes on the cell bottom. paxB- cells in suspension grew normally,
but on substrates, often failed to divide after regression of the furrow. The
efficient cytokinesis of paxB- cells in suspension is not due to shear
forces to assist abscission, since they divided normally in static suspension
culture as well. Double knockout strains lacking mhcA, which codes for
myosin II, and paxB or vinA displayed cytokinetic defects more severe than
each single knockout strain. In mitotic wild-type cells, GFP-PAXB was
diffusely distributed on the basal membrane, but was strikingly condensed
along the polar edges in mitotic mhcA- cells. These results are consistent
with our idea that Dictyostelium expresses two forms of cytokinesis, one
that is contractile ring-dependent and adhesion-independent, and the other
that is contractile ring-independent and adhesion-dependent, and that the
latter requires PAXB and VINA. Furthermore, that paxB- cells fail to divide
more frequently in the presence of substrate adhesion suggests additional
signaling roles of this adhesion molecule.
Submitted by: Taro Uyeda [t-uyeda@aist.go.jp]
--------------------------------------------------------------------------------
PI3-kinase signaling contributes to orientation in shallow gradients and
enhances speed in steep chemoattractant gradients
Leonard Bosgraaf, Ineke Keizer-Gunnink and Peter J.M. Van Haastert
J. Cell Sci, in press
Dictyostelium cells that chemotax toward cAMP produce phosphatidylinositol
(3,4,5) trisphosphate (PIP3) at the leading edge, which has been implicated
in actin reorganization and pseudopod extension. However, in the absence of
PIP3 signaling cells will chemotax via alternative pathways. Here we examined
the potential contribution of PIP3 to chemotaxis of wild type cells. The
results show that steep cAMP gradients (larger than 10% concentration
difference across the cell) induce strong PIP3 patches at the leading edge,
which has little effect on the orientation but strongly enhances the speed
of the cell. Using a new sensitive method for PIP3 detection that corrects
for the volume of cytosol in pixels at the boundary of the cell, we show
that in shallow cAMP gradient (less than 5% concentration difference across
the cell) PIP3 is still somewhat enriched at the leading edge. Cells lacking
PI3-kinase activity exhibit poor chemotaxis in these shallow gradients. Due
to the reduced speed and orientation in steep and shallow gradients,
respectively, cells lacking PIP3 signaling require 2 to 6 fold longer times
to reach a point source of chemoattractant compared to wild type cells.
These results show that although PI3K signaling is dispensable for
chemotaxis, it gives the wild type an advantage over mutant cells.
Submitted by: Peter Van Haastert [P.J.M.van.Haastert@rug.nl]
--------------------------------------------------------------------------------
Sorting of the v-SNARE VAMP7 in Dictyostelium discoideum: a role for more
than one Adaptor Protein (AP) complex.
Nelly Bennett a*, François Letourneur b, Michel Ragno a and Mathilde Louwagie c.
a Commissariat a l’Energie Atomique, iRTSV, Laboratoire de Biochimie et
Biophysique des Systemes Integres, CNRS UMR5092, Universite Joseph Fourier,
38054 Grenoble, France.
b Institut de Biologie et Chimie des Proteines, UMR5086-CNRS/Universite Lyon I,
IFR 128 BioSciences Lyon-Gerland, 69367 Lyon, France.
c Commissariat a l’Energie Atomique, iRTSV, Laboratoire d’Etude de la
Dynamique des Proteomes, INSERM U880, Universite Joseph Fourier, 38054
Grenoble, France.
* Corresponding author: nelly.bennett@cea.fr
Experimental Cell Research, in press
Soluble N-ethylmaleimide-sensitive-factor Attachment protein Receptors
(SNAREs) participate in the specificity of membrane fusions in the cell.
The mechanisms of specific SNARE sorting are still however poorly documented.
We investigated the possible role of Adaptor Protein (AP) complexes in sorting
of the Dictyostelium discoideum v-SNARE VAMP7. In live cells, GFP-VAMP7 is
observed in the membrane of endocytic compartments. It is also observed in
the plasma membrane of a small proportion of the cells. Mutation of a
potential dileucine motif dramatically increases the proportion of cells with
GFP-VAMP7 in their plasma membrane, strongly supporting the participation
of an AP complex in VAMP7 sorting to the endocytic pathway. A partial increase
occurs in knockout cells for the medium subunits of AP-2 and AP-3 complexes,
indicating a role for both AP-2 and AP-3. VAMP7, as well as its t-SNAREs
partners syntaxin 8 and Vti1, are co-immunoprecipitated with each of the
medium subunits of the AP-1, AP-2, AP-3 and AP-4 complexes. This result
supports the conclusion that VAMP7 directly interacts with both AP-2 and AP-3.
It also raises the hypothesis of an interaction with AP-1 and AP-4. GFP-VAMP7
is retrieved from the endocytic pathway at and/or before the late
post-lysosomal stage through an AP-independent mechanism.
Submitted by: Nelly Bennett [nelly.bennett@cea.fr]
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[End dictyNews, volume 31, number 6]
