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dictyNews Volume 38 Number 05
dictyNews
Electronic Edition
Volume 38, number 5
February 17, 2012
Please submit abstracts of your papers as soon as they have been
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Abstracts
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Rab8a regulates the exocyst-mediated kiss-and-run discharge of
the Dictyostelium contractile vacuole
Miriam Essid, Navin Gopaldass, Kunito Yoshida, Christien Merrifield,
and Thierry Soldati
Molecular Biology of the Cell, in press
Water expulsion by the contractile vacuole in Dictyostelium is carried
out by a giant kiss-and-run focal exocytic event during which the two
membranes are only transiently connected but do not completely merge.
We present a molecular dissection of the GTPase Rab8a and the
exocyst complex in tethering of the contractile vacuole to the plasma
membrane, fusion and final detachment. Right before discharge, the
contractile vacuole bladder sequentially recruits Drainin, a
Rab11a-effector, Rab8a, the exocyst complex and LvsA, a protein of
the Chediak-Higashi family. Rab8a recruitment precedes the
nucleotide-dependent arrival of the exocyst to the bladder by a few
seconds. A dominant-negative mutant of Rab8a strongly binds to the
exocyst and prevents recruitment to the bladder suggesting that a
Rab8a GEF activity is associated with the complex. Absence of Drainin
leads to over-tethering and blocks fusion, while expression of
constitutively active Rab8a allows fusion but blocks vacuole detachment
from the plasma membrane, inducing complete fragmentation of
tethered vacuoles. An indistinguishable phenotype is generated in cells
lacking LvsA, implicating this protein in post-fusion de-tethering.
Interestingly, overexpression of a constitutively active Rab8a mutant
reverses the lvsA-null CV phenotype.
Submitted by Thierry Soldati [thierry.soldati@unige.ch]
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Bestatin inhibits cell growth, division and spore cell differentiation
in Dictyostelium
Yekaterina Poloz1 , Andrew Catalano1 and Danton H. OÕDay1,2
1Department of Cell and Systems Biology, University of Toronto,
25 Harbord Street, Toronto, ON, Canada M5S 3G5
2Department of Biology, University of Toronto Mississauga,
3359 Mississauga Road North, Mississauga, ON, Canada L5L 1C6
Eukaryotic Cell, In press
Bestatin methyl ester (BME) is an inhibitor of Zn2+-binding
aminopeptidases that inhibits cell proliferation and induces apoptosis
in normal and cancer cells. We have used Dictyostelium as a model
organism to study the effects of BME. Only two Zn2+-binding
aminopeptidases have been identified in Dictyostelium to date,
puromycin sensitive aminopeptidase A and B (PsaA and PsaB). PSA
from other organisms is known to regulate cell division and
differentiation. Here we showed that PsaA is differentially expressed
throughout growth and development of Dictyostelium and its
expression is regulated by developmental morphogens. We present
evidence that BME specifically interacts with PsaA and inhibits its
aminopeptidase activity. Treatment of cells with BME inhibited the
rate of cell growth and the frequency of cell division in growing cells
and inhibited spore cell differentiation during late development.
Overexpression of PsaA-GFP also inhibited spore cell differentiation
but did not affect growth. Using chimeras, we have identified that
nuclear versus cytoplasmic localization of PsaA affects the choice
between stalk or spore cell differentiation pathway. Cells that
overexpressed PsaA-GFP (primarily nuclear) differentiated into stalk
cells, while cells that overexpressed PsaADeltaNLS2-GFP
(cytoplasmic) differentiated into spores. In conclusion, we have
identified that BME inhibits cell growth, division and differentiation
in Dictyostelium likely through inhibition of PsaA.
Submitted by: Danton OÕDay [danton.oday@utoronto.ca]
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CyrA, a matricellular protein that modulates cell motility in
Dictyostelium discoideum
Robert J. Huber1, Andres Suarez1, and Danton H. OÕDay1,2
1Department of Cell and Systems Biology, University of Toronto,
25 Harbord Street, Toronto, ON, Canada M5S 3G5
2Department of Biology, University of Toronto Mississauga,
3359 Mississauga Road North, Mississauga, ON, Canada L5L 1C6
Matrix Biology, in press
CyrA, an extracellular matrix (slime sheath), calmodulin (CaM)-binding
protein in Dictyostelium discoideum, possesses four tandem EGF-like
repeats in its C-terminus and is proteolytically cleaved during asexual
development. A previous study reported the expression and localization
of CyrA cleavage products CyrA-C45 and CyrA-C40. In this study, an
N-terminal antibody was produced that detected the full-length 63 kDa
protein (CyrA-C63). Western blot analyses showed that the intracellular
expression of CyrA-C63 peaked between 12 and 16 hours of
development, consistent with the time that cells are developing into a
motile, multicellular slug. CyrA immunolocalization and CyrA-GFP
showed that the protein localized to the endoplasmic reticulum,
particularly its perinuclear component. CyrA-C63 secretion began
shortly after the onset of starvation peaking between 8 and 16 hours
of development. A pharmacological analysis showed that CyrA-C63
secretion was dependent on intracellular Ca2+ release and active CaM,
PI3K, and PLA2. CyrA-C63 bound to CaM both intra- and extracellularly
and both proteins were detected in the slime sheath deposited by
migrating slugs. In keeping with its purported function, CyrA-GFP over-
expression enhanced cAMP-mediated chemotaxis and CyrA-C45 was
detected in vinculin B (VinB)-GFP immunoprecipitates, thus providing a
link between the increase in chemotaxis and a specific cytoskeletal
component. Finally, DdEGFL1-FITC was detected on the membranes
of cells capped with concanavalin A suggesting that a receptor exists
for this peptide sequence. Together with previous studies, the data
presented here suggests that CyrA is a bona fide matricellular protein
in Dictyostelium discoideum.
Submitted by: Danton OÕDay [danton.oday@utoronto.ca]
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TM9/Phg1 and SadA proteins control surface expression and stability
of SibA adhesion molecules in Dictyostelium.
Froquet R, le Coadic M, Perrin J, Cherix N, Cornillon S, Cosson P.
Dpartement de Physiologie Cellulaire et Metabolisme, Centre
Medical Universitaire, 1 rue Michel Servet, 1211 Geneva 4, Switzerland
Mol Biol Cell, in press
TM9 proteins form a family of conserved proteins with nine
transmembrane domains essential for cellular adhesion in many
biological systems, but their exact role in this process remains
unknown. Here we found that in Dictyostelium amoebae, genetic
inactivation of the TM9 protein Phg1A dramatically decreases the
surface levels of the SibA adhesion molecule. This is due to a decrease
in sibA mRNA levels, in SibA protein stability, and in SibA targeting to
the cell surface. A similar phenotype was observed in cells devoid of
SadA, a protein that does not belong to the TM9 family but also
exhibits 9 transmembrane domains and is essential for cellular
adhesion. A csA-SibA chimeric protein comprising only the
transmembrane and cytosolic domains of SibA and the extracellular
domain of csA, a Dictyostelium surface protein, also showed reduced
stability and relocalization to endocytic compartments in phg1A knockout
cells. These results indicate that TM9 proteins participate in cell adhesion
by controlling the levels of adhesion proteins present at the cell surface.
Submitted by Pierre Cosson [Pierre.Cosson@unige.ch]
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[End dictyNews, volume 38, number 5]
