dictyNews
Electronic Edition
Volume 38, number 5
February 17, 2012

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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 [[log in to unmask]]
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Bestatin inhibits cell growth, division and spore cell differentiation 
in Dictyostelium 

Yekaterina Poloz1 , Andrew Catalano1 and Danton H. O’Day1,2

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 O’Day [[log in to unmask]]
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CyrA, a matricellular protein that modulates cell motility in 
Dictyostelium discoideum

Robert J. Huber1, Andres Suarez1, and Danton H. O’Day1,2

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 O’Day [[log in to unmask]]
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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.

Département de Physiologie Cellulaire et Métabolisme, Centre 
Médical 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 [[log in to unmask]]
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[End dictyNews, volume 38, number 5]