dictyNews
Electronic Edition
Volume 33, number 10
October 16, 2009

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Abstracts
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Roles of an Unconventional Protein Kinase and  Myosin II in
Amoeba Osmotic Shock Responses

Venkaiah Betapudi and Thomas T. Egelhoff*


Traffic, in press

The contractile vacuole (CV) is a dynamic organelle that enables
Dictyostelium amoeba and other protist to maintain osmotic homeostasis
by expelling excess water. In the present study, we have uncovered a
mechanism that coordinates the mechanics of the CV with myosin II,
regulated by VwkA, an unconventional protein kinase that is conserved
in an array of protozoa. GFP-VwkA fusion proteins localize persistently
to the CV during both filling and expulsion phases of water. In vwkA  
null
cells, the established CV marker dajumin still localizes to the CV, but
these structures are large, spherical, and severely impaired for  
discharge.
Furthermore, myosin II cortical localization and assembly are abnormal  
in
vwkA null cells. Parallel analysis of wild type cells treated with  
myosin II
inhibitors or of myosin II null cells also results in enlarged CVs with
impaired dynamics. We suggest that the myosin II cortical cytoskeleton,
regulated by VwkA, serves a critical conserved role in the periodic
contractions of the CV, as part of the osmotic protective mechanism
of protozoa.


Submitted by Venkaiah Betapudi & Thomas Eegelhoff [[log in to unmask]]
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Functional characterisation of intracellular Dictyostelium discoideum
P2X receptors

Melanie J. Ludlow, Latha Durai and Steven J. Ennion

University of Leicester, United Kingdom


JBC (http://www.jbc.org/content/early/2009/10/15/jbc.M109.045674)

Indicative of cell surface P2X ion channel activation, extracellular ATP
evokes a rapid and transient calcium influx in the model eukaryote
Dictyostelium discoideum. Five P2X-like proteins (dP2XA-E) are present
in this organism. However, their roles in purinergic signalling are  
unclear
since dP2XA proved to have an intracellular localisation on the  
contractile
vacuole where it is thought to be required for osmoregulation. To  
determine
functional properties of the remaining four dP2X-like proteins and to  
assess
their cellular roles, we recorded membrane currents from expressed  
cloned
receptors and generated a quintuple knockout Dictyostelium strain  
devoid of
dP2X receptors. ATP evoked inward currents at dP2XB and dP2XE receptors
but not at dP2XC or dP2XD. β,γ-imido-ATP was more potent than ATP at
dP2XB but a weak partial agonist at dP2XE. Currents in dP2XB and dP2XE
were strongly inhibited by Na+ but insensitive to copper and the P2  
receptor
antagonists PPADS and suramin. Unusual for P2X channels, dP2XA and
dP2XB were also Cl- permeable. The extracellular purinergic response to
ATP persisted in p2xA/B/C/D/E quintuple knockout Dictyostelium
demonstrating that dP2X channels are not responsible. dP2XB,C,D, and E
were found to be intracellular localised to the contractile vacuole  
with the
ligand binding domain facing the lumen. However, quintuple p2xA/B/C/D/E
null cells were still capable of regulating cell volume in water  
demonstrating
that, contrary to previous findings, dP2X receptors are not required for
osmoregulation. Responses to the calmodulin antagonist calmidazolium
however were reduced in p2xA/B/C/D/E null cells suggesting that dP2X
receptors play a role in intracellular calcium signalling.


Submitted by Steven Ennion [[log in to unmask]
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[End dictyNews, volume 33, number 10]