dictyNews
Electronic Edition
Volume 38, number 30
November 30, 2012
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Abstracts
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Cyclical action of the WASH complex - FAM21 and Capping Protein
drive WASH recycling not initial recruitment
Laura Park, Peter A. Thomason, Tobias Zech, Jason S. King,
Douwe Veltman, Michael Carnell, Seiji Ura, Laura M. Machesky
and Robert H. Insall
Developmental Cell, in press
WASH causes retrograde traffic and exocytosis. It is found within a
five-protein regulatory complex in vivo, but the physiological roles of
the other members are unknown. Here we present a genetic
analysis of the subunits’ individual functions.
Mutants in each subunit are completely blocked in exocytosis, but
show distinct phenotypes. Loss of SWIP causes delocalisation of
WASH and strumpellin. Surprisingly, no other subunit is essential
for recruitment of the WASH complex to lysosomes. The C-terminal
repeat in FAM21 localises to lysosomes independently of other
complex members. There are therefore multiple localising signals,
acting through different subunits. Loss of Strumpellin and ccdc53
causes worse growth defects than loss of WASH, implying they
have additional roles.
All subunits except FAM21 are required for the complex to drive
actin assembly on lysosomes. Without actin, lysosomes never
recycle V-ATPase or form neutral postlysosomes. However, in
FAM21 knockout lysosomes, WASH generates excessive, dynamic
streams of actin. These successfully remove V-ATPase, forming
huge neutral postlysosomes that are still never exocytosed. The
difference between WASH and FAM21 phenotypes is conserved in
human cancer cells.
Thus FAM21 and WASH act at different steps of a cyclical pathway,
with FAM21 driving a recycling step that allows WASH to be
trafficked back to acidic lysosomes. Recycling is driven by capping
protein, which couples the WASH complex to the dynamic actin
on vesicles through the C-terminus of FAM21.
Submitted by Robert Insall [[log in to unmask]]
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Phosphorylation of Arp2 is required for normal development and cAMP
chemotaxis in Dictyostelium
Chang-Hoon Choi*, Peter A. Thomason*, Mehreen Zaki, Robert H. Insall
and Diane L. Barber
*Authors contributed equally to this work
JBC, in press
Phosphorylation of the Arp2 subunit of the Arp2/3 complex on evolutionarily
conserved threonine and tyrosine residues was recently identified and
shown to be necessary for nucleating activity of the Arp2/3 complex and
membrane protrusion of Drosophila cells. Here we use the Dictyostelium
diploid system to replace the essential Arp2 protein with mutants that cannot
be phosphorylated at T235/6 and Y200. We find that aggregation of the
resulting mutant cells after starvation is substantially slowed, with delayed
early developmental gene expression, and chemotaxis toward a cAMP
gradient is defective, with loss of polarity and attenuated F-actin assembly.
Chemotaxis toward cAMP is also diminished, with reduced cell speed and
directionality and shorter pseudopod lifetime, when Arp2 phosphorylation
mutant cells are allowed to develop longer, to a similar responsive state as
wild-type cells. However, clathrin- mediated endocytosis and chemotaxis
under agar to folate in vegetative cells are only subtly affected in Arp2
phosphorylation mutants. Thus phosphorylation of threonine and tyrosine is
important for a subset of the functions of the Arp2/3 complex, in particular
an unexpected major role in regulating development.
Submitted by Robert Insall [[log in to unmask]]
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[End dictyNews, volume 38, number 30]
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