dictyNews Electronic Edition Volume 38, number 30 November 30, 2012 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. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= 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 subunitsŐ 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 [r.insall@beatson.gla.ac.uk] --------------------------------------------------------------------------- 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 [r.insall@beatson.gla.ac.uk] ============================================================== [End dictyNews, volume 38, number 30]