Drosophila Vinculin Aggregation Assay

Drosophila melanogaster stocks used in this study: Mef2::Gal4, Act5C::Gal4, UAS::GFP-Lamp1, UAS::eGFP-huLC3, UAS::myr-GFP, UAS::mito-GFP (all sourced from the Bloomington Drosophila Stock Center, Indiana University); ΔVinc, βPS-GFP, GFP-talin (all Klapholz et al., 2015 (link)); mys^XG43 (Jannuzi et al., 2002 (link)); pax^Δ1 (Bataille et al., 2010 (link)); talin-mCherry (Venken et al., 2011 (link)); UAS::talin (Tanentzapf et al., 2006 (link)); UAS::GFP-IBS2 (Ellis et al., 2011 (link)); paxillin-GFP (Bataille et al., 2010 (link)); UAS::paxillin (Vakaloglou et al., 2012 (link)); tensin-GFP (Torgler et al., 2004 (link)); ILK-YFP (unpublished, made as ILK-GFP in Zervas et al., 2001 (link)); PINCH-GFP (Kadrmas et al., 2004 (link)); Fermitin1-GFP (a genomic fragment, −1579 to +3942 relative to the ATG, with mGFP6 inserted following residue 228 and with three-serine linkers on each side; a gift of Danelle Devenport, The Gurdon Institute, University of Cambridge, UK). To misexpress vinculin constructs, the UAS-Gal4 system was used (Brand and Perrimon, 1993 (link)). To remove maternal and zygotic contributions of talin and paxillin, the dominant female sterile technique was used (Chou and Perrimon, 1996 (link)). To generate follicle cell clones lacking βPS and expressing Vinc-CO-RFP, the MARCM system was used (Lee and Luo, 1999 (link)). hsFlp, Tub-Gal80^ts, FRT19A; Tub-Gal4, UAS cd8GFP/CyO flies were crossed to mys^XG43, FRT19A; UAS Vinc-CO-RFP/CyO flies, pupae were heat shocked for 2 h in a 37°C water bath, and non-CyO adult females selected and dissected. rhea and pax mutant clones expressing Vinc-CO were generated in the same manner with different MARCM lines for each FRT (FRT2A and FRT40A, respectively). To test the domains of talin required for vinc-CO aggregation, we employed a series of rhea deletions as well as a null allele rescued by construct lacking the head or having the internal deletion in the rod (Klapholz et al., 2015 (link)). For controls, wild-type FRT chromosomes were used in place of mutant chromosomes. To generate Flp-out clones (Struhl and Basler, 1993 (link)) expressing Vinc-CO or Vinc-Head in the follicle cells and wing, hsFlp; arm::FRTstopf+FRTGal4 (details available upon request) flies were crossed to UAS-vinc-X flies and progeny heat shocked either as L1 larvae (for clones in the wing) or pupae (for clones in the follicle cells). Fluorescence from the vinculin constructs was used to mark clones.