Novel roles of SCAR/WAVE subunits in the regulation of actin dynamics
Final Report Abstract
The SCAR/WAVE complex links upstream Rho-family GTPase signaling to the activation of the conserved ARP2/3 complex in different. SCAR/WAVE-induced and ARP2/3-complexmediated actin nucleation beneath the plasma membrane is crucial for the assembly of actin in protruding lamellipodia to drive cell migration. The SCAR/WAVE complex is composed of SCAR/WAVE, Abi, Nap, Pir and Brk1/HSPC300 and is believed to largely act as a molecular entity. We have determined the crystal structure and analyzed the function of the smallest complex subunit Brk1. Based on these results we suggest that a constant pool of Brk1 homotrimers allows for formation of a given number of monomers, which directly bind and stabilize de novo synthesized Scar/WAVE and Abi subunits. Since the activation of the Arp2/3-complex is a highly regulated process in vivo, we further assume that heterotrimeric Scar/AbiA/DdBrk1-complexes immediately associate with the heterodimeric Nap/Pir platform to mask the VCA domain of Scar/WAVE proteins to prevent the unregulated activation of the Arp2/3-complex. Notably, Brk1 forms the majority of the surface contacts between heterotrimeric HsBrk1/Abi2/WAVE1 and Nap/Pir platform within the trimeric coiled-coil region. We therefore proposed that in the final assembly step, Brk1 serves as an adapter protein to stabilize and coordinate the assembly of the heterotrimer and the binding to the Pir/Nap platform to assemble the mature Scar/WAVE complex. We further designed a novel extrachromosomal Cre-recombinase expression vector that enables stringent selection procedures and much higher transfection rates, resulting in highly efficient recycling of selection markers, which will be important for the generation of Dictyostelium mutants lacking combinations of SCAR/WAVE complex components in future work. Due to publication of highly overlapping work from a competing laboratory, we then had to deviate from our proposed programme and changed our strategy. In the following we therefore analyzed the function of Arpin and the I-BAR-domain protein IRSp53 in actin-based motility. In cooperation with our international cooperation partners we show that Arpin antagonizes membrane protrusions and allows cell turning by competitive inhibition Scar/WAVE-mediated activation of the Arp2/3 complex. The function of the protein seems to be evolutionary highly conserved as evidenced by perturbation of Arpin function in various experimental systems. As shown by biochemical and in vivo assays IRSp53 by itself counteract actin assembly. Binding to the small GTPase Cdc42 to IRSp53, however, relieves this inhibition and promotes IRSp53-dependent recruitment and clustering of VASP to the plasma membrane to initiate processive F-actin elongation. These events result in spatial restriction of VASP activity to initiate filopodia, drive cell migration, and promote tissue repair. The only I-BAR-domain protein member of Dictyostelium cells IBARa, reminiscent of IRSp53, also localizes to filopodia, but seem not to be required for their formation. Instead, it is critically involved in numerous processes including osmoregulation, phagocytosis, cell migration and cytokinesis, indicating that IBARa has an important role in processes that require a dynamic rearrangement of cellular membranes.
Publications
- „Identifizierung und Charakterisierung neuer an der Filopodienbildung beteiligter Proteine aus Dictyostelium discoideum“. Diss. Hannover, Gottfried Wilhelm Leibniz Universität, 2010. 142 Bl. : Ill., graph. Darst.
Jörn Linkner
- (2011). High-resolution X-ray structure of the trimeric Scar/WAVE-complex precursor Brk1. PLoS One 6:e21327
Linkner J, Witte G, Stradal T, Curth U, Faix J
- (2012). Highly effective removal of floxed Blasticidin S resistance cassettes from Dictyostelium discoideum mutants by extrachromosomal expression of Cre. European Journal of Cell Biology 156- 160
Linkner J, Nordholz B, Junemann A, Winterhoff M, Faix J
(See online at https://doi.org/10.1016/j.ejcb.2011.11.001) - (2013). CDC42 switches IRSp53 from inhibition of actin growth to elongation by clustering of VASP. EMBO Journal 32, 2735-2750
Disanza A, Bisi S, Winterhoff M, Milanesi F, Ushakov DS, Kast D, Marighetti P, Romet- Lemonne G, Müller HM, Nickel W, Linkner J, Waterschoot D, Ampè C, Cortellino S, Palamidessi A, Dominguez R, Carlier MF, Faix J, Scita G
(See online at https://doi.org/10.1038/emboj.2013.208) - (2013). Inhibitory signalling to the Arp2/3 complex steers cell migration. Nature 503, 281-284
Dang I, Gorelik R, Sousa-Blin C, Derivery E, Guérin C, Linkner J, Nemethova M, Dumortier JG, Giger FA, Chipysheva TA, Ermilova VD, Vacher S, Campanacci V, Herrada I, Planson AG, Fetics S, Henriot V, David V, Oguievetskaia K, Lakisic G, Pierre F, Steffen A, Boyreau A, Peyriéras N, Rottner K, Zinn-Justin S, Cherfils J, Bièche I, Alexandrova AY, David NB, Small JV, Faix J, Blanchoin L, Gautreau
(See online at https://doi.org/10.1038/nature12611) - (2013). The application of the Cre-loxP system for generating multiple knock-out and knock-in targeted loci. Methods Molecular Biology 983, 249-267
Faix J, Linkner J, Nordholz B, Platt JL, Liao XH, Kimmel AR
- (2014). The inverse BAR domain protein IBARa drives membrane remodeling to control osmoregulation, phagocytosis and cytokinesis. Journal of Cell Science 127, 1279-1292
Linkner J, Witte G, Zhao H, Junemann A, Nordholz B, Runge-Wollmann P, Lappalainen P, Faix J
(See online at https://doi.org/10.1242/jcs.140756)
