Poster: Cytoskeleton structure & dynamics

Abs # 687: SPIKE1-dependent cell morphogenesis: a direct and essential interaction with inactive ROP GTPase

Presenter:	Basu, Dipanwita , basu@purdue.edu
Authors	Basu, Dipanwita  (A)   Saad, Mohamed  (A)   Mallery, Eileen  (A)   Le, Jie  (A)   Szymanski, Daniel B. (A)
Affiliations:	(A): Purdue University

We use Arabidopsis trichome and pavement cell morphology mutants to understand how signaling pathways and cytoskeletal proteins control cell shape. Cytoskeleton localization, inhibitors studies and molecular genetic data indicate that microtubules are required to initiate trichome stalk and branch out growth, while actin filaments have more of a maintenance function. Mutations in SPIKE1 (SPK1) cause seedling lethality, reduced pavement cell lobing, sterility, and reduced trichome branching. From these phenotypes we hypothesize that SPK1 regulates microtubule-based functions, but we do not understand the mechanisms. SPK1 is a member of the DOCK family of signaling proteins. DOCK proteins integrate signaling information with actin based functions like cell migration and phagocytosis. All DOCK proteins encode a conserved C-terminal domain of ~400 amino acids called DOCK HOMOLOGY REGION2 (DHR2), which activates Rho-family GTPases by acting as a guanidine nucleotide exchange factor. Plants have a family of Rho-GTPases called ROPS (Rho Of Plants) that affect cytoskeleton. Our aim is to determine if SPK1 regulates ROP and to understand the consequence of this activity on cell morphogenesis. Several biochemical data demonstrated the ability of recombinant and native SPK1 to interact directly with the nucleotide-free ROP, a binding specificity that is shared by all known exchange factors. We identified six mutations that affect the DHR2 of SPK1. Mutant proteins accumulate in the different spk1 backgrounds, yet the plants display severe spk1 phenotypes. A C-terminally truncated DHR-2 that reconstitutes the spk1-3 allele failed to interact with ROPs. These data show that DHR-2 is essential for SPK1 binding to ROPs, and also contains an essential biochemical activity of SPK1.