Nuclear Localization of mDia2 Formin and its Possible Regulation by Rho Family GTPases

Abstract

Formin family proteins are potent activators of actin filament nucleation and elongation. While numerous studies indicate the possible roles of actin inside the nucleus and the tight association of the nucleus with cytoplasmic actin cytoskeleton, the role of actin-associated proteins and, in particular, regulators of actin polymerization in the organization of nuclear and perinuclear actin remains obscure. Nuclear localization of some formins, such as formin-1 and mDia2 was detected in some previous studies. Particularly, it was recently shown that mDia2 has both nuclear localization and nuclear export signals, and is accumulated in the nucleus upon inhibition of nuclear export by leptomycin B (LMB) (Miki et al, J Biol Chem. 2009). We confirmed and extended these results; in particular, we have measured the kinetics of nuclear accumulation of some formins upon LMB treatment and have found that mDia2 accumulates in the nucleus very fast while mDia1 does not accumulate. Since mDia2 activity is regulated by the Rho family GTPases, we further studied the nuclear localization of RhoA, Rac1, and Cdc42 and found that significant amounts of the wild type Rho GTPases were localized to the nucleus, while constitutively active Rho GTPases demonstrated essentially cytoplasmic localization. Moreover, active Cdc42 was shown to augment localization of mDia2 to cell plasma membrane and slow down its nuclear shuttling. Finally, we have found that even without LMB treatment, GFP-fused full length mDia2 is accumulated to the nuclear rim, where it colocalizes with nesprin, a protein involved in linking nucleus with actin cytoskeleton. Constitutively active Rac1 was also enriched in the nuclear rim. The experiments using cell treatment with digitonin, which permeabilizes plasma- but not the nuclear membrane, suggested that mDia2-enriched nuclear rim was located at the cytoplasmic side of the nuclear membrane.