659 Research Article Introduction Mitochondria play a unique role in cellular physiology. They are responsible for energy production, regulation of Ca 2+ concentration in the cytoplasm and programmed cell death. These important functions make the study of mitochondrial transport and localization an important topic in cell biology (Hollenbeck, 1996). Previous research has shown that mitochondria can move in cells along microtubules and actin filaments (Morris and Hollenbeck, 1995; Ligon and Steward, 2000) and several molecular motors that could be involved in this movement have been identified (Nangaku et al., 1994; Tanaka et al., 1998). In many types of cultured cells, mitochondria are distributed throughout the cytoplasm and this distribution depends on the integrity of cytoplasmic microtubules (Ball and Singer, 1982). However, often a considerable fraction of these organelles reside in the perinuclear area, whereas at the periphery mitochondria are distributed more sparsely (Tanaka et al., 1998; Trinczek et al., 1999; Collins et al., 2002). The majority of mitochondria at the periphery remains immobile or moves slowly, and only a smaller fraction moves with relatively high speed (Trinczek et al., 1999; De Vos et al., 2003). The presence of immobile organelles is probably explained by their anchoring at sites in the cytoplasm where local ATP supply is required. On the basis of ultrastructural and biochemical studies, several mechanisms for mitochondria ‘docking’ to microtubules and intermediate filaments have been proposed (Leterrier et al., 1994; Wagner et al., 2003). Thus, the control of the mitochondrial motility should be considered not only in terms of regulation of motor protein activity but also as a switching between stationary and motile phases. Substantial progress has been achieved in studies of mechanisms by which extracellular signals affect the cytoskeleton and change cell morphology. Members of the Rho family of small GTPases and their effectors were shown to regulate both actin cytoskeleton re-arrangements and microtubule dynamics (for reviews, see Bar-Sagi and Hall, 2000; Wittmann and Waterman-Storer, 2001). For example, Rac1 and Cdc42 induce formation of lamellipodia and filopodia, respectively, by activation of actin polymerization at the plasma membrane (Nobes and Hall, 1995). At the same time, Rac1 promotes microtubule growth into advancing lamellipodia of migrating cells (Wittmann et al., 2003), and Cdc42 is involved in the orientation of the microtubule- The distribution of mitochondria is strictly controlled by the cell because of their vital role in energy supply, regulation of cytosolic Ca 2+ concentration and apoptosis. We employed cultured mammalian CV-1 cells and Drosophila BG2-C2 neuronal cells with enhanced green fluorescent protein (EGFP)-tagged mitochondria to investigate the regulation of their movement and anchorage. We show here that lysophosphatidic acid (LPA) inhibits fast mitochondrial movements in CV-1 cells acting through the small GTPase RhoA. The action of RhoA is mediated by its downstream effectors: formin-homology family members mDia1 in mammalian cells and diaphanous in Drosophila. Overexpression of constitutively active mutant forms of formins leads to dramatic loss of mitochondrial motility and to their anchorage to actin microfilaments. Conversely, depletion of endogenous diaphanous protein in BG2-C2 cells by RNA interference (RNAi) stimulates the mitochondrial movement. These effects are not simply explained by increased cytoplasm viscosity resulting from an increased F-actin concentration since stimulators of Arp2/3-dependent actin polymerization and jasplakinolide do not cause inhibition. The observed effects are highly specific to mitochondria since perturbations of diaphanous or mDia1 have no effect on movement of other membrane organelles. Thus, mitochondrial movement is controlled by the small GTPase RhoA and this control is mediated by formins. Supplementary material available online at http://jcs.biologists.org/cgi/content/full/119/4/659/DC1 Key words: MDia1, Actin, Microtubules, Mitochondria Summary Regulation of mitochondria distribution by RhoA and formins Alexander A. Minin 1,2, * ,‡ , Alexander V. Kulik 1,3, *, Fatima K. Gyoeva 1 , Ying Li 2 , Gohta Goshima 4 and Vladimir I. Gelfand 5 1 Institute of Protein Research, Russian Academy of Sciences, Moscow 119988, Russia 2 Department of Cell and Structural Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA 3 Moscow Institute of Physics and Technology, Moscow 141700, Russia 4 Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94107, USA 5 Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA *These authors contributed equally to this work ‡ Author for correspondence (e-mail: minin@eimb.ru) Accepted 25 October 2005 Journal of Cell Science 119, 659-670 Published by The Company of Biologists 2006 doi:10.1242/jcs.02762 Journal of Cell Science