Cytoplasmic dynein regulates the subcellular distribution of mitochondria by controlling the recruitment of the fission factor dynamin-related protein-1

Aniko Varadi Linda I. Johnson-Cadwell Vincenzo Cirulli Yisang Yoon Victoria J. Allan Guy A. Rutter - While the subcellular organisation of mitochondria is likely to influence many aspects of cell physiology, its molecular control is poorly understood. Here, we have investigated the role of the retrograde motor protein complex, dyneindynactin, in mitochondrial localisation and morphology. Disruption of dynein function, achieved in HeLa cells either by over-expressing the dynactin subunit, dynamitin (p50), or by microinjection of an anti-dynein intermediate chain antibody, resulted in (a) the redistribution of mitochondria to the nuclear periphery, and (b) the formation of long and highly branched mitochondrial structures. Suggesting that an alteration in the balance between mitochondrial fission and fusion may be involved in both of these changes, overexpression of p50 induced the translocation of the Mitochondria are vital determinants of both the life and death of cells (Newmeyer and Ferguson-Miller, 2003). Thus, changes in mitochondrial morphology, and the spatial interaction of these organelles with other intracellular structures, seem likely to affect several aspects of cell physiology, including calcium homeostasis (Rutter and Rizzuto, 2000) and the regulation of apoptosis (Karbowski and Youle, 2003). However, knowledge of the mechanisms that control mitochondrial movement and dispersal within the cell remains fragmentary. Mitochondria appear to adopt a variety of different shapes in living cells, ranging from multiple small compartments (Collins and Bootman, 2003; Collins et al., 2002; Park et al., 2001) to elaborate tubular networks (Rizzuto et al., 1993; Rutter and Rizzuto, 2000; Legros et al., 2002). Suggesting an important role for the cytoskeleton in maintaining their intracellular distribution (Allan and Schroer, 1999), mitochondria interact with microfilaments, intermediate filaments and microtubules (Rappaport et al., 1998; Yaffe, 1999; Karbowski et al., 2001; Knowles et al., 2002) and fission factor dynamin-related protein (Drp1) from mitochondrial membranes to the cytosol and microsomes. Moreover, a dominant-negative-acting form of Drp1 mimicked the effects of p50 on mitochondrial morphology, while wild-type Drp1 almost completely restored normal mitochondrial distribution in p50 over-expressing cells. Thus, the dynein/dynactin complex plays an unexpected role in the regulation of mitochondrial morphology in living cells, by controlling the recruitment of Drp1 to these organelles. isolated mitochondria display both (+) and () end-directed movements along microtubules (Morris and Hollenbeck, 1995). Several members of the kinesin superfamily have been proposed to drive the anterograde movement of mitochondria (Hirokawa, 1998). Thus, Kif5b (also called kinesin I or conventional kinesin) (Tanaka et al., 1998) is localised to mitochondria in vivo, and function-blocking antibodies to Kif5b inhibit mitochondrial motility on microtubules in vitro (Nangaku et al., 1994). Similarly, inactivation of Kif5b alters mitochondrial distribution in undifferentiated extra-embryonic cells from mice (Tanaka et al., 1998), in Xenopus laevis oocytes (Heald et al., 1996) and in mammalian fibroblasts (Krylyshkina et al., 2002; Varadi et al., 2002). By contrast, the identity of the () end motor(s) involved is uncertain. Very recent studies have revealed the identity of some of the principal components of the mitochondrial fission/fusion machinery (Karbowski and Youle, 2003). A member of the dynamin family of GTPases, dynamin-related protein (Drp1, also known as DVLP, DLP1 or Dymple) has been shown to be involved in mitochondrial fission in both yeast and mammals (Karbowski and Youle, 2003). Thus, expression of a dominant negative mutant of Drp1 results in the formation of highly interconnected, fused mitochondria (Smirnova et al., 1998; Smirnova et al., 2001; Yoon et al., 2001; Pitts et al., 1999). Here, we have sought to determine the role of the dynein/dynactin complex in the retrograde movement of mitochondria. Unexpectedly, we show that disruption of dynein function in HeLa cells leads to the retreat of mitochondria from the cell periphery towards the nucleus, and the formation of long, interconnected mitochondria. This retrograde movement is associated with a large decrease in the association of Drp1 with mitochondria, and is reversed by overexpression of wild-type Drp1. Moreover, we also show that Drp1 interacts with the dynactin complex and provide evidence that this controls its recruitment to the mitochondrial outer membrane. cDNAs encoding dynamitin, p50 and p50-enhanced green fluorescent protein (EGFP) (Valetti et al., 1999) were kindly provided by Trina Schroer (Johns Hopkins University, Baltimore) and Vladimir Gelfand (Urbana, Illinois), respectively. Plasmid encoding a -tubulin.EGFP was from David Stephens (University of Bristol, UK). Cell culture reagents were from GibcoBRL (Life Science Research, Paisley, UK) and all molecular biologicals from Roche Diagnostics (Lewes, UK). Electron microscopy (EM) grade paraformaldehyde, glutaraldehyde and sodium cacodylate trihydrate were purchased from Electron Microscopy Sciences (Fort Washington, PA). Alexa Fluor goat antirabbit or anti-mouse 488 and 568 secondary antibodies, MitoTrackerRedTM and Oregon Green 488 BAPTA-1 dextran were from Molecular Probes (Eugene, USA). Mouse monoclonal anti-a tubulin and anti-dynein (Clones 70.1 and 74.1) antibodies and Annexin V-CY3 Apoptosis Detection Kit were obtained from Sigma (Poole, UK). Monoclonal anti-dynamitin p50 was purchased from BD Biosciences (Oxford, UK). Rabbit anti-human Drp1 polyclonal antibody was from AMS Biotechnology (Abingdon Oxon, UK). Mouse monoclonal trans-golgi network protein 38 (TGN38) and mouse monoclonal anti-human lysosome-associated membrane protein-1 (LAMP-1) specific antibodies were kindly provided by G. Banting (University of Bristol, UK) (Lee and Banting, 2002). HeLa cells were cultured in Dulbeccos modified Eagles medium (DMEM) tissue-culture medium supplemented with 10% (v/v) foetal calf serum (FCS) penicillin (100 units ml1), streptomycin (0.1 mg ml1) and L-glutamine (2 mM) at 37C in an atmosphere of humidified air (95%) and CO2 (5%) as described previously (Molnar et al., 1995). A plasmid encoding mitochondrially targeted Discoidium red fluorescent protein (mito.DsRed) was generated as described earlier (Varadi et al., 2002). Live cell imaging immunocytochemistry Cells were co-transfected with 1 m g of plasmids encoding mito.DsRed and p50, p50.EGFP, or empty vectors (pcDNA3 or pAdTrack-CMV, the latter encodes EGFP) (He et al., 1998), using 10 m g ml1 Lipofectamine in Optimem ITM medium (GibcoBRL, Life Science Research, Paisley, UK) for 4 hours. Alternatively, mitochondria were visualised in p50.EGFP-expressing live cells by staining with 100 nM MitoTrackerRedTM dye in growth medium for 30 minutes at 37C. Immunocytochemistry was performed (...truncated)