Significance is taken as *< 0.05. Ratio imaging was performed with energized mitochondria using decreased JC-1 concentrations, so that the amount of bound rhodamine B-labeled NFs or SAs (red fluorescence) on mitochondria (yellowish fluorescence) increases the red to green ratio. sites. In several cell lines, mitochondrial distribution is usually coordinated by both microtubules (MTs) and IFs (Summerhayes et al., 1983). In neurons, axonal mitochondrial transport can occur Rabbit polyclonal to HSP27.HSP27 is a small heat shock protein that is regulated both transcriptionally and posttranslationally. along both MTs and actin filaments, whereas neuronal cells lacking MT or actin filaments [but still retaining neurofilaments (NFs)] exhibit no mitochondrial motility (Morris and Hollenbeck, Big Endothelin-1 (1-38), human 1995). Mitochondrial distribution in cells is usually suggested to occur by a coordinated effect of MTs and actin filaments (Couchman and Rees, 1982; Krendel et al., 1998) with a role for IFs in muscle mass (Stromer and Bendayan, 1990; Reipert et al., 1999) and non-muscle cells (Toh et al., 1980; Mose-Larsen et al., 1982; Almahbobi et al., 1993; Collier et al., 1993). Regulation of organelle motility can be accounted for by several alternate or complementary mechanisms including the orchestration of motor activation (Sheetz, 1999), switching between plus and minus end-directed motors, and random or regulated static attachment to stationary cytoskeletal elements (Leterrier et al., 1994; Hollenbeck, 1996). In axons, motility of mitochondria, distributed to sites of high ATP demand (e.g., growth cones, synapses, and nodes of Ranvier), is usually altered by the apparent docking to NFs and MTs (Hollenbeck, 1996), consistent with electron microscopic studies exposing abundant mitochondria-NF and -MT Big Endothelin-1 (1-38), human interconnections (Hirokawa, 1982). In addition to frequent stops and starts, elastic recoil, and reversal of direction, axonal mitochondria spend a considerable amount of time stationary, although presumably bound to NFs and MTs (Martz et al., 1984; Forman et al., 1987; Morris and Hollenbeck, 1993; Ligon and Steward, Big Endothelin-1 (1-38), human 2000). Although an association between mitochondria and NFs is usually supported by many studies (Hirokawa, 1982; Leterrier et al., 1991, 1994), the regulation of mitochondria-NF interactions is usually uncharacterized. In fibroblasts, mitochondrial distribution can be managed by the IF network even after treatment with MT inhibitors. In contrast, treatment with a mitochondrial membrane potential inhibitor can redistribute mitochondria within the cell (Dudani et al., 1990). In yeast, which lacks IFs, mitochondrial position and movements depend around the actin cytoskeleton (Smith et al., 1995), and yeast mitochondria-actin interactions are ATP- but not membrane potential-sensitive (Lazzarino et al., 1994). In neurons, however, Overly et al. (1996) found a close relationship between metabolic activity and motility of mitochondria: highly active mitochondria in proximal dendrites were less motile compared with mitochondria with low metabolic activity in axons. To evaluate the potential regulation of mitochondria interactions with NFs, we analyzed the binding of isolated brain mitochondria and purified NFs for 1 hr at 4C. The supernatant was brought to 4 m glycerol and kept for 3 hr at 4C. The NF-glycerol combination was then centrifuged at 78,000 for 1 hr at 4C. The turbid-white NF pellet, contaminated with soluble proteins and membranes, was resuspended in RB buffer. To label filaments, a final concentration of 12 mm rhodamine B succinimide (kindly provided by Dr. Rolands Vegners, Latvian Institute of Organic Synthesis, Riga, Latvia) was added to the protein answer (30 min incubation at 4C). A protease inhibitor combination, as explained previously (Leterrier et al., 1996), was added to the NF answer after a gentle two-stroke homogenization step using a Teflon-glass homogenizer. The NFs were centrifuged over a discontinuous sucrose gradient (1.5 and 0.8 m sucrose in RB) at 100,000 for 14 hr at 4C to remove contaminating proteins and membranes. The crystal-clear NF pellet was softly homogenized with two strokes in RB made up of 0. 8 m sucrose and protease inhibitors. The purified NF suspension was dialyzed for 24 hr at 4C in RB made up of 0.8 m sucrose and 1 mm PMSF to remove NF-bound polypeptides (Gou et al., Big Endothelin-1 (1-38), human 1998). The purity of NF preparations was routinely checked by densitometry of Coomassie-stained gels after SDS-PAGE. For the preparations used in these studies, the NF triplet subunits [NF-heavy chain (NF-H), NF-medium chain (NF-M), and NF-light chain (NF-L)] accounted for 95% of the total protein (observe Results). Trace amounts of tightly associated proteins such as the dynein/dynactin complex (Shah et al., 2000) remain tenaciously bound to NFs.