Mutations in the human dynamin-related protein Drp1 cause mitochondria to form perinuclear clusters. We show here that these mitochondrial clusters consist of highly interconnected mitochondrial tubules. The increased connectivity between mitochondria indicates that the balance between mitochondrial division and fusion is shifted toward fusion. Such a shift is consistent with a block in mitochondrial division. Immunofluorescence and subcellular fractionation show that endogenous Drp1 is localized to mitochondria, which is also consistent with a role in mitochondrial division. A direct role in mitochondrial division is suggested by time-lapse photography of transfected cells, in which green fluorescent protein fused to Drp1 is concentrated in spots that mark actual mitochondrial division events. We find that purified human Drp1 can self-assemble into multimeric ring-like structures with dimensions similar to those of dynamin multimers. The structural and functional similarities between dynamin and Drp1 suggest that Drp1 wraps around the constriction points of dividing mitochondria, analogous to dynamin collars at the necks of budding vesicles. We conclude that Drp1 contributes to mitochondrial division in mammalian cells.

Mitochondria exist as a dynamic tubular network with projections that move, break, and reseal in response to local environmental changes. We present evidence that a human dynamin-related protein (Drp1) is specifically required to establish this morphology. Drp1 is a GTPase with a domain structure similar to that of other dynamin family members. To identify the function of Drp1, we transiently transfected cells with mutant Drp1. A mutation in the GTPase domain caused profound alterations in mitochondrial morphology. The tubular projections normally present in wild-type cells were retracted into large perinuclear aggregates in cells expressing mutant Drp1. The morphology of other organelles was unaffected by mutant Drp1. There was also no effect of mutant Drp1 on the transport functions of the secretory and endocytic pathways. By EM, the mitochondrial aggregates found in cells that were transfected with mutant Drp1 appear as clusters of tubules rather than a large mass of coalescing membrane. We propose that Drp1 is important for distributing mitochondrial tubules throughout the cell. The function of this new dynamin-related protein in organelle morphology represents a novel role for a member of the dynamin family of proteins.

Drosophila shibire and its mammalian homologue dynamin regulate an early step in endocytosis. We identified a Caenorhabditis elegans dynamin gene, dyn-1, based upon hybridization to the Drosophila gene. The dyn-1 RNA transcripts are trans-spliced to the spliced leader 1 and undergo alternative splicing to code for either an 830-or 838-amino acid protein. These dyn-1 proteins are highly similar in amino acid sequence, structure, and size to the Drosophila and mammalian dynamins: they contain an Nterminal GTPase, a pleckstrin homology domain, and a C-terminal proline-rich domain. We isolated a recessive temperature-sensitive dyn-1 mutant containing an alteration within the GTPase domain that becomes uncoordinated when shifted to high temperature and that recovers when returned to lower temperatures, similar to D. shibire mutants. When maintained at higher temperatures, dyn-1 mutants become constipated, egg-laying defective, and produce progeny that die during embryogenesis. Using a dyn-1::lacZ gene fusion, a high level of dynamin expression was observed in motor neurons, intestine, and pharyngeal muscle. Our results suggest that dyn-1 function is required during development and for normal locomotion.Dynamin is a 100-kDa GTPase that regulates an early stage of endocytosis (1). Although it was first isolated from bovine brain (2), much of our understanding of dynamin has come from the analysis of the Drosophila dynamin mutant, shibire (3, 4). shibire mutations confer rapid and reversible temperaturesensitive paralysis (5) that results from a block in endocytosis and the subsequent depletion of synaptic vesicles (6). The overexpression of a similar dynamin mutation in mammalian cells inhibits clathrin-mediated endocytosis at a point after coat proteins assemble at the plasma membrane but before the coated pits become deeply invaginated (7,8). Dynamin can self-assemble into rings in vitro, and dynamin rings are found at the neck of budding vesicles in the presence of GTP␥S (9, 10). These observations suggest that dynamin might act to pinch off clathrin-coated vesicles from the plasma membrane.Dynamin belongs to a growing family of GTP-binding proteins with diverse functions, such as the mammalian interferon-induced antiviral MX proteins (11), the yeast mitochondrial DNA replication protein MGM1 (12), the yeast membrane transport proteins VPS1 and DNM1 (13,14), and the plant cell plate formation protein phragmoplastin (15). These proteins possess a conserved amino-terminal GTPase domain (43-66% identity) but are typically much less related in sequence outside of this region (Ͻ30% identity). Dynamin contains a pleckstrin homology domain, which is thought to mediate protein-protein or protein-lipid interactions (16), and a C-terminal proline-rich domain (PRD). Although in vitro association of the dynamin PRD with a variety of ligands (microtubules, acidic phospholipids, SH3 domains, and even mAbs) increases the rate of GTP hydrolysis (17-20), it is unclear whether such interactions regulate dynamin activity in vi...

Dynamin is a 100-kDa GTPase that assembles into multimeric spirals at the necks of budding clathrin-coated vesicles. We describe three different intramolecular binding interactions that may account for the process of dynamin self-assembly. The first binding interaction is the dimerization of a 100-amino acid segment in the C-terminal half of dynamin. We call this segment the assembly domain, because it appears to be critical for multimerization.

Synthetic retinoid-related molecules, such as N-(4-hydroxyphenyl)retinamide (fenretinide) and 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) induce apoptosis in a variety of malignant cells. The mechanism(s) of action of these compounds does not appear to involve retinoic acid receptors (RARs) and retinoid X receptors (RXRs), although some investigators disagree with this view. To clarify whether some retinoid-related molecules can induce apoptosis without involving RARs and/or RXRs, we used 4-[3-(1-heptyl-4,4-dimethyl-2-oxo-1,2,3,4-tetrahydroquinolin-6-yl)-3-oxo-E-propenyl] benzoic acid (AGN193198) that neither binds effectively to RARs and RXRs nor transactivates in RAR-and RXR-mediated reporter assays. AGN193198 potently induced apoptosis in prostate, breast, and gastrointestinal carcinoma cells and in leukemia cells. AGN193198 also abolished growth (by 50% at 130 -332 nM) and induced apoptosis in primary cultures established from prostatic carcinoma (13 patients) and gastrointestinal carcinoma (1 patient). Apoptosis was induced rapidly, as indicated by mitochondrial depolarization and DNA fragmentation. Molecular events provoked by AGN193198 included activation of caspase-3, -8, -9, and -10 (by 4 -6 h) and the production of BID/p15 (by 6 h). These findings show that caspase-mediated induction of apoptosis by AGN193198 is RAR/RXR-independent and suggest that this compound may be useful in the treatment of prostate cancer.

These findings demonstrate that PPARγ agonists can have differential effects on RPE survival in response to oxidative stress. Oxidative stress leads to deregulation of a large set of genes in ARPE-19 cells. A specific subset of these genes can be selectively modulated by troglitazone and represent potential novel targets for cytoprotective therapies.

Caenorhabditis elegans dynamin is expressed at high levels in neurons and at lower levels in other cell types, consistent with the important role that dynamin plays in the recycling of synaptic vesicles. Indirect immunofluorescence showed that dynamin is concentrated along the dorsal and ventral nerve cords and in the synapse-rich nerve ring. Green fluorescent protein (GFP) fused to the N terminus of dynamin is localized to synapse-rich regions. Furthermore, this chimera was detected along the apical membrane of intestinal cells, in spermathecae, and in coelomocytes. Dynamin localization was not affected by disrupting axonal transport of synaptic vesicles in the unc-104 (kinesin) mutant. To investigate the alternative mechanisms that dynamin might use for translocation to the synapse, we systematically tested the localization of different protein domains by fusion to GFP. Localization of each chimera was measured in one specific neuron, the ALM. The GTPase, a middle domain, and the putative coiled coil each contribute to synaptic localization. Surprisingly, the pleckstrin homology domain and the proline-rich domain, which are known to bind to coated-pit constituents, did not contribute to synaptic localization. The GFP-GTPase chimera was most strongly localized, although the GTPase domain has no known interactions with proteins other than with dynamin itself. Our results suggest that different dynamin domains contribute to axonal transport and the sequestration of a pool of dynamin molecules in synaptic cytosol.

The human immunodeficiency virus (HIV) enhancer element is important in the regulation of HIV gene expression. A number of cellular proteins have been demonstrated to bind to the NF-KB motifs in this element. The genes encoding several of these proteins, including members of the rel family and PRDII-BF1, have been cloned. We characterized the binding of proteins encoded by the human c-rel and PRDII-BF1 genes to HIV NF-KB motifs and related enhancer elements. Both the human c-rel protein and two proteins derived from the PRDII-BF1 gene by alternative splicing bound specifically to the HIV NF-KB motif and related enhancer elements found in the immunoglobulin kappa, class I major histocompatibility complex, and interleukin-2 receptor genes. To determine the role of these factors in regulating HIV gene expression, we fused the cDNAs encoding either of the two proteins derived by alternative splicing of the PRDII-BF1 gene or the c-rel gene to the DNA binding region of the yeast transcription factor GAL4. GAL4 binding sites were inserted in place of the native HIV enhancer sequences in an HIV long terminal repeat chloramphenicol acetyltransferase construct. Cotransfection of these constructs revealed that c-rel was a strong activator of basal HIV gene expression but did not result in synergistic effects in the presence of tat. PRDII-BF1-derived cDNAs did not result in stimulation of either basal or tat-induced activated gene expression. These results indicate that multiple enhancer binding proteins may potentially regulate HIV in both a positive and negative manner.