Dynamin-related protein Drp1 is required for mitochondrial division in mammalian cells.

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.

The many shapes of mitochondrial membranes.

The roles of mitochondria in cell death and in aging have generated much excitement in recent years. At the same time, however, a quiet revolution in our thinking about mitochondrial ultrastructure has begun. This revolution started with the use of vital dyes and of green fluorescent protein fusion proteins, showing that mitochondria are very dynamic structures that constantly move, divide and fuse throughout the life of a cell. More recently, some of the first proteins contributing to these various processes have been discovered. Our view of the internal structures of mitochondria has also changed. Three-dimensional reconstructions obtained with high voltage electron microscopy show that cristae are often connected to the mitochondrial inner membrane by thin tubules. These new insights are brought to bear on the wealth of data collected by conventional electron microscopic analysis.

Localizing proteins in the cell from their phylogenetic profiles.

We introduce a computational method for identifying subcellular locations of proteins from the phylogenetic distribution of the homologs of organellar proteins. This method is based on the observation that proteins localized to a given organelle by experiments tend to share a characteristic phylogenetic distribution of their homologs-a phylogenetic profile. Therefore any other protein can be localized by its phylogenetic profile. Application of this method to mitochondrial proteins reveals that nucleus-encoded proteins previously known to be destined for mitochondria fall into three groups: prokaryote-derived, eukaryote-derived, and organism-specific (i.e., found only in the organism under study). Prokaryote-derived mitochondrial proteins can be identified effectively by their phylogenetic profiles. In the yeast Saccharomyces cerevisiae, 361 nucleus-encoded mitochondrial proteins can be identified at 50% accuracy with 58% coverage. From these values and the proportion of conserved mitochondrial genes, it can be inferred that approximately 630 genes, or 10% of the nuclear genome, is devoted to mitochondrial function. In the worm Caenorhabditis elegans, we estimate that there are approximately 660 nucleus-encoded mitochondrial genes, or 4% of its genome, with approximately 400 of these genes contributed from the prokaryotic mitochondrial ancestor. The large fraction of organism-specific and eukaryote-derived genes suggests that mitochondria perform specialized roles absent from prokaryotic mitochondrial ancestors. We observe measurably distinct phylogenetic profiles among proteins from different subcellular compartments, allowing the general use of prokaryotic genomes in learning features of eukaryotic proteins.

A model for dynamin self-assembly based on binding between three different protein domains.

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. The second binding interaction occurs between the assembly domain and the N-terminal GTPase domain. The strength of this interaction is controlled by the nucleotide-bound state of the GTPase domain, as shown with mutations in GTP binding motifs and in vitro binding experiments. The third binding interaction occurs between the assembly domain and a segment that we call the middle domain. This is the segment between the N-terminal GTPase domain and the pleckstrin homology domain. The three different binding interactions suggest a model in which dynamin molecules first dimerize. The dimers are then linked into a chain by a second binding reaction. The third binding interaction might connect adjacent rungs of the spiral.

Functional diversity in the dynamin family.

The function of the GTPase dynamin has been discussed for several years. It clearly plays a role in vesicle budding, but, despite recent insights, precisely how it functions in this process is still a matter of debate. In addition, it is now clear that dynamin is a member of a large protein family, present in a variety of cellular locations where members apparently perform a range of functions. This article describes current understanding of the structure and function of the various dynamin family members.

C. elegans dynamin-related protein DRP-1 controls severing of the mitochondrial outer membrane.

Little is known about the mechanism of mitochondrial division. We show here that mitochondria are disrupted by mutations in a C. elegans dynamin-related protein (DRP-1). Mutant DRP-1 causes the mitochondrial matrix to retract into large blebs that are both surrounded and connected by tubules of outer membrane. This indicates that scission of the mitochondrial outer membrane is inhibited, while scission of the inner membrane still occurs. Overexpressed wild-type DRP-1 causes mitochondria to become excessively fragmented, consistent with an active role in mitochondrial scission. DRP-1 fused to GFP is observed in spots on mitochondria where scission eventually occurs. These data indicate that wild-type DRP-1 contributes to the final stages of mitochondrial division by controlling scission of the mitochondrial outer membrane.

Contribution of the GTPase domain to the subcellular localization of dynamin in the nematode Caenorhabditis elegans.

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.

A human dynamin-related protein controls the distribution of mitochondria.

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.

A dynamin GTPase mutation causes a rapid and reversible temperature-inducible locomotion defect in C. elegans.

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 N-terminal 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.

Assignment of the dynamin-1 gene (DNM1) to human chromosome 9q34 by fluorescence in situ hybridization and somatic cell hybrid analysis.

The dynamins are recently discovered GTP-binding proteins postulated to mediate the scission of clathrin-coated vesicles at the plasma membrane. Of the three known mammalian dynamins, dynamin-1 (DNM1) appears to be particularly important for the formation of synaptic vesicles at presynaptic nerve termini. To investigate the possibility that mutations in the DNM1 gene cause a human disease, we determined the chromosomal localization of human DNM1. We conclude from fluorescence in situ hybridization and from the analysis of somatic cell hybrids that the map position in 9q34. This region has syntenic homology with mouse chromosome 2p, in agreement with the map position of the mouse DNM1 gene [see accompanying article by Klocke et al. (1997, Genomics 41:290-292)]. We discuss the potential relevance of the human DNM1 localization to diseases that were mapped genetically to the same chromosomal region.

Traffic of dynamin within individual Drosophila synaptic boutons relative to compartment-specific markers.

Presynaptic terminals contain several specialized compartments, which have been described by electron microscopy. We show in an identified Drosophila neuromuscular synapse that several of these compartments-synaptic vesicle clusters, presynaptic plasma membrane, presynaptic cytosol, and axonal cytoskeleton-labeled by specific reagents may be resolved from one another by laser scanning confocal microscopy. Using a panel of compartment-specific markers and Drosophila shibire(ts1) mutants to trap an intermediate stage in synaptic vesicle recycling, we have examined the localization and redistribution of dynamin within single synaptic varicosities at the larval neuromuscular junction. Our results suggest that dynamin is not a freely diffusible molecule in resting nerve terminals; rather, it appears localized to synaptic sites by association with yet uncharacterized presynaptic components. In shi(ts1) nerve terminals depleted of synaptic vesicles, dynamin is quantitatively redistributed to the plasma membrane. It is not, however, distributed uniformly over presynaptic plasmalemma; instead, fluorescence images show "hot spots" of dynamin on the plasma membrane of vesicle-depleted nerve terminals. We suggest that these dynamin-rich domains may mark the active zones for synaptic vesicle endocytosis first described at the frog neuromuscular junction.

Clathrin-independent pinocytosis is induced in cells overexpressing a temperature-sensitive mutant of dynamin.

A stable HeLa cell line expressing a dynamin mutant, dynts, exhibits a temperature-sensitive defect in endocytic clathrin-coated vesicle formation. Dynts carries a point mutation, G273D, corresponding to the Drosophila shibirets1 allele. The ts-defect in receptor-mediated endocytosis shows a rapid onset (< 5 min) and is readily reversible. At the nonpermissive temperature (38 degrees C) HRP uptake is only partially inhibited. Moreover, when cells are held at the nonpermissive temperature, fluid phase uptake fully recovers to wild-type levels within 30 min, while receptor-mediated endocytosis remains inhibited. The residual HRP uptake early after shift to the nonpermissive temperature and the induced HRP uptake that occurs after recovery are insensitive to cytosol acidification under conditions that potently inhibit receptor-mediated endocytosis of Tfn. Together, these results suggest that a dynamin- and clathrin-independent mechanism contributes to the total constitutive pinocytosis in HeLa cells and that dynts cells rapidly and completely compensate for the loss of clathrin-dependent endocytosis by inducing an alternate endocytic pathway.

Dynamin binds to SH3 domains of phospholipase C gamma and GRB-2.

Src homology 3 (SH3) domains are found in a variety of proteins that are involved in signal transduction or represent components of the cytoskeleton. These domains are thought to serve as modules that mediate specific protein-protein interactions that include proline-rich sequences on the target protein. We have identified proteins of 110, 80, 65, and 43 kDa in human embryonic fibroblasts that bind specifically to the SH3 domain of phospholipase C gamma, a primary substrate of receptor tyrosine kinases, and characterized the 110-kDa band as the microtubule-activated GTPase dynamin. In addition, dynamin binds the son of sevenless adaptor protein GRB-2 with even higher affinity. This interaction does not require the dynamin GTPase function and involves a proline-rich target sequence between residues 812 and 820 of dynamin.

Genetic studies on dynamin function in Drosophila.

The shibire(ts2) mutation of Drosophila melanogaster causes a temperature sensitive inhibition of endocytosis; this in turn leads to synaptic-vesicle depletion and consequent paralysis. Heat-pulses delivered during development of shibire(ts2) individuals affect the morphology of a number of adult structures. A simple screening protocol has been used to isolate several mutations that partially suppress the temperature-sensitive paralytic phenotype of shibire(ts2) mutant animals. All of these mutations very tightly linked to shibire and are likely to be second site intragenic mutations that restore partial activity to the shibire(ts2) product. The mutations suppress both behavioral, and easily-scored developmental phenotypes of shibire(ts2) characterized in this paper. Our results suggest that defects in endocytosis, and not in microtubule interactions, are responsible for all of the phenotypes of shibire(ts2) mutant Drosophila examined in this study.

Mutations in human dynamin block an intermediate stage in coated vesicle formation.

The role of human dynamin in receptor-mediated endocytosis was investigated by transient expression of GTP-binding domain mutants in mammalian cells. Using assays which detect intermediates in coated vesicle formation, the dynamin mutants were found to block endocytosis at a stage after the initiation of coat assembly and preceding the sequestration of ligands into deeply invaginated coated pits. Membrane transport from the ER to the Golgi complex was unaffected indicating that dynamin mutants specifically block early events in endocytosis. These results demonstrate that mutations in the GTP-binding domain of dynamin block Tfn-endocytosis in mammalian cells and suggest that a functional dynamin GTPase is required for receptor-mediated endocytosis via clathrin-coated pits.

Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic.

Temperature-sensitive paralysis is the most striking defect of adult Drosophila carrying the shibire mutation. This is believed to be due to a reversible block of endocytosis, which prevents membrane cycling and thus depletes synaptic vesicles. The shibire mutation also affects many tissues outside the nervous system. We have now mapped and characterized the shibire gene. A 275-kilobase yeast artificial chromosome was subcloned into cosmids, among which the gene was then located by analysing with restriction-fragment length polymorphisms. A 15-kilobase fragment of wild-type DNA rescues the mutant phenotype and the sequence of two mutant alleles show differences with wild type, demonstrating that we have isolated the shibire gene. The gene encodes a protein that is highly similar to rat dynamin, 69% of the amino-acid sequence is identical. Dynamin is a GTP-driven mechanochemical enzyme related to mammalian mx-proteins and to the yeast vps 1 gene product. Because the shibire gene product and dynamin have extensive similarity, we propose that they are cognate homologues. Dynamin causes microtubules to slide along each other in vitro and in extracts it is associated with a distinct, but so far uncharacterized, membrane fraction. In light of the shibire phenotype, we suggest that these proteins provide the motor for vesicular transport during endocytosis.

Multidrug resistance phenotype of human BRO melanoma cells transfected with a wild-type human mdr1 complementary DNA.

We have transfected a eukaryotic expression vector containing a mdr1 complementary DNA isolated from normal human liver into human BRO melanoma cells to study the drug-resistant phenotype produced by the exclusive overexpression of normal human mdr1 P-glycoprotein. The drug resistance pattern of mdr1-transfected clones includes relatively high resistance to gramicidin D (about 300-fold), vincristine (about 100-fold), and actinomycin D (about 100-fold) and a lower degree of resistance to doxorubicin (about 10-fold), VP16-213 (about 10-fold), and colchicine (about 6-fold). The transfectants did not exhibit resistance to trimetrexate, cis-platinum, mitomycin C, 1-beta-D-arabinofuranosylcytosine, bleomycin, G418, or magainin-2-amide; they were slightly more sensitive to verapamil (2-fold) but not to Triton X-100. As in other multidrug-resistant cell lines, resistance to vincristine could be reversed by verapamil and, more effectively, by cyclosporin A. Chloroquine only marginally increased drug sensitivity in mdr1-transfected cells. Gramicidin D resistance was also reversed by verapamil, suggesting that the mechanism of resistance to this polypeptide antibiotic is similar to that of other drugs transported by P-glycoprotein. Thus, expression of the wild-type mdr1 complementary DNA induces a drug-resistant phenotype similar to that induced by mdr1 complementary DNAs isolated from drug-resistant cell lines with relatively low colchicine resistance. As other cell lines may display a different pattern of drug resistance, it is clear that other resistance mechanisms or cell type-specific factors may modulate the resistance. mdr1-transfected cell lines provide a convenient tool for the identification of P-glycoprotein-mediated phenomena.