Mammalian dynamin-like protein DLP1 tubulates membranes

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) but not GTP. EM of the GTPgammaS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTPgammaS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.     

A functional link between dynamin and the actin cytoskeleton at podosomes

Cell transformation by Rous sarcoma virus results in a dramatic change of adhesion structures with the substratum. Adhesion plaques are replaced by dot-like attachment sites called podosomes. Podosomes are also found constitutively in motile nontransformed cells such as leukocytes, macrophages, and osteoclasts. They are represented by columnar arrays of actin which are perpendicular to the substratum and contain tubular invaginations of the plasma membrane. Given the similarity of these tubules to those generated by dynamin around a variety of membrane templates, we investigated whether dynamin is present at podosomes. Immunoreactivities for dynamin 2 and for the dynamin 2-binding protein endophilin 2 (SH3P8) were detected at podosomes of transformed cells and osteoclasts. Furthermore, GFP wild-type dynamin 2aa was targeted to podosomes. As shown by fluorescence recovery after photobleaching, GFP-dynamin 2aa and GFP-actin had a very rapid and similar turnover at podosomes. Expression of the GFP-dynamin 2aa(G273D) abolished podosomes while GFP-dynamin(K44A) was targeted to podosomes but delayed actin turnover. These data demonstrate a functional link between a member of the dynamin family and actin at attachment sites between cells and the substratum.     

A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.     

Big gulps: specialized membrane domains for rapid receptor-mediated endocytosis

Eukaryotic cells are well known to use an elaborate, clathrin-based endocytic machinery to internalize ligand-receptor complexes. Although the number of components identified as being used during this essential process has increased substantially over the past decade, an appreciation for how receptor-mediated endocytosis is organized at a broader, cellular scale is still mostly undefined. Here, some new insights into how cells cluster and organize the endocytic machinery at defined cytoplasmic domains to increase the speed and efficiency of ligand-receptor internalization are presented.     

Dynamin 3 is a component of the postsynapse,where it interacts with mGluR5 and Homer

The dynamins comprise a large family of mechanoenzymes known to participate in membrane modeling events. All three conventional dynamin genes (Dyn1, Dyn2, Dyn3) are expressed in mammalian brain and produce more than 27 different dynamin proteins as a result of alternative splicing. Past studies have suggested that Dyn1 participates in specialized neuronal functions such as rapid synaptic vesicle recycling, while Dyn2 may mediate the conventional clathrin-mediated uptake of surface receptors. Currently, the distribution, expression, and function of Dyn3 in neurons, or in any other cell type, are completely undefined. Here, we demonstrate that Dyn1 and Dyn3 localize differentially in the synapse. Dyn1 concentrates within the presynaptic compartment, while Dyn3 localizes to dendritic spine tips. Within the postsynaptic density (PSD), we found Dyn3, but not Dyn1, to be part of a biochemically isolated complex comprised of Homer and metabotropic glutamate receptors. Finally, although dominant-negative Dyn3 did not seem to inhibit receptor endocytosis, overexpression of a specific Dyn3 spliced variant in mature neurons caused a marked remodeling of dendritic spines. These data suggest that Dyn3 is a postsynaptic dynamin and, like its binding partner Homer, plays a significant role in dendritic spine morphogenesis and remodeling.