McNiven, Mark A.

Immature dendrites extend many actin-rich filopodial structures that can be replaced by synapse-containing dendritic spines as the neuron matures. The large GTPase dynamin-3 (Dyn3) is a component of the postsynapse in hippocampal neurons but its function is undefined. Here, we demonstrate that a specific Dyn3 variant (Dyn3baa) promotes the formation of immature dendritic filopodia in cultured neurons. This effect is dependent upon Dyn3 GTPase activity and a direct interaction with the F-actin-binding protein cortactin. Consistent with these findings, Dyn3baa binds to cortactin with a 200% higher affinity than Dyn3aaa, a near identical isoform that does not induce dendritic filopodia when expressed in cultured neurons. Finally, levels of Dyn3baa-encoding mRNA are tightly regulated during neuronal maturation and are markedly upregulated during synaptogenesis. Together, these findings provide the first evidence that an enhanced interaction between a specific Dyn3 splice variant and cortactin modulate actin-membrane dynamics in developing neurons to regulate the morphogenesis of dendritic spines.

Dynamins are a family of 100-kD GTPases comprised of at least three distinct gene products and multiple alternatively spliced variants. Homologies with the shibire gene product in Drosophila melanogaster and with Vps1p and Dnm1p in Saccharomyces cerevisiae suggest that dynamins play an important role in vesicular transport. Morphological studies have localized brain dynamin to coated pits and tubular invaginations at the plasma membrane, where it is believed to facilitate the formation of endocytic vesicles. Because similar membrane-budding events occur at the Golgi apparatus and multiple dynamin isoforms exist, we have studied the distribution of dynamins in mammalian cells. To this end, we generated and characterized peptide-specific antibodies directed against conserved regions of the dynamin family. By immunoblot analysis, these antibodies reacted specifically with a 100-kD protein in fibroblasts that sedimented with membranes and microtubules in vitro in a manner similar to brain dynamin. By immunofluorescence microscopy, these antibodies strongly labeled the Golgi complex in cultured fibroblasts and melanocytes, as confirmed by double labeling with a Golgi-specific antibody. Furthermore, Western blot analysis showed significant enrichment of a 100-kD dynamin band in Golgi fractions isolated from the liver. To substantiate these findings, we use a specific antidynamin antibody to immunoisolate Golgi membranes from subcellular Golgi fractions, as determined by EM and immunoblot analysis. This study provides the first morphological and biochemical evidence that a dynamin-like protein associates with the Golgi apparatus in mammalian cells, and suggests that dynamin-related proteins may have multiple cytoplasmic distributions. The potential contributions of dynamin to the secretory and endocytic pathways are discussed.

The mechanisms by which mammalian cells remodel the actin cytoskeleton in response to motogenic stimuli are complex and a topic of intense study. Dynamin 2 (Dyn2) is a large GTPase that interacts directly with several actin binding proteins, including cortactin. In this study, we demonstrate that Dyn2 and cortactin function to mediate dynamic remodeling of the actin cytoskeleton in response to stimulation with the motogenic growth factor platelet-derived growth factor. On stimulation, Dyn2 and cortactin coassemble into large, circular structures on the dorsal cell surface. These “waves” promote an active reorganization of actin filaments in the anterior cytoplasm and function to disassemble actin stress fibers. Importantly, inhibition of Dyn2 and cortactin function potently blocked the formation of waves and subsequent actin reorganization. These findings demonstrate that cortactin and Dyn2 function together in a supramolecular complex that assembles in response to growth factor stimulation and mediates the remodeling of actin to facilitate lamellipodial protrusion at the leading edge of migrating cells.

The degradation of extracellular matrix (ECM) by matrix metalloproteases is crucial in physiological and pathological cell invasion alike. Degradation occurs at specific sites where invasive cells make contact with the ECM via specialized plasma membrane protrusions termed invadopodia. Herein, we show that the dynamin 2 (Dyn2), a GTPase implicated in the control of actin-driven cytoskeletal remodeling events and membrane transport, is necessary for focalized matrix degradation at invadopodia. Dynamin was inhibited by using two approaches: 1) expression of dominant negative GTPase-impaired or proline-rich domain-deleted Dyn2 mutants; and 2) inhibition of the dynamin regulator calcineurin by cyclosporin A. In both cases, the number and extension of ECM degradation foci were drastically reduced. To understand the site and mechanism of dynamin action, the cellular structures devoted to ECM degradation were analyzed by correlative confocal light-electron microscopy. Invadopodia were found to be organized into a previously undescribed ECM-degradation structure consisting of a large invagination of the ventral plasma membrane surface in close spatial relationship with the Golgi complex. Dyn2 seemed to be concentrated at invadopodia.

The sperm acrosome is essential for sperm-egg fusion and is often defective in men with nonobstructive infertility. Here we report that male mice with a null mutation in Hrb are infertile and display round-headed spermatozoa that lack an acrosome. In wild-type spermatids, Hrb is associated with the cytosolic surface of proacrosomic transport vesicles that fuse to create a single large acrosomic vesicle at step 3 of spermiogenesis. Although proacrosomic vesicles form in spermatids that lack Hrb, the vesicles are unable to fuse, blocking acrosome development at step 2. We conclude that Hrb is required for docking and/or fusion of proacrosomic vesicles during acrosome biogenesis.

Dynamin guanosine triphosphatases support the scission of clathrin-coated vesicles from the plasmalemma during endocytosis. By fluorescence microscopy of cultured rat hepatocytes, a green fluorescent protein–dynamin II fusion protein localized with clathrin-coated vesicles at the Golgi complex. A cell-free assay was utilized to demonstrate the role of dynamin in vesicle formation at the trans-Golgi. Addition of peptide-specific anti-dynamin antibodies to the assay mixture inhibited both constitutive exocytic and clathrin-coated vesicle formation. Immunodepletion of dynamin proteins also inhibited vesicle formation, and budding efficiency was restored upon readdition of purified dynamin. These data suggest that dynamin participates in the formation of distinct transport vesicles from the trans-Golgi network.

Abstract. Dynamins are 100-kilodalton guanosine triphosphatases that participate in the formation of nascent vesicles during endocytosis. Here, we have tested if novel dynamin-like proteins are expressed in mammalian cells to support vesicle trafficking processes at cytoplasmic sites distinct from the plasma membrane. Immunological and molecular biological methods were used to isolate a cDNA clone encoding an 80-kilodalton novel dynamin-like protein, DLP1, that shares up to 42% homology with other dynamin-related proteins. DLP1 is expressed in all tissues examined and contains two alternatively spliced regions that are differentially expressed in a tissue-specific manner. DLP1 is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum. Morphological studies of DLP1 in cultured cells using either a specific antibody or an expressed green fluorescent protein (GFP)- DLP1 fusion protein revealed that DLP1 associates with punctate cytoplasmic vesicles that do not colocalize with conventional dynamin, clathrin, or endocytic ligands. Remarkably, DLP1-positive structures coalign with microtubules and, most strikingly, with endoplasmic reticulum tubules as verified by double labeling with antibodies to calnexin and Rab1 as well as by immunoelectron microscopy. These observations provide the first evidence that a novel dynamin-like protein is expressed in mammalian cells where it associates with a secretory, rather than endocytic membrane compartment.

The dynamin family of large GTPases has been implicated in the formation of nascent vesicles in both the endocytic and secretory pathways. It is believed that dynamin interacts with a variety of cellular proteins to constrict membranes. The actin cytoskeleton has also been implicated in altering membrane shape and form during cell migration, endocytosis, and secretion and has been postulated to work synergistically with dynamin and coat proteins in several of these important processes. We have observed that the cytoplasmic distribution of dynamin changes dramatically in fibroblasts that have been stimulated to undergo migration with a motagen/hormone. In quiescent cells, dynamin 2 (Dyn 2) associates predominantly with clathrin-coated vesicles at the plasma membrane and the Golgi apparatus. Upon treatment with PDGF to induce cell migration, dynamin becomes markedly associated with membrane ruffles and lamellipodia. Biochemical and morphological studies using antibodies and GFP-tagged dynamin demonstrate an interaction with cortactin. Cortactin is an actin-binding protein that contains a well defined SH3 domain. Using a variety of biochemical methods we demonstrate that the cortactin–SH3 domain associates with the proline-rich domain (PRD) of dynamin. Functional studies that express wild-type and mutant forms of dynamin and/or cortactin in living cells support these in vitro observations and demonstrate that an increased expression of cortactin leads to a significant recruitment of endogenous or expressed dynamin into the cell ruffle. Further, expression of a cortactin protein lacking the interactive SH3 domain (CortΔSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)ΔPRD) sequester little of this protein to the cortactin-rich ruffle. Interestingly, these mutant cells are viable, but display dramatic alterations in morphology. This change in shape appears to be due, in part, to a striking increase in the number of actin stress fibers. These findings provide the first demonstration that dynamin can interact with the actin cytoskeleton to regulate actin reorganization and subsequently cell shape.