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Actin reorganization at the immunological synapse is required for the amplification and generation of a functional immune response. Using small interfering RNA, we show here that dynamin 2 (Dyn2), a large GTPase involved in receptor-mediated internalization, did not alter antibody-mediated T cell receptor internalization but considerably affected T cell receptor-stimulated T cell activation by regulating multiple biochemical signaling pathways and the accumulation of F-actin at the immunological synapse. Moreover, Dyn2 interacted directly with the Rho family guanine nucleotide exchange factor Vav1, and this interaction was required for T cell activation. These data identify a functionally important interaction between Dyn2 and Vav1 that regulates actin reorganization and multiple signaling pathways in T lymphocytes.

An equine abuse policy was developed as an adjunct to an equine management survey. If at least 3 of 5 categories caused concern, a report to the authorities was indicated. The policy was not used but, in the absence of other guidelines, it might assist veterinarians considering potential abuse cases.

There are no detailed, representative, horse-level data about equine management practices in different parts of Canada. To help address this, the demographics, management, and welfare of 312 nonracing horses in Prince Edward Island were examined in a randomized, horse-level survey during summer 2002. Owners completed a pretested questionnaire, and a veterinarian examined each horse. Owners were experienced caregivers and the horses were generally in good condition. Areas for improvement included parasite control, dental and hoof care, and tail docking. The mean fecal egg count was 428 eggs per gram; 76% of owners never removed manure from the pasture. Sixty-two percent of horses had never had a veterinary dental examination. Many horses had hoof defects (excessively long hooves, 26.8%; hoof wall breaks, 32.0%; and white line disease, 8.5%). Many (54.9%) draft horses had docked tails. These results suggest owners might benefit their horses by receiving education in aspects of equine care.

The dynamin superfamily of large GTPases has been implicated in a variety of distinct intracellular membrane remodeling events. One of these family members, DLP1/Drp1, is similar to conventional dynamins as it contains an N-terminal GTPase domain followed by a middle region (MID), an unconserved region (UC), and a coiled-coil (CC) domain. DLP1 has been shown to function in membrane-based processes distinct from conventional dynamin, most notably mitochondrial fission. In this study, we tested whether the functional specificities of DLP1 and dynamin stems from differences in the individual domains of these proteins by generating dynamin/DLP1 chimeras in which correlate domains had been interchanged. Here we report that three consecutive C-terminal domains of DLP1 (MID-UC-CC) contain information necessary for DLP1-specific function and removing any one of these domains results in a loss of DLP1 function. Importantly, the coiled-coil (CC) domain of DLP1 alone targets specifically and exclusively to mitochondria, implicating its involvement in localizing DLP1 to this organelle in vivo. The mitochondrial targeting information within the DLP1 CC domain is not sufficient to retarget dynamin to mitochondria but is still able to adequately function as an assembly domain in a dynamin background. These data suggest that whereas the GTPase domain of DLP1 provides an enzymatic function, other domains contain information for intermolecular assembly and mitochondrial targeting.

The plasma membrane of many motile cells undergoes highly regulated protrusions and invaginations that support the formation of podosomes, invadopodia and circular dorsal ruffles. Although they are similar in appearance and in their formation--which is mediated by a highly conserved actin-membrane apparatus--these transient surface membrane distortions are distinct. Their function is to help the cell as it migrates, attaches and invades.

Cells make contact with the extracellular matrix (ECM) through extensions of the plasma membrane; these range from irregular dynamic structures, e.g. lamellipodia, ruffles and pseudopodia, to more localized and highly defined protrusions, e.g. podosomes and invadopodia. Both might be instruments through which cells sample the immediate extracellular environment and maintain polarized activities such as chemotaxis and focal degradation of the matrix. Podosomes are expressed in cells of the monocytic lineage, and most studies point to a role for podosomes in adhesion/motility. Invadopodia are prominent in certain aggressive cancer cells (or transformed cells) and appear to be directly responsible for focal ECM degradation. Recent studies have revived interest in these structures in terms of the actin regulation machinery. Within this framework, the atypical GTP-binding protein dynamin, a central modulator of protrusive events, has been associated to podosome and invadopodia structure and function. Here, we specifically discuss the role played by dynamin in controlling the activities and function of these structures.

Cryptosporidium parvum invasion of epithelial cells involves host cell membrane alterations which require a remodeling of the host cell actin cytoskeleton. In addition, an actin plaque, possibly associated with the dense-band region, forms within the host cytoplasm at the host-parasite interface. Here we show that Cdc42 and RhoA, but not Rac1, members of the Rho family of GTPases, are recruited to the host-parasite interface in an in vitro model of human biliary cryptosporidiosis. Interestingly, activation of Cdc42, but not RhoA, was detected in the infected cells. Neural Wiskott-Aldrich syndrome protein (N-WASP) and p34-Arc, actin-regulating downstream effectors of Cdc42, were also recruited to the host-parasite interface. Whereas cellular expression of a constitutively active mutant of Cdc42 promoted C. parvum invasion, overexpression of a dominant negative mutant of Cdc42, or depletion of Cdc42 mRNA by short interfering RNA-mediated gene silencing, inhibited C. parvum invasion. Expression of the WA fragment of N-WASP to block associated actin polymerization also inhibited C. parvum invasion. Moreover, inhibition of host cell Cdc42 activation by dominant negative mutation inhibited C. parvum-associated actin remodeling, membrane protrusion, and dense-band formation. In contrast, treatment of cells with a Rho inhibitor, exoenzyme C3, or cellular overexpression of dominant negative mutants of RhoA and Rac1 had no effect on C. parvum invasion. These data suggest that C. parvum invasion of target epithelia results from the organism's ability to activate a host cell Cdc42 GTPase signaling pathway to induce host cell actin remodeling at the attachment site.

Dynamin 2 (Dyn2) is a large GTPase involved in vesicle formation and actin reorganization. In this study, we report a novel role for Dyn2 as a component of the centrosome that is involved in centrosome cohesion. By light microscopy, Dyn2 localized aside centrin and colocalized with gamma-tubulin at the centrosome; by immunoelectron microscopy, however, Dyn2 was detected in the pericentriolar material as well as on centrioles. Exogenously expressed green fluorescent protein (GFP)-tagged Dyn2 also localized to the centrosome, whereas glutathione S-transferase (GST)-tagged Dyn2 pulled down a protein complex(es) containing actin, alpha-tubulin and gamma-tubulin from liver homogenate. Furthermore, gel overlay and immunoprecipitation indicated a direct interaction between gamma-tubulin and a 219-amino-acid middle domain of Dyn2. Reduction of Dyn2 protein levels with small-interfering RNA (siRNA) resulted in centrosome splitting, whereas microtubule nucleation from centrosomes was not affected, suggesting a role for Dyn2 in centrosome cohesion. Finally, fluorescence recovery after photobleaching (FRAP) analysis of a GFP-tagged Dyn2 middle domain indicated that Dyn2 is a dynamic exchangeable component of the centrosome. These findings suggest a novel function for Dyn2 as a participant in centrosome cohesion.

Bulk tank milk selenium (Se) concentration was compared with mean serum Se concentration in 15 herds and was found to be an accurate reflection of the herd Se status. The Se status of 109 Prince Edward Island (PEI) dairy herds was monitored for 1 year using bulk tank milk Se concentration. Fifty-nine percent of the herds surveyed were, at some point, found to be marginal or deficient in Se, putting them at risk of disease and suboptimal production. The periods of greatest risk of deficiency were fall and winter, at which time 5% and 4%, respectively, of herds sampled fell in the range considered truly deficient in Se. Herds in which Se supplementation was provided in the form of a commercial dairy concentrate were over 4 times more likely to be Se-adequate than herds not using this method, and adjusted average daily milk yield was 7.6% greater in herds determined to be Se-adequate when compared with Se-marginal herds. We conclude that many dairy producers in PEI are providing insufficient supplementary Se in the ration to meet the recommended Se intake for lactating cows.

The yeast protein Fis1p has been shown to participate in mitochondrial fission mediated by the dynamin-related protein Dnm1p. In mammalian cells, the dynamin-like protein DLP1/Drp1 functions as a mitochondrial fission protein, but the mechanisms by which DLP1/Drp1 and the mitochondrial membrane interact during the fission process are undefined. In this study, we have tested the role of a mammalian homologue of Fis1p, hFis1, and provided new and mechanistic information about the control of mitochondrial fission in mammalian cells. Through differential tagging and deletion experiments, we demonstrate that the intact C-terminal structure of hFis1 is essential for mitochondrial localization, whereas the N-terminal region of hFis1 is necessary for mitochondrial fission. Remarkably, an increased level of cellular hFis1 strongly promotes mitochondrial fission, resulting in an accumulation of fragmented mitochondria. Conversely, cell microinjection of hFis1 antibodies or treatment with hFis1 antisense oligonucleotides induces an elongated and collapsed mitochondrial morphology. Further, fluorescence resonance energy transfer and coimmunoprecipitation studies demonstrate that hFis1 interacts with DLP1. These results suggest that hFis1 participates in mitochondrial fission through an interaction that recruits DLP1 from the cytosol. We propose that hFis1 is a limiting factor in mitochondrial fission and that the number of hFis1 molecules on the mitochondrial surface determines fission frequency.

Background & Aims: Cryptosporidium parvum invasion of epithelia requires polymerization of host cell actin at the attachment site. We analyzed the role of host cell c-Src, a cytoskeleton-associated protein tyrosine kinase, in C. parvum invasion of biliary epithelia., Methods: In vitro models of biliary cryptosporidiosis using a human biliary epithelial cell line were used to assay the role of c-Src signaling pathway in C. parvum invasion., Results: c-Src and cortactin, an actin-binding protein and a substrate for c-Src, were recruited to the parasite-host cell interface during C. parvum invasion. Tyrosine phosphorylation of cortactin in infected cells was also detected. Inhibition of host cell c-Src significantly blocked C. parvum -induced accumulation and tyrosine phosphorylation of cortactin and actin polymerization at the attachment sites, thereby inhibiting C. parvum invasion of biliary epithelial cells. A triple mutation of tyrosine of cortactin in the epithelia also diminished C. parvum invasion. In addition, proteins originating from the parasite were detected within infected cells at the parasite-host cell interface. Antiserum against C. parvum membrane proteins blocked accumulation of c-Src and cortactin and significantly decreased C. parvum invasion. No accumulation of the endocytosis-related proteins, dynamin 2 and clathrin, was found at the parasite-host cell interface; also, inhibition of dynamin 2 did not block C. parvum invasion., Conclusions: C. parvum invasion of biliary epithelial cells requires host cell tyrosine phosphorylation of cortactin by a c-Src-mediated signaling pathway to induce actin polymerization at the attachment site, a process associated with microbial secretion but independent of host cell endocytosis.

We previously proposed that ductal bile formation is regulated by secretin-responsive relocation of aquaporin 1 (AQP1), a water-selective channel protein, from an intracellular vesicular compartment to the apical membrane of cholangiocytes. In this study, we immunoisolated AQP1-containing vesicles from cholangiocytes prepared from rat liver; quantitative immunoblotting revealed enrichment in these vesicles of not only AQP1 but also cystic fibrosis transmembrane regulator (CFTR) and AE2, a Cl- channel and a Cl-/HCO3- exchanger, respectively. Dual labeled immunogold electron microscopy of cultured polarized mouse cholangiocytes showed significant colocalization of AQP1, CFTR, and AE2 in an intracellular vesicular compartment; exposure of cholangiocytes to dibutyryl-cAMP (100 microm) resulted in co-redistribution of all three proteins to the apical cholangiocyte plasma membrane. After administration of secretin to rats in vivo, bile flow increased, and AQP1, CFTR, and AE2 co-redistributed to the apical cholangiocyte membrane; both events were blocked by pharmacologic disassembly of microtubules. Based on these in vitro and in vivo observations utilizing independent and complementary approaches, we propose that cholangiocytes contain an organelle that sequesters functionally related proteins that can account for ion-driven water transport, that this organelle moves to the apical cholangiocyte membrane in response to secretory agonists, and that these events account for ductal bile secretion at a molecular level.

Haddock (Melanogrammus aeglefinus) is a gadoid fish species that deposits dietary lipid mainly in the liver. The fatty acid (FA) beta-oxidation activity of various tissues was evaluated in juvenile haddock fed graded levels of lipid. The catabolism of a radiolabelled FA, [1-(14)C]palmitoyl-CoA, through peroxisomal and mitochondrial beta-oxidation was determined in the liver, red and white muscle of juvenile haddock fed 12, 18 and 24% lipid in the diet. There was no significant increase in the mitochondrial or peroxisomal beta-oxidation activity in the tissues tested as the dietary lipid level increased from 12 to 24%. Peroxisomes accounted for 100% of the beta-oxidation observed in the liver, whereas mitochondrial beta-oxidation dominated in the red (91%) and white muscle (97%) of juvenile haddock. Of the tissues tested, red muscle possessed the highest specific activity for beta-oxidation expressed on a per mg protein or per g wet weight basis. However, white muscle, which forms over 50% of the body mass in gadoid fish was the most important tissue in juvenile haddock for overall FA catabolism. The total lipid and FA composition of these tissues were also determined. This study confirmed that the liver was the major lipid storage organ in haddock. The hepatosomatic index (HSI; 10.0-15.2%) and lipid (73.8-79.3% wet wt.) in the liver increased significantly as dietary lipid was increased from 12 to 24% lipid. There was no significant increase in the lipid composition of the white muscle (0.8% wet wt.), red muscle (1.9% wet wt.) or heart (2.5% wet wt.).

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.

The mammalian dynamin-like protein 1 (DLP1), a member of the dynamin family of large GTPases, possesses mechanochemical properties known to constrict and tubulate membranes. In this study, we have combined two experimental approaches, induction of peroxisome proliferation by Pex11pbeta and expression of dominant-negative mutants, to test whether DLP1 plays a role in peroxisomal growth and division. We were able to localize DLP1 in spots on tubular peroxisomes in HepG2 cells. In addition, immunoblot analysis revealed the presence of DLP1 in highly purified peroxisomal fractions from rat liver and an increase of DLP1 after treatment of rats with the peroxisome proliferator bezafibrate. Expression of a dominant negative DLP1 mutant deficient in GTP hydrolysis (K38A) either alone or in combination with Pex11pbeta caused the appearance of tubular peroxisomes but had no influence on their intracellular distribution. In co-expressing cells, the formation of tubulo-reticular networks of peroxisomes was promoted, and peroxisomal division was completely inhibited. These findings were confirmed by silencing of DLP1 using siRNA. We propose a direct role for the dynamin-like protein DLP1 in peroxisomal fission and in the maintenance of peroxisomal morphology in mammalian cells.

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 actin cytoskeleton is believed to contribute to the formation of clathrin-coated pits, although the specific components that connect actin filaments with the endocytic machinery are unclear. Cortactin is an F-actin-associated protein, localizes within membrane ruffles in cultured cells, and is a direct binding partner of the large GTPase dynamin. This direct interaction with a component of the endocytic machinery suggests that cortactin may participate in one or several endocytic processes. Therefore, the goal of this study was to test whether cortactin associates with clathrin-coated pits and participates in receptor-mediated endocytosis. Morphological experiments with either anti-cortactin antibodies or expressed red fluorescence protein-tagged cortactin revealed a striking colocalization of cortactin and clathrin puncta at the ventral plasma membrane. Consistent with these observations, cells microinjected with these antibodies exhibited a marked decrease in the uptake of labeled transferrin and low-density lipoprotein while internalization of the fluid marker dextran was unchanged. Cells expressing the cortactin Src homology three domain also exhibited markedly reduced endocytosis. These findings suggest that cortactin is an important component of the receptor-mediated endocytic machinery, where, together with actin and dynamin, it regulates the scission of clathrin pits from the plasma membrane. Thus, cortactin provides a direct link between the dynamic actin cytoskeleton and the membrane pinchase dynamin that supports vesicle formation during receptor-mediated endocytosis.

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.

Many important cellular processes such as phagocytosis, cell motility and endocytosis require the participation of a dynamic and interactive actin cytoskeleton that acts to deform cellular membranes. The extensive family of non-traditional myosins has been implicated in linking the cortical actin gel with the plasma membrane. Recently, however, the dynamins have also been included in these cell processes as a second family of mechanochemical enzymes that self-associate and hydrolyze nucleotides to perform 'work' while linking cellular membranes to the actin cytoskeleton. The dynamins are believed to form large helical polymers from which extend many interactive proline-rich tail domains, and these domains bind to a variety of SH3-domain-containing proteins, many of which appear to be actin-binding proteins. Recent data support the concept that the dynamin family might act as a 'polymeric contractile scaffold' at the interface between biological membranes and filamentous actin.

Background/aims: The hepatocellular transport pathways and cellular proteins utilized during the packaging and secretion of hepatitis B virus are poorly understood. In this study, we tested if the large GTPase dynamin, a protein involved in vesicle formation and secretion at the trans-Golgi network in hepatocytes, is also used by hepatitis B virus (HBV) in secreting viral proteins., Methods: Using HepG2.2.15 cells expressing the full-length HBV genome, we tested the effects of wild-type and mutant dynamin on the localization and secretion of two hepatitis B antigens, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Distribution of these two antigens was analyzed morphologically in cells transiently transfected with wild-type or mutant dynamin constructs, whereas secretion of the antigens was measured by testing for antigen levels in the media of transfected cells., Results: Mutant dynamin was found to induce a striking redistribution of HBsAg and HBeAg to a perinuclear compartment, as well as a decrease in the levels of HBsAg and HBeAg present in cell culture media indicating a reduction in viral protein secretion. At the electron microscopy level, cells expressing the mutant dynamin showed a marked accumulation of viral particles in dilated cisternae of an uncharacterized cellular compartment., Conclusions: Intact dynamin function is required for secretion of HBV proteins from hepatocytes through an uncharacterized cellular compartment.

Cytokinesis involves the concerted efforts of the microtubule and actin cytoskeletons as well as vesicle trafficking and membrane remodeling to form the cleavage furrow and complete daughter cell separation. The exact mechanisms that support membrane remodeling during cytokinesis remain largely undefined. In this study, we report that the large GTPase dynamin, a protein involved in membrane tubulation and vesiculation, is essential for successful cytokinesis. Using biochemical and morphological methods, we demonstrate that dynamin localizes to the spindle midzone and the subsequent intercellular bridge in mammalian cells and is also enriched in spindle midbody extracts. In Caenorhabditis elegans, dynamin localized to newly formed cleavage furrow membranes and accumulated at the midbody of dividing embryos in a manner similar to dynamin localization in mammalian cells. Further, dynamin function appears necessary for cytokinesis, as C. elegans embryos from a dyn-1 ts strain, as well as dynamin RNAi-treated embryos, showed a marked defect in the late stages of cytokinesis. These findings indicate that, during mitosis, conventional dynamin is recruited to the spindle midzone and the subsequent intercellular bridge, where it plays an essential role in the final separation of dividing cells.

The objective of this study was to maintain the viability of chilled rainbow trout (Oncorhynchus mykiss) eyed eggs during storage using oxygenated perfluorochemical (PFC). Three trials were conducted using eggs at 161, 180 or 217 degree days (days from fertilization x incubation temperature in degrees C). A separate trial was conducted for 147 degree day eggs that were not at the eyed stage. For each trial, eggs were stored in a moisture-saturated atmosphere at 1 degrees C in PFC, water, and 1:1 combinations of PFC and PBS, PFC and 0.3 M glucose, PFC and mineral oil, or PFC and water. The PFC was oxygenated before each trial and all media were oxygenated at weekly intervals during the storage period. Eggs from each trial were also incubated without storage to provide Day 0 results. After 3 and 5 weeks of storage, eggs from each medium were incubated at 10 degrees C until hatch. Hatching percentage was expressed as a percentage of Day 0 results. The percentage of normal alevins that hatched was also determined. There were interactions (P < 0.01) between stage of development and treatment for hatching percentage after 3 and 5 weeks of storage. After 3 weeks of storage, eggs stored at 161, 180, or 217 degree days without PFC had hatching rates of 0-14.3% but eggs stored in any medium with PFC had hatching percentages from 75.1 to 106.4% of Day 0 values. After 5 weeks of storage, eggs stored at 161 degree days in PFC plus PBS or PFC plus water, and eggs stored at 217 degree days in PFC or PFC plus water, had higher (P < 0.05) hatching percentages than eggs stored in any of the other media. Eggs stored at 161 degree days for 5 weeks in PFC and water had a higher (P < 0.05) percentage of normal alevins hatching than eggs stored in PFC and PBS. Because of their early developmental stage, eggs stored at 147 degree days had low hatching percentages, except eggs stored for 3 weeks in PFC or PFC plus PBS. Chilling eyed eggs of rainbow trout to 1 degrees C and storing them in water with PFC as an oxygen carrier can preserve their viability for 5 weeks.

The Golgi apparatus is a dynamic organelle through which nascent secretory and transmembrane proteins are transported, post-translationally modified and finally packaged into carrier vesicles for transport along the cytoskeleton to a variety of destinations. In the past decade, studies have shown that a number of 'molecular motors' are involved in maintaining the proper structure and function of the Golgi apparatus. Here, we review just some of the many functions performed by these mechanochemical enzymes - dyneins, kinesins, myosins and dynamin - in relation to the Golgi apparatus.

Mx proteins are induced by type I interferon and inhibit a broad range of viruses by undefined mechanisms. They are included within the dynamin family of large GTPases, which are involved in vesicle trafficking and share common biophysical features. These properties include the propensity to self-assemble, an affinity for lipids, and the ability to tubulate membranes. In this report we establish that human MxA, despite sharing only 30% homology with conventional dynamin, possesses many of these properties. We demonstrate for the first time that MxA self-assembles into rings that tubulate lipids in vitro, and associates with a specific membrane compartment in cells, the smooth endoplasmic reticulum.

The large GTPase dynamin (Dyn2) has been demonstrated by us and others to interact with several different actin-binding proteins. To define how Dyn2 might participate in actin dynamics in livings cells we have expressed green fluorescent protein (GFP)-tagged Dyn2 in cultured cells and observed labeling of comet-like vesicles and macropinosomes. The comet structures progressed with a constant velocity and were reminiscent of actin comets associated with motile vesicles in cells expressing type I phosphatidylinositol phosphate 5-kinases. Based on these observations we sought to determine whether Dyn2 is an integral component of actin comets. Cells expressing type I phosphatidylinositol phosphate 5-kinase and Dyn2-GFP revealed a prominent colocalization of Dyn2 and actin in comet structures. Interestingly, comet formation and motility were normal in cells expressing wild-type Dyn2-GFP but altered markedly in Dyn2 mutant-expressing cells. Dyn2K44A-GFP mutant cells displayed a significant reduction in comet number, length, velocity, and efficiency of movement. In contrast, comets in cells expressing Dyn2DeltaPRD-GFP appeared dark and did not incorporate the mutant Dyn2 protein, indicating that the proline-rich domain (PRD) is required for Dyn2 recruitment. Further, these comets were significantly longer and slower than those in control cells. These findings demonstrate a role for Dyn2 in actin-based vesicle motility.

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.

Background & Aims: The molecular mechanisms that contribute to the cholestatic condition in hepatocytes are poorly defined. It has been postulated that a disruption of normal vesicle-based protein trafficking may lead to alterations in hepatocyte polarity., Methods: To determine if vesicle motility is reduced by cholestasis, hepatocytes cultured from livers of bile duct ligation (BDL)- or ethinyl estradiol (EE)-injected rats, were viewed and recorded by high-resolution video microscopy. Cholestatic hepatocytes were analyzed by phalloidin staining and electron microscopy. Functional analysis was done by the sodium fluorescein sequestration assay., Results: In cholestatic hepatocytes, there was a significant decrease in the number of motile cytoplasmic vesicles observed compared with control cells. Further examination of cells from BDL- or EE-treated livers revealed the presence of numerous large intracellular lumina. More than 24% of cells in BDL-treated livers and 19% of cells in EE-treated livers displayed these structures, compared with 1.1% found in control hepatocytes. Phalloidin staining of hepatocytes showed a prominent sheath of actin surrounding the lumina, reminiscent of those seen about bile canaliculi. Electron microscopy revealed that these structures were lined by actin-filled microvilli. Further, these pseudocanaliculi perform many of the functions exhibited by bona fide canaliculi, such as sequestering sodium fluorescein., Conclusions: Both mechanically and chemically induced cholestasis have substantial effects on vesicle-based transport, leading to marked disruption of hepatocellular polarity.

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.

Mitochondrial division is a complex process requiring the synergistic actions of multiple factors, including mechanical enzymes and accessory proteins. Recent studies have identified a number of these factors and started to elucidate how they act together to bring about mitochondrial fission.

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 (CortDeltaSH3) significantly reduces dynamin localization to the ruffle. Accordingly, transfected cells expressing Dyn 2 lacking the PRD (Dyn 2(aa)DeltaPRD) 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.

Rab3 isotypes are expressed in regulated secretory cells. Here, we report that rab3D is also expressed in rat hepatocytes, classic models for constitutive secretion. Using reverse transcriptase polymerase chain reaction (RT-PCR) with primers specific for rat rab3D, we amplified a 151 base pair rab3D fragment from total RNA extracted from primary cultures of rat hepatocytes. Immunoblot analysis using polyclonal antibodies to peptides representing the N- and C-terminal hypervariable regions of murine rab3D recognized a protein of approximately 25 kd in hepatocyte lysates, hepatic subcellular fractions, and tissue extracts. The distribution of rab3D was primarily cytosolic; however, only membrane-associated rab3D significantly bound guanosine triphosphate (GTP) in overlay assays. Several lines of investigation indicate that rab3D is associated with the transcytotic pathway. First, rab3D was enriched in a crude vesicle carrier fraction (CVCF), which includes transcytotic carriers. Vesicular compartments immunoisolated from the CVCF on magnetic beads coated with anti-rab3D antibody were enriched in the transcytosed form of the polymeric IgA receptor (pIgA-R), but lacked not only the pIgA-R precursor form associated with the secretory pathway, but also a Golgi marker protein. Second, indirect immunofluorescence on frozen liver sections and in polarized cultured hepatocytes localized rab3D-positive sites at or near the apical plasma membrane and to the pericanalicular cytoplasm. Finally, cholestasis induced by bile duct ligation (BDL), a manipulation known to slow transcytosis, caused rab3D to accumulate in the pericanalicular cytoplasm of cholestatic hepatocytes. Our results indicate that rab3D plays a role in the regulation of apically directed transcytosis in rat hepatocytes.

The large GTPase dynamin is a mechanoenzyme that participates in the scission of nascent vesicles from the plasma membrane. Recently, dynamin has been demonstrated to associate with the Golgi apparatus in mammalian cells by morphological and biochemical methods. Additional studies using a well characterized, cell-free assay have supported these findings by demonstrating a requirement for dynamin function in the formation of clathrin-coated, and non-clathrin-coated vesicles from the trans-Golgi network (TGN). In this study, we tested if dynamin participates in Golgi function in living cells through the expression of a dominant negative dynamin construct (K44A). Cells co-transfected to express this mutant dynamin and a GFP-tagged Golgi resident protein (TGN38) exhibit Golgi structures that are either compacted, vesiculated, or tubulated. Electron microscopy of these mutant cells revealed large numbers of Golgi stacks comprised of highly tubulated cisternae and an extraordinary number of coated vesicle buds. Cells expressing mutant dynamin and GFP-tagged VSVG demonstrated a marked retention (8- to 11-fold) of the nascent viral G-protein in the Golgi compared to control cells. These observations in living cells are consistent with previous morphological and in vitro studies demonstrating a role for dynamin in the formation of secretory vesicles from the TGN.

The large GTPase dynamin is a mechanoenzyme that mediates the liberation of nascent clathrin-coated pits from the plasma membrane during endocytosis. Recently, this enzyme has been demonstrated to comprise an extensive family of related proteins that have been implicated in a large variety of vesicle trafficking events during endocytosis, secretion and even maintenance of mitochondrial form. The potential contributions by the dynamin family to these diverse but related functions are discussed.

The actin cytoskeleton plays an important role in the mediation of exocytosis and the determination of cell shape. Experimentally induced changes in cell shape have been shown to affect stimulated secretion in pancreatic acini. In this study, we have examined whether physiologic agonists induce changes in acinar cell shape to modulate secretion. Computer-enhanced video microscopy, immunofluorescence confocal microscopy, and quantitative Western blotting were used to study cell shape changes and cytoskeletal dynamics in rat pancreatic acini. Amylase assays were performed to study the effect of the actin-myosin cytoskeletal antagonists latrunculin A, BDM, and ML-9 on secretion. We found that pancreatic acini underwent a prominent and reversible shape change in response to the physiologic secretory agonist cholecystokinin. This was accompanied by an apical activation of myosin II as well as a basolateral redistribution of both actin and myosin II. Cytoskeletal antagonists inhibited this shape change and attenuated stimulated amylase secretion. Therefore, in addition to acting as a barrier at the apex, the actin-myosin cytoskeleton may also function to modulate cell shape to further regulate stimulated secretion., (Copyright 2000 Wiley-Liss, Inc.)

Cells internalize soluble ligands through endocytosis and large particles through actin-based phagocytosis. The dynamin family of GTPases mediates the scission of endocytic vesicles from the plasma membrane. We report here that dynamin 2, a ubiquitously expressed dynamin isoform, has a role in phagocytosis in macrophages. Dynamin 2 is enriched on early phagosomes, and expression of a dominant-negative mutant of dynamin 2 significantly inhibits particle internalization at the stage of membrane extension around the particle. This arrest in phagocytosis resembles that seen with inhibitors of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K prevents the recruitment of dynamin to the site of particle binding. Although expression of mutant dynamin in macrophages inhibited particle internalization, it had no effect on the production of inflammatory mediators elicited by particle binding.

Dynamin is a 100-kDa GTPase that has been implicated in endocytosis. To extend our understanding of its cellular functions, we have microinjected specific affinity-purified anti-dynamin antibodies into cultured mammalian epithelial cells. Using this approach, dynamin function can be inhibited specifically and rapidly in single cells. Effects of microinjected inhibitory antibodies on distinct endocytic processes and plasmalemmal morphology were then assayed by fluorescence microscopy (FM) and ultrastructural analysis. Micro-injected antibodies inhibit the clathrin-mediated endocytosis of fluorophore-labeled transferrin and cause a marked invagination of the plasma membrane. Many of these long plasmalemmal invaginations had clathrin-coated pits along their cytoplasmic surface. A number of distinct noncoated pits resembling plasmalemmal caveolae also accumulated in anti-dynamin antibody-injected cells. Further, the cellular uptake of cholera toxin B, which normally occurs by the internalization of caveolae, was inhibited in these cells. In support of these observations, immunoisolation techniques, double-label immuno-FM, and immunoelectron microscopy (immuno-EM) provided biochemical and morphological evidence that dynamin associates with plasmalemmal caveolae. Together, these observations indicate that dynamin mediates scission from the plasma membrane of both clathrin-coated pits and caveolae during distinct endocytic processes. These results demonstrate that dynamin isoforms are involved in an additional endocytic process that is distinct from clathrin-mediated endocytosis and provide significant insights into the molecular mechanisms governing the GTP-mediated internalization of caveolae. Evidence is provided demonstrating that dynamin isoforms have a differential distribution in mammalian cells. Targeting information for these isoforms is provided at least in part by regions of alternative splicing. Thus, the different dynamin isoforms may be localized to distinct cellular compartments but provide a similar scission function during the biogenesis of nascent cytoplasmic vesicles.

The dynamin family of large GTPases has been implicated in vesicle formation from both the plasma membrane and various intracellular membrane compartments. The dynamin-like protein DLP1, recently identified in mammalian tissues, has been shown to be more closely related to the yeast dynamin proteins Vps1p and Dnm1p (42%) than to the mammalian dynamins (37%). Furthermore, DLP1 has been shown to associate with punctate vesicles that are in intimate contact with microtubules and the endoplasmic reticulum (ER) in mammalian cells. To define the function of DLP1, we have transiently expressed both wild-type and two mutant DLP1 proteins, tagged with green fluorescent protein, in cultured mammalian cells. Point mutations in the GTP-binding domain of DLP1 (K38A and D231N) dramatically changed its intracellular distribution from punctate vesicular structures to either an aggregated or a diffuse pattern. Strikingly, cells expressing DLP1 mutants or microinjected with DLP1 antibodies showed a marked reduction in ER fluorescence and a significant aggregation and tubulation of mitochondria by immunofluorescence microscopy. Consistent with these observations, electron microscopy of DLP1 mutant cells revealed a striking and quantitative change in the distribution and morphology of mitochondria and the ER. These data support very recent studies by other authors implicating DLP1 in the maintenance of mitochondrial morphology in both yeast and mammalian cells. Furthermore, this study provides the first evidence that a dynamin family member participates in the maintenance and distribution of the ER. How DLP1 might participate in the biogenesis of two presumably distinct organelle systems is discussed.

Suprastimulation of pancreatic acinar cells with specific agonists inhibits zymogen secretion and induces the formation of large basolateral blebs. Currently the molecular mechanisms that mediate this dramatic morphologic response are undefined. Further, it is unclear if blebbing represents a terminal or reversible event. Using computer-enhanced video microscopy of living acini we have found that these large blebs form rapidly (within 2-3 minutes) and exhibit ameboid undulations. They are induced by small increases in agonist concentration and require an energy-dependent phosphorylation event. Remarkably, the blebs are rapidly absorbed when agonist levels are reduced, indicating that blebbing is a reversible response to a physiological stimulus, not a terminal event. Morphological methods show that these dramatic changes in cell shape are accompanied by a marked reorganization of actin and myosin II at the basolateral domain. During 30 minutes of suprastimulation, both basolateral actin and myosin II gradually increase to form a ring centered at the necks of the blebs. Immunocytochemical and biochemical studies with a phospho-specific antibody to the myosin regulatory light chain reveal an activation of myosin II in suprastimulated acini that is completely absent in resting cells. Studies using cytoskeletal antagonistic drugs indicate that bleb formation and motility require actin remodeling concomitant with an activation of myosin II. This aberrant activation and reorganization of the actin-myosin cytoskeleton is likely to have detrimental effects on acinar cell function. Additionally, this mechanism of bleb formation may be conserved among other forms of physiological blebbing events.

While the clinical features of sclerosing cholangitis secondary to opportunistic infections of the biliary tree in patients with acquired immunodeficiency syndrome (AIDS) are well known, the mechanisms by which microbial pathogens such as Cryptosporidium parvum associated with this syndrome actually cause disease are obscure. We established an in vitro model of biliary cryptosporidiosis employing a human biliary epithelial cell line. Using morphological and biochemical techniques, we examined the interaction of C. parvum with cultured human cholangiocytes. When the apical plasma membrane of polarized, confluent monolayers of human biliary epithelial cells was exposed to C. parvum oocysts that had been excysted in vitro, sporozoites attached to and invaded the cells in a time-, dose-, temperature-, and pH-dependent manner. The infectious process was both plasma membrane domain- and cell-specific, because no attachment or invasion occurred when the basolateral membrane of cholangiocytes was exposed to the parasite, or when a human hepatocyte cell line (HepG2) was used. Time-lapse video microscopy and scanning electron microscopy (SEM) showed that sporozoite attachment was rapid, involved extensive cholangiocyte membrane ruffling, and culminated in parasite penetration into a tight-fitting vacuole formed by invagination of the plasma membrane similar to those found in naturally occurring infection in vivo. Transmission electron microscopy (TEM) showed that C. parvum organisms formed parasitophorus vacuoles and were able to undergo a complete reproductive cycle, forming both asexual and sexual reproductive stages. Unexpectedly, direct cytopathic effects were noted in infected monolayers, with widespread programmed cell death (i.e., apoptosis) of biliary epithelial cells as assessed both morphologically and biochemically beginning within hours after exposure to the organism. The novel finding of specific cytopathic invasion of biliary epithelia by C. parvum may be relevant to the pathogenesis and possible therapy of the secondary sclerosing cholangitis seen in AIDS patients with biliary cryptosporidiosis.

Dynamins are 100-kDa GTPases that are essential for clathrin-coated vesicle formation during receptor-mediated endocytosis. To date, three different dynamin genes have been identified, with each gene expressing at least four different alternatively spliced forms. Currently, it is unclear whether these different dynamin gene products perform distinct or redundant cellular functions. Therefore, the focus of this study was to identify additional spliced variants of dynamin from rat tissues and to define the distribution of the dynamin family members in a cultured rat epithelial cell model (Clone 9 cells). After long-distance reverse transcription (RT)-PCR of mRNA from different rat tissues, the full-length cDNAs encoding the different dynamin isoforms were sequenced and revealed four additional spliced variants for dynamin I and nine for dynamin III. Thus, in rat tissues there are a total of at least 25 different mRNAs produced from the three dynamin genes. Subsequently, we generated stably transfected Clone 9 cells expressing full-length cDNAs of six different spliced forms tagged with green fluorescent protein. Confocal or fluorescence microscopy of these transfected cells revealed that many of the dynamin proteins associate with distinct membrane compartments, which include clathrin-coated pits at the plasma membrane and the Golgi apparatus, and several undefined vesicle populations. These results indicate that the dynamin family is more extensive than was originally predicted and suggest that the different dynamin proteins are localized to distinct cytoplasmic or membrane compartments.

Dynamin's role in clathrin-mediated endocytosis is now well established. Here we review new evidence from the past two years for the function of dynamin and related GTPases in other Intracellular trafficking events. We then summarize current information on the domain structure and function of this multidomain GTPase. Finally, we describe dynamin partners and their function in the context of clathrin-mediated endocytosis.

The dynamins comprise an expanding family of ubiquitously expressed 100-kD GTPases that have been implicated in severing clathrin-coated pits during receptor-mediated endocytosis. Currently, it is unclear whether the different dynamin isoforms perform redundant functions or participate in distinct endocytic processes. To define the function of dynamin II in mammalian epithelial cells, we have generated and characterized peptide-specific antibodies to domains that either are unique to this isoform or conserved within the dynamin family. When microinjected into cultured hepatocytes these affinity-purified antibodies inhibited clathrin-mediated endocytosis and induced the formation of long plasmalemmal invaginations with attached clathrin-coated pits. In addition, clusters of distinct, nonclathrin-coated, flask-shaped invaginations resembling caveolae accumulated at the plasma membrane of antibody-injected cells. In support of this, caveola-mediated endocytosis of labeled cholera toxin B was inhibited in antibody-injected hepatocytes. Using immunoisolation techniques an anti-dynamin antibody isolated caveolar membranes directly from a hepatocyte postnuclear membrane fraction. Finally, double label immunofluorescence microscopy revealed a striking colocalization between dynamin and the caveolar coat protein caveolin. Thus, functional in vivo studies as well as ultrastructural and biochemical analyses indicate that dynamin mediates both clathrin-dependent endocytosis and the internalization of caveolae in mammalian cells.

Ethanol has been predicted to alter vesicle-based protein traffic in hepatocytes, in part, via a disruption of the microtubule (MT) cytoskeleton. However, information on the effects of chronic ethanol exposure on MT function in vivo is sparse. Therefore the goal of this study was to test for ethanol-induced changes in rat liver tubulin expression, assembly, and cellular organization, using molecular, biochemical and morphological methods. The results of this study showed that tubulin mRNA and protein levels were not altered by ethanol. Tubulin, isolated from control and ethanol-fed rats, showed similar polymerization characteristics as assessed by calculation of the critical concentration for assembly and morphological structure. In contrast, the total amount of assembly-competent tubulin was reduced in livers from ethanol-fed rats compared with control rats when assessed by quantitative immunoblot analysis using a tubulin antibody. In addition, we observed that MT regrowth and organization in cultured hepatocytes treated with cold and nocodazole was markedly impaired by chronic ethanol exposure. In summary, these results indicate that tubulin levels in liver are not reduced by ethanol exposure. While there is a substantial amount of tubulin protein capable of assembling into functional MTs in ethanol-damaged livers, a marked portion of this tubulin is polymerization incompetent. This may explain why these hepatocytes exhibit a reduced number of MTs with an altered organization.