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PubMed2003

Real-time analysis of clathrin-mediated endocytosis during cell migration.

Simultaneous dual-color total-internal-reflection fluorescence microscopy (TIR-FM) was performed to analyze the internalization and distribution of markers for clathrin-mediated endocytosis (clathrin, dynamin1, dynamin2 and transferrin) in migrating cells. In MDCK cells, which endogenously express dynamin2, the dynamin2-EGFP fluorescence demonstrated identical spatial and temporal behavior as clathrin both prior to and during internalization. By contrast, in the same cells, the neuronal dynamin1 only localized with clathrin just prior to endocytosis. In migrating cells, each endocytic marker was polarized towards the leading edge, away from the lagging edge. These observations suggest a re-evaluation of the functional differences between dynamin1 and dynamin2, and of the role of clathrin-mediated endocytosis in cell migration.

Animals↗
PubMed2003

Agonist-induced endocytosis of lysophosphatidic acid-coupled LPA1/EDG-2 receptors via a dynamin2- and Rab5-dependent pathway.

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that exerts a pleiotropic range of effects on cells through activation of three closely related G-protein-coupled receptors termed LPA1/EDG-2, LPA2/EDG-4 and LPA3/EDG-7. Of these receptors, the LPA1 receptor is the most widely expressed. In this study, we investigated the agonist-induced endocytosis of the human LPA1 receptor, bearing an N-terminal FLAG epitope tag, in stably transfected HeLa cells. Treatment with LPA induced the rapid endocytosis of approximately 40% of surface LPA1 within 15 minutes. Internalization was both dose dependent and LPA specific since neither lysophophatidylcholine nor sphingosine-1-phosphate induced LPA1 endocytosis. Removal of agonist following 30 minutes incubation resulted in recycling of LPA1 back to the cell surface. LPA1 internalization was strongly inhibited by dominant-inhibitory mutants of both dynamin2 (K44A) and Rab5a (S34N). In addition, both dynamin2 K44A and Rab5 S34N mildly inhibited LPA1-dependent activation of serum response factor. Finally, our results also indicate that LPA1 exhibits basal, LPA-dependent internalization in the presence of serum-containing medium.

DNA↗
PubMed2003

Inhibition of GTP-dependent vesicle trafficking impairs internalization of plasmalemmal eNOS and cellular nitric oxide production.

The Ca2+ mobilizing peptide, bradykinin (BK), stimulates endothelial nitric oxide synthase (eNOS)-derived cellular nitric oxide (NO) production in association with altering the subcellular distribution of the enzyme. In the present study we examine the influence of cellular GTPases, particularly the large GTPase dynamin, on BK-mediated eNOS localization and cellular NO production. BK stimulation of ECV cells, which were stably transfected with eNOS-GFP (eNOS-GFP ECV304), increased NO production. This was associated with the mobilization of eNOS-GFP protein into Triton X-100-insoluble fractions of cell lysates, and an internalization of plasmalemmal eNOS-GFP in live and fixed ECV 304 cells. Incubation of digitonin-permeabilized ECV304 cells with the non-hydrolyzed GTP analog, GTP-gamma-S, abrogated the BK-mediated internalization of eNOS-GFP as assessed by confocal microscopy. Conversely, inhibition of clathrin-dependent endocytosis, via overexpression of AP 180 or pretreatment of cells with chlorpromazine, did not influence BK-mediated eNOS redistribution. Furthermore, specific inhibition of dynamin-2 GTPase function by overexpression of a dominant negative construct, K44A, prevented the BK-mediated enrichment of eNOS-GFP within low buoyant density, caveolin-enriched fractions of eNOS-GFP ECV304 cell lysates. Dynamin-2 K44A overexpression also markedly impaired BK-dependent, L-NAME-inhibited NO production as did incubation of permeabilized cells with GTP-gamma-s. These studies demonstrate that disruption of dynamin- and GTP-dependent, but clathrin-independent, vesicle trafficking pathways impairs BK-dependent cellular NO production, via inhibition of the internalization of eNOS-containing plasmalemmal vesicles.

Animals↗
PubMed2003

Caveosomes and endocytosis of lipid rafts.

Endocytosis of various endogenous plasma membrane molecules, including signalling receptors, glycosphingolipids and glycosylphosphatidylinositol (GPI)-linked proteins, occurs in the absence of functional clathrin-coated pits. Most of these molecules are found in biochemically defined lipid rafts, which suggests that at least some clathrin-independent endocytosis may be raft specific or raft mediated. However, recent studies of the uptake of raft markers have revealed a diversity of internalization methods. Although lipid rafts may somehow be recognized by endocytic machinery, at this stage the data do not readily fit with the idea of a single raft-specific or raft-dependent endocytic pathway. Many studies report uptake of raft molecules by caveolar endocytosis (defined by sensitivity to cholesterol depletion and to overexpression of a specific mutant of dynamin 2). It is now apparent that this is a highly regulated process, and caveolin 1, one of the characteristic protein components of caveolae, might in fact act to slow or inhibit endocytosis. The molecular details of caveolar endocytosis have yet to be elucidated. Several sources indicate that clathrin-independent uptake to a distinct class of caveolin-1-containing endosome, termed the caveosome, allows different types of endocytic mechanisms to have different functional consequences for the cell. It is likely that there are mechanisms that allow recruitment and targeting of specific molecules to caveosomes.

Animals↗
PubMed2005

Molecular dissection of internalization of Porphyromonas gingivalis by cells using fluorescent beads coated with bacterial membrane vesicle.

Porphyromonas gingivalis is one of the causative agents of adult periodontitis, and has been reported to be internalized by nonphagocytic epithelial cells. However, the mechanism for the internalization remains unclear. In the present study, we addressed this issue using fluorescent beads coated with bacterial membrane vesicles (MVs) that retain surface components of P. gingivalis. We established an assay system in which we could easily quantify the bead internalization to cells. MVs-coated beads were internalized by HeLa cells in kinetics similar to that of living bacteria. The internalization depended on dynamin but not clathrin. The beads were internalized through the actin-mediated pathway that is controlled by phosphatidylinositol (PI) 3-kinase. The dynamics of microtubule assembly and disassembly was also required. Further, the treatment of cells with cholesterol-binding reagents significantly inhibited bead internalization, and the internalized beads were apparently colocalized with ganglioside GM1 and caveolin-1, which suggest the involvement of the lipid raft in the process. These results suggest that P. gingivalis accomplishes its internalization utilizing membrane lipid raft and cytoskeletal functions of the target cells.

Actin Cytoskeleton↗
PubMed2006

Pathomechanisms of mutant proteins in Charcot-Marie-Tooth disease.

We review the putative functions and malfunctions of proteins encoded by genes mutated in Charcot-Marie-Tooth disease (CMT; inherited motor and sensory neuropathies) in normal and affected peripheral nerves. Some proteins implicated in demyelinating CMT, peripheral myelin protein 22, protein zero (P0), and connexin32 (Cx32/GJB1) are crucial components of myelin. Periaxin is involved in connecting myelin to the surrounding basal lamina. Early growth response 2 (EGR2) and Sox10 are transcriptional regulators of myelin genes. Mutations in the small integral membrane protein of lysosome/late endosome, the myotubularin-related protein 2 (MTMR2), and MTMR13/set-binding factor 2 are involved in vesicle and membrane transport and the regulation of protein degradation. Pathomechanisms related to alterations of these processes are a widespread phenomenon in demyelinating neuropathies because mutations of myelin components may also affect protein biosynthesis, transport, and/or degradation. Related disease mechanisms are also involved in axonal neuropathies although there is considerably more functional heterogeneity. Some mutations, most notably in P0, GJB1, ganglioside-induced differentiation-associated protein 1 (GDAP1), neurofilament light chain (NF-L), and dynamin 2 (DNM2), can result in demyelinating or axonal neuropathies introducing additional complexity in the pathogenesis. Often, this relates to the intimate connection between Schwann cells and neurons/axons leading to axonal damage even if the mutation-caused defect is Schwann-cell-autonomous. This mechanism is likely for P0 and Cx32 mutations and provides the basis for the unifying hypothesis that also demyelinating neuropathies develop into functional axonopathies. In GDAP1 and DNM2 mutants, both Schwann cells and axons/neurons might be directly affected. NF-L mutants have a primary neuronal defect but also cause demyelination. The major challenge ahead lies in determining the individual contributions by neurons and Schwann cells to the pathology over time and to delineate the detailed molecular functions of the proteins associated with CMT in health and disease.

Cell Cycle Proteins↗
PubMed2002

Localization of dynamin 2 in rat seminiferous tubules during the spermatogenic cycle.

Dynamin is a protein essential to endocytosis. Dynamin 2, a dynamin isoform, is expressed most intensely in testicular tissue; however, precise localization has never been studied. Therefore, we investigated the expression of dynamin 2 in rat testicular tissue using immunohistochemical methods, and discuss here the physiological function of this protein. Testicular tissues were obtained from Wistar rats at 10, 21 and 63 days of age. Immunohistochemistrical examination and Western blot analysis were conducted using dynamin 2 specific antibody. Western blot analysis showed that expression in 21- and 63-day-old rats was more intense than that in 10-day-old rats. Dynamin 2 expression was observed using immunohistochemical method in the seminiferous tubules of all rats. In the 63-day-old rats, the expression was intense, especially in spermatids in the earlier maturation stages and in spermatocytes, and was observed in Sertoli cells. However, in spermatids, the expression gradually declined as spermatids matured to spermatozoa. In the 21-day-old rats, the expression was evident in spermatocytes and Sertoli cells, but that in the 10-day-old rats was weak. Intense expression of dynamin 2 during spermatogenesis suggests that this protein plays an important role in this process.

Aging↗
PubMed2006

Platelet-activating factor-induced clathrin-mediated endocytosis requires beta-arrestin-1 recruitment and activation of the p38 MAPK signalosome at the plasma membrane for actin bundle formation.

Clathrin-mediated endocytosis (CME) is a common pathway used by G protein-linked receptors to transduce extracellular signals. We hypothesize that platelet-activating factor (PAF) receptor (PAFR) ligation requires CME and causes engagement of beta-arrestin-1 and recruitment of a p38 MAPK signalosome that elicits distinct actin rearrangement at the receptor before endosomal scission. Polymorphonuclear neutrophils were stimulated with buffer or 2 microM PAF (1 min), and whole cell lysates or subcellular fractions were immunoprecipitated or slides prepared for colocalization and fluorescent resonance energy transfer analysis. In select experiments, beta-arrestin-1 or dynamin-2 were neutralized by intracellular introduction of specific Abs. PAFR ligation caused 1) coprecipitation of the PAFR and clathrin with beta-arrestin-1, 2) fluorescent resonance energy transfer-positive interactions among the PAFR, beta-arrestin-1, and clathrin, 3) recruitment and activation of the apoptosis signal-regulating kinase-1/MAPK kinase-3/p38 MAPK (ASK1/MKK3/p38 MAPK) signalosome, 4) cell polarization, and 5) distinct actin bundle formation at the PAFR. Neutralization of beta-arrestin-1 inhibited all of these cellular events, including PAFR internalization; conversely, dynamin-2 inhibition only affected receptor internalization. Selective p38 MAPK inhibition globally abrogated actin rearrangement; however, inhibition of MAPK-activated protein kinase-2 and its downstream kinase leukocyte-specific protein-1 inhibited only actin bundle formation and PAFR internalization. In addition, ASK1/MKK3/p38 MAPK signalosome assembly appears to occur in a novel manner such that the ASK1/p38 MAPK heterodimer is recruited to a beta-arrestin-1 bound MKK3. In polymorphonuclear neutrophils, leukocyte-specific protein-1 may play a role similar to fascin for actin bundle formation. We conclude that PAF signaling requires CME, beta-arrestin-1 recruitment of a p38 MAPK signalosome, and specific actin bundle formation at the PAFR for transduction before endosomal scission.

Actins↗
PubMed1997

Identification and characterization of a nerve terminal-enriched amphiphysin isoform.

Amphiphysin is a nerve terminal-enriched protein thought to function in synaptic vesicle endocytosis, in part through Src homology 3 (SH3) domain-mediated interactions with dynamin and synaptojanin. Here, we report the characterization of a novel amphiphysin isoform (termed amphiphysin II) that was identified through a homology search of the data base of expressed sequence tags. Antibodies specific to amphiphysin II recognize a 90-kDa protein on Western blot that is brain-specific and highly enriched in nerve terminals. Like amphiphysin (now referred to as amphiphysin I), amphiphysin II binds to dynamin and synaptojanin through its SH3 domain. Further, both proteins bind directly to clathrin in an SH3 domain-independent manner. Taken together, these data suggest that amphiphysin II may participate with amphiphysin I in the regulation of synaptic vesicle endocytosis.

Amino Acid Sequence↗
PubMed2007

Dynamin 2 associates with complexins and is found in the acrosomal region of mammalian sperm.

Previous data showed that complexin I, a SNARE regulatory protein, is localized in and/or around the acrosome and is necessary for the acrosome reaction in sperm. To understand how complexin I regulates the acrosome reaction, we used complexin-GST pulldown assays to identify interacting proteins. We showed that both complexins I and II bound mouse sperm dynamin 2. Dynamin 2 is a 100 kDa GTPase essential to many aspects of endocytosis but its potential role in exocytosis is unknown. Dynamin 2 is expressed in rat testis and widely expressed in other tissues; however, the function of dynamin 2 in germ cells is uncertain. Dynamin 2 protein was detected in mouse testis and was most abundant in or around the developing acrosome of spermatids. In addition, dynamin 2 was co-localized with complexin I in the acrosomal region of mammalian sperm. Its co-localization and interaction with complexin I suggest that dynamin 2 may play a role during acrosome formation and/or acrosomal exocytosis.

Acrosome↗
PubMed1993

Dynamin GTPase regulated by protein kinase C phosphorylation in nerve terminals.

Dynamin is a microtubule-binding protein with a microtubule-activated GTPase activity. The gene encoding dynamin is mutated in shibire, a Drosophila mutant defective in endocytosis in nerve terminals and other cells. These observations place dynamin into two distinct functional contexts, suggesting roles in microtubule-based motility or in endocytosis. We report here that dynamin is identical to the neuronal phosphoprotein dephosphin (P96), originally identified by its stimulus-dependent dephosphorylation in nerve terminals. Dynamin is a protein doublet of M(r) 94 and 96K arising by alternative splicing of its primary transcript. In the nerve terminal, both forms of dynamin are phosphorylated by protein kinase C (PKC) and are quantitatively dephosphorylated on excitation. In vitro, dynamin is also phosphorylated by casein kinase II which inhibits PKC phosphorylation. Phosphorylation by PKC but not by casein kinase II enhances the GTPase activity of dynamin 12-fold. The dynamins are therefore a group of nerve terminal phosphoproteins whose GTPase is regulated by phosphorylation in parallel with synaptic vesicle recycling. The regulation of dynamin GTPase could serve as the trigger for the rapid endocytosis of synaptic vesicles after exocytosis.

Alternative Splicing↗
PubMed2001

Fission of biological membranes: interplay between dynamin and lipids.

Membrane budding and fission are the key stages of ubiquitous processes of formation of intracellular transport vesicles. We present a theoretical consideration of one of the most important types of fission machinery, which is mediated by GTPase dynamin and controlled by lipid composition of the membrane. We suggest a mechanism for collapse of a membrane neck driven by interplay between the dynamin collar and the bending elastic energy of the neck membrane. The collar plays a role of a rigid external skeleton, which imposes mechanical constraints on the neck. We show that in certain conditions the membrane of the neck loses its stability and collapses. Collapse can result from: (i) shifting of the spontaneous curvature of the neck membrane towards negative values, (ii) stretching of the dynamin collar, (iii) tightening of the dynamin collar. The three factors can act separately or concertedly. The suggested model accounts for the major experimental knowledge on membrane fission mediated by dynamin. It includes the elements of all previous models of dynamin action based on different sets of experimental results [Sever et al., Traffic 2000; 1: 385-392]. It reconciles, at least partially, the apparent contradictions between the existing alternative views on biomembrane fission machinery.

Cell Membrane↗
PubMed2006

Selective loss of synaptic proteins in Alzheimer's disease: evidence for an increased severity with APOE varepsilon4.

A pathological feature of Alzheimer's disease (AD) is an area-specific neuronal loss that may be caused by excitotoxicity-related synaptic dysfunction. Relative expression levels of synaptophysin, dynamin I, complexins I and II, N-cadherin, and alphaCaMKII were analysed in human brain tissue from AD cases and controls in hippocampus, and inferior temporal and occipital cortices. Synaptophysin and dynamin I are presynaptic terminal proteins not specific to any neurotransmitter system whereas complexin II, N-cadherin, and alphaCaMKII are specific for excitatory synapses. Complexin I is a presynaptic protein localised to inhibitory synapses. There were no significant differences in synaptophysin, dynamin I, N-cadherin, or alphaCaMKII protein levels between AD cases and controls. The complexin proteins were both markedly lower in AD cases than in controls (P < 0.01). Cases were also categorised by APOE genotype. Averaged across areas there was a 36% lowering of presynaptic proteins in AD cases carrying at least one epsilon4 allele compared with in AD cases lacking the epsilon4 allele. We infer that synaptic protein level is not indicative of neuronal loss, but the synaptic dysfunction may result from the marked relative loss of the complexins in AD, and lower levels of presynaptic proteins in AD cases with the APOE epsilon4 allele.

Adaptor Proteins, Vesicular Transport↗
PubMed1999

Modulation of gene expression following long-term synaptic depression in the striatum.

A number of behavioural and cellular studies have suggested that activity-dependent synaptic plasticity associated with learning and memory may lead to the expression of various genes whose protein products can play a critical role in memory acquisition and consolidation. Long-term potentiation (LTP) and long-term depression (LTD) represent two forms of synaptic plasticity which have been widely studied by electrophysiological techniques. However, the molecular mechanisms at target gene involved in the generation of long term depression remain to be determined. To elucidate the molecular mechanism underlying activity dependent synaptic remodeling in striatal long term depression, we used the mRNA differential display technology to isolate genes that are induced or modulated by high frequency stimulation of the corticostriatal pathway in a rat brain slice preparation. We have differentially displayed, by means of reverse transcriptase-polymerase chain reaction, mRNA species isolated from striatal slices in which long term depression was induced by tetanic stimuli as well as from slices stimulated at low frequency. We then compared radio-labeled RT-PCR banding patterns to isolate cDNAs that are differentially expressed. Three independent cDNAs were isolated and identified whose mRNA level were enhanced by tetanic stimulation inducing long term depression. We provide evidence that two of these genes encode proteins involved in synaptic vesicle trafficking (dynamin I and amphiphysin II). Moreover, expression of tissue plasminogen activator (t-PA) gene was also increased following striatal long term depression. Our data suggest that a complex pattern of genes acting at presynaptic level and extracellularly may be involved in LTD-associated synaptic remodeling.

Animals↗
PubMed2000

Amphiphysin IIm, a novel amphiphysin II isoform, is required for macrophage phagocytosis.

Phagocytosis of pathogens by macrophages initiates the innate immune response, which in turn orchestrates the adaptive immune response. Amphiphysin II participates in receptor-mediated endocytosis, in part, by recruiting the GTPase dynamin to the nascent endosome. We demonstrate here that a novel isoform of amphiphysin II associates with early phagosomes in macrophages. We have ablated the dynamin-binding site of this protein and shown that this mutant form of amphiphysin II inhibits phagocytosis at the stage of membrane extension around the bound particles. We define a signaling cascade in which PI3K is required to recruit amphiphysin II to the phagosome, and amphiphysin II in turn recruits dynamin. Thus, amphiphysin II facilitates a critical initial step in host response to infection.

Amino Acid Sequence↗
PubMed2004

Localized expression of amphiphysin Ir, a retina-specific variant of amphiphysin I, in the ribbon synapse and its functional implication.

In the vertebrate retina, presynaptic terminals of photoreceptors and bipolar cells form ribbon synapses and release neurotransmitter continuously. Endocytic machinery in the ribbon synapse is likely to differ from that in conventional synapses because of the much higher rate of synaptic vesicle recycling. However, protein components of the ribbon synapse identified so far are quite similar to those of the conventional synapses. Recently we identified amphiphysin I splice variants, termed amphiphysin Ir, that are transcribed specifically in retina from the authentic amphiphysin I gene [Y. Terada et al. (2002) FEBS Lett., 519, 185-190]. Amphiphysin I is a nerve terminal-enriched protein, and involved in synaptic vesicle endocytosis as heterodimer with amphiphysin II, an isoform of amphiphysin I. We report here that the retina-specific amphiphysin Ir is expressed exclusively in the ribbon synapse and not in conventional synapses. This is the first endocytosis-related, ribbon synapse-specific protein identified in the retina. By immunoprecipitation and double-immunolabelling, amphiphysin Ir was shown to be associated not only with amphiphysin II, but also with dynamin, clathrin and alpha-adaptin that are involved in synaptic vesicle recycling. The results suggest that endocytosis of the synaptic vesicle membrane in retinal ribbon synapses proceeds through a pathway similar to the one that is used in conventional synapses, although amphiphysin Ir is substituted for amphiphysin I. Amphiphysin Ir may play an essential role in the avid endocytic activity of ribbon synapses by associating with yet unknown protein partner(s) through its large insertional domain, which is absent from the conventional amphiphysin I.

Animals↗
PubMed1998

In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly.

Profilins are thought to be essential for regulation of actin assembly. However, the functions of profilins in mammalian tissues are not well understood. In mice profilin I is expressed ubiquitously while profilin II is expressed at high levels only in brain. In extracts from mouse brain, profilin I and profilin II can form complexes with regulators of endocytosis, synaptic vesicle recycling and actin assembly. Using mass spectrometry and database searching we characterized a number of ligands for profilin I and profilin II from mouse brain extracts including dynamin I, clathrin, synapsin, Rho-associated coiled-coil kinase, the Rac-associated protein NAP1 and a member of the NSF/sec18 family. In vivo, profilins co-localize with dynamin I and synapsin in axonal and dendritic processes. Our findings strongly suggest that in brain profilin I and profilin II complexes link the actin cytoskeleton and endocytic membrane flow, directing actin and clathrin assembly to distinct membrane domains.

Actins↗
PubMed2006

Protein repair in the brain, proteomic analysis of endogenous substrates for protein L-isoaspartyl methyltransferase in mouse brain.

Protein L-isoaspartyl methyltransferase (PIMT) catalyzes repair of L-isoaspartyl peptide bonds, a major source of protein damage under physiological conditions. PIMT knock-out (KO) mice exhibit brain enlargement and fatal epileptic seizures. All organs accumulate isoaspartyl proteins, but only the brain manifests an overt pathology. To further explore the role of PIMT in brain function, we undertook a global analysis of endogenous substrates for PIMT in mouse brain. Extracts from PIMT-KO mice were subjected to two-dimensional gel electrophoresis and blotted onto membranes. Isoaspartyl proteins were radiolabeled on-blot using [methyl-(3)H]S-adenosyl-L-methionine and recombinant PIMT. Fluorography of the blot revealed 30-35 (3)H-labeled proteins, 22 of which were identified by peptide mass fingerprinting. These isoaspartate-prone proteins represent a wide range of cellular functions, including neuronal development, synaptic transmission, cytoskeletal structure and dynamics, energy metabolism, nitrogen metabolism, pH homeostasis, and protein folding. The following five proteins, all of which are rich in neurons, accumulated exceptional levels of isoaspartate: collapsin response mediator protein 2 (CRMP2/ULIP2/DRP-2), dynamin 1, synapsin I, synapsin II, and tubulin. Several of the proteins identified here are prone to age-dependent oxidation in vivo, and many have been identified as autoimmune antigens, of particular interest because isoaspartate can greatly enhance the antigenicity of self-peptides. We propose that the PIMT-KO phenotype results from the cumulative effect of isoaspartate-related damage to a number of the neuron-rich proteins detected in this study. Further study of the isoaspartate-prone proteins identified here may help elucidate the molecular basis of one or more developmental and/or age-related neurological diseases.

Animals↗