The Experts below are selected from a list of 1392 Experts worldwide ranked by ideXlab platform

Phillip J. Robinson - One of the best experts on this subject based on the ideXlab platform.

  • Pharmacological Inhibition of Dynamin II Reduces Constitutive Protein Secretion from Primary Human
    2016
    Co-Authors: Maaike Kockx, Phillip J. Robinson, Denuja Karunakaran, Mathew Traini, Jing Xue, Kuan Yen Huang, Diana Nawara, Katharina Gaus, Wendy Jessup, Leonard Kritharides
    Abstract:

    Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether Dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of Dynamin decreases secretion of apolipoprotein E (apoE) and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of Dynamin to membranes (MiTMABs) or directly target the GTPase domain (Dyngo or Dynole series), dose- and time- dependently reduced the secretion of apoE. SiRNA oligo’s targeting all isoforms of Dynamin II confirmed the involvement of Dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of Dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of Dynamin II modulate the constitutive secretion of macrophage apoE as a clas

  • Pharmacological Inhibition of Dynamin II Reduces Constitutive Protein Secretion from Primary Human Macrophages
    PloS one, 2014
    Co-Authors: Maaike Kockx, Phillip J. Robinson, Denuja Karunakaran, Mathew Traini, Jing Xue, Kuan Yen Huang, Diana Nawara, Katharina Gaus, Wendy Jessup, Leonard Kritharides
    Abstract:

    Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether Dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of Dynamin decreases secretion of apolipoprotein E (apoE) and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of Dynamin to membranes (MiTMABs) or directly target the GTPase domain (Dyngo or Dynole series), dose- and time- dependently reduced the secretion of apoE. SiRNA oligo’s targeting all isoforms of Dynamin II confirmed the involvement of Dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of Dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of Dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes.

  • Dynamin II function is required for egf mediated stat3 activation but not erk1 2 phosphorylation
    Growth Factors Journal, 2012
    Co-Authors: Rodney B. Luwor, Andrew B. Mcgeachie, Phillip J. Robinson, Xiaowen Chin, Hong-jian Zhu
    Abstract:

    Signalling from receptor tyrosine kinases is elicited by ligand binding which initiates the activation of many downstream signalling cascades. Endocytosis has been widely accepted as one mechanism in which cells inactivate signalling by internalising and subsequently degrading activated receptors. However, it is now evident that endocytosis of signalling receptors is important in initiation and sustaining downstream signalling. We and others have previously shown that epidermal growth factor receptor (EGFR) overexpression and activation of signal transducer and activator of transcription 3 (Stat3) are associated with tumourigenicity. Here, we examine the role of endocytosis in EGFR signal attenuation and differential signalling. Inhibition of Dynamin II (Dyn II), a GTPase required for endocytosis, with a small molecular weight inhibitor, led to reduced EGF-mediated Stat3 phosphorylation and transcriptional activity in the A431 and HN5 human tumour cell lines. However, Dyn II inhibition had minimal effect ...

  • Dynamin II function is required for EGF-mediated Stat3 activation but not Erk1/2 phosphorylation
    Growth factors (Chur Switzerland), 2012
    Co-Authors: Rodney B. Luwor, Andrew B. Mcgeachie, Phillip J. Robinson, Xiaowen Chin, Hong-jian Zhu
    Abstract:

    Signalling from receptor tyrosine kinases is elicited by ligand binding which initiates the activation of many downstream signalling cascades. Endocytosis has been widely accepted as one mechanism in which cells inactivate signalling by internalising and subsequently degrading activated receptors. However, it is now evident that endocytosis of signalling receptors is important in initiation and sustaining downstream signalling. We and others have previously shown that epidermal growth factor receptor (EGFR) overexpression and activation of signal transducer and activator of transcription 3 (Stat3) are associated with tumourigenicity. Here, we examine the role of endocytosis in EGFR signal attenuation and differential signalling. Inhibition of Dynamin II (Dyn II), a GTPase required for endocytosis, with a small molecular weight inhibitor, led to reduced EGF-mediated Stat3 phosphorylation and transcriptional activity in the A431 and HN5 human tumour cell lines. However, Dyn II inhibition had minimal effect ...

  • Calcineurin activity is required for the completion of cytokinesis
    Cellular and Molecular Life Sciences, 2010
    Co-Authors: Megan Chircop, Chandra S Malladi, Christopher P Gordon, Adam Mccluskey, Michael Zavortink, Audrey T. Lian, Scott L. Page, Phillip J. Robinson
    Abstract:

    Successful completion of cytokinesis requires the spatio-temporal regulation of protein phosphorylation and the coordinated activity of protein kinases and phosphatases. Many mitotic protein kinases are well characterized while mitotic phosphatases are largely unknown. Here, we show that the Ca^2+- and calmodulin-dependent phosphatase, calcineurin (CaN), is required for cytokinesis in mammalian cells, functioning specifically at the abscission stage. CaN inhibitors induce multinucleation in HeLa cells and prolong the time cells spend connected via an extended intracellular bridge. Upon Ca^2+ influx during cytokinesis, CaN is activated, targeting a set of proteins for dephosphorylation, including Dynamin II (dynII). At the intracellular bridge, phospho-dynII and CaN are co-localized to dual flanking midbody rings (FMRs) that reside on either side of the central midbody ring. CaN activity and disassembly of the FMRs coincide with abscission. Thus, CaN activity at the midbody plays a key role in regulating the completion of cytokinesis in mammalian cells.

Ari Helenius - One of the best experts on this subject based on the ideXlab platform.

  • clathrin and caveolin 1 independent endocytosis entry of simian virus 40 into cells devoid of caveolae
    Journal of Cell Biology, 2005
    Co-Authors: Evamaria Damm, Jürgen Kartenbeck, Lucas Pelkmans, Anna Mezzacasa, Teymuras V Kurzchalia, Ari Helenius
    Abstract:

    Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)–deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1–independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, Dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.

  • Clathrin- and caveolin-1–independent endocytosis: entry of simian virus 40 into cells devoid of caveolae
    The Journal of cell biology, 2005
    Co-Authors: Evamaria Damm, Jürgen Kartenbeck, Lucas Pelkmans, Anna Mezzacasa, Teymuras V Kurzchalia, Ari Helenius
    Abstract:

    Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)–deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1–independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, Dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.

  • echovirus 1 endocytosis into caveosomes requires lipid rafts Dynamin II and signaling events
    Molecular Biology of the Cell, 2004
    Co-Authors: Vilja Pietiainen, Lucas Pelkmans, Varpu Marjomaki, Paula Upla, Ari Helenius, Timo Hyypia
    Abstract:

    Binding of echovirus 1 (EV1, a nonenveloped RNA virus) to the α2β1 integrin on the cell surface is followed by endocytic internalization of the virus together with the receptor. Here, video-enhanced live microscopy revealed the rapid uptake of fluorescently labeled EV1 into mobile, intracellular structures, positive for green fluorescent protein-tagged caveolin-1. Partial colocalization of EV1 with SV40 (SV40) and cholera toxin, known to traffic via caveosomes, demonstrated that the vesicles were caveosomes. The initiation of EV1 infection was dependent on Dynamin II, cholesterol, and protein phosphorylation events. Brefeldin A, a drug that prevents SV40 transport, blocked the EV1 infection cycle, whereas drugs that disrupt the cellular cytoskeleton had no effect. In situ hybridization revealed the localization of viral RNA with endocytosed viral capsid proteins in caveosomes before initiation of viral replication. Thus, both the internalization of EV1 to caveosomes and subsequent events differ clearly from caveolar endocytosis of SV40 because EV1 uptake is fast and independent of actin and EV1 is not sorted further to sER from caveosomes. These results shed further light on the cell entry of nonenveloped viral pathogens and illustrate the use of viruses as probes to dissect caveolin-associated endocytic pathways.

  • Echovirus 1 Endocytosis into Caveosomes Requires Lipid Rafts, Dynamin II, and Signaling Events
    Molecular biology of the cell, 2004
    Co-Authors: Vilja Pietiainen, Lucas Pelkmans, Varpu Marjomaki, Paula Upla, Ari Helenius, Timo Hyypia
    Abstract:

    Binding of echovirus 1 (EV1, a nonenveloped RNA virus) to the alpha2beta1 integrin on the cell surface is followed by endocytic internalization of the virus together with the receptor. Here, video-enhanced live microscopy revealed the rapid uptake of fluorescently labeled EV1 into mobile, intracellular structures, positive for green fluorescent protein-tagged caveolin-1. Partial colocalization of EV1 with SV40 (SV40) and cholera toxin, known to traffic via caveosomes, demonstrated that the vesicles were caveosomes. The initiation of EV1 infection was dependent on Dynamin II, cholesterol, and protein phosphorylation events. Brefeldin A, a drug that prevents SV40 transport, blocked the EV1 infection cycle, whereas drugs that disrupt the cellular cytoskeleton had no effect. In situ hybridization revealed the localization of viral RNA with endocytosed viral capsid proteins in caveosomes before initiation of viral replication. Thus, both the internalization of EV1 to caveosomes and subsequent events differ clearly from caveolar endocytosis of SV40 because EV1 uptake is fast and independent of actin and EV1 is not sorted further to sER from caveosomes. These results shed further light on the cell entry of nonenveloped viral pathogens and illustrate the use of viruses as probes to dissect caveolin-associated endocytic pathways.

  • local actin polymerization and Dynamin recruitment in sv40 induced internalization of caveolae
    Science, 2002
    Co-Authors: Lucas Pelkmans, Daniel Puntene, Ari Helenius
    Abstract:

    Simian virus 40 (SV40) utilizes endocytosis through caveolae for infectious entry into host cells. We found that after binding to caveolae, virus particles induced transient breakdown of actin stress fibers. Actin was then recruited to virus-loaded caveolae as actin patches that served as sites for actin “tail” formation. Dynamin II was also transiently recruited. These events depended on the presence of cholesterol and on the activation of tyrosine kinases that phosphorylated proteins in caveolae. They were necessary for formation of caveolae-derived endocytic vesicles and for infection of the cell. Thus, caveolar endocytosis is ligand-triggered and involves extensive rearrangement of the actin cytoskeleton.

Lucas Pelkmans - One of the best experts on this subject based on the ideXlab platform.

  • ameobal pathogen mimivirus infects macrophages through phagocytosis
    PLOS Pathogens, 2008
    Co-Authors: Eric Ghigo, Pham Thi Kim Lien, Jürgen Kartenbeck, Lucas Pelkmans, Christian Capo, Jean-louis Mege, Didier Raoult
    Abstract:

    Mimivirus, or Acanthamoeba polyphaga mimivirus (APMV), a giant double-stranded DNA virus that grows in amoeba, was identified for the first time in 2003. Entry by phagocytosis within amoeba has been suggested but not demonstrated. We demonstrate here that APMV was internalized by macrophages but not by non-phagocytic cells, leading to productive APMV replication. Clathrin- and caveolin-mediated endocytosis pathways, as well as degradative endosome-mediated endocytosis, were not used by APMV to invade macrophages. Ultrastructural analysis showed that protrusions were formed around the entering virus, suggesting that macropinocytosis or phagocytosis was involved in APMV entry. Reorganization of the actin cytoskeleton and activation of phosphatidylinositol 3-kinases were required for APMV entry. Blocking macropinocytosis and the lack of APMV colocalization with rabankyrin-5 showed that macropinocytosis was not involved in viral entry. Overexpression of a dominant-negative form of Dynamin-II, a regulator of phagocytosis, inhibited APMV entry. Altogether, our data demonstrated that APMV enters macrophages through phagocytosis, a new pathway for virus entry in cells. This reinforces the paradigm that intra-amoebal pathogens have the potential to infect macrophages.

  • clathrin and caveolin 1 independent endocytosis entry of simian virus 40 into cells devoid of caveolae
    Journal of Cell Biology, 2005
    Co-Authors: Evamaria Damm, Jürgen Kartenbeck, Lucas Pelkmans, Anna Mezzacasa, Teymuras V Kurzchalia, Ari Helenius
    Abstract:

    Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)–deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1–independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, Dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.

  • Clathrin- and caveolin-1–independent endocytosis: entry of simian virus 40 into cells devoid of caveolae
    The Journal of cell biology, 2005
    Co-Authors: Evamaria Damm, Jürgen Kartenbeck, Lucas Pelkmans, Anna Mezzacasa, Teymuras V Kurzchalia, Ari Helenius
    Abstract:

    Simian Virus 40 (SV40) has been shown to enter host cells by caveolar endocytosis followed by transport via caveosomes to the endoplasmic reticulum (ER). Using a caveolin-1 (cav-1)–deficient cell line (human hepatoma 7) and embryonic fibroblasts from a cav-1 knockout mouse, we found that in the absence of caveolae, but also in wild-type embryonic fibroblasts, the virus exploits an alternative, cav-1–independent pathway. Internalization was rapid (t1/2 = 20 min) and cholesterol and tyrosine kinase dependent but independent of clathrin, Dynamin II, and ARF6. The viruses were internalized in small, tight-fitting vesicles and transported to membrane-bounded, pH-neutral organelles similar to caveosomes but devoid of cav-1 and -2. The viruses were next transferred by microtubule-dependent vesicular transport to the ER, a step that was required for infectivity. Our results revealed the existence of a virus-activated endocytic pathway from the plasma membrane to the ER that involves neither clathrin nor caveolae and that can be activated also in the presence of cav-1.

  • echovirus 1 endocytosis into caveosomes requires lipid rafts Dynamin II and signaling events
    Molecular Biology of the Cell, 2004
    Co-Authors: Vilja Pietiainen, Lucas Pelkmans, Varpu Marjomaki, Paula Upla, Ari Helenius, Timo Hyypia
    Abstract:

    Binding of echovirus 1 (EV1, a nonenveloped RNA virus) to the α2β1 integrin on the cell surface is followed by endocytic internalization of the virus together with the receptor. Here, video-enhanced live microscopy revealed the rapid uptake of fluorescently labeled EV1 into mobile, intracellular structures, positive for green fluorescent protein-tagged caveolin-1. Partial colocalization of EV1 with SV40 (SV40) and cholera toxin, known to traffic via caveosomes, demonstrated that the vesicles were caveosomes. The initiation of EV1 infection was dependent on Dynamin II, cholesterol, and protein phosphorylation events. Brefeldin A, a drug that prevents SV40 transport, blocked the EV1 infection cycle, whereas drugs that disrupt the cellular cytoskeleton had no effect. In situ hybridization revealed the localization of viral RNA with endocytosed viral capsid proteins in caveosomes before initiation of viral replication. Thus, both the internalization of EV1 to caveosomes and subsequent events differ clearly from caveolar endocytosis of SV40 because EV1 uptake is fast and independent of actin and EV1 is not sorted further to sER from caveosomes. These results shed further light on the cell entry of nonenveloped viral pathogens and illustrate the use of viruses as probes to dissect caveolin-associated endocytic pathways.

  • Echovirus 1 Endocytosis into Caveosomes Requires Lipid Rafts, Dynamin II, and Signaling Events
    Molecular biology of the cell, 2004
    Co-Authors: Vilja Pietiainen, Lucas Pelkmans, Varpu Marjomaki, Paula Upla, Ari Helenius, Timo Hyypia
    Abstract:

    Binding of echovirus 1 (EV1, a nonenveloped RNA virus) to the alpha2beta1 integrin on the cell surface is followed by endocytic internalization of the virus together with the receptor. Here, video-enhanced live microscopy revealed the rapid uptake of fluorescently labeled EV1 into mobile, intracellular structures, positive for green fluorescent protein-tagged caveolin-1. Partial colocalization of EV1 with SV40 (SV40) and cholera toxin, known to traffic via caveosomes, demonstrated that the vesicles were caveosomes. The initiation of EV1 infection was dependent on Dynamin II, cholesterol, and protein phosphorylation events. Brefeldin A, a drug that prevents SV40 transport, blocked the EV1 infection cycle, whereas drugs that disrupt the cellular cytoskeleton had no effect. In situ hybridization revealed the localization of viral RNA with endocytosed viral capsid proteins in caveosomes before initiation of viral replication. Thus, both the internalization of EV1 to caveosomes and subsequent events differ clearly from caveolar endocytosis of SV40 because EV1 uptake is fast and independent of actin and EV1 is not sorted further to sER from caveosomes. These results shed further light on the cell entry of nonenveloped viral pathogens and illustrate the use of viruses as probes to dissect caveolin-associated endocytic pathways.

Peter Westermann - One of the best experts on this subject based on the ideXlab platform.

  • Complexes of syndapin II with Dynamin II promote vesicle formation at the trans-Golgi network
    Journal of Cell Science, 2006
    Co-Authors: Michael M. Kessels, Peter Westermann, Jiaxin Dong, Wibke Leibig, Britta Qualmann
    Abstract:

    The role of Dynamin and so-called accessory proteins in endocytosis is well established. However, molecular details of the function(s) of Dynamin II at the Golgi are largely unclear. We demonstrate that the ubiquitously expressed syndapin II isoform interacts with the proline-rich domain (PRD) of Dynamin II through its Src-homology 3 (SH3) domain. Co-immunoprecipitation of endogenous syndapin II and Dynamin II, and successful reconstitutions of such complexes at membranes in COS-7 cells, show the in vivo relevance of the interaction. Syndapin II can associate with Golgi membranes and this association increases upon Golgi exit block. Brefeldin A treatment clearly shows that the observed perinuclear localization of syndapin II co-localizing with syntaxin 6 reflects the Golgi complex and that it requires functional integrity of the Golgi. Syndapins are crucial for Golgi vesicle formation because anti-syndapin antibodies, used either in in vitro reconstitutions or in living cells, inhibited this process. Both types of assays additionally revealed the essential role of syndapin II SH3 interactions with the Dynamin II PRD in vesicle formation. An excess of the syndapin SH3 domain strongly inhibited budding from Golgi membranes in vitro. Likewise, overexpression of the syndapin SH3 domain or of a Dynamin II variant incapable of associating with syndapin II (Dynamin IIDeltaPRD) impaired trafficking of vesicular stomatitis virus glycoprotein (VSVG)-GFP in vivo. By contrast, full-length syndapin II-l had no negative effect, and instead promoted VSVG-GFP export from the Golgi. Importantly, a cytosolic fraction containing endogenous syndapin-Dynamin complexes was sufficient to promote vesicle formation from Golgi membranes in a syndapin-dependent manner. Thus, syndapin-Dynamin complexes are crucial and sufficient to promote vesicle formation from the trans-Golgi network.

  • Profilin I attached to the Golgi is required for the formation of constitutive transport vesicles at the trans-Golgi network
    Biochimica et Biophysica Acta, 2000
    Co-Authors: Jiaxin Dong, Boris Radau, Albrecht Otto, Eva-christina Müller, Carsten Lindschau, Peter Westermann
    Abstract:

    Abstract Profilin I was identified, by mass spectrometric sequencing and immunoblotting, as a component of purified Golgi cisternae from HepG2 cells. Binding to the Golgi was verified by indirect immunofluorescence in MT-1 cells showing that a fraction of profilin I colocalizes with TGN38, a marker of the trans-Golgi network (TGN). Studying the formation of constitutive exocytic vesicles at the TGN in a cell-free system demonstrated that cytosolic profilin I has no effect, while incubation of Golgi cisternae with a profilin I-specific antibody reduced vesicle formation by about 50%. Notably, the antibody displaces a fraction of the Golgi-bound Dynamin II indicating that profilin I may indirectly promote vesicle formation by supporting the binding of Dynamin II to the Golgi membrane. The impact of Dynamin II on vesicle formation is demonstrated by incubating the Golgi with the proline-rich domain of Dynamin II which concomitantly displaces Dynamin II and inhibits vesicle formation. The data provide evidence that profilin I attaches to the Golgi apparatus and is required for the formation of constitutive transport vesicles.

  • Expression and purification of Dynamin II domains and initial studies on structure and function.
    Protein expression and purification, 2000
    Co-Authors: Jiaxin Dong, Rolf Misselwitz, Heinz Welfle, Peter Westermann
    Abstract:

    Abstract Dynamin II, a large GTP-binding protein, is involved in endocytosis and in vesicle formation at the trans-Golgi network. To further elucidate functions of Dynamin II, the pleckstrin homology domain (PHD), the proline-rich domain (PRD), and the C-terminal part of Dynamin II (Dynamin 500–870 ) were expressed in Escherichia coli. The PHD, tagged C-terminally by a (His) 6 peptide, was expressed to 15% of cellular proteins and could be purified on nickel-chelating agarose. On the contrary, the PRD and Dynamin 500–870 had to be tagged with a (His) 6 peptide at the N-terminus to bind to nickel-chelating agarose. Additional tagging with the S-peptide, which forms a stable complex with immobilized S-protein, allowed removal of strongly interacting E. coli proteins. Circular dichroic spectra indicate a structured recombinant PHD with a secondary structure content similar to that of the known PHD from Dynamin I. The N-terminally tagged, recombinant PRD is unfolded but nevertheless binds specifically to the SH3 domain of amphiphysin II as well as to proteins extracted from rat brain. The described methods are suitable to isolate functionally active domains of Dynamin II in sufficient amount and purity for further studies.

  • Dynamin II Binds to theTrans-Golgi Network☆☆☆
    Biochemical and biophysical research communications, 1996
    Co-Authors: Olaf Maier, Maria Knoblich, Peter Westermann
    Abstract:

    Abstract Two Dynamin II - specific antibodies were used to detect Dynamin II attached to highly purified Golgi membranes of HepG2 cells. Within the Golgi apparatus Dynamin II is predominantly localized to the trans-Golgi network, as shown by immunoelectron microscopy. Increased binding of Dynamin II to the trans-Golgi network in the presence of GTP-γ-S and a reduced binding upon stimulation of secretion by phorbolester indicates that Dynamin II may have a function in post-Golgi vesicle transport.

Ngoc Chau - One of the best experts on this subject based on the ideXlab platform.

  • Pyrimidyn Compounds: Dual-Action Small Molecule Pyrimidine-Based Dynamin Inhibitors
    2016
    Co-Authors: Andrew B. Mcgeachie, Luke R Odell, Timothy A. Hill, Ngoc Chau, Nick N Gorgani, Annie Quan, Michael A Cousin, James A. Daniel, Damien J. Keating, Ellen M. Van Dam
    Abstract:

    Dynamin is required for clathrin-mediated endocytosis (CME). Its GTPase activity is stimulated by phospholipid binding to its PH domain, which induces helical oligomerization. We have designed a series of novel pyrimidine-based “Pyrimidyn” compounds that inhibit the lipid-stimulated GTPase activity of full length Dynamin I and II with similar potency. The most potent analogue, Pyrimidyn 7, has an IC50 of 1.1 μM for Dynamin I and 1.8 μM for Dynamin II, making it among the most potent Dynamin inhibitors identified to date. We investigated the mechanism of action of the Pyrimidyn compounds in detail by examining the kinetics of Pyrimidyn 7 inhibition of Dynamin. The compound competitively inhibits both GTP and phospholipid interactions with Dynamin I. While both mechanisms of action have been previously observed separately, this is the first inhibitor series to incorporate both and thereby to target two distinct domains of Dynamin. Pyrimidyn 6 and 7 reversibly inhibit CME of both transferrin and EGF in a number of non-neuronal cell lines as well as inhibiting synaptic vesicle endocytosis (SVE) in nerve terminals. Therefore, Pyrimidyn compounds block endocytosis by directly competing with GTP and lipid binding to Dynamin, limiting both the recruitment of Dynamin to membranes and its activation. This dual mode of action provides an important new tool for molecular dissection of Dynamin’s role in endocytosis

  • Phenothiazine‐Derived Antipsychotic Drugs Inhibit Dynamin and Clathrin‐Mediated Endocytosis
    Traffic (Copenhagen Denmark), 2015
    Co-Authors: James A. Daniel, Ngoc Chau, Ainslie Whiting, Mohammed K. Abdel-hamid, Lisa Von Kleist, Sai Krishnan, Peter Maamary, Shannon R. Joseph, Fiona Simpson
    Abstract:

    Chlorpromazine is a phenothiazine-derived antipsychotic drug (APD) that inhibits clathrin-mediated endocytosis (CME) in cells by an unknown mechanism. We examined whether its action and that of other APDs might be mediated by the GTPase activity of Dynamin. Eight of eight phenothiazine-derived APDs inhibited Dynamin I (dynI) in the 2-12 µm range, the most potent being trifluoperazine (IC50 2.6 ± 0.7 µm). They also inhibited Dynamin II (dynII) at similar concentrations. Typical and atypical APDs not based on the phenothiazine scaffold were 8- to 10-fold less potent (haloperidol and clozapine) or were inactive (droperidol, olanzapine and risperidone). Kinetic analysis showed that phenothiazine-derived APDs were lipid competitive, while haloperidol was uncompetitive with lipid. Accordingly, phenothiazine-derived APDs inhibited dynI GTPase activity stimulated by lipids but not by various SH3 domains. All Dynamin-active APDs also inhibited transferrin (Tfn) CME in cells at related potencies. Structure-activity relationships (SAR) revealed Dynamin inhibition to be conferred by a substituent group containing a terminal tertiary amino group at the N2 position. Chlorpromazine was previously proposed to target AP-2 recruitment in the formation of clathrin-coated vesicles (CCV). However, neither chlorpromazine nor thioridazine affected AP-2 interaction with amphiphysin or clathrin. Super-resolution microscopy revealed that chlorpromazine blocks neither clathrin recruitment by AP-2, nor AP-2 recruitment, showing that CME inhibition occurs downstream of CCV formation. Overall, potent Dynamin inhibition is a shared characteristic of phenothiazine-derived APDs, but not other typical or atypical APDs, and the data indicate that Dynamin is their likely in-cell target in endocytosis.

  • Pyrimidyn Compounds: Dual-Action Small Molecule Pyrimidine-Based Dynamin Inhibitors
    ACS Chemical Biology, 2013
    Co-Authors: Andrew B. Mcgeachie, Damien J Keating, Luke R Odell, Timothy A. Hill, Ngoc Chau, Nick N Gorgani, Annie Quan, James A. Daniel, Michael A Cousin
    Abstract:

    Dynamin is required for clathrin-mediated endocytosis (CME). Its GTPase activity is stimulated by phospholipid binding to its PH domain, which induces helical oligomerization. We have designed a series of novel pyrimidine-based "Pyrimidyn" compounds that inhibit the lipid-stimulated GTPase activity of full length Dynamin I and II with similar potency. The most potent analogue, Pyrimidyn 7, has an IC50 of 1.1 μM for Dynamin I and 1.8 μM for Dynamin II, making it among the most potent Dynamin inhibitors identified to date. We investigated the mechanism of action of the Pyrimidyn compounds in detail by examining the kinetics of Pyrimidyn 7 inhibition of Dynamin. The compound competitively inhibits both GTP and phospholipid interactions with Dynamin I. While both mechanisms of action have been previously observed separately, this is the first inhibitor series to incorporate both and thereby to target two distinct domains of Dynamin. Pyrimidyn 6 and 7 reversibly inhibit CME of both transferrin and EGF in a number of non-neuronal cell lines as well as inhibiting synaptic vesicle endocytosis (SVE) in nerve terminals. Therefore, Pyrimidyn compounds block endocytosis by directly competing with GTP and lipid binding to Dynamin, limiting both the recruitment of Dynamin to membranes and its activation. This dual mode of action provides an important new tool for molecular dissection of Dynamin's role in endocytosis.

  • Phosphorylation of Dynamin II at serine-764 is associated with cytokinesis.
    Biochimica et biophysica acta, 2010
    Co-Authors: Megan Chircop, Chandra S Malladi, Ngoc Chau, Annie Quan, Boris Sarcevic, Martin R. Larsen, Michael Zavortink, Charlotte M. Smith, Victor Anggono, Peter G Hains
    Abstract:

    Abstract Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates Dynamin II during cytokinesis at the midbody of dividing cells. However, Dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether Dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that Dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO 2 chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length Dynamin II and Glutathione-S-Transferase-tagged–Dynamin II–proline-rich domain in vitro , and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-Dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous Dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of Dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.

  • the pthaladyns gtp competitive inhibitors of Dynamin i and II gtpase derived from virtual screening
    Journal of Medicinal Chemistry, 2010
    Co-Authors: Luke R Odell, Dian Howan, Mark J Robertson, Ngoc Chau, Ainslie Whiting, James A. Daniel, Ruben Abagyan, Anna Mariana, Christopher P Gordon, Nick N Gorgani
    Abstract:

    We report the development of a homology model for the GTP binding domain of human Dynamin I based on the corresponding crystal structure of Dictyostelium discoidum Dynamin A. Virtual screening identified 2-[(2-biphenyl-2-yl-1,3-dioxo-2,3-dihydro-1H-isoindole-5-carbonyl)amino]-4-chlorobenzoic acid (1) as a ∼170 μM potent inhibitor. Homology modeling- and focused library-led synthesis resulted in development of a series of active compounds (the “pthaladyns”) with 4-chloro-2-(2-(4-(hydroxymethyl)phenyl)-1,3-dioxoisoindoline-5-carboxamido)benzoic acid (29), a 4.58 ± 0.06 μM Dynamin I GTPase inhibitor. Pthaladyn-29 displays borderline selectivity for Dynamin I relative to Dynamin II (∼5−10 fold). Only pthaladyn-23 (Dynamin I IC50 17.4 ± 5.8 μM) was an effective inhibitor of Dynamin I mediated synaptic vesicle endocytosis in brain synaptosomes with an IC50 of 12.9 ± 5.9 μM. This compound was also competitive with respect to Mg2+·GTP. Thus the pthaladyns are the first GTP competitive inhibitors of Dynamin I and ...