Browsing by Author "Newpher, Thomas M"

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    Calmodulin dissociation regulates Myo5 recruitment and function at endocytic sites.
    (EMBO J, 2010-09-01) Grötsch, Helga; Giblin, Jonathan P; Idrissi, Fatima-Zahra; Fernández-Golbano, Isabel-María; Collette, John R; Newpher, Thomas M; Robles, Virginia; Lemmon, Sandra K; Geli, María-Isabel
    Myosins-I are conserved proteins that bear an N-terminal motor head followed by a Tail Homology 1 (TH1) lipid-binding domain. Some myosins-I have an additional C-terminal extension (C(ext)) that promotes Arp2/3 complex-dependent actin polymerization. The head and the tail are separated by a neck that binds calmodulin or calmodulin-related light chains. Myosins-I are known to participate in actin-dependent membrane remodelling. However, the molecular mechanisms controlling their recruitment and their biochemical activities in vivo are far from being understood. In this study, we provided evidence suggesting the existence of an inhibitory interaction between the TH1 domain of the yeast myosin-I Myo5 and its C(ext). The TH1 domain prevented binding of the Myo5 C(ext) to the yeast WIP homologue Vrp1, Myo5 C(ext)-induced actin polymerization and recruitment of the Myo5 C(ext) to endocytic sites. Our data also indicated that calmodulin dissociation from Myo5 weakened the interaction between the neck and TH1 domains and the C(ext). Concomitantly, calmodulin dissociation triggered Myo5 binding to Vrp1, extended the myosin-I lifespan at endocytic sites and activated Myo5-induced actin polymerization.
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    Clathrin is important for normal actin dynamics and progression of Sla2p-containing patches during endocytosis in yeast.
    (Traffic (Copenhagen, Denmark), 2006-05) Newpher, Thomas M; Lemmon, Sandra K
    Clathrin is a major vesicle coat protein involved in receptor-mediated endocytosis. In yeast and higher eukaryotes, clathrin is recruited to the plasma membrane during the early stage of endocytosis along with clathrin-associated adaptors. As coated pits undergo maturation, a burst of actin polymerization accompanies and helps drive vesicle internalization. Here, we investigate the dynamics of clathrin relative to the early endocytic patch protein Sla2p. We find that clathrin is recruited to the cortex prior to Sla2p. In the absence of clathrin, normal numbers of Sla2p patches form, but many do not internalize or are dramatically delayed in completion of endocytosis. Patches that do internalize receive Sla1p late, which is followed by Abp1, which appears near the end of Sla2p lifetime. In addition, clathrin mutants develop actin comet tails, suggesting an important function in actin patch organization/dynamics. Similar to its mammalian counterparts, the light chain (LC) subunit of yeast clathrin interacts directly with the coiled-coil domain of Sla2p. A mutant of Sla2p that no longer interacts with LC (sla2Delta376-573) results in delayed progression of endocytic patches and aberrant actin dynamics. These data demonstrate an important role for clathrin in organization and progression of early endocytic patches to the late stages of endocytosis.
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    Glutamate receptor dynamics in dendritic microdomains.
    (Neuron, 2008-05) Newpher, Thomas M; Ehlers, Michael D
    Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity.
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    In vivo dynamics of clathrin and its adaptor-dependent recruitment to the actin-based endocytic machinery in yeast.
    (Developmental cell, 2005-07) Newpher, Thomas M; Smith, Robin P; Lemmon, Vance; Lemmon, Sandra K
    Clathrin-mediated transport is a major pathway for endocytosis. However, in yeast, where cortical actin patches are essential for endocytosis, plasma membrane-associated clathrin has never been observed. Using live cell imaging, we demonstrate cortical clathrin in association with the actin-based endocytic machinery in yeast. Fluorescently tagged clathrin is found in highly mobile internal trans-Golgi/endosomal structures and in smaller cortical patches. Total internal reflection fluorescence microscopy showed that cortical patches are likely endocytic sites, as clathrin is recruited prior to a burst of intensity of the actin patch/endocytic marker, Abp1. Clathrin also accumulates at the cortex with internalizing alpha factor receptor, Ste2p. Cortical clathrin localizes with epsins Ent1/2p and AP180s, and its recruitment to the surface is dependent upon these adaptors. In contrast, Sla2p, End3p, Pan1p, and a dynamic actin cytoskeleton are not required for clathrin assembly or exchange but are required for the mobility, maturation, and/or turnover of clathrin-containing endocytic structures.
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    Novel function of clathrin light chain in promoting endocytic vesicle formation.
    (Molecular biology of the cell, 2006-10) Newpher, Thomas M; Idrissi, Fatima-Zahra; Geli, Maria Isabel; Lemmon, Sandra K
    Clathrin-mediated endocytosis is a major pathway for uptake of lipid and protein cargo at the plasma membrane. The lattices of clathrin-coated pits and vesicles are comprised of triskelions, each consisting of three oligomerized heavy chains (HC) bound by a light chain (LC). In addition to binding HC, LC interacts with members of the Hip1/R family of endocytic proteins, including the budding yeast homologue, Sla2p. Here, using in vivo analysis in yeast, we provide novel insight into the role of this interaction. We find that overexpression of LC partially restores endocytosis to cells lacking clathrin HC. This suppression is dependent on the Sla2p binding region of LC. Using live cell imaging techniques to visualize endocytic vesicle formation, we find that the N-terminal Sla2p binding region of LC promotes the progression of arrested Sla2p patches that form in the absence of HC. We propose that LC binding to Sla2p positively regulates Sla2p for efficient endocytic vesicle formation.
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    Parallel on-axis holographic phase microscopy of biological cells and unicellular microorganism dynamics
    (APPLIED OPTICS, 2010-05-20) Shaked, Natan T; Newpher, Thomas M; Ehlers, Michael D; Wax, Adam
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    Postsynaptic positioning of endocytic zones and AMPA receptor cycling by physical coupling of dynamin-3 to Homer.
    (Neuron, 2007-09) Lu, Jiuyi; Helton, Thomas D; Blanpied, Thomas A; Rácz, Bence; Newpher, Thomas M; Weinberg, Richard J; Ehlers, Michael D
    Endocytosis of AMPA receptors and other postsynaptic cargo occurs at endocytic zones (EZs), stably positioned sites of clathrin adjacent to the postsynaptic density (PSD). The tight localization of postsynaptic endocytosis is thought to control spine composition and regulate synaptic transmission. However, the mechanisms that situate the EZ near the PSD and the role of spine endocytosis in synaptic transmission are unknown. Here, we report that a physical link between dynamin-3 and the postsynaptic adaptor Homer positions the EZ near the PSD. Disruption of dynamin-3 or its interaction with Homer uncouples the PSD from the EZ, resulting in synapses lacking postsynaptic clathrin. Loss of the EZ leads to a loss of synaptic AMPA receptors and reduced excitatory synaptic transmission that corresponds with impaired synaptic recycling. Thus, a physical link between the PSD and the EZ ensures localized endocytosis and recycling by recapturing and maintaining a proximate pool of cycling AMPA receptors.
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    Regulation of spine structural plasticity by Arc/Arg3.1.
    (Seminars in cell & developmental biology, 2018-05) Newpher, Thomas M; Harris, Scott; Pringle, Jasmine; Hamilton, Colleen; Soderling, Scott
    Dendritic spines are actin-rich, postsynaptic protrusions that contact presynaptic terminals to form excitatory chemical synapses. These synaptic contacts are widely believed to be the sites of memory formation and information storage, and changes in spine shape are thought to underlie several forms of learning-related plasticity. Both membrane trafficking pathways and the actin cytoskeleton drive activity-dependent structural and functional changes in dendritic spines. A key molecular player in regulating these processes is the activity-regulated cytoskeleton-associated protein (Arc), a protein that has diverse roles in expression of synaptic plasticity. In this review, we highlight important findings that have shaped our understanding of Arc's functions in structural and functional plasticity, as well as Arc's contributions to memory consolidation and disease.
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    Role of Scd5, a protein phosphatase-1 targeting protein, in phosphoregulation of Sla1 during endocytosis.
    (J Cell Sci, 2012-10-15) Chi, Richard J; Torres, Onaidy T; Segarra, Verónica A; Lansley, Tanya; Chang, Ji Suk; Newpher, Thomas M; Lemmon, Sandra K
    Phosphorylation regulates assembly and disassembly of proteins during endocytosis. In yeast, Prk1 and Ark1 phosphorylate factors after vesicle internalization leading to coat disassembly. Scd5, a protein phosphatase-1 (PP1)-targeting subunit, is proposed to regulate dephosphorylation of Prk1/Ark1 substrates to promote new rounds of endocytosis. In this study we analyzed scd5-PP1Δ2, a mutation causing impaired PP1 binding. scd5-PP1Δ2 caused hyperphosphorylation of several Prk1 endocytic targets. Live-cell imaging of 15 endocytic components in scd5-PP1Δ2 revealed that most factors arriving before the invagination/actin phase of endocytosis had delayed lifetimes. Severely affected were early factors and Sla2 (Hip1R homolog), whose lifetime was extended nearly fourfold. In contrast, the lifetime of Sla1, a Prk1 target, was extended less than twofold, but its cortical recruitment was significantly reduced. Delayed Sla2 dynamics caused by scd5-PP1Δ2 were suppressed by SLA1 overexpression. This was dependent on the LxxQxTG repeats (SR) of Sla1, which are phosphorylated by Prk1 and bind Pan1, another Prk1 target, in the dephosphorylated state. Without the SR, Sla1ΔSR was still recruited to the cell surface, but was less concentrated in cortical patches than Pan1. sla1ΔSR severely impaired endocytic progression, but this was partially suppressed by overexpression of LAS17, suggesting that without the SR region the SH3 region of Sla1 causes constitutive negative regulation of Las17 (WASp). These results demonstrate that Scd5/PP1 is important for recycling Prk1 targets to initiate new rounds of endocytosis and provide new mechanistic information on the role of the Sla1 SR domain in regulating progression to the invagination/actin phase of endocytosis.
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    Spine microdomains for postsynaptic signaling and plasticity.
    (Trends in cell biology, 2009-05) Newpher, Thomas M; Ehlers, Michael D
    Changes in the molecular composition and signaling properties of excitatory glutamatergic synapses onto dendritic spines mediate learning-related plasticity in the mammalian brain. This molecular adaptation serves as the most celebrated cell biological model for learning and memory. Within their micron-sized dimensions, dendritic spines restrict the diffusion of signaling molecules and spatially confine the activation of signal transduction pathways. Much of this local regulation occurs by spatial compartmentalization of glutamate receptors. Here, we review recently identified cell biological mechanisms regulating glutamate receptor mobility within individual dendritic spines. We discuss the emerging functions of glutamate receptors residing within sub-spine microdomains and propose a model for distinct signaling platforms with specialized functions in synaptic plasticity.