Browsing by Subject "Clathrin"

Now showing 1 - 5 of 5

Open Access
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.
Open Access
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.
Open Access
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.
Open Access
Role of endocytosis in the activation of the extracellular signal-regulated kinase cascade by sequestering and nonsequestering G protein-coupled receptors.
(Proc Natl Acad Sci U S A, 2000-02-15) Pierce, KL; Maudsley, S; Daaka, Y; Luttrell, LM; Lefkowitz, RJ
Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the alpha(2A) adrenergic receptor (alpha(2A) AR), with ERK activation mediated by the beta(2) adrenergic receptor (beta(2) AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the alpha(2A) AR, like that mediated by both the beta(2) AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant beta-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both alpha(2A) and beta(2) AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the beta(2) AR, the alpha(2A) AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although beta(2) AR stimulation leads to redistribution of both the beta(2) AR and EGFR, activation of the alpha(2A) AR leads to redistribution of the EGFR but the alpha(2A) AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.
Open Access
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.