Andrew Wilde

EGF Receptor Signaling Stimulates SRC Kinase Phosphorylation of Clathrin, Influencing Clathrin Redistribution and EGF Uptake

Epidermal growth factor (EGF) binding to its receptor causes rapid phosphorylation of the clathrin heavy chain at tyrosine 1477, which lies in a domain controlling clathrin assembly. EGF-mediated clathrin phosphorylation is followed by clathrin redistribution to the cell periphery and is the product of downstream activation of SRC kinase by EGF receptor (EGFR) signaling. In cells lacking SRC kinase, or cells treated with a specific SRC family kinase inhibitor, EGF stimulation of clathrin phosphorylation and redistribution does not occur, and EGF endocytosis is delayed. These observations demonstrate a role for SRC kinase in modification and recruitment of clathrin during ligand-induced EGFR endocytosis and thereby define a novel effector mechanism for regulation of endocytosis by receptor signaling.

BST-2/HM1.24 is a raft-associated apical membrane protein with an unusual topology

An expression screen of a rat cDNA library for sequences encoding Golgi‐localized integral membrane proteins identified a protein with an apparent novel topology, i.e. with both an N‐terminal transmembrane domain and a C‐terminal glycosyl‐phosphatidylinositol (GPI) anchor. Our data are consistent with this. Thus, the protein would have a topology that, in mammalian cells, is shared only by a minor, but pathologically important, topological isoform of the prion protein (PrP). The human orthologue of this protein has been described previously (BST‐2 or HM1.24 antigen) as a cell surface molecule that appears to be involved in early pre‐B‐cell development and which is present at elevated levels at the surface of myeloma cells. We show that rat BST‐2/HM1.24 has both a cell surface and an intracellular (juxtanuclear) location and is efficiently internalized from the cell surface. We also show that the cell surface pool of BST‐2/HM1.24 is predominantly present in the apical plasma membrane of polarized cells. The fact that rat BST‐2/HM1.24 apparently possesses a GPI anchor led us to speculate that it might exist in cholesterol‐rich lipid microdomains (lipid rafts) at the plasma membrane. Data from several experiments are consistent with this localization. We present a model in which BST‐2/HM1.24 serves to link adjacent lipid rafts within the plasma membrane.

Ran stimulates spindle assembly by altering microtubule dynamics and the balance of motor activities.

The guanosine tri-phosphatase Ran stimulates assembly of microtubule spindles. However, it is not known what aspects of the microtubule cytoskeleton are subject to regulation by Ran in mitosis. Here we show that Ran–GTP stimulates microtubule assembly by increasing the rescue frequency of microtubules three- to eightfold. In addition to changing microtubule dynamics, Ran–GTP also alters the balance of motor activities, partly as a result of an increase in the amount of motile Eg5, a plus-end-directed microtubule motor that is essential for spindle formation. Thus, Ran regulates multiple processes that are involved in spindle assembly.

NGF Signals through TrkA to Increase Clathrin at the Plasma Membrane and Enhance Clathrin-Mediated Membrane Trafficking

Neurotrophin (NT) signals may be moved from axon terminals to neuron cell bodies via signaling endosomes—organelles in which NTs continue to be bound to their activated receptors. Suggesting that clathrin-coated membranes serve as one source of signaling endosomes, in earlier studies we showed that nerve growth factor (NGF) treatment increased clathrin at the plasma membrane and resulted in colocalization of clathrin with TrkA, the receptor tyrosine kinase for NGF. Strikingly, however, we also noted that most clathrin puncta at the surface of NGF-treated cells did not colocalize with TrkA, raising the possibility that NGF induces a general increase in clathrin-coated membrane formation. To explore this possibility further, we examined the distribution of clathrin in NGF- and BDNF-treated cells. NGF signaling in PC12 cells robustly redistributed the adaptor protein AP2 and the clathrin heavy chain (CHC) to surface membranes. Using confocal and epifluorescence microscopy, as well as biochemical assays, we showed the redistribution of clathrin to be attributable to the activation of TrkA. Significantly, NGF signaled through TrkA to induce an increase in clathrin-mediated membrane trafficking, as revealed in the increased endocytosis of transferrin. In that BDNF treatment increased AP2 and clathrin at the surface membranes of hippocampal neurons, these findings may represent a physiologically significant response to NTs. We conclude that NT signaling increases clathrin-coated membrane formation and clathrin-mediated membrane trafficking and speculate that this effect contributes to their trophic actions via the increased internalization of receptors and other proteins that are present in clathrin-coated membranes.

Ran modulates spindle assembly by regulating a subset of TPX2 and Kid activities including Aurora A activation.

Ran, a GTPase in the Ras superfamily, is proposed to be a spatial regulator of microtubule spindle assembly by maintaining key spindle assembly factors in an active state close to chromatin. RanGTP is hypothesized to maintain the spindle assembly factors in the active state by binding to importin β, part of the nuclear transport receptor complex, thereby preventing the inhibitory binding of the nuclear transport receptors to spindle assembly factors. To directly test this hypothesis, two putative downstream targets of the Ran spindle assembly pathway, TPX2, a protein required for correct spindle assembly and Kid, a chromokinesin involved in chromosome arm orientation on the spindle, were analyzed to determine if their direct binding to nuclear transport receptors inhibited their function. In the amino-terminal domain of TPX2 we identified nuclear targeting information, microtubule-binding and Aurora A binding activities. Nuclear transport receptor binding to TPX2 inhibited Aurora A binding activity but not the microtubule-binding activity of TPX2. Inhibition of the interaction between TPX2 and Aurora A prevented Aurora A activation and recruitment to microtubules. In addition we identified nuclear targeting information in both the amino-terminal microtubule-binding domain and the carboxy-terminal DNA binding domain of Kid. However, the binding of nuclear transport receptors to Kid only inhibited the microtubule-binding activity of Kid. Therefore, by regulating a subset of TPX2 and Kid activities, Ran modulates at least two processes involved in spindle assembly.

A bacterial acetyltransferase destroys plant microtubule networks and blocks secretion.

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption.

Cleavage Furrow Organization Requires PIP2-Mediated Recruitment of Anillin

Cell division is achieved by a plasma membrane furrow that must ingress between the segregating chromosomes during anaphase [1-3]. The force that drives furrow ingression is generated by the actomyosin cytoskeleton, which is linked to the membrane by an as yet undefined molecular mechanism. A key component of the membrane furrow is anillin. Upon targeting to the furrow through its pleckstrin homology (PH) domain, anillin acts as a scaffold linking the actomyosin and septin cytoskeletons to maintain furrow stability (reviewed in [4, 5]). We report that the PH domain of anillin interacts with phosphatidylinositol phosphate lipids (PIPs), including PI(4,5)P(2), which is enriched in the furrow. Reduction of cellular PI(4,5)P(2) or mutations in the PH domain of anillin that specifically disrupt the interaction with PI(4,5)P(2), interfere with the localization of anillin to the furrow. Reduced expression of anillin disrupts symmetric furrow ingression that can be restored by targeting ectopically expressed anillin to the furrow using an alternate PI(4,5)P(2) binding module, a condition where the septin cytoskeleton is not recruited to the plasma membrane. These data demonstrate that the anillin PH domain has two functions: targeting anillin to the furrow by binding to PI(4,5)P(2) to maintain furrow organization and recruiting septins to the furrow.

Poleward Transport of TPX2 in the Mammalian Mitotic Spindle Requires Dynein, Eg5, and Microtubule Flux

TPX2 is a spindle assembly factor that is required for MT assembly near chromosomes. Using photoactivation of fluorescence, we report that TPX2 is transported poleward in the half-spindle. Poleward transport of TPX2 is sensitive to inhibition of dynein or Eg5, and to suppression of MT flux.

γ-Tubulin complexes and their role in microtubule nucleation

Publisher Summary This chapter discusses the purification and characterization of γ-tubulin ring complex (γTuRC) and γ-tubulin small complex (γTuSC) and the function of γTuRC at the centrosome. The proposed mechanism of γTuRC-mediated microtubule nucleation is also reviewed. The identification of γ-tubulin and its essential role in microtubule nucleation is a major breakthrough in the study of microtubules and centrosomes. The subsequent purification of γTuRC provides the much-needed tool to investigate the role of γ-tubulin in microtubule nucleation and centrosome assembly. γTuRC is essential for both microtubule nucleation and formation of a functional centrosome. Further characterization of the γTuRC components helps to study the assembly of γTuRC and its recruitment to the centrosome. It provides an understanding of centrosome assembly, which is an essential process for proper cell division. The mechanism of microtubule nucleation by γ-tubulin comes from both biochemical and structural studies. γ-Tubulin exists as a complex with other proteins inside the cell; in certain cell types, multiple γ-tubulin complexes of different sizes co-exist. Antibody affinity production of γ-tubulin complexes from Drosophila embryos and Xenopus egg extracts is diagrammatically represented in the chapter.

The Fowler Syndrome-Associated Protein FLVCR2 Is an Importer of Heme

ABSTRACT Mutations in FLVCR2, a cell surface protein related by homology and membrane topology to the heme exporter/retroviral receptor FLVCR1, have recently been associated with Fowler syndrome, a vascular disorder of the brain. We previously identified FLVCR2 to function as a receptor for FY981 feline leukemia virus (FeLV). However, the cellular function of FLVCR2 remains unresolved. Here, we report the cellular function of FLVCR2 as an importer of heme, based on the following observations. First, FLVCR2 binds to hemin-conjugated agarose, and binding is competed by free hemin. Second, mammalian cells and Xenopus laevis oocytes expressing FLVCR2 display enhanced heme uptake. Third, heme import is reduced after the expression of FLVCR2-specific small interfering RNA (siRNA) or after the binding of the FY981 FeLV envelope protein to the FLVCR2 receptor. Finally, cells overexpressing FLVCR2 are more sensitive to heme toxicity, a finding most likely attributable to enhanced heme uptake. Tissue expression analysis indicates that FLVCR2 is expressed in a broad range of human tissues, including liver, placenta, brain, and kidney. The identification of a cellular function for FLVCR2 will have important implications in elucidating the pathogenic mechanisms of Fowler syndrome and of phenotypically associated disorders.