Rab11 regulates exocytosis of recycling vesicles at the plasma membrane

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.102913 Senye Takahashi, Keiji Kubo, Satoshi Waguri, Atsuko Yabashi, Hye-Won Shin, Yohei Katoh and Kazuhisa Nakayama*?*Corresponding author: Kazuhisa Nakayama, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan; Phone: +81-75-753-4527; Fax: +81-75-753-4557; E-mail: kazunaka{at}pharm.kyoto-u.ac.jp. Rab11 is known to associate primarily with perinuclear recycling endosomes and regulate recycling of endocytosed proteins. However, the recycling step in which Rab11 participates remains unknown. We here show that, in addition to causing tubulation of recycling endosomes, Rab11 depletion gives rise to accumulation of recycling carriers containing endocytosed transferrin and transferrin receptor beneath the plasma membrane. We also show that the carriers are transported from perinuclear recycling endosomes to the cell periphery along microtubules. Total internal reflection fluorescence microscopy of cells expressing EGFP-tagged transferrin receptor revealed that Rab11 depletion inhibits tethering and fusion of recycling carriers to the plasma membrane. Depletion of a component of the exocyst tethering complex, Sec15 or Exo70, the former which interacts with Rab11, leads to essentially the same phenotypes as those of Rab11 depletion. Thus, in addition to its role in recycling processes at perinuclear recycling endosomes, Rab11 is transported along microtubules to the cell periphery through association with recycling carriers, and directly regulates vesicle exocytosis at the plasma membrane in concert with the exocyst.

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APPL1 regulates basal NF-{kappa}B activity by stabilizing NIK

Advance Online Publication June 8, 2012 doi: 10.1242/?jcs.105171 APPL1 is a multifunctional adaptor protein that binds membrane receptors, signaling proteins and nuclear factors, thereby acting in endosomal trafficking and in different signaling pathways. Here we uncover a novel role of APPL1 as a positive regulator of transcriptional activity of NF-?B under basal but not TNFa-stimulated conditions. APPL1 was found to directly interact with TRAF2, an adaptor protein known to activate the canonical NF-?B signaling. APPL1 synergized with TRAF2 to induce NF-?B activation and both proteins were necessary for this process by functioning upstream of the IKK complex. Although TRAF2 was not detectable on APPL endosomes, endosomal recruitment of APPL1 was required for its function in the NF-?B pathway. Importantly, in the canonical pathway APPL1 appeared to regulate the proper spatial distribution of p65 in the absence of cytokine stimulation, since its overexpression enhanced and its depletion reduced the nuclear accumulation of p65. Analyzing the patterns of gene transcription upon APPL1 overproduction or depletion we found altered expression of NF-?B target genes encoding cytokines. At the molecular level, overexpressed APPL1 markedly increased the level of NIK, the key component of the noncanonical NF-?B pathway, by reducing its association with the degradative complex containing TRAF2, TRAF3 and cIAP1. In turn, high levels of NIK triggered nuclear translocation of p65. Collectively, we propose that APPL1 regulates basal NF-?B activity by modulating the stability of NIK, which affects the activation of p65. This places APPL1 as a novel link between the canonical and noncanonical machineries of NF-?B activation.

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Toxofilin upregulates the host cortical actin cytoskeleton dynamics facilitating Toxoplasma invasion

Advance Online Publication May 28, 2012 doi: 10.1242/?jcs.103648 Violaine Delorme-Walker, Marie Abrivard, Vanessa Lagal, Karen Anderson, Audrey Perazzi, Virginie Gonzalez, Christopher Page, Juliette Chauvet, Wendy Ochoa, Niels Volkmann, Dorit Hanein and Isabelle Tardieux*?5 Correspondence should be addressed to I. T. (email : isabelle.tardieux{at}inserm.fr) Toxoplasma, a human pathogen and a model apicomplexan parasite, actively and rapidly invades host cells. To initiate invasion, the parasite induces the formation of a parasite-cell junction, progressively propels itself through the junction inside a newly formed vacuole that encloses the entering parasite. Litle is known how a few micron-large diameter parasite overcome the host cell cortical actin barrier to support these remarkably rapid process of internalization (< few seconds). Correlative light and electron microscopy in conjunction with electron tomography and three-dimensional image analysis indicate that toxofilin an actin-binding protein, secreted by invading parasites correlates with localized sites of disassembly of the host cell actin meshwork. Moreover, quantitative fluorescence speckle microscopy in cells expressing toxofilin indicates that toxofilin regulates actin filament disassembly and turnover. Furthermore, Toxoplasma tachyzoites lacking toxofilin, are impaired in cortical actin disassembly and exhibit delayed invasion kinetics. We propose that toxofilin locally upregulates actin turnover thus increasing depolymerization events at the site of entry that, in turn loosens the local host cell actin meshwork, facilitating parasite internalization and vacuole folding.

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