Rac controls PIP5K localisation and PtdIns(4,5)<i>P</i>2 synthesis, which modulates vinculin localisation and neurite dynamics

Halstead, Jonathan R.; Savaskan, Nicolai Ε.; Bout, Iman van den; Horck, Francis van; Hajdo-Milašinović, Amra; Snell, Mireille; Keune, Willem Jan; Klooster, Jean Paul ten; Hordijk, Peter L.; Divecha, Nullin

doi:10.1242/jcs.062679

Cited by 42 publications

(40 citation statements)

References 45 publications

(55 reference statements)

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“…PA also activates certain protein kinases, such as phosphatidylinositol 4-phosphate 5-kinase (PIP5K) through direct interaction (Moritz et al, 1992;Jenkins et al, 1994). Overexpression of PIP5K was sufficient to induce neurite retraction (Halstead et al, 2010), which is similar with the dendritic phenotypes caused by overexpression of PLD1. Moreover, the activity of PIP5K is essential for neurite retraction in response to LPA (van Horck et al, 2002;Yamazaki et al, 2002), one metabolite of PA.…”

Section: Discussionsupporting

confidence: 60%

PLD1 Negatively Regulates Dendritic Branching

Zhu

Kang

Zhang

et al. 2012

Journal of Neuroscience

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Neurons have characteristic dendritic arborization patterns that contribute to information processing. One essential component of dendritic arborization is the formation of a specific number of branches. Although intracellular pathways promoting dendritic growth and branching are being elucidated, the mechanisms that negatively regulate the branching of dendrites remain enigmatic. In this study, using gain-of-function and loss-of-function studies, we show that phospholipase D1 (PLD1) acts as a negative regulator of dendritic branching in cultured hippocampal neurons from embryonic day 18 rat embryos. Overexpression of wild-type PLD1 (WT-PLD1) decreases the complexity of dendrites, whereas knockdown or inhibition of PLD1 increases dendritic branching. We further demonstrated that PLD1 acts downstream of RhoA, one of the small Rho GTPases, to suppress dendritic branching. The restriction of dendritic branching by constitutively active RhoA (V14-RhoA) can be partially rescued by knockdown of PLD1. Moreover, the inhibition of dendritic branching by V14-RhoA and WT-PLD1 can be partially ameliorated by reducing the level of phosphatidic acid (PA), which is the enzymatic product of PLD1. Together, these results suggest that RhoA-PLD1-PA may represent a novel signaling pathway in the restriction of dendritic branching and may thus provide insight into the mechanisms of dendritic morphogenesis.

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Section: Discussionsupporting

confidence: 60%

PLD1 Negatively Regulates Dendritic Branching

Zhu

Kang

Zhang

et al. 2012

Journal of Neuroscience

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“…However, Rac1 regulates many signaling pathways in T cells. Indeed, on one hand, Rac1 can activate the phosphatidylinositol 4-phosphate-5-kinase, which induces PI(4,5)P 2 synthesis (69,70). On the other hand, Rac1 can also regulate actin cytoskeleton dynamics (49,50) through several signaling pathways, such as Rac1-Pak2-cofilin or Rac1-IRSp53-Wave2-Arp3 (Fig.…”

Section: Rac1 Knockdown Modulates Hiv-1 Gag Vlp Release and Gag Intramentioning

confidence: 99%

Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells

Thomas

Mariani-Floderer

López-Huertas

et al. 2015

J Virol

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During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes. IMPORTANCEDuring HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes.

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“…This construct was then used to generate mutants where the Glu-61 residue was replaced with either a Met or Leu residue. These mutations comprise a Rac1 binding site and make the enzyme mostly cytoplasmic (39), although the constructs still bind to the PM at higher expression levels or longer transfections times (Ͼ16 -20 h). The wild-type and E61L mutant enzymes were then subcloned into the mRFP-FKBP12 backbone (40) with PvuI/KpnI enzymes to generate the recruitable versions of the proteins.…”

Section: Methodsmentioning

confidence: 99%