Novel Coronin7 interactions with Cdc42 and N-WASP regulate actin organization and Golgi morphology

Abstract

The contribution of the actin cytoskeleton to the unique architecture of the Golgi complex is manifold. An important player in this process is Coronin7 (CRN7), a Golgi-resident protein that stabilizes F-actin assembly at the trans-Golgi network (TGN) thereby facilitating anterograde trafficking. Here, we establish that CRN7-mediated association of F-actin with the Golgi apparatus is distinctly modulated via the small Rho GTPase Cdc42 and N-WASP. We identify N-WASP as a novel interaction partner of CRN7 and demonstrate that CRN7 restricts spurious F-actin reorganizations by repressing N-WASP ‘hyperactivity’ upon constitutive Cdc42 activation. Loss of CRN7 leads to increased cellular F-actin content and causes a concomitant disruption of the Golgi structure. CRN7 harbours a Cdc42- and Rac-interactive binding (CRIB) motif in its tandem β-propellers and binds selectively to GDP-bound Cdc42N17 mutant. We speculate that CRN7 can act as a cofactor for active Cdc42 generation. Mutation of CRIB motif residues that abrogate Cdc42 binding to CRN7 also fail to rescue the cellular defects in fibroblasts derived from CRN7 KO mice. Cdc42N17 overexpression partially rescued the KO phenotypes whereas N-WASP overexpression failed to do so. We conclude that CRN7 spatiotemporally influences F-actin organization and Golgi integrity in a Cdc42- and N-WASP-dependent manner.

Wiskott-Aldrich syndrome protein senses irradiation-induced DNA damage to coordinate the cell-protective Golgi dispersal response in human T and B lymphocytes

“…Multiple F-actin nucleation-promoting factors (NPFs), including members of the WASp family (WHAMM, N-WASp, and WASP family member 1 [Wave]) are located at the Golgi apparatus and are involved in Golgi biology. [22][23][24][25][26][27][28] We now show that WASp, the founding member of the WASp family, is also located at the Golgi apparatus in human primary CD4 T H cells, as evidenced by colocalization of a portion of WASp with GOLPH3 by using confocal immunofluorescence imaging (Fig 1, A).…”

“…Co-IP: Co-immunoprecipitation CPT: Camptothecin DDR: DNA damage repair and response DNA-PK: DNA-dependent protein kinase DNA-PKcs: DNA-dependent protein kinase catalytic subunit DSB: Double-strand break GDR: Golgi-dispersal response GFP: Green fluorescent protein GKO: GOLPH3 gene knockout GOLPH3: Given that WASp family proteins (neural Wiskott-Aldrich syndrome protein [N-WASp] and WASP homolog-associated protein with actin, membranes and microtubules [WHAMM]) that affect F-actin polymerization have a role in regulating Golgi apparatus morphology, [22][23][24][25][26][27][28] we wondered whether WASp, a founding member of this family, has a role in the IR-induced DDR-mediated GDR. We hypothesized that WASp deficiency, by disrupting the GDR, contributes to lymphopenia and immune dysregulation in patients with WAS.…”

Depletion of Nesprin-2 is associated with an embryonic lethal phenotype in mice

“…~ 50,000 cells were deposited per side of the well[40]. Cell migration was followed by time lapse video microscopy (37°C, 5% CO 2 ) using a Leica DMIRE2 microscope.…”

Combinatorial deployment of F-actin regulators to build complex 3D actin structuresin vivo

“…As we have examined the localization and function of the different ANRPs in cortical cap formation, and as the Arp2/3 complex is the major regulator of new actin in the apical cortex, we wanted to test the strength of Arp2/3 recruitment by each ANRP in vivo. Although, as discussed above, the ANRPs have been implicated in several different mechanisms of actin-regulation, many of the ANRPs have been shown to either activate or stabilize Arp2/3 complex function (Uruno et al, 2001;Weaver et al, 2002;Uetrecht and Bear, 2006;Pollitt and Insall, 2009;Bhattacharya et al, 2016). We therefore adapted a mitochondrial-tagging assay (Wong and Munro, 2014) to recruit ANRPs to the mitochondria and then tested the degree to which Arp3 and F-actin become ectopically localized.…”