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

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.

Migration Index towards the y-axis, FMI, for primary fibroblasts using ImageJ 'Manual Tracking and Chemotaxis' tool (n = 12 cells, 2 independent experiments; n.s., not significant).    In WT (left), CRN7 might act to positively facilitate Cdc42 activation leading to N-WASP activation downstream in the pathway. Cdc42 in its GTP bound form can recruit N-WASP to the Golgi complex and trigger actin polymerization. As a checkpoint, CRN7 directly binds N-5 WASP and competitively (?) inhibits its activity upon constitutive Cdc42 activation to restrict spurious F-actin content. However, in KO (right), any Cdc42-GEF can still activate Cdc42 and lead to N-WASP "hyperactivity" (in the absence of inhibition by CRN7) which then promotes F-actin formation leading to an enhancement of actin-driven processes. In WT, controlled F-actin levels at the Golgi helps to maintain its structure, while in KO, increased F-    The antibodies used for western blot and immunofluorescence were as follows: GFP, mouse mAb K3-184-2 [2], CRN7, mAb K37-142-1, Myc, mAb 9E10, GST, mAb K84-913-0 were prepared in our laboratory, Rabbit GFP, polyclonal, was obtained from Dr. M. Schleicher (München), 58K, mAb 58K-9, Pericentrin, rabbit polyclonal, and Ki-67, rabbit polyclonal, were from Abcam, Mouse mAb GAPDH coupled to peroxidase (POD) and N-WASP, rabbit pAbs were from Sigma, Cdc42, mouse mAb was from Santa Cruz Biotechnology, mouse mAb vinculin was from Sigma, and GM130, mouse mAb was from BD Biosciences. All secondary antibodies were procured from Invitrogen.

Isolation of genomic DNA and PCR genotyping
Mouse tail tips were incubated overnight in lysis buffer (50 mM Tris-HCl, pH 8.5, 100 mM EDTA, 1% SDS, 100 mM NaCl) including proteinase K at 55°C. The DNA was precipitated After appropriate washing steps a film was exposed to the membrane and developed after 2-10 days of exposure at -80°C depending on the signal intensity.

RNA isolation, reverse transcriptase (RT) PCR and northern blotting
Total RNA was extracted from primary fibroblasts using Trizol reagent and following the instruction manual (Invitrogen). The concentration and quality of the total RNA was determined on a NanoDrop Agilent Bioanalyzer.

Cell adhesion and proliferation assay
Equal numbers of cells were seeded in 96-well plates that were pre-coated with 20 µg/ml fibronectin and pre-blocked with 1% BSA, and incubated for 30 min at 37°C. 30 minutes post-plating the plates were shaken and rinsed to remove the detached cells. The attached cells were treated with 4% PFA and thereafter stained with Neutral Red (Sigma) solution, a dye which stains live cells only, and incubated at 37°C for 30 min. Following incubation, the stain was removed, wells rinsed and solubilisation solution (50% ethanol + 1% acetic acid) added to solubilise the dye from the stained cells and left to stand for 10 min with intermittent pipetting to enhance mixing of the solubilised dye. The absorbance was measured using a TECAN plate-reader at a wavelength of 540 nm. The background absorbance of multi-well plates was measured at 690 nm and subtracted from 540 nm measurement as described in [3].
Cell proliferation was quantified from cells of equal density which were allowed to proliferate for 24 h, and were then stained for the proliferation marker Ki-67 (rabbit polyclonal antibodies, Abcam) for visual counting under the microscope.

Western blotting
Proteins were transferred from polyacrylamide gels onto nitrocellulose membranes by wet blotting or semi-dry method. The transfer was confirmed by PonceauS staining of the membrane. Blocking was performed with 5% milk powder in TBS-T (50 mM Tris, 150 mM NaCl, 0.05% Tween 20 adjusted to pH 7.6). For protein detection, the membrane was incubated with primary antibodies (purified or hybridoma supernatant) diluted in TBS-T for 1-2 h at room temperature or overnight at 4°C. After washes the membrane was incubated with the corresponding secondary peroxidase-conjugated antibodies. The membranes were incubated with the detection reagent (ChemiGlowTM, Alpha Innotech Corporation) and chemiluminescence signals were monitored with the FlourChemTMSP imaging system (Alpha Innotech) or by classical photographic film exposure. Antibodies were removed from nitrocellulose membranes by stripping of the membranes with 0.1 M NaOH for 10 minutes followed by washing steps with TBST.