Black lines indicate that intervening lanes happen to be spliced out. IP
Black lines indicate that intervening lanes have already been spliced out. IP, immunoprecipitation; WB, Western blotting. (B) Cingulin domain evaluation for its association with -tubulin. -Tubulin binds to the head domain of cingulin. FL, full length. (C) Coimmunoprecipitation of endogenous cingulin with -tubulin. Eph4 extracts were pulled down with anti-cingulin or antitubulin antibody. (D) Generation of cingulin knockdown (KD) Eph4 cells. (E) Immunofluorescence for -tubulin in wild type, cingulin KD cells, and KD cells expressing an exogenous RNAi-resistant cingulin sequence (cingulin revertant [CGN] Rev.). Bar, 5 . The relative signal intensity of immunofluorescence was quantified for -tubulin (major line) and ZO-1 (bottom line) for ten cells.JCB VOLUME 203 Number 4 KD, RNAi-resistant cingulin was transfected into cingulin KD cells, which restored the MT J association. Also, the MT J association was disrupted in ZO-1 knockout Eph4 cells, in which cingulin is identified to be dissociated from TJs (Fig. S1 D; Umeda et al., 2004). These findings collectively indicated that cingulin plays a major role inside the side-by-side association of MTs with TJs. To examine the dynamics in the PAN-MTs, we transfected RFP-EB1 into Eph4 and cingulin KD cells, to trace the EB1 signals because the plus-end marker of MTs. In Eph4 cells, the EB1 signals had been situated parallel to the TJs. Alternatively, in cingulin KD cells, EB1 signals tended to be located end on with respect to the membranes at points of cell ell adhesion (Videos 4 and 5). Cingulin is also reported to associate with actin filaments (D’Atri and Citi, 2001) at the same time as with guanine nucleotide exchange issue (GEF) 1 and p114 RhoGEF, as shown in MDCK and Caco-2 cells, respectively (Aijaz et al., 2005; Terry et al., 2011). There was no difference in actin filament arrangement, myosin light chain phosphorylation, p114 RhoGEF, or GEF-H1 among wild-type Eph4 and cingulin KD Eph4 cells (Fig. S2, B ). We also didn’t detect differences in Rho activity, as shown in fluorescence BRPF3 Formulation resonance power transfer (FRET) analyses, in between the wild-type and cingulin KD cells (Videos six and 7). These results collectively indicated that cingulin mediates the lateral association of MTs with TJs, inside a manner that does not involve Rho-related signaling.Role of AMPK-mediated phosphorylation of cingulin in its association with MTsWe subsequent examined the mechanism regulating cingulin’s association with TJs. Cingulin is phosphorylated on its serine residues, comparable to other TJ proteins, like occludin and JAM-A (Citi and Denisenko, 1995; Seth et al., 2007; Raleigh et al., 2011; Iden et al., 2012). Cingulin has two AMPK target motifs L/SXXRXS/ T at its serine-132 and -150 residues (Fig. three A), and TJ assembly is reported to be facilitated by the AMPK activator AICAR (5-aminoimidazole-4-carboxamide ribonucleotide; Zhang et al., 2006; Zheng and Cantley, 2007). We for that reason examined no matter if cingulin can be a substrate of AMPK. We initially analyzed the binding of AMPK to cingulin, by coimmunoprecipitation experiments with exogenous H-cingulin and V5-AMPK1 expressed in HEK293 cells. The results showed that both KDM5 web proteins had been coimmunoprecipitated by an anti-HA antibody, indicating that they bound each and every other (Fig. three B). Next, to examine irrespective of whether cingulin was a substrate of AMPK, we generated dephosphomimetic mutants of GST-cingulin, consisting of single (S132A or S150A) and double (S132A/S150A) dephosphomimetic mutants of cingulin fused to GST. GS.