PMC:64779 / 11708-17667 JSONTXT

{"project":"2_test","denotations":[{"id":"11806752-10077590-9188075","span":{"begin":148,"end":150},"obj":"10077590"},{"id":"11806752-11030761-9188076","span":{"begin":243,"end":244},"obj":"11030761"},{"id":"11806752-10644436-9188077","span":{"begin":245,"end":247},"obj":"10644436"},{"id":"11806752-10400985-9188078","span":{"begin":248,"end":250},"obj":"10400985"},{"id":"11806752-10077590-9188079","span":{"begin":251,"end":253},"obj":"10077590"}],"text":"Alpha 4 does not co-localise with MID1 or MID2 proteins harboring in-frame B-box deletions\nBoth endogenous mutant MID1 protein in OS patient cells [18] and various transiently expressed mutant MID1-GFP fusion proteins form cytoplasmic clumps [5,15,17,18]. We chose to exploit this previous observation by co-transfecting GFP-tagged MID1ΔCTD (or MID2ΔCTD) with a construct expressing a myc-tagged Alpha 4 protein in order to investigate whether Alpha 4 still remained bound to MID1 within such aggregates. The results clearly showed a distribution of myc-Alpha 4 that was indistinguishable from the clumped MID1ΔCTD and MID2ΔCTD truncated proteins, indicating that Alpha 4 indeed aggregates with the truncated MID1 and MID2 proteins (Fig 2B; m-o).\nFigure 2 MID1/Alpha 4 and MID2/Alpha 4 interactions are maintained in all MID domain-specific deletions except for those involving the B-boxes. (A) Yeast two-hybrid analysis shows that the MID B-boxes are required for interaction with Alpha 4. The interaction of Alpha 4 with MID1ΔBB (21), or MID2ΔBB (23), is compared to its interaction with MID1ΔCC (22), or MID2ΔCC (24). (B) Subcellular localisation of myc tagged-Alpha 4 when co-expressed in Cos-1 cells with MID1 domain-specific deletions as GFP fusion proteins. Fluorescence detection of GFP-MID1ΔRF (a), GFP-MID1ΔBB (d), GFP-MID1ΔCC (g), GFP-MID1ΔFNIII (j), and GFP-MID1ΔCTD (m). Anti-myc antibody detection of myc-Alpha 4 in the same cells as expressing the various MID1 domain-specific deletions (b,e,h,k,n). Overlay of the GFP and anti-myc images of the same cells merged with DAPI stain of nuclei (c,f,i,l,o). All merged images, with the exception of (f) show co-localisation of myc-Alpha 4 with the various MID1 domain deletions. In (f), myc-Alpha 4 fails to co-localise with GFP-MID1ΔBB in small cytoplasmic aggregates. To further define the motif in MID1 (and MID2) responsible for the interaction with Alpha 4, we generated in-frame deletions of all other motifs (Table 1) and fused the resulting clones to GFP. We initially transfected each construct alone to examine the effect of each deletion on the intracellular localisation of the proteins. The results showed that each motif, or at least their conserved spacing or arrangement, was essential for the distribution of MID1 (and MID2) along the length of the microtubules. The distribution of the individual domain deleted MID proteins when transfected alone was indistinguishable from their distributions when transfected along with myc-Alpha 4 (see below). Consequently, the GFP fluorescence images in Fig 2B can also be considered a representation of each distribution pattern in the absence of co-transfected myc-Alpha 4. Like the ΔCTD constructs, deletion of either the B-boxes or the FNIII domain also resulted in cytoplasmic clumps or speckles although these usually appeared smaller and greater in number and the speckles still appeared to co-localise with microtubules (data not shown). Both ΔRING proteins, in contrast, showed variability in their distribution with most transfected cells still showing association along the length of the microtubules. However, the microtubule association of these ΔRING proteins often did not extend to the cell periphery. Notably, deletion of the coiled-coil motif in each protein resulted a diffuse cytoplasmic distribution, suggesting that these proteins had lost their ability to associate with microtubules (see Fig 2B; g-i).\nTable 1 The MID1 and MID2 deletion constructs used in pEGFP-C2 for cellular co-localisation analysis, co-immunoprecipitation and in pPC86/pDBLeu for interaction analysis with Alpha 4. Terms: RF denotes a C3HC4 RING finger; BB denotes C2H2 B-Boxes; CC denotes a Coiled-coil motif; FNIII denotes a Fibronectin Type III domain and CTD denotes a C-terminal domain (encompassing a SPRY domain). We then individually co-transfected each MID1 (and MID2) deletion construct with myc-Alpha 4 in an attempt to define the domain responsible for the interaction with Alpha 4. Co-transfection of either the ΔRING or ΔFNIII proteins with myc-Alpha 4 (Fig 2B; a-c and j-l, respectively) resulted in co-localisation of Alpha 4 with the abnormally distributed MID1 and MID2 proteins, as seen with the ΔCTD proteins. Co-expression of either ΔCC construct with Alpha 4 resulted in both proteins exhibiting a diffuse cytoplasmic distribution although the pattern of each was still suggestive of the two proteins being able to interact (Fig 2B; g-i). Strikingly, when either of the ΔBB constructs was co-expressed with Alpha 4, the mutant MID1 and MID2 proteins still formed cytoplasmic clumps but, in both cases, Alpha 4 remained diffuse in the cytoplasm (Fig 2B; d-f). These results imply that the B-boxes (and/or the linker residues between the B-boxes and RING motifs) are primarily responsible for the interaction with Alpha 4.\nTo confirm these results, we cloned the MID1 and MID2 domain-deletion constructs in-frame into pPC86 and co-transformed into MaV203 with pDBLeu-Alpha 4. As expected from the immunofluorescence data, activation of all two-hybrid reporter genes, at a level similar to that seen with the full-length MID proteins, was observed with the ΔRING, ΔFNIII and ΔCTD proteins (results not shown), confirming that neither of these domains mediated binding to Alpha 4. Notably, both ΔCC proteins also interacted with Alpha 4 (Fig 2A) suggesting that the two proteins were indeed still interacting in the cytoplasm in the immunofluorescence experiments despite not being associated with microtubules. However, the strength of the interaction between the ΔCC proteins and Alpha 4 appeared to be reduced compared to the full-length, ΔRING, ΔFNIII and ΔCTD (data not shown). In contrast, but again confirming the immunofluorescence result, the ΔBB constructs did not activate reporter gene expression indicating the interaction between Alpha 4 and either MID protein was abolished by removal of this motif (Fig 2A)."}