SGC-CBP30 Our results suggest that formation of DDR b
Our results suggest that formation of DDR1b clusters may be important for and precede receptor phosphorylation. Indeed, while DDR1b clustering was readily detected (by YFP signal) 30 min after collagen administration, phosphorylated DDR1 species at Y513 (present in the IJXM) were evident after 4 h of collagen stimulation. Furthermore, the observation that not all DDR1b clusters were positive for Y513 signal lends support to the hypothesis that receptor clustering may be a prerequisite for receptor phosphorylation. Moreover, our findings that DDR1b clusters were positive for Y513 and not Y792 (present in the KD) suggest that differentially phosphorylated DDR1b receptor subpopulations may be segregated to various subcellular sites. However, whether the stronger detection of DDR1b/c-Y513 versus DDR1-Y792 signals in DDR1b clusters is due to differences in antibody affinity or differences in epitope availability and/or phosphorylation/dephosphorylation kinetics needs to be determined. Despite the differences in the clustering abilities of DDR1b versus DDR2 upon ligand administration, we also noted striking similarities in the spatial distribution and phosphorylation of DDR1b-YFP and DDR2-GFP. We show for the first time that in MC3T3-E1 cells, after 4 h of collagen administration, both DDR1b-YFP and DDR2-GFP assemble into filamentous structures. A fraction of these filamentous structures was positively highlighted by DDR1b/c-Y513, DDR1-Y792, and DDR2-Y740 antibodies. Our observations that not all filamentous structures were positive for these SGC-CBP30 suggest that, like DDR1b clustering, the assembly of DDRs into filamentous structures may precede receptor phosphorylation. Recent studies have reported the co-localization of total and pDDRs in cells with collagen , , . Our results expand these observations by showing that higher-order assembly of DDRs into receptor clusters and/or filamentous structures may associate with different morphological states of collagen. While the DDR1b clusters (formed at early time points after collagen stimulation) associated with non-fibrillar collagen, the DDR1b/DDR2 filamentous structures (formed after prolonged collagen stimulation) associated with collagen fibrils. It should be noted that in this study soluble collagen was presented to the cells, which could spontaneously assemble into fibrils and/or undergo a cell-mediated assembly into fibrils. These processes could dictate if the formation of collagen fibrils is a prerequisite for, is synchronous with, or succeeds the assembly of DDRs into filamentous structures in cells. Our observations that the filamentous structures formed by DDRs do not evolve with time but only appear after prolonged collagen stimulation and are contiguous with collagen fibrils even in the peri-cellular regions suggest that a fibrillar form of collagen may be required to assemble DDRs. Further studies using multi-modal high-resolution microscopy to monitor DDRs as well as collagen morphology on the cell surface at various time points or presenting the cells with preformed collagen fibrils instead of soluble collagen will be required to understand the role of collagen fibrillogenesis and the fibrillar state of collagen in clustering, spatio-temporal distribution, and phosphorylation of DDRs. Our current study using soluble collagen I holds relevance primarily for extracellular matrix remodeling when newly synthesized soluble collagen is presented to the cells. Further studies are needed to fully comprehend the role of DDRs in health and disease by investigating how DDRs bind and respond to fibrils of collagen I as well as to other collagen types present in natural tissues. Besides collagen, other cytoskeletal and/or intracellular proteins could also have a putative role in modulating the spatial distribution and assembly of DDRs into clusters or filamentous structures. Our initial investigations to identify such proteins yielded limited success. Both DDR1b clusters as well as DDR1b and DDR2 filamentous structures did not co-localize with either vimentin, vinculin (Fig. S8), or f-actin (Fig. S9). This is consistent with an earlier report showing the lack of co-localization between DDR1 with these cytoskeletal proteins in cells cultured on immobilized collagen fibrils . However, DDR1 has been reported to co-localize with non-muscle myosin II . Along similar lines, we found that the filamentous structures formed by DDR1b-YFP and DDR2-GFP were enriched with the actin-binding protein cortactin (Fig. S10). It is tempting to speculate that the higher-order assembly of DDRs into filamentous structures may serve as a scaffold for recruiting proteins like cortactin and myosin II to communicate with the cell cytoskeleton. In this regard, it is important to note that DDRs have reported to play a role in cell-mediated traction forces and mechano-transduction  and in formation of linear invadosomes . Further studies are required to understand how the DDR–collagen assembly and ensuing receptor phosphorylation may be involved in these processes.