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Cell extensions are critical structures that enable matrix remodeling in wound healing and cancer invasion but the regulation of their formation is not well-defined. We searched for new proteins that mediated cell extension formation over collagen by tandem mass tagged mass spectrometry analysis of purified extensions in 3T3 fibroblasts. Unexpectedly, importin-5, ENH isoform 1b (PDLIM5) and 26 S protease regulatory subunit 6B (PSMC4) were more abundant (> 10-fold) in membrane-penetrating cell extensions than cell bodies, which was confirmed by immunostaining and immunoblotting and also observed in human gingival fibroblasts. After siRNA knockdown of these proteins and plating cells on grid-supported floating collagen gels for 6 h, formation of cell extensions and collagen remodeling were examined. Knockdown of importin-5 reduced collagen compaction (1.9-fold), pericellular collagen degradation (˜ 1.8-fold) and number of cell extensions (˜ 69%). Knockdown of PSMC4 reduced collagen compaction (˜ 1.5-fold), pericellular collagen degradation (˜ 1.7-fold) and number of cell extensions (˜ 42%). Knockdown of PDLIM5 reduced collagen compaction (˜ 1.6-fold) and number of cell extensions (˜ 21%). Inhibition of the TGF-β RI kinase, Smad3 or ROCK-II signaling pathways reduced the abundance of PDLIM5 in cell extensions but PSMC4 and importin-5 were reduced only by Smad3 or ROCK-II inhibitors.We conclude that these novel proteins are required for cell extension formation and their recruitment into extensions involves the Smad3 and ROCK signaling pathways.