A sequence of kymographs was obtained using ImageJ software. bar = 200 m. (B) The transfection efficiencies of GFP or ACTN1-GFP expression were decided STO-609 acetate as the ratio of GFP-positive cells to total cells at 48 h post-transfection. The results represent the mean SD of three impartial experiments. n.s., not significant.(TIF) pone.0120616.s002.tif (3.9M) GUID:?38CC4E55-0C11-414E-A29F-196A69423302 S1 Movie: Focal adhesion dynamics of a control mCherry-expressing cell. Representative movie of GFP-VCL fluorescence of an mCherry-expressing DLD-1 cell. Live imaging was performed by TIRF microscopy at a frame rate of 1 1 min/frame. Time is usually indicated in h:min.(AVI) pone.0120616.s003.avi (8.6M) GUID:?201CA36E-1982-4550-826B-66E0CE173F41 S2 Movie: Focal adhesion dynamics of an LRRFIP1 antibody ACTN1-mCherry-expressing cell. Representative movie of GFP-VCL fluorescence STO-609 acetate of an ACTN1-mCherry-expressing DLD-1 cell. Time is usually indicated in h:min.(AVI) pone.0120616.s004.avi (8.6M) GUID:?2829C552-2287-4711-B371-4AA83622FD2A S3 Movie: Focal adhesion dynamics of an ACTN4-mCherry-expressing cell. Representative movie of GFP-VCL fluorescence of an ACTN4-mCherry-expressing DLD-1 cell. Time is usually indicated in h:min.(AVI) pone.0120616.s005.avi (8.6M) GUID:?13E1650F-FED9-4D1A-B596-CF0DB656B89A Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract -Actinins (ACTNs) are known to crosslink actin filaments at focal adhesions in migrating cells. Among the four isoforms of mammalian ACTNs, ACTN1 and ACTN4 are ubiquitously expressed. Recently, ACTN4 was reported to enhance malignancy cell motility, invasion, and metastasis. However, the mechanism by which ACTN4 drives these malignant phenotypes remains unclear. Here, we show that ACTN4, but not ACTN1, induces the formation of immature focal adhesions in DLD-1 cells, leading to the quick turnover of focal adhesions. Interestingly, zyxin (ZYX) assembly to focal adhesions STO-609 acetate was markedly decreased in ACTN4-expressing DLD-1 cells, while the recruitment of paxillin (PAX) occurred normally. On the other hand, in ACTN1-expressing DLD-1 cells, PAX and ZYX were normally recruited to focal adhesions, suggesting that ACTN4 specifically impairs focal adhesion maturation by inhibiting the recruitment of ZYX to focal complexes. Using purified recombinant proteins, we found that ZYX binding to ACTN4 was defective under conditions where ZYX binding to ACTN1 was observed. Furthermore, Matrigel invasion of SW480 cells that express high endogenous levels of ACTN4 protein was inhibited STO-609 acetate by ectopic expression of ACTN1. Altogether, our results suggest that ZYX defective binding to ACTN4, which occupies focal adhesions instead of STO-609 acetate ACTN1, induces the formation of immature focal adhesions, resulting in the enhancement of cell motility and invasion. Introduction -Actinins (ACTNs) are ubiquitously expressed cytoskeleton proteins that crosslink actin filaments at adherence junctions in epithelial cells and focal adhesions in polarized migrating cells [1,2]. In focal adhesions, ACTNs interact with a variety of other focal adhesion-associated proteins such as vinculin (VCL) [3,4] and integrins [5,6], and then link actin filaments to focal adhesions [7C9]. You will find four isoforms of ACTNs in mammalian cells [10C12]. ACTN1 and ACTN4 are ubiquitously expressed and are called non-muscle isoforms, while ACTN2 and ACTN3 are specifically expressed in muscle tissues. Among ACTNs, ACTN4 is usually primarily involved in cell motility and malignancy invasion [12C21]. During cell movement, ACTN4 protein expression level is usually markedly increased and ACTN4 concentrates at the leading edge of migrating cells . ACTN4 knockdown suppresses the migration and invasion of malignancy cells [15C18,20C22], whereas its overexpression in colorectal malignancy cells induces lymph node metastasis in immunodeficient mice . Furthermore, ACTN4 protein expression is usually closely related to poor end result in patients with breast , colorectal , pancreatic [20,23], ovarian , bladder , and lung  malignancy. However, the reason why ACTN4, rather than.