**P<0.01. == Reduction of ANG enhances stress fiber assembly and reduces cytoskeleton dynamics == The interactions between ANG and the stress fiber components suggest that ANG regulates the organization of actin cytoskeleton. significantly decreased FGFR1 in ANG-deficient cells. Taken together, our data demonstrated that the presence of ANG in the cytoplasm optimizes stress fiber assembly and focal adhesion formation to accommodate cell migration. The finding that ANG promoted cancer cell migration might provide new clues for tumor metastasis research. == Introduction == Angiogenin (ANG) is up-regulated in various types of human cancer, including breast, cervical, colon, colorectal, endometrial, gastric, liver, kidney, ovarian, pancreatic, prostate, and urothelial cancers, as well as astrocytoma, leukemia, lymphoma, melanoma, osteosarcoma, and Wilms’ tumor[1], indicating a close relationship between ANG and tumor development. Traditionally, ANG has been recognized as an angiogenic factor which promotes angiogenesis by activating endothelial and easy muscle cells and inducing the formation of tubular structures[2][4]. Recently, ANG has been reported to directly enhance the proliferation of cancer cells such as HeLa cells and PC-3 cells, indicating that ANG plays dual roles in cancer progression by acting on both vascular and cancer cells[1],[2],[5],[6]. ANG exerts its functions both extracellularly and intracellularly. Extracellular ANG activates signal-related kinase1/2 (ERK1/2) in human umbilical vein endothelial cells (HUVECs) or stress-associated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in human umbilical artery easy muscle cells (HuASMCs)[3],[4]. Meanwhile, ANG can be internalized and translocated to the nucleolus where it enhances rRNA transcription and ribosome biogenesis to meet the high demand for protein synthesis during cell proliferation[7]. Evidence shows that ANG also localizes in the cytoplasm[1],[8],[9], but the role of the cytosolic ANG is largely unknown. ANG has been reported to promote endothelial cell migration[10],[11]. Cell migration is a highly complex and regulated process which Midodrine requires the integrated activities of cytoskeleton reorganization and cell-matrix interaction. During migration, cells attach to the matrixviafocal adhesions (FAs)[12], while stress fibers anchor to FAs at their ends and generate forces to move and reshape the cell[13]. The assembly and disassembly of FAs, and the movement of stress fibers coordinately lead the cells to migrate[14]. It was reported that this secreted ANG attaches to the extracellular matrix (ECM) and serves as a substratum to facilitate endothelial cell adhesion and spreading[15],[16]. ANG binds to a smooth muscle type -actin around the endothelial cell surface[17], and the subsequently dissociated ANG-actin complex promotes the degradation of the basement membrane to enhance cell invasion and migration[10]. On the other hand, ANG activates the protein kinase B/Akt signaling pathway to promote HUVEC migration[11]. A recent study showed that ANG inhibits actin polymerization at sub-physiological KCl concentrationsin vitro[18], suggesting that ANG influences cytoskeletal organization directly. However, the precise role of ANG in cytoskeletal organization and cell migration remains to be elucidated. To better understand the intracellular roles of ANG, we have performed a co-immunoprecipitation coupled mass spectrometry (MS) analysis to identify potential ANG-interacting proteins. Among the obtained 14 candidate ANG-binding proteins, -actin, -actinin 4, and Midodrine non-muscle myosin heavy chain 9 are stress fiber components. After confirmation of the interactions between ANG and the three proteins, we explored the biological role of ANG in stress fiber formation, focal adhesion dynamics, and cell migration. == Results == == Identification and functional classification Midodrine of ANG-interacting proteins == To screen potential ANG-interacting proteins, we used a co-immunoprecipitation combined with MS approach. The extracellular ANG can be internalized by its target cells such as HeLa cells and human umbilical vein endothelial cells (HUVECs)[5],[7][9],[19],[20], possibly through an endocytosis pathway[19]. After treating the HeLa cells with exogenous ANG, the intracellular level of this protein increased (Determine 1Blower panel). Accordingly, the immunoprecipitated complex from exogenous ANG-treated cells contained more ANG-interacting proteins than that from the untreated HeLa cells, shown as enhanced bands in silver-staining gel (Determine 1B, upper panel). Therefore, seven obviously enhanced bands were subjected to protein identification by MS (Determine 1Bmarked ag). The MS data were applied to NCBI database searching. Three types of protein were filtered out during the analysis: keratins; proteins in both the control (ANG) and ANG-treated (ANG+) groups (considered to be nonspecifically trapped by the protein A agarose beads); and proteins that did not contain any peptides with >95% confidence. After organizing the data, we finally identified 14 putative ANG-associated proteins (Determine 1B,Table 1). == Determine 1. Identification of ANG-associated proteins. == (A) Schematic illustration of the strategy used to screen ANG-associated proteins. (B) Proteins immunoprecipitated with anti-ANG antibodies from total lysates of HeLa cells with (+ANG) or without (ANG) ANG were fractionated by 12% SDS-PAGE gel. The gels were either visualized by silver staining (upper panel) Midodrine or blotted with anti-ANG antibodies (lower panel). The differential bands (marked ag) were subjected to trypsin digestion and Q-TOF MS analysis. The identified proteins.