Shear Stress-induced Breast Cancer Cell Migration Is Mediated By ARAP1-dependent Circular Dorsal Ruffles Formation

Shear stress(SS)is pervasive in egress routes of malignancy,yet the intrinsic effects of force on breast cancer cells remain poorly understood.Circular dorsal ruffles(CDRs)are actin-rich structures that form on the dorsal surface of many mammalian cells in response to growth factor stimulation.CDRs appear at the dorsal surface where they expand and then constrict toward their center in a coordinated fashion to maintain a circular appearance,until they finally close,typically within 5-60 minutes following stimulation and are involved in diverse processes,such as integrin recycling,internalization of receptor tyrosine kinases and cell migration.However,the effect and the biological consequences of shear stress on CDRs formation remained incompletely understood.Here,we identified ARAP1-dependent CDRs formation as a critical role in low shear stress-induced cell migration.In this study,using confocal fluorescence microscope,we for the first time demonstrated that shear stress could induce the production of circular ruffle structures in MDA-MB-231 cells.Confocal image stacks analysis of F-actin-stained cells revealed that the CDRs formation after shear stress exposure.We found that shear stress stimulation induced localization of ARAP1 to an area of the plasma membrane ring structure of CDRs.Moreover,ARAP1 regulated the size of the CDRs filamentous-actin ring in an Arf GAP activity–dependent manner,when smaller CDRs were formed by ARAP1 knockdown.In addition,expression of a dominant-negative mutant of Arf1,the downstream of ARAP1,changed the size of CDRs.Knockdown of ILK(integrin-linked-kinase)decreased the formation of CDRs and cell migration.Expression of ARAP1 or Arf1 rescued the effect of ILK knockdown on CDRs but ARAP1-R578 K or Arf1-Q71 L did not rescue ILK knockdown defect in CDRs.As dorsal ruffles collapse,ARAP1 and Arf1 are internalized into macropinosomes and colocalized with macropinosome marker-Dextran.The internalized ARAP1 and Arf1 then transit through Rab5-positive early endosomal compartments to form the new lamellipodium on the ventral surface.The results of typical lag phase showed that CDRs formation promoted the extension of lamellipodium.Furthermore,we showed that ARAP1 and Arf1 are required for the formation of CDRs in migrating cells.Finally,our results revealed ARAP1 and Arf1 are required for the shear stress-induced cell migration,in an ILK-dependent manner.In summary,these results defined a novel mechanism by which shear stress improved ARAP1-dependent CDRs formation in ILK-dependent manner,thereby promoting extension of lamellipodium and cell migration.