To Study the Role of Calcium Signaling on Podosome Formation

Abstract

Store-operated calcium entry (SOCE) is a predominant Ca2+ entry pathway in non-excitable cells Stromal interaction molecule 1 (STIM1) is an endoplasmic reticulum (ER) Ca2+ sensor that is specifically located on the ER membrane and STIM1-mediated Ca2+ signaling is necessary for cell proliferation migration and angiogenesis Podosome is a dynamic structure composed of a core of F-actin which is surrounded by scaffold proteins and kinases During cell migration podosome represents the protrusions of ventral plasma membrane which plays an important role in cell invasion and extracellular matrix (ECM) degradation Little information is available on the role of Ca2+ signaling in the regulation of podosome rosette formation Here I used v-Src transformed mouse embryonic fibroblasts (MEFs) as a model to study the role of Ca2+ signaling on podosome formation Results from immunofluorescence assay showed that podosome rosette formation was dose-dependently decreased by SKF-96365 and 2-APB the specific inhibitors of SOCE Consistently the podosome rosette formation was suppressed by silencing STIM1 Moreover blockade of STIM1-mediated Ca2+ signaling changed the podosome dynamics and shortened the maintenance phase of podosome rosettes In the maintenance phase of mature podosome rosettes the actomyosin surrounded the podosome rosettes to affect the structure of podosme in the STIM1 knockdown group On the other hand results from Western blotting indicated that the vimentin expression significantly increased by the specific inhibitor and siRNA of SOCE Consistently the results of immunofluorescent assay showed that the network of vimentin became more complete after SKF-96365 treatment Furthermore I used the atomic force microscopy (AFM) to investigate the cell stiffness change and found that the stiffness was increased by SKF-96365 treatment Taken together my results suggest that STIM1-dependent Ca2+ signaling plays an important role in podosome rosette formation through the actomysin affects the maintenance phase of podosome rosettes In addition the STIM1-mediated Ca2+ signaling are also involved in the regulation of invadopodia formation in breast cancer cells and osteosarcoma U2OS cells

Date of Award	2014 Aug 5
Original language	English
Supervisor	Meng-Ru Shen (Supervisor)