在非興奮性細胞中，鈣池調控鈣離子流入 (SOCE) 在調控鈣離子進入細胞中以及維持細胞內鈣離子恆定扮演很重要的角色。鈣離子基質交互分子1 (STIM1) 是位於內質網上感測鈣離子濃度之蛋白，STIM1所調控的鈣離子流入對於細胞的增生、移動以及血管新生都是必要的。足體環 (podosome rosette) 是以F-actin為主體並且圍繞許多支持蛋白的動態結構。在細胞移動的過程中，足體在細胞的腹部形成，對於細胞侵襲以及細胞外基質降解皆扮演重要角色。目前的研究對於鈣離子訊號和足體環之間的關係並不清楚，因此我的研究目標在於探討其兩者之間的關係。我以v-Src transformed小鼠胚胎成纖維細胞作為研究平台來研究鈣離子訊號在足體形成過程中扮演的角色。我利用了藥理和基因方法來研究SOCE對於足體環形成的影響。從中發現兩種SOCE抑制劑皆會顯著抑制足體環的形成，並與劑量呈現正相關的趨勢。此外，在降低STIM1表現後，發現足體環的形成也相同受到抑制。除了會影響足體環的形成外，也發現對於足體環的動態以及整體結構會有影響。而在其中發現，肌動蛋白 (actomyosin) 在足體環形成中的維持期分布會被改變。同時，也發現在抑制STIM1所調控的鈣離子流入後，細胞中的中間絲蛋白 (vimentin) 表現會顯著的上升，且會促進其中間絲蛋白網絡的形成，進而提高細胞的硬度。綜合以上結果，證明由STIM1所調控的鈣離子流入在足體環形成過程中扮演重要的角色，且肌動蛋白以及中間絲蛋白似乎也參與其中。此外，在乳癌細胞以及骨肉瘤細胞中也發現，在抑制STIM1所調控的鈣離子流入後，其侵襲性偽足 (invadopodia) 的形成比例也會被抑制。

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.

Artym, V.V., Y. Zhang, F. Seillier-Moiseiwitsch, K.M. Yamada, and S.C. Mueller. 2006. Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function. Cancer Res. 66:3034-3043.
Berridge, M.J., M.D. Bootman, and H.L. Roderick. 2003. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol. 4:517-529.
Burns, S., A.J. Thrasher, M.P. Blundell, L. Machesky, and G.E. Jones. 2001. Configuration of human dendritic cell cytoskeleton by Rho GTPases, the WAS protein, and differentiation. Blood. 98:1142-1149.
Buschman, M.D., P.A. Bromann, P. Cejudo-Martin, F. Wen, I. Pass, and S.A. Courtneidge. 2009. The novel adaptor protein Tks4 (SH3PXD2B) is required for functional podosome formation. Mol Biol Cell. 20:1302-1311.
Carafoli, E. 2003. The calcium-signalling saga: tap water and protein crystals. Nat Rev Mol Cell Biol. 4:326-332.
Chabadel, A., I. Banon-Rodriguez, D. Cluet, B.B. Rudkin, B. Wehrle-Haller, E. Genot, P. Jurdic, I.M. Anton, and F. Saltel. 2007. CD44 and beta3 integrin organize two functionally distinct actin-based domains in osteoclasts. Mol Biol Cell. 18:4899-4910.
Chen, Y.F., W.T. Chiu, Y.T. Chen, P.Y. Lin, H.J. Huang, C.Y. Chou, H.C. Chang, M.J. Tang, and M.R. Shen. 2011. Calcium store sensor stromal-interaction molecule 1-dependent signaling plays an important role in cervical cancer growth, migration, and angiogenesis. Proc Natl Acad Sci U S A. 108:15225-15230.
Chen, Y.T., Y.F. Chen, W.T. Chiu, K.Y. Liu, Y.L. Liu, J.Y. Chang, H.C. Chang, and M.R. Shen. 2013a. Microtubule-associated histone deacetylase 6 supports the calcium store sensor STIM1 in mediating malignant cell behaviors. Cancer Res. 73:4500-4509.
Chen, Y.T., Y.F. Chen, W.T. Chiu, Y.K. Wang, H.C. Chang, and M.R. Shen. 2013b. The ER Ca2+ sensor STIM1 regulates actomyosin contractility of migratory cells. J Cell Sci. 126:1260-1267.
Clapham, D.E., L.W. Runnels, and C. Strubing. 2001. The TRP ion channel family. Nat Rev Neurosci. 2:387-396.
Clark, E.S., and A.M. Weaver. 2008. A new role for cortactin in invadopodia: regulation of protease secretion. Eur J Cell Biol. 87:581-590.
Clark, E.S., A.S. Whigham, W.G. Yarbrough, and A.M. Weaver. 2007. Cortactin is an essential regulator of matrix metalloproteinase secretion and extracellular matrix degradation in invadopodia. Cancer Res. 67:4227-4235.
Davis, F.M., I. Azimi, R.A. Faville, A.A. Peters, K. Jalink, J.W. Putney, Jr., G.J. Goodhill, E.W. Thompson, S.J. Roberts-Thomson, and G.R. Monteith. 2014. Induction of epithelial-mesenchymal transition (EMT) in breast cancer cells is calcium signal dependent. Oncogene. 33:2307-2316.
Destaing, O., S.M. Ferguson, A. Grichine, C. Oddou, P. De Camilli, C. Albiges-Rizo, and R. Baron. 2013. Essential function of dynamin in the invasive properties and actin architecture of v-Src induced podosomes/invadosomes. PLoS One. 8:e77956.
Destaing, O., F. Saltel, J.C. Geminard, P. Jurdic, and F. Bard. 2003. Podosomes display actin turnover and dynamic self-organization in osteoclasts expressing actin-green fluorescent protein. Mol Biol Cell. 14:407-416.
Gimona, M., I. Kaverina, G.P. Resch, E. Vignal, and G. Burgstaller. 2003. Calponin repeats regulate actin filament stability and formation of podosomes in smooth muscle cells. Mol Biol Cell. 14:2482-2491.
Harburger, D.S., M. Bouaouina, and D.A. Calderwood. 2009. Kindlin-1 and -2 directly bind the C-terminal region of beta integrin cytoplasmic tails and exert integrin-specific activation effects. J Biol Chem. 284:11485-11497.
Kinley, A.W., S.A. Weed, A.M. Weaver, A.V. Karginov, E. Bissonette, J.A. Cooper, and J.T. Parsons. 2003. Cortactin interacts with WIP in regulating Arp2/3 activation and membrane protrusion. Curr Biol. 13:384-393.
Lafuente, E.M., A.A. van Puijenbroek, M. Krause, C.V. Carman, G.J. Freeman, A. Berezovskaya, E. Constantine, T.A. Springer, F.B. Gertler, and V.A. Boussiotis. 2004. RIAM, an Ena/VASP and Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion. Dev Cell. 7:585-595.
Lehto, V.P., T. Hovi, T. Vartio, R.A. Badley, and I. Virtanen. 1982. Reorganization of cytoskeletal and contractile elements during transition of human monocytes into adherent macrophages. Lab Invest. 47:391-399.
Linder, S. 2007. The matrix corroded: podosomes and invadopodia in extracellular matrix degradation. Trends Cell Biol. 17:107-117.
Linder, S., and P. Kopp. 2005. Podosomes at a glance. J Cell Sci. 118:2079-2082.
Linder, S., D. Nelson, M. Weiss, and M. Aepfelbacher. 1999. Wiskott-Aldrich syndrome protein regulates podosomes in primary human macrophages. Proc Natl Acad Sci U S A. 96:9648-9653.
Luxenburg, C., S. Winograd-Katz, L. Addadi, and B. Geiger. 2012. Involvement of actin polymerization in podosome dynamics. J Cell Sci. 125:1666-1672.
Mader, C.C., M. Oser, M.A. Magalhaes, J.J. Bravo-Cordero, J. Condeelis, A.J. Koleske, and H. Gil-Henn. 2011. An EGFR-Src-Arg-cortactin pathway mediates functional maturation of invadopodia and breast cancer cell invasion. Cancer Res. 71:1730-1741.
Marchisio, P.C., D. Cirillo, L. Naldini, M.V. Primavera, A. Teti, and A. Zambonin-Zallone. 1984. Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures. J Cell Biol. 99:1696-1705.
McAndrew, D., D.M. Grice, A.A. Peters, F.M. Davis, T. Stewart, M. Rice, C.E. Smart, M.A. Brown, P.A. Kenny, S.J. Roberts-Thomson, and G.R. Monteith. 2011. ORAI1-mediated calcium influx in lactation and in breast cancer. Mol Cancer Ther. 10:448-460.
Minke, B., and B. Cook. 2002. TRP channel proteins and signal transduction. Physiol Rev. 82:429-472.
Montell, C., L. Birnbaumer, and V. Flockerzi. 2002. The TRP channels, a remarkably functional family. Cell. 108:595-598.
Moreau, V., F. Tatin, C. Varon, and E. Genot. 2003. Actin can reorganize into podosomes in aortic endothelial cells, a process controlled by Cdc42 and RhoA. Mol Cell Biol. 23:6809-6822.
Mueller, S.C., and W.T. Chen. 1991. Cellular invasion into matrix beads: localization of beta 1 integrins and fibronectin to the invadopodia. J Cell Sci. 99 ( Pt 2):213-225.
Murphy, D.A., and S.A. Courtneidge. 2011. The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function. Nat Rev Mol Cell Biol. 12:413-426.
Nagasawa, M., and I. Kojima. 2012. Translocation of calcium-permeable TRPV2 channel to the podosome: Its role in the regulation of podosome assembly. Cell Calcium. 51:186-193.
Oikawa, T., T. Itoh, and T. Takenawa. 2008. Sequential signals toward podosome formation in NIH-src cells. J Cell Biol. 182:157-169.
Oikawa, T., and T. Takenawa. 2009. PtdIns(3,4)P2 instigates focal adhesions to generate podosomes. Cell Adh Migr. 3:195-197.
Osiak, A.E., G. Zenner, and S. Linder. 2005. Subconfluent endothelial cells form podosomes downstream of cytokine and RhoGTPase signaling. Exp Cell Res. 307:342-353.
Pan, Y.R., C.L. Chen, and H.C. Chen. 2011. FAK is required for the assembly of podosome rosettes. J Cell Biol. 195:113-129.
Parekh, A.B. 2010. Store-operated CRAC channels: function in health and disease. Nat Rev Drug Discov. 9:399-410.
Peterson, F.C., Q. Deng, M. Zettl, K.E. Prehoda, W.A. Lim, M. Way, and B.F. Volkman. 2007. Multiple WASP-interacting protein recognition motifs are required for a functional interaction with N-WASP. J Biol Chem. 282:8446-8453.
Prevarskaya, N., R. Skryma, and Y. Shuba. 2011. Calcium in tumour metastasis: new roles for known actors. Nat Rev Cancer. 11:609-618.
Putney, J.W., Jr. 2005. Capacitative calcium entry: sensing the calcium stores. J Cell Biol. 169:381-382.
Ridley, A.J., M.A. Schwartz, K. Burridge, R.A. Firtel, M.H. Ginsberg, G. Borisy, J.T. Parsons, and A.R. Horwitz. 2003. Cell migration: integrating signals from front to back. Science. 302:1704-1709.
Salido, G.M., I. Jardin, and J.A. Rosado. 2011. The TRPC ion channels: association with Orai1 and STIM1 proteins and participation in capacitative and non-capacitative calcium entry. Adv Exp Med Biol. 704:413-433.
Siddiqui, T.A., S. Lively, C. Vincent, and L.C. Schlichter. 2012. Regulation of podosome formation, microglial migration and invasion by Ca2+-signaling molecules expressed in podosomes. J Neuroinflammation. 9:250.
Soboloff, J., B.S. Rothberg, M. Madesh, and D.L. Gill. 2012. STIM proteins: dynamic calcium signal transducers. Nat Rev Mol Cell Biol. 13:549-565.
Tarone, G., D. Cirillo, F.G. Giancotti, P.M. Comoglio, and P.C. Marchisio. 1985. Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes. Exp Cell Res. 159:141-157.
Thiery, J.P., H. Acloque, R.Y. Huang, and M.A. Nieto. 2009. Epithelial-mesenchymal transitions in development and disease. Cell. 139:871-890.
van Helden, S.F., M.M. Oud, B. Joosten, N. Peterse, C.G. Figdor, and F.N. van Leeuwen. 2008. PGE2-mediated podosome loss in dendritic cells is dependent on actomyosin contraction downstream of the RhoA-Rho-kinase axis. J Cell Sci. 121:1096-1106.
Varon, C., F. Tatin, V. Moreau, E. Van Obberghen-Schilling, S. Fernandez-Sauze, E. Reuzeau, I. Kramer, and E. Genot. 2006. Transforming growth factor beta induces rosettes of podosomes in primary aortic endothelial cells. Mol Cell Biol. 26:3582-3594.
Vennekens, R., T. Voets, R.J. Bindels, G. Droogmans, and B. Nilius. 2002. Current understanding of mammalian TRP homologues. Cell Calcium. 31:253-264.
Wang, N., and D. Stamenovic. 2000. Contribution of intermediate filaments to cell stiffness, stiffening, and growth. Am J Physiol Cell Physiol. 279:C188-194.
Weaver, A.M., J.E. Heuser, A.V. Karginov, W.L. Lee, J.T. Parsons, and J.A. Cooper. 2002. Interaction of cortactin and N-WASp with Arp2/3 complex. Curr Biol. 12:1270-1278.
Weaver, A.M., A.V. Karginov, A.W. Kinley, S.A. Weed, Y. Li, J.T. Parsons, and J.A. Cooper. 2001. Cortactin promotes and stabilizes Arp2/3-induced actin filament network formation. Curr Biol. 11:370-374.
Yamaguchi, H., and T. Oikawa. 2010. Membrane lipids in invadopodia and podosomes: key structures for cancer invasion and metastasis. Oncotarget. 1:320-328.
Yang, S., J.J. Zhang, and X.Y. Huang. 2009. Orai1 and STIM1 are critical for breast tumor cell migration and metastasis. Cancer Cell. 15:124-134.
Yilmaz, M., and G. Christofori. 2009. EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev. 28:15-33.