Focal adhesions (FAs) 是一由很多蛋白質所組成的且具動態的結構，並可以整合和傳遞外界的機械受力訊息。根據文獻上指出，小 GTPase RhoA 以及肌凝蛋白II 所調控的肌動蛋白纖維收縮力會調控 FAs的成熟。當細胞種在較硬的基質上時，細胞的攤覆能力以及其內的RhoA活性會比較高，伴隨著的是細胞本身的硬度增加，對於這種細胞因外界基質軟硬度不同而有不同的行為特性，將此現象定義為細胞對於基質軟硬度的適應性。在先前實驗室的研究發現：當細胞種於較軟的基質上時，caveolin-1 (Cav-1) 蛋白質的表現量會降低，同時其 FAs的成熟度也會降低。因此本實驗的目的在了解是否軟性基質所造成的Cav-1降解，進而導致細胞的FAs不成熟以及細胞變軟。從本論文我們得知，利用MβCD破壞Cav-1的功能或是利用shRNA抑制Cav-1的表現都會使細胞變軟，此外由shRNA抑制細胞的Cav-1表現能有效的降低其FAs的成熟度且喪失其對基質軟硬度的適應性。相反的，大量表現Cav-1能有效的減少軟性基質所造成的FAs不成熟以及喪失細胞對於基質軟硬度的適應性。最後也發現到Cav-1 的表現和RhoA的活性呈現正相關；另一方面，我們也發現到Cav-1-RhoA 的訊息傳遞會抑制Pak-interacting exchange factor β (β-Pix)，Rac1的guanine nucleotide exchange factor (GEF)聚集在FAs上。綜合以上的實驗結果可以得知，Cav-1-RhoA 的訊息傳遞可以調控受基質軟硬度所影響的FAs的成熟以及細胞本身的軟硬度。而Cav-1-RhoA 和 β-Pix-Rac1 之間的關係可能就是扮演調控現象的重要角色。

Focal adhesions, the large and dynamic protein complexes, integrated and transmitted both the mechanical force and the regulatory signals. Accumulated data indicated that small GTPase RhoA and Myosin II mediated-actomyosin tension played an important role in focal adhesions maturation. When cultured on substrate of varying rigidity, the normal cells increased their spread area, RhoA activity, and cell stiffness as a function of substrate stiffness. Such phenomenon is defined as cell adaptability to substrate stiffness. Our previous studies showed that soft substrate downregulated caveolin-1 (Cav-1) and hindered focal adhesions maturation. Cav-1, the major component of caveolae, has been linked to mechanotransduction for its role in focal adhesions regulation and integrin-mediated actin remodeling. We are interested in how the downregulation of Cav-1 by soft substrate impairs focal adhesions maturation and result in cell softening. Both disruption of Cav-1 function by MβCD and knockdown of Cav-1 by shRNA resulted in cell softening. Moreover, Cav-1 knockdown also impaired focal adhesions maturation on stiff substrate and cell adaptability to substrate stiffness. On the contrary, Cav-1 overexpression in NIH3T3 cell rescued soft substrate-impaired focal adhesions maturation. Ha-RasV12 overexpressed NIH3T3 cells (7-4) exhibited cell softening, loss of cell adaptability to substrate stiffness, and downregulation of Cav-1. The overexpression of Cav-1 in 7-4 cell restored not only cell stiffness but also cell adaptability to substrate stiffness. The expression of Cav-1 is also positively correlated with the RhoA activity. Finally, we found that Cav-1-RhoA signal inversely regulated the accumulation of Pak-interacting exchange factor β (β-Pix), the Rac1 activator, at focal adhesions. Taken together, our data suggest that the Cav-1-RhoA signal regulates substrate stiffness regulated-focal adhesions maturation and subsequently cell stiffness. The seesaw relationship of Cav-1-RhoA and β-Pix-Rac1 may play an important role for the maturation of focal adhesions.

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