Simple SummaryThe mechanical cues in the extracellular cell matrix (ECM) regulate multiple cell behaviors by dominating the membrane tension through the cell membrane–cytoskeleton–focal adhesions (FAs) complex. However, the mechanism still needs clarification, due to the lack of technical means to alter the cytoskeleton arrangement and FAs distribution artificially. This study utilizes polydimethylsiloxane stamps with specific shapes to solve the issue and quantifies the order degree of cytoskeleton and membrane tension by defining the concept of information entropy. The results showed that the actin filaments arrangement and FAs distribution in the cells patterned by stamps were changed significantly and further regulated the membrane tension. The actin filaments accumulated in the zone where FAs were rare to maintain the stability of the overall membrane tension. In this process, the actin filaments act as shock absorbers to cushion the alternation in membrane tension without changing the steady state plasma membrane tension. Generally, this study offers an effective tool for the in-depth analysis of cell shape and function and a new method for the artificial regulation of cell function changes.AbstractThe extracellular stress signal transmits along the cell membrane–cytoskeleton–focal adhesions (FAs) complex, regulating the cell function through membrane tension. However, the mechanism of the complex regulating membrane tension is still unclear. This study designed polydimethylsiloxane stamps with specific shapes to change the actin filaments’ arrangement and FAs’ distribution artificially in live cells, visualized the membrane tension in real time, and introduced the concept of information entropy to describe the order degree of the actin filaments and plasma membrane tension. The results showed that the actin filaments’ arrangement and FAs’ distribution in the patterned cells were changed significantly. The hypertonic solution resulted in the plasma membrane tension of the pattern cell changing more evenly and slowly in the zone rich in cytoskeletal filaments than in the zone lacking filaments. In addition, the membrane tension changed less in the adhesive area than in the non-adhesive area when destroying the cytoskeletal microfilaments. This suggested that patterned cells accumulated more actin filaments in the zone where FAs were difficult to generate to maintain the stability of the overall membrane tension. The actin filaments act as shock absorbers to cushion the alternation in membrane tension without changing the final value of membrane tension.