... continued from Introduction to Cell Mechanobiology (1).
Among the three cytoskeletal filaments, actin filaments assume the most crucial role in shaping the overall mechanics of the cell. They assemble and disassemble rapidly, switching between G-actin (the monomeric form) and F-actin (the polymeric form) flexibly. Therefore, they are apt for reorganization, which drives cell behaviors such as cell spreading. Furthermore, polymerization and depolymerization occur more intensely at one end, which usually orients toward the plasma membrane and is referred to as the barbed end [5]. This feature allows for anisotropic cell spreading, in which actin stress fibers polarize and consistently grow in one direction.
The actin cytoskeleton also binds to the nucleus through the perinuclear actin cap, a dome comprised of parallel stress fibers covering the top of the nucleus [6]. According to S.B. Khatau et al. (2006), because the stress fibers play a role in determining cell shape by anchoring to the extracellular matrix, cell shape may regulate nuclear shape through the perinuclear actin cap.
To modulate cell mechanics, actin filaments change their structure and organization by cooperating with other proteins. The resulting protein complexes produce various forces that regulate “cell shape, adhesion, and motility” [7] [8]. The actomyosin complex is ubiquitous in the actin cytoskeleton. The movement of myosin induces the actin filament to contract; the contraction force is conducted along F-actin to other parts of the cell, where it may be converted into other forces. For instance, it is converted into cortical tension at the actin cortex, maintaining the shape of the plasma membrane. The ARP2/3 complex creates branched actin filaments by joining daughter F-actin to a single mother F-actin [9]; by adjusting the number of branches, it can optimize the cortical tension to maintain cellular integrity. Contraction force is also converted into traction force at the ends of stress fibers. There, F-actin connects to ECM proteins via a transmembrane integrin dimer and several other proteins, all of which assemble into a large focal adhesion complex. With traction, cell shape and mechanical properties may stabilize on the ECM. While traction force is directed toward the cell’s interior, stretching force at the filopodium is directed outwards. As F-actin continues to elongate at the cell lamellipodia, it creates filopodia, small protrusions on the plasma membrane that lead in cell spreading [10]. Unique in the filopodia, fascin bundles single actin fibers together; the ENA/VASP complex increases the actin elongation rate approximately two- to threefold; and formin, cooperating with ENA/VASP, inhibits capping proteins from terminating actin polymerization [11]. Every possible cell behavior indicates a delicate balance between such forces, as well as an actin configuration that create these forces. Furthermore, gradual changes in the cell form as seen in cell spreading was correlated to gradual actin reorganization: the time necessary for actin to complete reorganization implies certain mechanical limitations of the cell. Therefore, it is worthwhile to observe patterns of actin reorganization, from which the cell’s adaptive and self-regulatory abilities may be understood.
In physiological environments such as the human body, cells spread in spaces within the ECM with unequal lengths and widths. It is known that when a cell anchors to the ECM, it must undergo a global reorganization of the actin cytoskeleton to polarize and thus spread anisotropically. Previous research confirms that in spreading behaviors, focal adhesion formation, F-actin alignment, and the generation of stretch and traction forces precede cell reshaping [12][13]. A 2018 study discovered that in an anisotropically developing cell, cell shape, defined by the actin configuration, can be modeled by near-congruent ellipses; the eccentricity of the ellipses is correlated to the degree of anisotropy [14]. The work reinforces the interconnection between cell structure, shape, and mechanics. Still, the precise impact of anisotropic confinement on the actin cytoskeleton—whether visually or mechanically exhibited by the cell—remains unknown. The process of actin reorganization also has not been examined, as most research focuses on its result. Hence, in this study, we comprehensively investigated cell mechanics of protein micropatterns—including shape modifications, actin reorganizations, and mechanical variations. To achieve anisotropic confinement, we designed rectangular protein micropatterns with different aspect ratios (length-to-width): 1:1 (AR1), 3:1 (AR3), and 5:1 (AR5). They had the same area (1200 μm2), however, so that cytoplasmic growth among the cells was equal and irrelevant to the results. 3T3 cells were used in the experiment due to their satisfactory adhesiveness to ECM proteins. To characterize anisotropic confinement and cell behavior more conveniently, we established an x-y-z axial system, in which the micropattern’s length on the x-axis elongated with respect to its length on the y-axis. The z-axis was reserved to describe three-dimensional cell structures.
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