Cell size and actin architecture determine force generation in optogenetically activated cells.

Area of Science:

• Cell biology
• Biophysics
• Mechanobiology

Background:

• Adherent cells utilize actomyosin contractility for force generation and environmental sensing, impacting crucial cellular processes.
• The Rho family of small GTPases regulates actomyosin system assembly and function, but how their activation translates to cell-scale forces is complex.
• Understanding cell mechanics and force generation is vital for comprehending cell migration, division, differentiation, and fate.

Purpose of the Study:

• To investigate how RhoA activation influences cell-scale force generation in response to varying physical environments.
• To elucidate the dynamics of actomyosin contractility following RhoA activation using optogenetics and mechanical measurements.
• To develop a mathematical model to decouple the biochemical and mechanical aspects of the RhoA-mediated cellular response.

Main Methods:

• Utilized a CRY2/CIBN optogenetic system for whole-cell RhoA activation.
• Employed micropatterning and traction force microscopy on soft elastic substrates to measure cell-generated forces.
• Developed a mathematical model parameterized by substrate deformation energy dynamics.

Main Results:

• Single cells exhibited rapid contraction (minute timescale) proportional to initial traction force upon RhoA activation, returning to a precise tension setpoint over several minutes.
• The RhoA response showed rapid buildup (20 s) and slow decay (50 s).
• Larger cells and those with more polarized actin cytoskeletons generated greater substrate deformation energy, with RhoA activation saturating above 50 ms pulse length.

Conclusions:

• Adherent cells establish tensional homeostasis via the RhoA system.
• Cell size and actin cytoskeleton architecture, influenced by the extracellular environment, critically determine the tension setpoint and its dynamics.
• Optogenetic control of RhoA activation reveals intrinsic biochemical activation limits and precise mechanical feedback mechanisms in cellular force generation.