Development and Characterization of a FRET-based Formin Tension Sensor in Living Cells

Area of Science:

• Cellular mechanics
• Cytoskeletal dynamics
• Biophysics

Background:

• Mechanotransduction via the actin cytoskeleton regulates vital cellular processes.
• Non-muscle myosin 2 (NM2) controls tensile forces, while formins like mDia1 elongate actin under tension.
• Experimental quantification of tension in formin-elongated actin filaments has been challenging.

Purpose of the Study:

• To develop and validate a tension-sensitive probe for measuring forces in formin-elongated actin filaments.
• To quantify subcellular tension in live cells with pN precision.
• To investigate the role of NM2 paralogs in generating cytoskeletal tension.

Main Methods:

• Development of a Förster resonance energy transfer (FRET)-based tension-sensitive probe (mDia1TS).
• Quantification of tension in live U2OS cells using fluorescence lifetime imaging microscopy (FLIM).
• Application of EGF stimulation, NM2 inhibition (para-nitroblebbistatin), and siRNA knockdowns of NM2 paralogs.

Main Results:

• The mDia1TS probe measured an average tension of approximately 3.5 pN in live cells.
• Mitogenic stimulation with EGF altered tension distribution, increasing it at the periphery and relaxing the center.
• NM2 inhibition reduced tension by 2 pN, and NM2-A was identified as the primary contributor to tensile force.

Conclusions:

• The mDia1TS probe enables direct, precise, and subcellular measurement of tension in active mDia1 within cells.
• This method provides new insights into the dynamic regulation of cytoskeletal forces during cellular processes.
• The findings highlight the significant role of NM2-A in generating mechanical tension essential for cell function.