Dynamic interaction between actin and nesprin2 maintain the cell nucleus in a prestressed state

Area of Science:

• Cell Biology
• Biophysics
• Mechanobiology

Background:

• Mechanical coupling between the nucleus and cytoskeleton is vital for force transduction from the extracellular matrix to chromatin.
• This physical connection is crucial for nuclear positioning and function, but its quantification has been lacking.

Purpose of the Study:

• To investigate and quantify the nature of the nuclear-cytoskeleton connection using advanced fluorescence spectroscopy techniques.
• To elucidate the role of nesprin2G and actin stress fibers in nuclear morphology and mechanotransduction.

Main Methods:

• Quantitative fluorescence spectroscopy techniques, including high-resolution 3D imaging.
• Fluorescence Recovery After Photobleaching (FRAP) to assess nesprin2G alignment.
• Fluorescence Cross-Correlation Spectroscopy (FCCS) to analyze actin-nesprin2G interaction dynamics.
• Fluorescence Resonance Energy Transfer (FRET) to determine physical proximity.
• Over-expression of Dominant Negative Klarsicht, ANC-1, Syne Homology (DNKASH) to disrupt actin-nucleus connections.

Main Results:

• Nesprin2G forms linear structures along actin stress fibers (ASFs) in the apical nucleus.
• Nesprin2G alignment becomes heterogeneous with altered cell shape.
• Actin and nesprin2G exhibit transient interactions (12ms timescale).
• Apical ASFs and nesprin2G are in close, spatially heterogeneous proximity.
• Disrupting the actin-nucleus connection increases nuclear height.

Conclusions:

• The study characterizes the actin-nesprin2G interaction and its role in regulating nuclear morphology.
• Quantitative fluorescence techniques are validated for studying physical connections essential for mechanotransduction.