Genetically Encoded Microtubule Binders for Single-Cell Interrogation of Cytoskeleton Dynamics and Protein Activity

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Microtubule (MT) dynamics are crucial for cellular functions and are regulated by microtubule-associated proteins (MAPs) and tubulin post-translational modifications (PTMs).
• Existing methods for observing MTs in living cells often have limitations in real-time visualization and manipulation.

Purpose of the Study:

• To introduce genetically encoded oligomeric MT binders, OligoMT and OligoTIP, for real-time visualization and manipulation of MT behaviors in living cells.
• To demonstrate the utility of these binders in studying MT network dynamics, cell division, and the impact of tubulin acetylation on MT stability.

Main Methods:

• Development of genetically encoded oligomeric proteins (OligoMT and OligoTIP) that bind to microtubules.
• Application of these binders for live-cell imaging to label the MT cytoskeleton and track MT plus-ends.
• Utilizing OligoMT and OligoTIP to assess cellular processes like cell division and to quantify the effects of tubulin acetylation.

Main Results:

• OligoMT successfully labeled the MT cytoskeleton, providing a reliable marker for MT network visualization.
• OligoTIP enabled live monitoring of growing MT plus-ends, offering insights into MT dynamics.
• These tools were used to assess MT stability under different conditions, including the effect of tubulin acetylation, and demonstrated potential as biosensors for drug actions and enzymatic activity.

Conclusions:

• OligoMT and OligoTIP are versatile, genetically encoded tools for real-time observation and manipulation of microtubule dynamics in living cells.
• These engineered binders facilitate mechanistic dissection of MT biology and offer translational applications in cell-based high-throughput drug discovery.