Optogenetic Control of Microtubule Dynamics

Area of Science:

• Cell Biology
• Optogenetics
• Biophysics

Background:

• Microtubules (MTs) are crucial for cell structure and function, with dynamic growth regulated by +TIP proteins.
• EB1 is a key +TIP protein that binds to growing MT plus ends, organizing the +TIP network.
• Existing methods for modulating MT dynamics are slow compared to cellular processes.

Purpose of the Study:

• To develop and characterize a photo-inactivatable EB1 (π-EB1) for rapid, light-controlled modulation of microtubule dynamics.
• To demonstrate the utility of π-EB1 for investigating +TIP functions with high spatial and temporal resolution.
• To provide protocols for implementing π-EB1 optogenetics with live-cell microscopy and patterned illumination.

Main Methods:

• Engineering of photo-inactivatable π-EB1 by incorporating a blue light-sensitive LOV2/Zdk1 module.
• High-resolution live-cell microscopy combined with optogenetic control.
• Patterned illumination techniques for micrometer-scale control of π-EB1 activity.
• Development of a DIY LED cube for multiwell plate microscopy.

Main Results:

• Blue light-induced π-EB1 photodissociation rapidly disassembles +TIP complexes.
• π-EB1 optogenetics effectively attenuates microtubule growth in mammalian cells.
• Patterned illumination enables precise spatial control over π-EB1 activity and MT dynamics.

Conclusions:

• π-EB1 serves as a powerful optogenetic tool for acute and spatially resolved investigation of microtubule plus-end dynamics.
• The developed techniques are broadly applicable to other optogenetic tools based on blue light-sensitive modules.
• The DIY LED cube facilitates accessible implementation of optogenetic microscopy in various experimental settings.