Precise control of microtubule disassembly in living cells

Abstract

Microtubules (MTs) are components of the evolutionarily conserved cytoskeleton, which tightly regulates various cellular activities. Our understanding of MTs is largely based on MT-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific MT populations due to their slow effects on the entire pool of MTs in cells. To address this limitation, we have used chemogenetics and optogenetics to disassemble specific MT subtypes by rapid recruitment of engineered MT-cleaving enzymes. Acute MT disassembly swiftly halted vesicular trafficking and lysosome dynamics. We also used this approach to disassemble MTs specifically modified by tyrosination and several MT-based structures including primary cilia, mitotic spindles, and intercellular bridges. These effects were rapidly reversed by inhibiting the activity or MT association of the cleaving enzymes. The disassembly of targeted MTs with spatial and temporal accuracy enables to uncover new insights of how MTs precisely regulate cellular architectures and functions.

Actin filaments form a size‐dependent diffusion barrier around centrosomes

“…YFP‐FKBP, YFP‐FKBP‐Grp1, YFP‐FKBP‐Luciferase, YFP‐FKBP‐∆Nβ‐Gal, YFP‐FKBP‐β‐Gal, and Lyn‐CFP‐FRB were generated previously (Lin et al , 2013a; Hong et al , 2018; Liu et al , 2022). For generating CEP120C‐GFP, we used the pcDNA4‐CEP120‐myc (Tsai et al , 2019) as a template to amplify C‐terminus of CEP120 (a.a. 416–986) and subcloned it into pEGFP‐C1 backbone (BD Biosciences Clontech).…”

In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M

“…However, until recently, only two optogenetic tools to modulate MT dynamics and network architecture were known: Wittmann’s photo-inactivatable MT-polymerizing tool π-EB1, and Slep’s photoactivated plus tip recruitment system . Two further optogenetic tools to photocontrol enzymatic severing of MTs have recently appeared as preprints, , highlighting the interest in methods to photocontrol microtubule dynamics, network organization, and interaction partners.…”

Photoswitchable Epothilone‐Based Microtubule Stabilisers Allow GFP‐Imaging‐Compatible, Optical Control over the Microtubule Cytoskeleton**

“…[13] However, they are not widely used, likely due to slow kinetics of the hydrolytic step in the photouncaging cascades, and photolability of the CA4 stilbene. [14] Two optogenetic tools to depress MT polymerisation under light are reported: Wittmann's photo-inactivat-able π-EB1, [15] and Slep's photoactivated plus tip recruitment system; [16] and in recent weeks, two preprints of photocontrolled MT-severing enzymes have also appeared, [17,18] highlighting broad interest in methods to photocontrol microtubule dynamics and network organisation.…”