Precise control of microtubule disassembly in living cells

Liu, Grace Y.; Chen, Shiau‐Chi; Lee, Gang Hui; Shaiv, Kritika; Chen, Pin‐Yu; Cheng, Hsuan; Hong, Shi‐Rong; Yang, Wen‐Ting; Huang, Shih-Wei; Chang, Yaw-Jen; Wang, Hsien‐Chu; Kao, Ching‐Lin; Sun, Pin‐Chiao; Chao, Ming‐Hong; Lee, Yian‐Ying; Tang, Ming Jer; Lin, Yu

doi:10.15252/embj.2021110472

Cited by 13 publications

(9 citation statements)

References 80 publications

(112 reference statements)

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“…There is a drastic decrease in the relative tubulin mass when the severing action is started, as observed in figure A10. This has not been observed in the recent experiments using photoexcitation [30,31] as oligomerization of the severing enzyme has not been considered in the proposed models similar to the experiment [28].…”

Section: Discussionmentioning

confidence: 84%

“…In a few in-vitro experiments, a reduction of tubulin mass was observed as stabilized MTs without free tubulins were used [21,23,29]. Overexpression of severing enzymes in the interphase cells causes a reduction of tubulin mass and MT number [14,30,31]. In an experiment, the knockdown of Katanin caused a reduction of axonal MTs in neurons, suggesting the importance of Katanin in the growth of axonal MTs.…”

Section: Introductionmentioning

confidence: 99%

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Computer simulation reveals the effect of severing enzymes on dynamic and stabilized microtubules

Sen

Kunwar

2023

Phys. Biol.

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Microtubule severing enzymes Katanin and Spastin cut the microtubule (MT) into smaller fragments and are being studied extensively using in-vitro experiments due to their crucial role in different cancers andneurodevelopmental disorders. It has been reported that the severing enzymes are either involved in increasing or decreasing the tubulin mass. Currently, there are a few analytical and computational models for MT amplification and severing. However, these models do not capture the action of microtubule severing explicitly, as these are based on partial differential equations in one dimension. On the other hand, a few discrete lattice-based models were used earlier to understand the activity of severing enzymes only on stabilized MTs. Hence, in this study, discrete lattice-based Monte Carlo models that included microtubule dynamics and severing enzyme activity have been developed to understand the effect of severing enzymes on tubulin mass, MT number, and MT length. It was found that the action of severing enzyme reduces average MT length while increasing their number; however, the total tubulin mass can decrease or increase depending on the concentration of GMPCPP (Guanylyl-(α, β)-methylene-diphosphonate) - which is a slowly hydrolyzable analogue of GTP (Guanosine triphosphate). Further, relative tubulin mass also depends on the detachment ratio of GTP/GMPCPP and GDP (Guanosine diphosphate) tubulin dimers and the binding energies oftubulin dimers covered by the severing enzyme.

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Section: Discussionmentioning

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Computer simulation reveals the effect of severing enzymes on dynamic and stabilized microtubules

Sen

Kunwar

2023

Phys. Biol.

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“…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).…”

Section: Methodsmentioning

confidence: 99%

Actin filaments form a size‐dependent diffusion barrier around centrosomes

et al. 2022

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The centrosome, a non-membranous organelle, constrains various soluble molecules locally to execute its functions. As the centrosome is surrounded by various dense components, we hypothesized that it may be bordered by a putative diffusion barrier. After quantitatively measuring the trapping kinetics of soluble proteins of varying size at centrosomes by a chemically inducible diffusion trapping assay, we find that centrosomes are highly accessible to soluble molecules with a Stokes radius of less than 5.8 nm, whereas larger molecules rarely reach centrosomes, indicating the existence of a size-dependent diffusion barrier at centrosomes. The permeability of this barrier is tightly regulated by branched actin filaments outside of centrosomes and it decreases during anaphase when branched actin temporally increases. The actinbased diffusion barrier gates microtubule nucleation by interfering with c-tubulin ring complex recruitment. We propose that actin filaments spatiotemporally constrain protein complexes at centrosomes in a size-dependent manner.

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“…Recently, others have designed optogenetic tools to depolymerize MTs, including photostatins (Borowiak et al ., 2015), a construct based on the depolymerizing activity of kinesin 13 (Lu et al ., 2020) or the MT severing enzymes spastin (Liu et al ., 2022) or katanin (Meiring et al ., 2022). Although these are useful tools to study the function of MTs, they do not lend themselves to the study of MT invasion of dendritic spines because MT invasions are infrequent and transient (Hu et al ., 2008; Jaworski et al ., 2009).…”

Section: Introductionmentioning

confidence: 99%

A Methodology for Specific Disruption of Microtubules in Dendritic Spines

Holland,

Miller,

Millette

et al. 2024

Preprint

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Dendritic spines, the mushroom-shaped extensions along dendritic shafts of excitatory neurons, are critical for synaptic function and are one of the first neuronal structures disrupted in neurodevelopmental and neurodegenerative diseases. Microtubule (MT) polymerization into dendritic spines is an activity-dependent process capable of affecting spine shape and function. Studies have shown that MT polymerization into spines occurs specifically in spines undergoing plastic changes. However, discerning the function of MT invasion of dendritic spines requires the specific inhibition of MT polymerization into spines, while leaving MT dynamics in the dendritic shaft, synaptically connected axons and associated glial cells intact. This is not possible with the unrestricted, bath application of pharmacological compounds. To specifically disrupt MT entry into spines we coupled a MT elimination domain (MTED) from the Efa6 protein to the actin filament-binding peptide LifeAct. LifeAct was chosen because actin filaments are highly concentrated in spines and are necessary for MT invasions. Temporally controlled expression of this LifeAct-MTED construct inhibits MT entry into dendritic spines, while preserving typical MT dynamics in the dendrite shaft. Expression of this construct will allow for the determination of the function of MT invasion of spines and more broadly, to discern how MT-actin interactions affect cellular processes.Significance StatementThe LifeAct-MTED construct provides spatial and temporal control of microtubule depolymerization within individual cellsTargeting this construct directly to spines allows for the specific inhibition of microtubule dynamics into dendritic spines, without affecting microtubule dynamics in the dendritic shaftImplementation of the construct will allow for the testing of microtubule contributions to specific biological processes, without global disruption of microtubule dynamics

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Precise control of microtubule disassembly in living cells

Cited by 13 publications

References 80 publications

Computer simulation reveals the effect of severing enzymes on dynamic and stabilized microtubules

Computer simulation reveals the effect of severing enzymes on dynamic and stabilized microtubules

Actin filaments form a size‐dependent diffusion barrier around centrosomes

A Methodology for Specific Disruption of Microtubules in Dendritic Spines

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