Genetically encoded live-cell sensor for tyrosinated microtubules

Kesarwani, Shubham; Lama, Prakash; Chandra, Anchal; Reddy, P. Purushotham; Jijumon, A S; Bodakuntla, Satish; Rao, Balaji M.; Janke, Carsten; Das, Ranabir; Sirajuddin, Minhajuddin

doi:10.1083/jcb.201912107

Cited by 26 publications

(21 citation statements)

References 83 publications

(112 reference statements)

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“…Addressing these questions would greatly benefit from tools that enable the direct visualization of MT subsets in live cells. Dynamic MTs can be readily visualized in live cells using plus-end markers or the recently developed sensor for tyrosinated tubulin (Kesarwani et al, 2020); however, this is not the case for stable MTs, which can currently only be visualized upon cold treatment or exposure to MT-depolymerizing drugs, or approximated using immunocytochemistry for different PTMs. To address this, we set out to develop a live-cell marker for stable MTs.…”

Section: Introductionmentioning

confidence: 99%

A live-cell marker to visualize the dynamics of stable microtubules throughout the cell cycle

Jansen

Iwanski

Burute

et al. 2023

Journal of Cell Biology

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The microtubule (MT) cytoskeleton underlies processes such as intracellular transport and cell division. Immunolabeling for posttranslational modifications of tubulin has revealed the presence of different MT subsets, which are believed to differ in stability and function. Whereas dynamic MTs can readily be studied using live-cell plus-end markers, the dynamics of stable MTs have remained obscure due to a lack of tools to directly visualize these MTs in living cells. Here, we present StableMARK (Stable Microtubule-Associated Rigor-Kinesin), a live-cell marker to visualize stable MTs with high spatiotemporal resolution. We demonstrate that a rigor mutant of Kinesin-1 selectively binds to stable MTs without affecting MT organization and organelle transport. These MTs are long-lived, undergo continuous remodeling, and often do not depolymerize upon laser-based severing. Using this marker, we could visualize the spatiotemporal regulation of MT stability before, during, and after cell division. Thus, this live-cell marker enables the exploration of different MT subsets and how they contribute to cellular organization and transport.

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Section: Introductionmentioning

confidence: 99%

A live-cell marker to visualize the dynamics of stable microtubules throughout the cell cycle

Jansen

Iwanski

Burute

et al. 2023

Journal of Cell Biology

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“…We next turned our efforts to precisely disrupt microtubules with specific PTMs. Very recently, an elegant work has established a biosensor, A1AY1, which specifically targets highly tyrosinated microtubules in living cells (Kesarwani et al , 2020 ). To apply it in our system, A1AY1 was tagged with FRB and a red fluorescent protein, TagRFP.…”

Section: Resultsmentioning

confidence: 99%

“…With this capability, the translocation of engineered Spastins onto several acetylated microtubule structures including primary cilia, mitotic spindles, and intercellular bridges (Janke & Magiera, 2020 ) efficiently disrupted the corresponding structures (Fig 2 ). In addition to long‐lived microtubules, the dynamic microtubules with tyrosination modification can be targeted and disrupted specifically by the combination of a tyrosinated microtubule biosensor and our microtubule disruption system (Fig 3 ; Kesarwani et al , 2020 ). To our knowledge, this is the first system that permits the rapid disassembly of targeted microtubule subtypes and microtubule‐based structures.…”

Section: Discussionmentioning

confidence: 99%

Precise control of microtubule disassembly in living cells

et al. 2022

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Microtubules tightly regulate various cellular activities. Our understanding of microtubules is largely based on experiments using microtubule-targeting agents, which, however, are insufficient to dissect the dynamic mechanisms of specific microtubule populations, due to their slow effects on the entire pool of microtubules. To overcome this technological limitation, we have used chemo and optogenetics to disassemble specific microtubule subtypes, including tyrosinated microtubules, primary cilia, mitotic spindles, and intercellular bridges, by rapidly recruiting engineered microtubule-cleaving enzymes onto target microtubules in a reversible manner. Using this approach, we show that acute microtubule disassembly swiftly halts vesicular trafficking and lysosomal dynamics. It also immediately triggers Golgi and ER reorganization and slows the fusion/fission of mitochondria without affecting mitochondrial membrane potential. In addition, cell rigidity is increased after microtubule disruption owing to increased contractile stress fibers. Microtubule disruption furthermore prevents cell division, but does not cause cell death during interphase. Overall, the reported tools facilitate detailed analysis of how microtubules precisely regulate cellular architecture and functions.

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“…In the future, it will be interesting to employ life cell sensors to analyze the role of PTMs in a detailed spatiotemporal manner during processes such as leukocyte migration and immune synapse formation. The tools are currently limited; however, recent advances now enable monitoring of tyrosinated microtubules in living cells ( Kesarwani et al, 2020 ).…”

Section: Adaptive Migration Strategies Determine the Dependence On MImentioning

confidence: 99%

Dynamic Microtubule Arrays in Leukocytes and Their Role in Cell Migration and Immune Synapse Formation

Kopf

Kiermaier

2021

Front. Cell Dev. Biol.

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The organization of microtubule arrays in immune cells is critically important for a properly operating immune system. Leukocytes are white blood cells of hematopoietic origin, which exert effector functions of innate and adaptive immune responses. During these processes the microtubule cytoskeleton plays a crucial role for establishing cell polarization and directed migration, targeted secretion of vesicles for T cell activation and cellular cytotoxicity as well as the maintenance of cell integrity. Considering this large spectrum of distinct effector functions, leukocytes require flexible microtubule arrays, which timely and spatially reorganize allowing the cells to accommodate their specific tasks. In contrast to other specialized cell types, which typically nucleate microtubule filaments from non-centrosomal microtubule organizing centers (MTOCs), leukocytes mainly utilize centrosomes for sites of microtubule nucleation. Yet, MTOC localization as well as microtubule organization and dynamics are highly plastic in leukocytes thus allowing the cells to adapt to different environmental constraints. Here we summarize our current knowledge on microtubule organization and dynamics during immune processes and how these microtubule arrays affect immune cell effector functions. We particularly highlight emerging concepts of microtubule involvement during maintenance of cell shape and physical coherence.

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Genetically encoded live-cell sensor for tyrosinated microtubules

Cited by 26 publications

References 83 publications

A live-cell marker to visualize the dynamics of stable microtubules throughout the cell cycle

A live-cell marker to visualize the dynamics of stable microtubules throughout the cell cycle

Precise control of microtubule disassembly in living cells

Dynamic Microtubule Arrays in Leukocytes and Their Role in Cell Migration and Immune Synapse Formation

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