Analysis of microtubule dynamic instability using a plus-end growth marker

Matov, Alexandre; Applegate, Kathryn T.; Kumar, Praveen; Thoma, Claudio R.; Krek, Wilhelm; Danuser, Gaudenz; Wittmann, Torsten

doi:10.1038/nmeth.1493

Cited by 218 publications

(281 citation statements)

References 29 publications

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“…Because of the density of microtubules in cells, microtubule dynamics can only be reliably measured in the cell periphery, which can bias measurements of microtubule dynamics and does not allow monitoring of microtubule behavior toward the center of the cell (27). To overcome this bias, recent studies have monitored microtubule dynamics by imaging fluorescently labeled microtubule plus-end tracking proteins, such as EB1ΔC (referred to throughout the manuscript as EB1, for simplicity) (27,28). Imaging of EB1-GFP fluorescence typically results in the observation of "comets," which reflects EB1-GFP binding to the rapidly growing plus ends of microtubule polymers.…”

Section: Nad + Prevents Microtubule Polymer Disassembly Elicited Bymentioning

confidence: 99%

“…Imaging EB1-GFP enables imaging of both microtubule growth rates as well as the duration of growth phases. The frequency and duration of both pause and shrink rates can also be inferred using this approach (27).…”

Section: Nad + Prevents Microtubule Polymer Disassembly Elicited Bymentioning

confidence: 99%

“…This moderate effect may reflect the ability of SIRT1 to induce the transcriptional up-regulation of Example of EB1 comet track overlays in an MCF-7 cell. To quantify microtubule dynamics based on the movement of EB1-GFP comets, we used an image analysis algorithm described previously (27). Briefly, this algorithm detects EB1-GFP comets and calculates the microtubule growth rates, shown here as heatmapped lines representing growth rate and trajectory.…”

Section: Nad + Prevents Microtubule Polymer Disassembly Elicited Bymentioning

confidence: 99%

“…To determine microtubule shrink rate, we used MCF-7 cells expressing EB1-GFP, a microtubule end-binding protein that tracks the tips of growing microtubules. In contrast to the directly observed growth rates (E), microtubule shrink rates were inferred computationally, as described previously (27). The cells were treated with NAD + (5 mM) for 2 h, and time-lapse images were acquired every 0.5 s for 1 min.…”

Section: Nad + Prevents Microtubule Polymer Disassembly Elicited Bymentioning

confidence: 99%

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NAD ⁺ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

Harkcom¹,

Ghosh²,

Sung³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Nicotinamide adenine dinucleotide (NAD + ) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD + -regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD + . Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD + levels. We find that these effects of NAD + are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD + on microtubule polymers. Taken together, these data demonstrate that NAD + and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents.N icotinamide adenine dinucleotide (NAD + ) is an endogenous dinucleotide that is present in the cytosol, nucleus, and mitochondria. Athough it serves an important role as a redox cofactor in metabolism, NAD + is also a substrate for several families of enzymes, including the poly(ADP ribose) polymerases and the sirtuin deacetylase enzymes (reviewed in refs. 1 and 2). The level of intracellular NAD + is regulated by many factors, including diet and energy status (3), axonal injury (4), DNA damage (5), and certain disease states (6), suggesting that NAD + -dependent signaling is dynamically modulated in diverse contexts.NAD + -dependent signaling can be induced by treatment of cells with exogenous NAD + , which increases intracellular NAD + levels and results in diverse effects in cells and animals. These effects include enhanced oxygen consumption and ATP production (7), as well as protection from genotoxic stress and apoptosis (3). Mice treated with nicotinamide riboside, a NAD + precursor that is metabolized into NAD + , have enhanced oxidative metabolism, increased insulin sensitivity, and protection from high-fat diet-induced obesity (8). These results demonstrate that NAD + -dependent pathways can enhance metabolic function and improve a variety of disease phenotypes.An NAD + -regulated pathway also inhibits axonal degeneration elicited by axonal transection (4). Treatment of axons with 5-20 mM NAD + markedly delays the axon degenerative process (9). Additionally, animals that express the Wallerian degeneration slow (Wld S ) protein, a fusion of the NAD + biosynthetic enzyme Nicotinamide mononucleotide adenylyl transferase 1 and Ube4a, exhibit markedly delayed degeneration of the distal axonal fragment after axonal transection (10), and expression of Wld S mitigates disease phenotypes in several neurodegenerative disease models (11)(12)(13)(14). Thus, understanding the intracellular pathways regulated by NAD + may be important for understanding the pa...

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Section: Nad + Prevents Microtubule Polymer Disassembly Elicited Bymentioning

confidence: 99%

Section: Nad + Prevents Microtubule Polymer Disassembly Elicited Bymentioning

confidence: 99%

Section: Nad + Prevents Microtubule Polymer Disassembly Elicited Bymentioning

confidence: 99%

Section: Nad + Prevents Microtubule Polymer Disassembly Elicited Bymentioning

confidence: 99%

See 2 more Smart Citations

NAD ⁺ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

Harkcom¹,

Ghosh²,

Sung³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

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“…4A). GFP-fused EB1 (EB1-GFP) has been widely used to monitor microtubule growth dynamics as a microtubule-growth marker (24). We attempted to visualize EB1-GFP in a fruit fly embryo in its 13th developmental stage (Fig.…”

Section: Application Of Csu Models To Animals Expressing Gfp-fusion Pmentioning

confidence: 99%

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging

Togo

Yamagata

Kondo

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to “pinhole cross-talk,” which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.

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Noncentrosomal microtubules regulate autophagosome transport through CAMSAP2‐EB1 cross‐talk

Wei

Meng

2017

FEBS Letters

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Microtubules (MTs) play essential roles in many steps of autophagy, an important degradation pathway in the maintenance of cellular homoeostasis. In many cells, MT networks are comprised of centrosomal MTs and noncentrosomal MTs. However, it is unknown whether noncentrosomal MTs and its binding proteins are involved in autophagy. Here, we show in HeLa cells that calmodulin-regulated spectrin-associated protein 2 (CAMSAP2), a noncentrosomal MT minus-end stabilizing protein, regulates retrograde transport of autophagosomes through MT dynamics. CAMSAP2 cooperates with EB1 to regulate end-binding protein 1 (EB1) behaviour at MT plus ends, MT growth directions and autophagosome transport. An association between CAMSAP2 and EB1 in the cytosol may modulate EB1 binding to MT plus ends. Collectively, our data indicate that noncentrosomal MTs regulate autophagy through a cross-talk between CAMSAP2 and EB1.

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Analysis of microtubule dynamic instability using a plus-end growth marker

Cited by 218 publications

References 29 publications

NAD ⁺ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

NAD ⁺ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging

Noncentrosomal microtubules regulate autophagosome transport through CAMSAP2‐EB1 cross‐talk

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Analysis of microtubule dynamic instability using a plus-end growth marker

Cited by 218 publications

References 29 publications

NAD + and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

NAD + and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging

Noncentrosomal microtubules regulate autophagosome transport through CAMSAP2‐EB1 cross‐talk

Contact Info

Product

Resources

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NAD ⁺ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

NAD ⁺ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents