TrackMate: An open and extensible platform for single-particle tracking

Tinévez, Jean-Yves; Perry, Nick; Schindelin, Johannes; Hoopes, Genevieve M.; Reynolds, Gregory D.; Laplantine, Emmanuel; Bednarek, Sebastian Y.; Shorte, Spencer; Eliceiri, Kevin W.

doi:10.1016/j.ymeth.2016.09.016

Cited by 2,933 publications

(2,556 citation statements)

References 59 publications

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“…For single-cell tracking, images were processed as described above and were analysed in batch mode using the TrackMate plug-in available in the Fiji package (Tinevez et al, 2016). This plug-in operated on a userspecified framework that enabled automation of spot segmentation and frame-to-frame spot-tracking.…”

Section: Automated Single-cell Tracking and Analysismentioning

confidence: 99%

A quantitative FastFUCCI assay defines cell cycle dynamics at a single-cell level

Koh

Mascalchi

Rodríguez

et al. 2017

Journal of Cell Science

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The fluorescence ubiquitination-based cell cycle indicator (FUCCI) is a powerful tool for use in live cells but current FUCCI-based assays have limited throughput in terms of image processing and quantification. Here, we developed a lentiviral system that rapidly introduced FUCCI transgenes into cells by using an all-in-one expression cassette, FastFUCCI. The approach alleviated the need for sequential transduction and characterisation, improving labelling efficiency. We coupled the system to an automated imaging workflow capable of handling large datasets. The integrated assay enabled analyses of single-cell readouts at high spatiotemporal resolution. With the assay, we captured in detail the cell cycle alterations induced by antimitotic agents. We found that treated cells accumulated at G2 or M phase but eventually advanced through mitosis into the next interphase, where the majority of cell death occurred, irrespective of the preceding mitotic phenotype. Some cells appeared viable after mitotic slippage, and a fraction of them subsequently re-entered S phase. Accordingly, we found evidence that targeting the DNA replication origin activity sensitised cells to paclitaxel. In summary, we demonstrate the utility of the FastFUCCI assay for quantifying spatiotemporal dynamics and identify its potential in preclinical drug development.

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Section: Automated Single-cell Tracking and Analysismentioning

confidence: 99%

A quantitative FastFUCCI assay defines cell cycle dynamics at a single-cell level

Koh

Mascalchi

Rodríguez

et al. 2017

Journal of Cell Science

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“…For TERRA-TRF1 colocalisation analysis and TERRA cluster expression counts, GFP and mCherry image Z-stacks were acquired at 30 second intervals. Displacements of the TERRA RNA clusters and single particles were tracked with the ImageJ TrackMate plugin [40]. Tracks containing five or more points were selected for diffusion coefficient analysis as described in [35].…”

Section: Methodsmentioning

confidence: 99%

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells

et al. 2018

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Telomeres cap the ends of eukaryotic chromosomes, protecting them from degradation and erroneous recombination events which may lead to genome instability. Telomeres are transcribed giving rise to telomeric repeat-containing RNAs, called TERRA. The TERRA long noncoding RNAs have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. While TERRA RNAs are predominantly nuclear and localize at telomeres, little is known about the dynamics and function of TERRA molecules expressed from individual telomeres. Herein, we developed an assay to image endogenous TERRA molecules expressed from a single telomere in living human cancer cells. We show that single-telomere TERRA can be detected as TERRA RNA single particles which freely diffuse within the nucleus. Furthermore, TERRA molecules aggregate forming TERRA clusters. Three-dimensional size distribution and single particle tracking analyses revealed distinct sizes and dynamics for TERRA RNA single particles and clusters. Simultaneous time lapse confocal imaging of TERRA particles and telomeres showed that TERRA clusters transiently co-localize with telomeres. Finally, we used chemically modified antisense oligonucleotides to deplete TERRA molecules expressed from a single telomere. Single-telomere TERRA depletion resulted in increased DNA damage at telomeres and elsewhere in the genome. These results suggest that single-telomere TERRA transcripts participate in the maintenance of genomic integrity in human cancer cells.

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“…Microtubule dynamics were quantified by using ImageJ's TrackMate plugin (Tinevez et al, 2017), following live acquisition of high-resolution images of neurons at 6−9 hours in vitro (HIV) expressing EB1::GFP, a marker of MT plus-ends (Morrison et al, 2002;Stepanova et al, 2003) (Fig. 3A,B and Fig.…”

Section: Daam Regulates Axonal Microtubule Dynamicsmentioning

confidence: 99%

The formin DAAM is required for coordination of the actin and microtubule cytoskeleton in axonal growth cones

Szikora¹,

Földi²,

Tóth³

et al. 2017

Development

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Directed axonal growth depends on correct coordination of the actin and microtubule cytoskeleton in the growth cone. However, despite the relatively large number of proteins implicated in actin−microtubule crosstalk, the mechanisms whereby actin polymerization is coupled to microtubule stabilization and advancement in the peripheral growth cone remained largely unclear. Here, we identified the formin Dishevelled-associated activator of morphogenesis (DAAM) as a novel factor playing a role in concerted regulation of actin and microtubule remodeling in Drosophila melanogaster primary neurons. In vitro, DAAM binds to F-actin as well as to microtubules and has the ability to crosslink the two filament systems. Accordingly, DAAM associates with the neuronal cytoskeleton, and a significant fraction of DAAM accumulates at places where the actin filaments overlap with that of microtubules. Loss of DAAM affects growth cone and microtubule morphology, and several aspects of microtubule dynamics; and biochemical and cellular assays revealed a microtubule stabilization activity and binding to the microtubule tip protein EB1. Together, these data suggest that, besides operating as an actin assembly factor, DAAM is involved in linking actin remodeling in filopodia to microtubule stabilization during axonal growth.

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TrackMate: An open and extensible platform for single-particle tracking

Cited by 2,933 publications

References 59 publications

A quantitative FastFUCCI assay defines cell cycle dynamics at a single-cell level

A quantitative FastFUCCI assay defines cell cycle dynamics at a single-cell level

Live-cell imaging reveals the dynamics and function of single-telomere TERRA molecules in cancer cells

The formin DAAM is required for coordination of the actin and microtubule cytoskeleton in axonal growth cones

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