Automated tracking of <i>S. pombe</i> spindle elongation dynamics

Uzsoy, Ana Sofía M.; Zareiesfandabadi, Parsa; Jennings, Jamie A.; Kemper, A. F.; Elting, Mary Williard

doi:10.1111/jmi.13044

Cited by 6 publications

(5 citation statements)

References 48 publications

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“…We performed live-cell spinning disk confocal imaging with laser ablation at room temperature of 22°C as previously described ( 46 , 47 , 48 , 49 ). Briefly, we performed GFP imaging on a Nikon Ti-E stand on an Andor Dragonfly spinning disk confocal fluorescence microscope; spinning disk dichroic Chroma ZT405/488/561/640rpc; 488-nm (50 mW) diode laser (75 ms–120 ms exposures) with Borealis attachment (Andor); emission filter Chroma ET525/50m; and an Andor iXon3 camera.…”

Section: Methodsmentioning

confidence: 99%

Force by minus-end motors Dhc1 and Klp2 collapses the S. pombe spindle after laser ablation

Zareiesfandabadi

Elting

2022

Biophysical Journal

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A microtubule-based machine called the mitotic spindle segregates chromosomes when eukaryotic cells divide. In the fission yeast Schizosaccharomyces pombe, which undergoes closed mitosis, the spindle forms a single bundle of microtubules inside the nucleus. During elongation, the spindle extends via antiparallel microtubule sliding by molecular motors. These extensile forces from the spindle are thought to resist compressive forces from the nucleus. We probe the mechanism and maintenance of this force balance via laser ablation of spindles at various stages of mitosis. We find that spindle pole bodies collapse toward each other after ablation, but spindle geometry is often rescued, allowing spindles to resume elongation. Although this basic behavior has been previously observed, many questions remain about the phenomenon's dynamics, mechanics, and molecular requirements. In this work, we find that previously hypothesized viscoelastic relaxation of the nucleus cannot explain spindle shortening in response to laser ablation. Instead, spindle collapse requires microtubule dynamics and is powered by the minus-end-directed motor proteins dynein Dhc1 and kinesin-14 Klp2, but it does not require the minusend-directed kinesin Pkl1.

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Section: Methodsmentioning

confidence: 99%

Force by minus-end motors Dhc1 and Klp2 collapses the S. pombe spindle after laser ablation

Zareiesfandabadi

Elting

2022

Biophysical Journal

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“…During anaphase, motor proteins can crosslink and slide apart antiparallel microtubule neighbors, creating extensile force within the spindle and driving spindle elongation 6,8 . In S. pombe, this elongation is quite dramatic with spindles elongating from hundreds of nanometers in length to roughly 10 μm over the course of mitosis 9,10 . Furthermore, these spindles are quite spatiotemporally stereotyped, adhering to a strict protocol throughout mitosis 11 , suggesting that fission yeasts can precisely tune their mitotic forces.…”

Section: S Pombe Mitotic Spindles Are Highly Crosslinkedmentioning

confidence: 99%

“…During anaphase, motor proteins can crosslink and slide apart antiparallel microtubule neighbors, creating extensile force within the spindle and driving spindle elongation 7,9 . In S. pombe, this elongation is quite dramatic, with spindles elongating from hundreds of nanometers in length to roughly 10 μm over the course of mitosis 10,11 . Accompanying spindle elongation, the closed nuclear envelope undergoes a series of shape transitions, from a spheroid, through a peanut-shaped intermediate, to a dumbbell that will ultimately separate into two daughter nuclei (Figure 1A).…”

Section: S Pombe Mitotic Spindles Are Highly Crosslinkedmentioning

confidence: 99%

“…Spinning disk confocal live imaging and laser ablation experiments were performed similar to those described previously 10,13,15 . Live videos were captured using a Nikon Ti-E stand on an Andor Dragonfly spinning disk confocal fluorescence microscope; spinning disk dichroic Chroma ZT405/488/561/640rpc; 488 nm (50 mW) diode laser (240 ms exposures) with Borealis attachment (Andor); emission filter Chroma Chroma ET525/50m; and an Andor iXon3 camera.…”

Section: Live Cell Imaging Laser Ablation and Fluorescence Correlatio...mentioning

confidence: 99%

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Mechanical coupling with the nuclear envelope shapes theS. pombemitotic spindle

Begley

Medina

Zareiesfandabadi

et al. 2022

Preprint

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The fission yeast S. pombe divides via closed mitosis. In short, mitotic spindle elongation and chromosome segregation transpire entirely within the complete nuclear envelope. Both the spindle and nuclear envelope must undergo significant conformation changes and are subject to varying external forces during this process. While the mechanical relationship between the two mitotic structures has been explored previously, much is still left to be discovered. Here, we investigate this relationship by observing the behaviors of spindles and nuclei in live mitotic fission yeasts following laser ablation. First, we characterize these dynamics in molecularly typical S. pombe spindles, finding them to be stabilized by dense crosslinking, before demonstrating that the compressive force acting on the spindle poles is higher in mitotic cells with greater nuclear envelope tension and that spindle compression can be relieved by lessening nuclear envelope tension. We finally examine the differences between the mitotic apparatus in S. pombe and S. japonicus, an evolutionary relative of S. pombe that undergoes semi-open mitosis, and show that S. japonicus mitotic spindles appear to both splay and bend more easily than those of their S. pombe relatives. Altogether, these data suggest that fission yeast spindle crosslinking may be tuned to support spindle extension and oppose nuclear envelope tension.

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“…Microtubules with fluorescent labels on tubulin dimers appear in images as Gaussian lines rather than spots, meaning that tools for their automated analysis must work differently. There has been considerable development in tools for microtubule tracking both for systems in vitro [ 14 , 15 , 16 , 17 ], and in cells [ 18 , 19 , 20 , 21 ]. However, fully automated tools for microtubule tracking in 3-dimensional (3D) live cell data are lacking.…”

Section: Introductionmentioning

confidence: 99%

Quantifying Yeast Microtubules and Spindles Using the Toolkit for Automated Microtubule Tracking (TAMiT)

et al. 2023

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Fluorescently labeled proteins absorb and emit light, appearing as Gaussian spots in fluorescence imaging. When fluorescent tags are added to cytoskeletal polymers such as microtubules, a line of fluorescence and even non-linear structures results. While much progress has been made in techniques for imaging and microscopy, image analysis is less well-developed. Current analysis of fluorescent microtubules uses either manual tools, such as kymographs, or automated software. As a result, our ability to quantify microtubule dynamics and organization from light microscopy remains limited. Despite the development of automated microtubule analysis tools for in vitro studies, analysis of images from cells often depends heavily on manual analysis. One of the main reasons for this disparity is the low signal-to-noise ratio in cells, where background fluorescence is typically higher than in reconstituted systems. Here, we present the Toolkit for Automated Microtubule Tracking (TAMiT), which automatically detects, optimizes, and tracks fluorescent microtubules in living yeast cells with sub-pixel accuracy. Using basic information about microtubule organization, TAMiT detects linear and curved polymers using a geometrical scanning technique. Images are fit via an optimization problem for the microtubule image parameters that are solved using non-linear least squares in Matlab. We benchmark our software using simulated images and show that it reliably detects microtubules, even at low signal-to-noise ratios. Then, we use TAMiT to measure monopolar spindle microtubule bundle number, length, and lifetime in a large dataset that includes several S. pombe mutants that affect microtubule dynamics and bundling. The results from the automated analysis are consistent with previous work and suggest a direct role for CLASP/Cls1 in bundling spindle microtubules. We also illustrate automated tracking of single curved astral microtubules in S. cerevisiae, with measurement of dynamic instability parameters. The results obtained with our fully-automated software are similar to results using hand-tracked measurements. Therefore, TAMiT can facilitate automated analysis of spindle and microtubule dynamics in yeast cells.

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Automated tracking of S. pombe spindle elongation dynamics

Cited by 6 publications

References 48 publications

Force by minus-end motors Dhc1 and Klp2 collapses the S. pombe spindle after laser ablation

Force by minus-end motors Dhc1 and Klp2 collapses the S. pombe spindle after laser ablation

Mechanical coupling with the nuclear envelope shapes theS. pombemitotic spindle

Quantifying Yeast Microtubules and Spindles Using the Toolkit for Automated Microtubule Tracking (TAMiT)

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