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

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 minus-end-directed kinesin Pkl1.

Automated tracking of S. pombe spindle elongation dynamics.

The mitotic spindle is a microtubule-based machine that pulls the two identical sets of chromosomes to opposite ends of the cell during cell division. The fission yeast Schizosaccharomyces pombe is an important model organism for studying mitosis due to its simple, stereotyped spindle structure and well-established genetic toolset. S. pombe spindle length is a useful metric for mitotic progression, but manually tracking spindle ends in each frame to measure spindle length over time is laborious and can limit experimental throughput. We have developed an ImageJ plugin that can automatically track S. pombe spindle length over time and replace manual or semi-automated tracking of spindle elongation dynamics. Using an algorithm that detects the principal axis of the spindle and then finds its ends, we reliably track the length of the spindle as the cell divides. The plugin integrates with existing ImageJ features, exports its data for further analysis outside of ImageJ and does not require any programming by the user. Thus, the plugin provides an accessible tool for quantification of S. pombe spindle length that will allow automatic analysis of large microscopy data sets and facilitate screening for effects of cell biological perturbations on mitotic progression.

The mitotic spindle is a biological machine that pulls the two identical sets of DNA to opposite ends of the cell during cell division. Incorrect cell division can result in serious issues like cancer and miscarriages. Schizosaccharomyces pombe (S. pombe), a kind of yeast, is commonly used to study cell division because its mitotic spindle is essentially linear in shape and its DNA sequence is well known, allowing for more complex experiments. To measure how well a cell divides, we measure the length of the spindle over time, but this can be tedious to do by hand for many cell images. We have developed software that interfaces with ImageJ (a common image analysis tool) that automatically tracks the length of S. pombe spindles over time and can replace manual tracking. Our software calculates the spindle's lines of symmetry, while allows us to accurately measure the length and track the ends over time. It integrates with existing ImageJ features, exports its data for further analysis outside of ImageJ, and does not require any programming by the user. Thus, the plugin provides an accessible tool for measuring S. pombe spindle length that will allow automatic analysis of large microscopy data sets and facilitate screening for effects of defects in cell division. This will facilitate the study of the basic fundamental process of how cells divide, and could have significant long term medical impacts.

Viscoelastic Relaxation of the Nuclear Envelope Does Not Cause the Collapse of the Spindle After Ablation in S. pombe.

A large molecular machine called the mitotic spindle is responsible for accurate chromosome segregation in eukaryotic cells. The spindle consists of protein filaments known as microtubules and microtubule-associated proteins such as motors and crosslinkers, which help impart its organization. In the case of the fission yeast S. pombe, these form a single bundle inside the nucleus. During spindle elongation, sliding by motor proteins provides an internal source of extensile forces, which are resisted by the compressive forces of the nuclear envelope. To probe the sources of this force balance, we cut the spindle using focused laser light at various stages of spindle elongation. We find that the spindle pole bodies collapse toward each other post-ablation. While this basic behavior has been previously observed, many questions remain about the timing, mechanics, and molecular requirements of this phenomenon. Here, we quantify the time scale of the relaxation and probe its underlying mechanism. We demonstrate that viscoelastic relaxation of the nuclear envelope cannot explain this phenomenon and provide evidence of active forces as the underlying mechanism.