A method for astral microtubule tracking in fluorescence images of cells doped with taxol and nocodazole

In this paper we describe an algorithm that performs automatic detection and tracking of astral microtubules in fluorescence confocal images. This sub-population of microtubules only exists during and immediately before mitosis and aids in the spindle orientation by connecting it to the cell cortex. Anomalies in their dynamic behaviour play a causal role in many diseases, such as development disorders and cancer. The main novelty of the proposed algorithm lies in the fact it provides a fully automated estimation of parameters related to microtubule dynamic instability (growth velocity, track length and track lifetime), and helps in understanding the effects of intermediate drug concentrations. Its performance has been objectively assessed using publicly available synthetic data and largely employed metrics. Moreover, we present experiments addressing cell cultures doped with different concentrations of taxol and nocodazole. Such drugs are known to suppress the microtubule dynamic instability, but their effects at intermediate concentrations are not completely assessed. The algorithm been compared with other stateof- the-art approaches, tested on consistent real datasets. The results are encouraging in terms of performance, robustness and simplicity of use, and the algorithm is now routinely employed in our Department of Molecular Biotechnology

Mitotic spindle scaling during Xenopus development by kif2a and importin α.

Early development of many animals is characterized by rapid cleavages that dramatically decrease cell size, but how the mitotic spindle adapts to changing cell dimensions is not understood. To identify mechanisms that scale the spindle during Xenopus laevis embryogenesis, we established an in vitro system using cytoplasmic extracts prepared from embryos that recapitulates in vivo spindle size differences between stage 3 (4 cells, 37 µm) and stage 8 (∼4000 cells, 18 µm). We identified the kinesin-13 kif2a as a driver of developmental spindle scaling whose microtubule-destabilizing activity is inhibited in stage 3 spindles by the transport receptor importin α, and activated in stage 8 when importin α partitions to a membrane pool. Altering spindle size in developing embryos impaired spindle orientation during metaphase, but chromosome segregation remained robust. Thus, spindle size in Xenopus development is coupled to cell size through a ratiometric mechanism controlling microtubule destabilization.DOI:http://dx.doi.org/10.7554/eLife.00290.001

A mitotic SKAP isoform regulates spindle positioning at astral microtubule plus ends

The Astrin/SKAP complex plays important roles in mitotic chromosome alignment and centrosome integrity, but previous work found conflicting results for SKAP function. Here, we demonstrate that SKAP is expressed as two distinct isoforms in mammals: a longer, testis-specific isoform that was used for the previous studies in mitotic cells and a novel, shorter mitotic isoform. Unlike the long isoform, short SKAP rescues SKAP depletion in mitosis and displays robust microtubule plus-end tracking, including localization to astral microtubules. Eliminating SKAP microtubule binding results in severe chromosome segregation defects. In contrast, SKAP mutants specifically defective for plus-end tracking facilitate proper chromosome segregation but display spindle positioning defects. Cells lacking SKAP plus-end tracking have reduced Clasp1 localization at microtubule plus ends and display increased lateral microtubule contacts with the cell cortex, which we propose results in unbalanced dynein-dependent cortical pulling forces. Our work reveals an unappreciated role for the Astrin/SKAP complex as an astral microtubule mediator of mitotic spindle positioning.Leukemia & Lymphoma Society of America (Scholar Award)National Institute of General Medical Sciences (U.S.) (GM088313)American Cancer Society (121776

Astral Microtubule Pivoting Promotes Their Search for Cortical Anchor Sites during Mitosis in Budding Yeast

Positioning of the mitotic spindle is crucial for proper cell division. In the budding yeast Saccharomyces cerevisiae, two mechanisms contribute to spindle positioning. In the Kar9 pathway, astral microtubules emanating from the daughter-bound spindle pole body interact via the linker protein Kar9 with the myosin Myo2, which moves the microtubule along the actin cables towards the neck. In the dynein pathway, astral microtubules off-load dynein onto the cortical anchor protein Num1, which is followed by dynein pulling on the spindle. Yet, the mechanism by which microtubules target cortical anchor sites is unknown. Here we quantify the pivoting motion of astral microtubules around the spindle pole bodies, which occurs during spindle translocation towards the neck and through the neck. We show that this pivoting is largely driven by the Kar9 pathway. The microtubules emanating from the daughter-bound spindle pole body pivot faster than those at the mother-bound spindle pole body. The Kar9 pathway reduces the time needed for an astral microtubule inside the daughter cell to start pulling on the spindle. Thus, we propose a new role for microtubule pivoting: By pivoting around the spindle pole body, microtubules explore the space laterally, which helps them search for cortical anchor sites in the context of spindle positioning in budding yeast

Dissecting Molecular Pathways that ensure Proper Chromosome Segregation and Cell Division

Equal segregation of the genome is a prerequisite for cell survival. During cell division the duplicated DNA is compacted into chromosomes and a multi-protein macrostructure, known as the kinetochore (Kt), is assembled on each copy of compacted DNA. Simultaneously, the mitotic spindle, which is made up of microtubules (MTs), is built to facilitate the equal distribution of the chromosomes between the resulting daughter cells. Kinetochores mediate the interaction between the MTs and the chromosomes, properly positioning them for segregation. To ensure that the DNA is equally divided in every cell division, cells have built a surveillance system to detect any errors that may occur. An important checkpoint during mitosis is the spindle assembly checkpoint (SAC), which ensures that all chromosomes are forming the appropriate attachments with MTs. In cases of erroneous attachment, mitotic progression is halted and an error correction pathway is activated to give chromosomes a second change to establish proper attachment. When all the chromosomes have achieved biorientation, cells progress into anaphase, where each copy of the DNA is pulled toward opposite axis of the cell. It is critical that chromosome are properly attached and aligned to ensure equal segregation, as misegragation is the leading cause of cell death or cancer. Finally, a cleavage furrow is formed at the cell equator that constricts and severs the cell, giving rise to two daughters. It is remarkable how cells are able to go through many rounds of division without any errors. Although a lot of work has been done to further understand the mechanisms in which cells detect and correct potential catastrophic error, many more mystery remains to be uncovered. In the last six years, I tried to make my contribution to field by exploring how a previously classified as a non-motile kinesin generates force to move chromosomes, how much force is being exerted on a bioriented kinetochore, the roles aurora kinases play to ensure proper kinetochore-microtubule attachment and proper cytokinesis

Polarized light microscopy in reproductive and developmental biology

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of John Wiley & Sons for personal use, not for redistribution. The definitive version was published in Molecular Reproduction and Development (2013), doi:10.1002/mrd.22221.The polarized light microscope reveals orientational order in native molecular structures inside living cells, tissues, and whole organisms. Therefore, it is a powerful tool to monitor and analyze the early developmental stages of organisms that lend themselves to microscopic observations. In this article we briefly discuss the components specific to a traditional polarizing microscope and some historically important observations on chromosome packing in sperm head, first zygote division of the sea urchin, and differentiation initiated by the first uneven cell division in the sand dollar. We then introduce the LC-PolScope and describe its use for measuring birefringence and polarized fluorescence in living cells and tissues. Applications range from the enucleation of mouse oocytes to analyzing the polarized fluorescence of the water strider acrosome. We end by reporting first results on the birefringence of the developing chick brain, which we analyzed between developmental stages of days 12 through 20.This work was supported by funds from the National Institute of General Medical Sciences (grant 1R01GM100160-01A1 awarded to TT) and the National Institute of Biomedical Imaging and Bioengineering (grant EB002045 awarded to RO)