A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis

Anastasia Chasapi, Daniel Sage, Daniel Andreas Schmitter, Viesturs Simanis, Michaël Unser, Ioannis Xenarios
Biomed Central Ltd, 2013
Article

Résumé

Background: The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. Results: We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. Conclusions: "RodCellJ" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells.

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https://infoscience.epfl.ch/record/189210?ln=fr

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Anastasia Chasapi, Daniel Sage, Daniel Andreas Schmitter, Viesturs Simanis, Michaël Unser, Ioannis Xenarios
Biomed Central Ltd, 2013
Article

Résumé

Official source

https://infoscience.epfl.ch/record/189210?ln=fr

À propos de ce résultat

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Publications associées (78)

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Recruitment of NIMA kinase shows that maturation of the S. pombe spindle-pole body occurs over consecutive cell cycles and reveals a role for NIMA in modulating SIN activity

Andrea Krapp, Viesturs Simanis

Mitotic exit in Saccharomyces cerevisiae and septation in Schizosaccharomyces pombe are regulated by a conserved signaling network called the mitotic exit and septum initiation networks (SIN), respectively. The network is active on one of the two anaphase B spindle-pole bodies (SPBs). Whereas the inherent asymmetry of growth by budding accounts for elements of the asymmetry in S. cerevisiae, it has been unclear how, or why, the pathway is asymmetric in S. pombe. We show that elements of SPB duplication in S. pombe are conservative, and that the SIN is active on the new SPB. SIN association with the new SPB persists after transient depolymerization of microtubules. The localization of the NIMA-related kinase, Fin1, reveals further complexity in SPB inheritance. Fin1 associates with the SPB bearing the older components in all cells and with the "new" SPB in half of the population. Fin1 only binds the new SPB when this new SPB has arisen from the duplication of an SPB that is two or more cycles old. Thus, each of the four SPBs generated over two consecutive cell cycles are different, because they have distinct fates in the next cell cycle. Fin1 binds the SPB once the SIN is active and the association requires the SIN inhibitors Byr4 and Cdc16. Fin1 physically associates with Byr4. Compromising Fin1 function leads to SIN activation on both anaphase B SPBs and promotes septation, indicating that Fin1 restrains SIN activity on the old SPB.

2004

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During the division of a mother cell into two daughter cells the genetic material of the mother cell is replicated and segregated to the daughter cells before the cytoplasm divides into two portions in a process called cytokinesis. In order to maintain the genetic stability of the daughter cells it is crucial that the bisection of the cells only occurs once the genetic material has been faithfully partitioned. In the model organism S. pombe the timely coordination of cytokinesis is regulated by a cellular signaling cascade called the Septation Initiation Network (SIN). The SIN is required for the formation and contraction of the contractile actomyosin ring (CAR) that assembles in the cell centre and guides the centripetal deposition of septum material during its contraction. Most of the regulators of the SIN are located to the SPB at some stage of the cell cycle. Interestingly, in anaphase B, when the SIN is supposed to be fully active, positive and negative regulators of the SIN localize to opposite SPBs. This asymmetric configuration of the SIN is lost at the end of cytokinesis, when the SIN is turned off; the positive regulators dissociate from the new SPB and the negative regulators assemble on this SPB. In order to understand this process in more detail we analyzed the localization of Cdc7p, a positive regulator of the SIN. We found that the disappearance of Cdc7p from the new SPB at the end of cytokinesis correlated with the separation of the two halves of the cytoplasm. Moreover, our data suggested that the separation of the two SPBs resulting from the completion of septum formation is important for the resetting of the SIN. Interestingly, we observed that Sid2p, the downstream effector of the SIN, was important for the maintenance of asymmetry in anaphase B and for the timing of the resetting of the SIN by acting in a negative feedback. Taken together, we suggested a model where the downstream effector of the SIN may promote the loading of the negative regulators to the SPBs; in anaphase to the old SPB (due to higher affinity of the negative regulators for this SPB) and upon cell separation to both SPBs, because the old SPB cannot buffer the negative feedback. In a separate study we designed a screen to isolate new regulators of the SIN. Mutagenesis of a strain that over-expressed spg1, the core activator of the SIN, led to the isolation of 28 mutants that are dependent on high levels of Spg1p for viability. The characterization of the nine heat-sensitive mutants among them revealed that they are all new alleles of known SIN genes. Interestingly, two mutants that mapped to the SIN effector sid2, lysed at the time of cytokinesis, when shifted to the restrictive temperature. This observation points to a possible role of the SIN in regulating cell separation. Surprisingly, one of the mutants that mapped to the essential positive SIN regulator sid1 contained a premature STOP codon after 243 nucleotides. The analysis of this mutant revealed that translational read-through level of the STOP codon is sufficient for successful cytokinesis. Similarly, high levels of sid1 expression did not interfere with cytokinesis. This indicated that the regulation of the SIN by Sid1p does not depend on its levels but on a precise control of its action. Since over expression of sid1 led to the accumulation of the protein in the nucleus this may hint that Sid1p recruitment to the SPB is regulated by nuclear proteins.

EPFL2012

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Analysis of S. pombe SIN protein association to the SPB reveals two genetically separable states of the SIN

Anastasia Chasapi, Andrea Krapp, Jacques Rougemont, Daniel Sage, Daniel Andreas Schmitter, Viesturs Simanis, Michaël Unser, Ioannis Xenarios

The Schizosaccharomyces pombe septation initiation network (SIN) regulates cytokinesis, and asymmetric association of SIN proteins with the mitotic spindle pole bodies (SPBs) is important for its regulation. Here, we have used semi-automated image analysis to study SIN proteins in large numbers of wild-type and mutant cells. Our principal conclusions are: first, that the association of Cdc7p with the SPBs in early mitosis is frequently asymmetric, with a bias in favour of the new SPB; second, that the early association of Cdc7p-GFP to the SPB depends on Plo1p but not Spg1p, and is unaffected by mutations that influence its asymmetry in anaphase; third, that Cdc7p asymmetry in anaphase B is delayed by Pom1p and by activation of the spindle assembly checkpoint, and is promoted by Rad24p; and fourth, that the length of the spindle, expressed as a fraction of the length of the cell, at which Cdc7p becomes asymmetric is similar in cells dividing at different sizes. These data reveal that multiple regulatory mechanisms control the SIN in mitosis and lead us to propose a two-state model to describe the SIN.

Company Of Biologists Ltd2015

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EPFL2012