Abstract
Microtubules play multiple roles in a wide range of cellular phenomena, including cell polarity establishment and chromosome segregation. A number of microtubule regulators have been identified, including microtubule-associated proteins and kinases, and knowledge of these factors has contributed to our molecular understanding of microtubule regulation of each relevant cellular process. The known regulators, however, are insufficient to explain how those processes are linked to one another, underscoring the need to identify additional regulators. To find such novel mechanisms and microtubule regulators, we performed a screen that combined genetics and microscopy for fission yeast mutants defective in microtubule organization. We isolated approximately 900 mutants showing defects in either microtubule organization or the nuclear envelope, and these mutants were classified into 12 categories. We particularly focused on one mutant, kis1, which displayed spindle defects in early mitosis. The kis1 mutant frequently failed to assemble a normal bipolar spindle. The responsible gene encoded a kinetochore protein, Mis19 (also known as Eic1), which localized to the interface of kinetochores and spindle poles. We also found that the inner kinetochore proteins Mis6/CENP-I and Cnp1/CENP-A were delocalized from kinetochores in the kis1 cells and that kinetochore-microtubule attachment was defective. Another mutant, mis6, also displayed similar spindle defects. We conclude that Kis1 is required for inner kinetochore organization, through which Kis1 ensures kinetochore-microtubule attachment and spindle integrity. Thus, we propose an unexpected relationship between inner kinetochore organization and spindle integrity.
Highlights
Microtubules are cylindrical polymers found in all eukaryotic cells and are involved in a number of cellular processes during the cell cycle [1,2]
The latter strain appeared suitable for use in combination with severe mutation backgrounds that affect microtubule formation because the former green fluorescent protein (GFP)-Atb2 construct occasionally showed synthetic growth defects when combined with some mutations
Mis6/CENP-I and Cnp1/CENP-A of the inner kinetochore proteins were delocalized in the kis1 mutant
Summary
Microtubules are cylindrical polymers found in all eukaryotic cells and are involved in a number of cellular processes during the cell cycle [1,2]. Cytoplasmic microtubules are arranged in a mesh-like array in animal cells or in a cylindrical array along the longitudinal axis in fission yeast. Centrosomes in animal cells and spindle pole bodies (SPBs) in yeast play central roles in the assembly of a microtubulebased bipolar spindle. The spindle elongates in anaphase using the antiparallel bundling of interpolar microtubules to further separate segregated chromosomes, and the spindle is required for faithful chromosome segregation and cell division. All eukaryotic cells must alter microtubule organization between the cytoplasmic array and the spindle when entering or exiting mitosis
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