Role of Molecular Motors and Maps in Spindle Dynamics and Chromosome Segregation in the Fission Yeast Schizosaccharomyces Pombe

Mitosis is a key event in the life of a cell, where duplicated chromosomes are separated into the daughter cells. Defects associated with chromosome segregation can lead to aneuploidy, a hallmark of cancer. Chromosome segregation is achieved by the mitotic spindle, which is composed of microtubules (MTs), motors and microtubule associated protein (MAPs). Motors such as kinesins generate forces within the spindle while MAPs perform functions such as organize the spindle pole and maintain the bipolar spindle. Both motors and MAPs contribute to spindle mechanics. Here I used the relatively simple fission yeast to address how defects in spindle mechanics affect chromosome segregation. The metaphase spindle is maintained at a constant length by an antagonistic force-balance model yet how the regulation of metaphase spindle length contribute to subsequent chromosome segregation remains unexplored. To test the force-balance model, I applied gene deletion and fast microfluidic temperature-control with live-cell imaging to monitor the effect of deleting or switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. I show that kinesin-5 cut7p and MT bundler ase1p contribute to outward pushing forces, and kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Motors and MAPs cooperate to focus MTs at the spindle pole. Defects in MT focusing lead to defects in chromosome segregation, resulting in aneuploidy. The mechanism behind these observations is not well understood. Here I identified a new mechanism for aneuploidy in fission yeast. Kinesin-14 pkl1p and MAP msd1p localize to the spindle poles and focus the MT minus ends. Their absence leads to pole and MT defocusing, resulting in protrusion of MT minus ends due to cut7p-dependent pushing forces at the spindle midzone. Infrequent long MT minus end protrusions can push the already separated chromosome mass back to the cell center, where cytokinesis will `cut\u27 the chromosome mass, creating two daughter cells with unequal chromosome content

Antagonistic spindle motors and MAPs regulate metaphase spindle length and chromosome segregation

SummaryMetaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at characteristic constant length [1–3]. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules (MTs) and their interactions with motors and MT-associated proteins (MAPs). Spindle length is further proposed to be important for chromosome segregation fidelity, as cells with shorter- or longer-than-normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force-balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature control with live-cell imaging to monitor the effect of deleting or switching off different combinations of antagonistic force contributors in the fission yeast metaphase spindle. We show that the spindle midzone proteins kinesin-5 cut7p and MT bundler ase1p contribute to outward-pushing forces and that the spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward-pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and in some combinations also partially rescued chromosome segregation defects

Mcp1p tracks microtubule plus ends to destabilize microtubules at cell tips

Microtubule plus ends are dynamically regulated by a wide variety of proteins for performing diverse cellular functions. Here, we show that the fission yeast Schizosaccharomyces pombe uncharacterized protein mcp1p is a microtubule plus-end tracking protein which depends on the kinesin-8 klp6p for transporting along microtubules towards microtubule plus ends. In the absence of mcp1p, microtubule catastrophe and rescue frequencies decrease, leading to an increased dwell time of microtubule plus ends at cell tips. Thus, these findings suggest that mcp1p may synergize with klp6p at microtubule plus-ends to destabilize microtubules

DANGER, a novel regulatory protein of inositol 1,4,5-trisphosphate-receptor activity

We report the cloning and characterization of DANGER, a novel protein which physiologically binds to inositol 1,4,5-trisphosphate receptors (IP 3R). DANGER is a membrane-associated protein predicted to contain a partial MAB-21 domain. It is expressed in a wide variety of neuronal cell lineages where it localizes to membranes in the cell periphery together with IP3R. DANGER interacts with IP3R in vitro and co-immunoprecipitates with IP3R from cellular preparations. DANGER robustly enhances Ca2+-mediated inhibition of IP3R Ca 2+ release without affecting IP3 binding in microsomal assays and inhibits gating in single-channel recordings of IP3R. DANGER appears to allosterically modulate the sensitivity of IP3R to Ca2+ inhibition, which likely alters IP3R-mediated Ca 2+ dynamics in cells where DANGER and IP3R are co-expressed. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.link_to_subscribed_fulltex