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The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

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Stu2p is a member of a conserved family of microtubule-binding proteins and an essential protein in yeast. Here, we report the first in vivo analysis of microtubule dynamics in cells lacking a member of this protein family. For these studies, we have used a conditional Stu2p depletion strain expressing alpha-tubulin fused to green fluorescent protein. Depletion of Stu2p leads to fewer and less dynamic cytoplasmic microtubules in both G1 and preanaphase cells. The reduction in cytoplasmic microtubule dynamics is due primarily to decreases in both the catastrophe and rescue frequencies and an increase in the fraction of time microtubules spend pausing. These changes have significant consequences for the cell because they impede the ability of cytoplasmic microtubules to orient the spindle. In addition, recovery of fluorescence after photobleaching indicates that kinetochore microtubules are no longer dynamic in the absence of Stu2p. This deficiency is correlated with a failure to properly align chromosomes at metaphase. Overall, we provide evidence that Stu2p promotes the dynamics of microtubule plus-ends in vivo and that these dynamics are critical for microtubule interactions with kinetochores and cortical sites in the cytoplasm.

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Using green fluorescent protein probes and rapid acquisition of high-resolution fluorescence images, sister centromeres in budding yeast are found to be separated and oscillate between spindle poles before anaphase B spindle elongation. The rates of movement during these oscillations are similar to those of microtubule plus end dynamics. The degree of preanaphase separation varies widely, with infrequent centromere reassociations observed before anaphase. Centromeres are in a metaphase-like conformation, whereas chromosome arms are neither aligned nor separated before anaphase. Upon spindle elongation, centromere to pole movement (anaphase A) was synchronous for all centromeres and occurred coincident with or immediately after spindle pole separation (anaphase B). Chromatin proximal to the centromere is stretched poleward before and during anaphase onset. The stretched chromatin was observed to segregate to the spindle pole bodies at rates greater than centromere to pole movement, indicative of rapid elastic recoil between the chromosome arm and the centromere. These results indicate that the elastic properties of DNA play an as of yet undiscovered role in the poleward movement of chromosome arms.

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Microtubule assembly in Saccharomyces cerevisiae is initiated from sites within spindle pole bodies (SPBs) in the nuclear envelope. Microtubule plus ends are thought to be organized distal to the SPBs, while minus ends are proximal. Several hypotheses for the function of microtubule motor proteins in force generation and regulation of microtubule assembly propose that assembly and disassembly occur at minus ends as well as at plus ends. Here we analyse microtubule assembly relative to the SPBs in haploid yeast cells expressing green fluorescent protein fused to alpha-tubulin, a microtubule subunit. Throughout the cell cycle, analysis of fluorescent speckle marks on cytoplasmic astral microtubules reveals that there is no detectable assembly or disassembly at minus ends. After laser-photobleaching, metaphase spindles recover about 63% of the bleached fluorescence, with a half-life of about 1 minute. After anaphase onset, photobleached marks in the interpolar spindle are persistent and do not move relative to the SPBs. In late anaphase, the elongated spindles disassemble at the microtubule plus ends. These results show for astral and anaphase interpolar spindle microtubules, and possibly for metaphase spindle microtubules, that microtubule assembly and disassembly occur at plus, and not minus, ends.

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We have used local fluorescence photoactivation to mark the lattice of spindle microtubules during anaphase A in Xenopus extract spindles. We find that both poleward spindle microtubule flux and anaphase A chromosome movement occur at similar rates ( approximately 2 microm/min). This result suggests that poleward microtubule flux, coupled to microtubule depolymerization near the spindle poles, is the predominant mechanism for anaphase A in Xenopus egg extracts. In contrast, in vertebrate somatic cells a "Pacman" kinetochore mechanism, coupled to microtubule depolymerization near the kinetochore, predominates during anaphase A. Consistent with the conclusion from fluorescence photoactivation analysis, both anaphase A chromosome movement and poleward spindle microtubule flux respond similarly to pharmacological perturbations in Xenopus extracts. Furthermore, the pharmacological profile of anaphase A in Xenopus extracts differs from the previously established profile for anaphase A in vertebrate somatic cells. The difference between these profiles is consistent with poleward microtubule flux playing the predominant role in anaphase chromosome movement in Xenopus extracts, but not in vertebrate somatic cells. We discuss the possible biological implications of the existence of two distinct anaphase A mechanisms and their differential contributions to poleward chromosome movement in different cell types.

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