Centrosomes and Spindle Pole Bodies: 67 (Methods in Cell Biology)

Centrosomes and Spindle Pole Bodies: Volume 67

The SPB incorporates additional Spcp during cell-cycle arrests in G2 and M phases and the growth does not depend on microtubules.

The Saccharomyces cerevisiae Spindle Pole Body Is a Dynamic Structure

Taking these observations into account, we can build a model of the SPB life cycle Figure 9. In G2, the SPB structure does not exchange. Late in the cell cycle the SPB grows and then shrinks again early in the next G1. Model of the SPB life cycle.

Centrosomes and Spindle Pole Bodies, Volume 67 Methods in Cell Biology

The SPBs are depicted with shorter cytoplasmic microtubules and longer nuclear microtubules. The satellite forms at the distal tip of the half-bridge in G1. The satellite enlarges to form the duplication plaque. In G2, the bridge cleaves and the SPBs move apart to form a short spindle.

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In anaphase, the spindle elongates. A SPB core growth during the cell cycle. In an unperturbed cell cycle, the SPB core grows late in the cell cycle. B Exchange of components during the cell cycle. Old components are gray, new components are black.

Centrosomes and Spindle Pole Bodies

Furthermore, because overexpression of Mps1p leads to activation of the mitotic checkpoint Hardwick et al. Thus, the amount of Spcp at the SPB is a measure of the size of the core structure.

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We found that growth and shrinkage of the core SPB are regulated in the cell cycle. Findings by Bullitt et al. SPBs in Schizosaccharomyces pombe also grow but start out large in G1. After duplication, the new SPBs are small and equal in diameter but quickly grow to full size Ding et al. A growth phase seems to be a conserved part of the SPB life cycle in S. Clb1p and Clb2p activity becomes dominant in late G2 Lew, In synchronized cultures, no SPB growth was observed at the beginning of G2.

SPBs also grew during all G2 and M arrests tested, including an arrest at the DNA damage checkpoint, at the mitotic checkpoint, and in telophase. This diverse collection of arrests all shares the common feature of high levels of Clb2p. The old Spcp that comes from the old SPB could be either degraded or introduced into the new pole. Two lines of evidence indicate that the fluorescence recovery we observed at the bleached SPB was due to dynamic rearrangement of Spcp-GFP molecules rather than to return of fluorescence to individual bleached GFP molecules.

First, although GFP molecules can show reversible photobleaching, this process occurs very rapidly, on the order of a few milliseconds Swaminathan et al. Second, two different experiments have shown that fluorescence recovery depends on the active metabolism of the cell. The dynamic turnover of FtsZ cytoskeletal molecules in the Z-ring in bacteria was not observed when cells were treated with paraformaldehyde before photobleaching Stricker et al. The extent of Spc42p exchange is not known.

A deeper appreciation of the exchange of SPB components can be gained by comparing it to subunit exchange in other cellular structures.

INTRODUCTION

Spindle microtubules in yeast are much more dynamic than Spcp, with an average half-time of recovery of 52 s Maddox et al. In higher eukaryotic cells, Mad2p and Cdc20p transiently associate with the kinetochore, with average half-time of recovery of 28 and 5. Thus far, the SPB has many dynamic properties but is still more stable than other components examined.

Subunit exchange in other budding yeast mitotic structures such as the core kinetochore and cohesin complexes have not yet been reported. The fact that the old SPB contains less new material than the new SPB can explain the phenotype of many temperature-sensitive mutants in structural and regulatory SPB components. These mutants often contain one apparently normal SPB and one defective or missing SPB, as expected, if during assembly at the nonpermissive temperature, the new SPB components are not evenly dispersed.

This asymmetric SPB mutant phenotype has been observed in spc, spc42, spc29, mps1, msp2, ndc1 , and cdc37 mutants Winey et al. Also, several proteins localize asymmetrically on the cytoplasmic portion of the spindle. It is not known whether asymmetries in the spindle are established by the difference between the old and new SPBs or by an independent mechanism such as differential contact with the polarized cell cortex. Spindle asymmetry in the nucleus has also been reported. In ipl1, sli15 , and spc34 mutants, the chromosomes fail to biorient and are preferentially attached to the old SPB Janke et al.

Kinetochore microtubules in S. The mechanism for achieving biorientation of sister chromatids is not known, but several lines of evidence suggest that the chromatids are actively rearranged by the time of sister separation. First, replicatively older chromatids are randomly distributed between mother and daughter upon division Neff and Burke, The rearrangement of chromatids could be achieved by breaking the attachment at one or more of four possible interfaces: Exchange of Spcp during duplication could be a mechanism for dispersing the kinetochores randomly between the two SPBs.

The combination of quantifying levels of proteins during the cell cycle and live-cell quantitative FRAP analysis was a powerful way to investigate assembly of the SPB. We found several dynamic aspects of assembly: These dynamic characteristics were not compatible with the conservative-dispersive model of duplication, which assumes that the duplicated entities are static. Instead, we found that assembly and duplication could be well characterized by examining exchange and growth.

This methodology could be applied to other inherited structures in the cell to discern universal and unique properties of the assembly of macromolecular structures. We find that Spcp is very dynamic. This dynamic behavior may be integral to the dynamic process of building a mitotic spindle and provides a mechanism to respond to changes in cellular conditions. Article published online ahead of print. Article and publication date are available at www.

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Journal List Mol Biol Cell v. Tim Stearns, Monitoring Editor. This article has been cited by other articles in PMC. Yeast strains used in this study. Open in a separate window. Strains have the W genotype, with the exceptions noted. A mechanism for coupling exit from mitosis to partitioning of the nucleus. Cell , 21— Rapidly maturing variants of the Discosoma red fluorescent protein DsRed. The yeast spindle pole body is assembled around a central crystal of Spc42p. Cell 89 , — Duplication of spindle plaques and integration of the yeast cell cycle.

Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae. The budding yeast Cdc15 localizes to the spindle pole body in a cell-cycle-dependent manner. Commun 2 , — A temperature-sensitive calmodulin mutant loses viability during mitosis. The spindle pole body of Schizosaccharomyces pombe enters and leaves the nuclear envelope as the cell cycle proceeds.

Cell 8 , — Spc29p is a component of the Spcp subcomplex and is essential for spindle pole body duplication. USA 96 , — The kD spindle pole body component of Saccharomyces cerevisiae is a phosphoprotein that is modified in a cell cycle-dependent manner. Can calmodulin function without binding calcium? Cell 65 , — Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption.

Science , — Behavior of kinetochores during mitosis in the fungus Saprolegnia ferax. Visualization of Mad2 dynamics at kinetochores, along spindle fibers, and at spindle poles in living cells. Genes involved in sister chromatid separation are needed for B-type cyclin proteolysis in budding yeast. Cell 81 , — Four new subunits of the Dam1-Duo1 complex reveal novel functions in sister kinetochore biorientation.

A late mitotic regulatory network controlling cyclin destruction in Saccharomyces cerevisiae. Cell 9 , — Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells. The sudden recruitment of gamma-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules. A spacer protein in the Saccharomyces cerevisiae spindle poly body whose transcript is cell cycle-regulated.

Mode of centriole duplication and distribution. Cell cycle control in Saccharomyces cerevisiae. Cold Spring Harbor Laboratory Press, — The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae. Analysis of Tub4p, a yeast gamma-tubulin-like protein: Cdc53p acts in concert with Cdc4p and Cdc34p to control the G1-to-S-phase transition and identifies a conserved family of proteins.

High levels of chromosome instability in polyploids of Saccharomyces cerevisiae. Asymmetric spindle pole localization of yeast Cdc15 kinase links mitotic exit and cytokinesis. Random segregation of chromatids at mitosis in Saccharomyces cerevisiae. Genetics , — Three-dimensional analysis and ultrastructural design of mitotic spindles from the cdc20 mutant of Saccharomyces cerevisiae. Cell 8 , 1— High-voltage electron tomography of spindle pole bodies and early mitotic spindles in the yeast Saccharomyces cerevisiae.

Cell 10 , — The Bub2p spindle checkpoint links nuclear migration with mitotic exit. Cell 6 , 1— Modes of spindle pole body inheritance and segregation of the Bfa1p-Bub2p checkpoint protein complex. New alleles of the yeast MPS1 gene reveal multiple requirements in spindle pole body duplication.

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The molecular function of Ase1p: Cold Spring Harbor Laboratory Press. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics , 19— Opposing the action of these microtubule-stabilizing proteins are a number of microtubule-depolymerizing factors which permit the dynamic remodeling of the mitotic spindle to promote chromosome congression and attainment of bipolarity.

The activities of these MAPs are carefully regulated to maintain proper microtubule dynamics during spindle assembly, with many of these proteins serving as Aurora and Polo-like kinase substrates. In a properly formed mitotic spindle, bi-oriented chromosomes are aligned along the equator of the cell with spindle microtubules oriented roughly perpendicular to the chromosomes, their plus-ends embedded in kinetochores and their minus-ends anchored at the cell poles. The precise orientation of this complex is required to ensure accurate chromosome segregation and to specify the cell division plane.

However, it remains unclear how the spindle becomes organized. Two models predominate the field, which are synergistic and not mutually exclusive. In the search-and-capture model , the spindle is predominantly organized by the poleward separation of centrosomal microtubule organizing centers MTOCs. Spindle microtubules emanate from centrosomes and 'seek' out kinetochores; when they bind a kinetochore they become stabilized and exert tension on the chromosomes.

In an alternative self assembly model, microtubules undergo acentrosomal nucleation among the condensed chromosomes.

Constrained by cellular dimensions, lateral associations with antiparallel microtubules via motor proteins, and end-on attachments to kinetochores, microtubules naturally adopt a spindle-like structure with chromosomes aligned along the cell equator. In this model, microtubules are nucleated at microtubule organizing centers and undergo rapid growth and catastrophe to 'search' the cytoplasm for kinetochores.

Once they bind a kinetochore, they are stabilized and their dynamics are reduced. The newly mono-oriented chromosome oscillates in space near the pole to which it is attached until a microtubule from the opposite pole binds the sister kinetochore. This second attachment further stabilizes kinetochore attachment to the mitotic spindle. Gradually, the bi-oriented chromosome is pulled towards the center of the cell until microtubule tension is balanced on both sides of the centromere ; the congressed chromosome then oscillates at the metaphase plate until anaphase onset releases cohesion of the sister chromatids.

In this model, microtubule organizing centers are localized to the cell poles, their separation driven by microtubule polymerization and 'sliding' of antiparallel spindle microtubules with respect to one another at the spindle midzone mediated by bipolar, plus-end-directed kinesins. In contrast to the search-and-capture mechanism in which centrosomes largely dictate the organization of the mitotic spindle, this model proposes that microtubules are nucleated acentrosomally near chromosomes and spontaneously assemble into anti-parallel bundles and adopt a spindle-like structure.

Glass beads coated with RCC1 induce microtubule nucleation and bipolar spindle formation in Xenopus egg extracts, revealing that the Ran GTP gradient alone is sufficient for spindle assembly. The unbound SAFs then promote microtubule nucleation and stabilization around mitotic chromatin, and spindle bipolarity is organized by microtubule motor proteins. Spindle assembly is largely regulated by phosphorylation events catalyzed by mitotic kinases. Cyclin dependent kinase complexes CDKs are activated by mitotic cyclins, whose translation increases during mitosis. Aurora kinases are required for proper spindle assembly and separation.

Aurora B is a member of the chromosomal passenger complex and mediates chromosome-microtubule attachment and sister chromatid cohesion. Polo-like kinase, also known as PLK, especially PLK1 has important roles in the spindle maintenance by regulating microtubule dynamics. By the end of DNA replication, sister chromatids are bound together in an amorphous mass of tangled DNA and protein that would be virtually impossible to partition into each daughter cell. To avoid this problem, mitotic entry triggers a dramatic reorganization of the duplicated genome.

Centrosomes as signalling centres

Sister chromatids are disentangled and resolved from one another. Chromosomes also shorten in length, up to 10, fold in animal cells, [30] in a process called condensation. Condensation begins in prophase and chromosomes are maximally compacted into rod-shaped structures by the time they are aligned in the middle of the spindle at metaphase. While these dynamic rearrangements are vitally important to ensure accurate and high-fidelity segregation of the genome, our understanding of mitotic chromosome structure remains largely incomplete.

A few specific molecular players have been identified, however: The completion of spindle formation is a crucial transition point in the cell cycle called the spindle assembly checkpoint. If chromosomes are not properly attached to the mitotic spindle by the time of this checkpoint, the onset of anaphase will be delayed. Cell division orientation is of major importance for tissue architecture, cell fates and morphogenesis.

Cells tend to divide along their long axis according to the so-called Hertwig rule. The axis of cell division is determined by the orientation of the spindle apparatus. Cells divide along the line connecting two centrosomes of the spindle apparatus. After formation, the spindle apparatus undergoes rotation inside the cell. The astral microtubules originating from centrosomes reach the cell membrane where they are pulled towards specific cortical clues.