Genetic Analysis of Duplication

Genetic analysis of SPB duplication has identified a number of genes required for the duplication of this organelle. All of the genes are essential and are studied primarily using conditional alleles. The duplication genes have been identified in genetic screens (i.e. [18, 19]) or in the course of the analysis of genes encoding components of the SPB (i.e. [23, 25]). The general phenotype of cells harboring these mutations is a mitotic arrest with a monopolar spindle instead of the normal bipolar spindle. This phenotype was first recognized for cdc31-1 [16] and indicates that cells will proceed into the cell cycle growing a bud and replicating chromosomes in the absence of SPB duplication. The mutant cells arrest in mitosis, because monopolar spindles cannot capture chromosomes correctly and the spindle assembly checkpoint is activated [86]. The cells can eventually exit the arrest (i. e.[87, 88]), but how this happens is not understood. Finally, the observation that aberrant microtubule arrays seen in these mutants arise from a single, unduplicated SPB revealed that these SPBs exhibit different morphologies in different mutant strains (e.g. [18]). These different morphologies indicate that the genes act at different steps in SPB duplication, an interpretation that has been supported by execution-point experiments and epistasis tests (e. g. [18]). Such work has led to the identification of genes required for each of the steps of SPB duplication that had been identified previously by EM analysis.

As mentioned above, CDC31 was the first gene known to be required for SPB duplication in yeast. The single SPB in cdc31 mutants was observed to have little or no bridge structure, suggesting that Cdc31p is necessary for forming or maintaining the bridge and, therefore, would be required early in SPB duplication. In deed, execution-point experiments showed that Cdc31p is not required after the formation of the satellite [18]. Furthermore, Cdc31p is found at the halfbridge [89]. Importantly, CDC31 encodes the yeast homolog of centrin. Centrins are small, conserved calcium binding proteins that are found at MTOCs and are involved in the duplication of centrioles [90] and basal bodies [91]. Cdc31p is not a membrane protein, yet it is associated with the halfbridge. Cdc31p is tethered to the halfbridge by the membrane proteins Kar1p and Mps3p. Furthermore, Kar1p interacts with Mps3p indicating that there is a complex of these two proteins that binds Cdc31p. Another halfbridge binding partner, Sfi1p, has been discovered quite recently [13]. Much like those with cdc31 mutations, cells containing temperature-sensitive mutations in KAR1, MPS3, and SFI1 fail in SPB duplication at the restrictive temperature and have unduplicated SPBs with little or no halfbridge. How these proteins in various complexes act to initiate SPB duplication leading to the formation of the satellite is unknown, but is likely to be conserved in that centrin is required for the duplication of SPBs, centrioles and basal bodies. Moreover, Sfi1p is widely conserved [13].

Analysis of the SPB components Spc42p and Spc29p reveals that the two genes responsible for these components are also required for SPB duplication. These proteins are required after formation of the satellite, because when strains mutant in either gene are released from mating factor arrest (after satellite formation) at the restrictive temperature, they still fail in SPB duplication [25, 28]. These findings are well explained by the discovery that both these proteins are components of the satellite, and would be added to the satellite as the duplication plaque is formed. Interestingly, overexpression of Spc42p leads to a large lateral expansion of the central plaque that has been used for structural analysis and as the basis of assembly assays [25]. The protein kinase Mps1p, discussed further below, is also required for the maturation of the satellite, and its activity is required for proper assembly of Spc42p, most likely because Spc42p is a substrate of Mps1p [26]. The other satellite components Cnm67p and Nud1p have other cellular functions which complicate experiments to determine if they have roles in SPB duplication [15, 8].

The final step in SPB duplication, identified by mutation, is the insertion of the nascent SPB, or duplication plaque, into a pore in the nuclear envelope. Mutants in two membrane proteins of the nuclear envelope, Mps2p and Ndc1p, appear to fail in this step, giving rise to an unusual phenotype [18, 92]. In these mutants, two SPB-like organelles are observed, but only one of them has normal nuclear microtubules and is associated with the chromatin. The other SPB is on the cytoplasmic side of the nuclear envelope and only has microtubules in the cytoplasm, which will serve to move this defective SPB away from the other SPB. As it is moved, the defective SPB will only bring along the nuclear envelope but not the chromosomes. The defective SPB in these mutants appears to be quite similar to a duplication plaque that has not been inserted into the nuclear envelope. It is not known how these proteins function, but Mps2p is known to bind Bbp1p [23]. Mutations in BBP1 have a similar phenotype to those of MPS2 and NDC1 mutants, suggesting that Bbp1p participates in the insertion event. Furthermore, Bbp1p binds Spc29p, suggesting a role for anchoring the SPB in the nuclear envelope [23]. Upon inser tion into the pore in the nuclear envelope, the new SPB has access to components present in the nucleoplasm and can thus form the inner plaque. As mentioned, components of the inner plaque include Spc110p and its binding partner calmodulin which, in turn, bind the y-tubulin complex (Spc97/98p and Tub4p). Mutations in genes encoding these components can show defects in the inner plaque or its complete absence [57, 66]. Completion of the inner plaque marks the completion of SPB duplication, resulting in duplicated side-by-side SPBs (Figure 4.3d).

Critical for SPB duplication is the Mpslp protein kinase. Mpslp is found at both SPBs and kinetochores, where it functions in both SPB duplication and in the spindle assembly checkpoint [18, 26, 86, 93]. The original allele of MPS1, mps1-1, exhibits unduplicated SPBs with a large halfbridge, indicating that the satellite was not formed or was not functional for duplication [18]. Execution-point experiments revealed that Mpslp is required upon release from mating factor arrest and the likely substrate for this requirement may be Spc42p, an Mpslp binding partner and in vitro substrate [26]. Further analysis of MPS1 led to the identification of additional mutant alleles that appear to be defective earlier in SPB duplication similar to CDC31/KAR1/MPS3 mutants (mps1-8, [26]) or later in duplication similar to NDC1/MPS2/BBP1 mutants (mpsl-737, [94]). These findings indicate that Mpslp is required at all known steps of SPB duplication either because it has multiple substrates and/or some substrate(s) needs multiple phosphorylation events to act at all steps during duplication. No other regulator of SPB duplication behaves this way. Mpslp protein kinase is conserved, and vertebrate orthologs have been shown to be involved in the spindle assembly checkpoint [95-97]. Localization of Mpsl at centrosomes [96, 98, 99] and Mpsl control of centrosome duplication [98, 99] has been controversial [97].

Several other important regulators of SPB duplication have been identified and include the proteosome subunit Pcslp [100], the ubiquitin-like proteins Rad23p and Dsk2p [101], the chaperone Cdc37p [102] and the heat shock transcription factor Hsflp [103]. Which steps in duplication require these genes are not known, but it is clear that a diverse collection of protein activities is required for SPB duplication. In many cases, the vertebrate orthologs of genes required for SPB duplication are clearly required for centrosome duplication. More detailed analysis of SPB duplication will yield insights into the control of this process, which will also shed light on the control of centrosome duplication.

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