Michael E. Dresser

Meiotic Recombination in C. elegans Initiates by a Conserved Mechanism and Is Dispensable for Homologous Chromosome Synapsis

Chromosome segregation at meiosis I depends on pairing and crossing-over between homologs. In most eukaryotes, pairing culminates with formation of the proteinaceous synaptonemal complex (SC). In budding yeast, recombination initiates through double-strand DNA breaks (DSBs) and is thought to be essential for SC formation. Here, we examine whether this mechanism for initiating meiotic recombination is conserved, and we test the dependence of homologous chromosome synapsis on recombination in C. elegans. We find that a homolog of the yeast DSB-generating enzyme, Spo11p, is required for meiotic exchange in this metazoan, and that radiation-induced breaks partially alleviate this dependence. Thus, initiation of recombination by DSBs is apparently conserved. However, homologous synapsis is independent of recombination in the nematode, since it occurs normally in a C. elegans spo-11 null mutant.

MPS3 mediates meiotic bouquet formation in Saccharomyces cerevisiae

In meiotic prophase, telomeres associate with the nuclear envelope and accumulate adjacent to the centrosome/spindle pole to form the chromosome bouquet, a well conserved event that in Saccharomyces cerevisiae requires the meiotic telomere protein Ndj1p. Ndj1p interacts with Mps3p, a nuclear envelope SUN domain protein that is required for spindle pole body duplication and for sister chromatid cohesion. Removal of the Ndj1p-interaction domain from MPS3 creates an ndj1Δ-like separation-of-function allele, and Ndj1p and Mps3p are codependent for stable association with the telomeres. SUN domain proteins are found in the nuclear envelope across phyla and are implicated in mediating interactions between the interior of the nucleus and the cytoskeleton. Our observations indicate a general mechanism for meiotic telomere movements.

Morphogenetic Pathway of Spore Wall Assembly in Saccharomyces cerevisiae

ABSTRACT The Saccharomyces cerevisiae spore is protected from environmental damage by a multilaminar extracellular matrix, the spore wall, which is assembled de novo during spore formation. A set of mutants defective in spore wall assembly were identified in a screen for mutations causing sensitivity of spores to ether vapor. The spore wall defects in 10 of these mutants have been characterized in a variety of cytological and biochemical assays. Many of the individual mutants are defective in the assembly of specific layers within the spore wall, leading to arrests at discrete stages of assembly. The localization of several of these gene products has been determined and distinguishes between proteins that likely are involved directly in spore wall assembly and probable regulatory proteins. The results demonstrate that spore wall construction involves a series of dependent steps and provide the outline of a morphogenetic pathway for assembly of a complex extracellular structure.

Budding yeast PDS5 plays an important role in meiosis and is required for sister chromatid cohesion.

Budding yeast PDS5 is an essential gene in mitosis and is required for chromosome condensation and sister chromatid cohesion. Here we report that PDS also is required in meiosis. Pds5p localizes on chromosomes at all stages during meiotic cycle, except anaphase I. PDS5 plays an important role at first meiotic prophase. Failure in function of PDS5 causes premature separation of chromosomes. The loading of Pds5p onto chromosome requires the function of REC8, but the association of Rec8p with chromosome is independent of PDS5. Mutant analysis and live cell imaging indicate that PDS5 play a role in meiosis II as well.

Interaction between the F plasmid TraA (F‐pilin) and TraQ proteins

Elaboration of conjugative (F) pili by F+ strains of Escherichia coli requires the activities of over a dozen F‐encoded DNA transfer (Tra) proteins. The organization and functions of these proteins are largely unknown. Using the yeast two‐hybrid assay, we have begun to analyse binary interactions among the Tra proteins required for F‐pilus formation. We focus here on interactions involving F‐pilin, the only known F‐pilus subunit. Using a library of F tra DNA fragments that contained all the F genes required for F pilus formation in a yeast GAL4 activation domain vector (pACTII), we transformed yeast containing a plasmid (pAS1CYH2traA) encoding a GAL4 DNA‐binding domain–F‐pilin fusion. Doubly transformed cells were screened for GAL4‐dependent gene expression. This screen repeatedly identified only a single Tra protein, TraQ, previously identified as a likely F‐pilin chaperone. The F‐pilin–TraQ interaction appeared to be specific, as no transcriptional activation was detected in yeast transformants containing pACTIItraQ plasmids and the Salmonella typhi pED208 traA gene cloned in pAS1CYH2. Two traQ segments isolated in the screen against F‐pilin were tested for complementation of a traQ null allele in E. coli. One, lacking the first 11 (of 94) TraQ amino acids, restored DNA donor activity, donor‐specific bacteriophage sensitivity and membrane F‐pilin accumulation to wild‐type levels. The second, lacking the first 21 amino acids, was much less effective in these assays. Both TraQ polypeptides accumulated in E. coli as transmembrane proteins. The longer, biologically active segment was fused to the GAL4 DNA‐binding domain gene of pAS1CYH2 and used to screen the tra fragment library. The only positives from this screen identified traA segments. The fusion sites between the traA and GAL4 segments identified the hydrophobic, C‐terminal domain IV of F‐pilin as sufficient for the interaction. As TraQ is the only Tra protein required for the accumulation of inner membrane F‐pilin, the interaction probably reflects a specific, chaperone‐like function for TraQ in E. coli. Attempts to isolate an F‐pilin–TraQ complex from E. coli were unsuccessful, suggesting that the interaction between the two is normally transient, as expected from previous studies of the kinetics of TraA membrane insertion and processing to F‐pilin.

Chromosome mechanics and meiotic engine maintenance.

The behavior of chromosomes during meiosis has been likened to a middle school dance, where partners find one another, form couples that move about and trade information, and then separate to opposite sides of the dance hall. With chromosomes, as with the dancers, forming exclusive couples often is difficult—individuals can be attracted to more than one partner or find themselves trapped behind or between other couples—and, failing to form a couple effectively, end up on the wrong side of the dance hall. For chromosomes, this failure of pairing and segregation leads to an unbalanced chromosome complement (aneuploidy), with its attendant problems of sterility and genetic disease. Two papers in this issue of PLoS Genetics [1],[2] demonstrate that telomere-promoted movements influence nearly every step in chromosome pairing and meiotic recombination, opening a new avenue to address questions that have intrigued biologists and vexed clinicians for over a hundred years. Chromosome movement is implicit in the classically recognized stages of meiotic prophase, but descriptions of directly visualized movements have been rare (see [3]). Early in prophase, chromosomes transition from having their centromeres clustered near the spindle pole (the Rabl orientation) to having their telomeres clustered at the nuclear periphery adjacent to the spindle pole (the bouquet orientation; see Figure 1). The bouquet stage ends with dispersal of telomeres across the inner nuclear envelope as chromosomes finalize their intimate pairing by forming synaptonemal complexes (SCs) that link chromosome pairs closely along their lengths. The formation of these intimate, exclusive partnerships would seem to finish the task at hand and to end the need for active, whole-chromosome movements, but this turns out not to be the case. Figure 1 Chromosomes Pass through Distinct Organizational Phases as They Negotiate Meiotic Prophase. Movements that persist throughout meiotic prophase were first described by Hiraokas group in the fission yeast Schizosaccharomyces pombe where, following bouquet formation, telomeres remain at the spindle pole while it leads the nucleus along microtubules, back and forth through the cell, until just before the first meiotic division [4]. Meiotic prophase is noncanonical in S. pombe in that synaptonemal complexes are not formed and recombination is not regulated to avoid forming crossovers near one another (i.e., there is no positive crossover interference). This has led some to question the generality of persistent movements. Recently, however, similarly persistent rapid prophase movements (RPMs) have been described in the budding yeast Saccharomyces cerevisiae [5]–[7]. Although these movements are of individual chromosomes rather than of the whole genomic complement, and although they appear to be promoted by actin rather than by microtubules, each system involves SUN domain–containing proteins that are known to mediate transnuclear envelope linkages, in the present case tethering telomeres to the cytoskeleton [8]–[10]. Such linkages also are present in mammalian meiotic nuclei [11],[12], indicating a widely conserved mechanism and suggesting conserved function(s). Before its role in these movements was recognized, the budding yeast Ndj1 protein was known to promote bouquet formation, the normal kinetics of SC formation, and the usual pattern of meiotic recombination; to maintain low levels of ectopic recombination (genetic exchanges between homologous DNA sequences in nonallelic locations); and, ultimately, to reduce the frequency of aneuploidy [13]–[17]. Ndj1 also plays a role in anchoring telomeres to the inner nuclear envelope [15], apparently by stabilizing the association of telomeres with the transmembrane SUN protein, Mps3 [10]. Reports that the meiosis-specific budding yeast protein Csm4 is similar to Ndj1 in being required to prevent aneuploidy [18] led the authors of the two current papers to ask, in remarkable molecular detail, whether the meiotic requirements for Csm4 are similar to those for Mps3 and Ndj1. The simple answer is “Yes,” but the angel is in the details. The authors find that Csm4, unlike Mps3 and Ndj1, is not required to anchor telomeres to the nuclear envelope but is required for telomeres to engage in the RPMs (see [7],[8]). Nevertheless, the impact on the progress of recombination, in all its currently understood molecular intricacies, is similar—delays in the appearances of recombination intermediates begin very early in prophase and persist or lengthen as prophase progresses. The implication of these observations is that the RPMs are the critical factor rather than telomere tethering to the nuclear envelope per se. So then, what is the role of the RPMs? Here, the authors diverge somewhat in their answers. The paper from the Shinohara lab proposes that RPMs promote the biochemistry of recombination more or less directly, perhaps by affecting chromosome structure [1]. The paper from the Alani and Kleckner labs proposes that RPMs function during an early phase when the cell determines which early recombination intermediates will become crossovers. They suggest that delays in this phase, perhaps due to a requirement for Ndj1 and Csm4 to resolve chromosome interlocks (at zygotene, see Figure 1), generates the subsequent defects [2]. Tests of these hypotheses will require considerable ingenuity in experimental design. A simple and striking conclusion from these papers is that mechanical energy, pumped into the nucleus via the telomeres, contributes critically to the work of genetic recombination. Identification of the MNC complex (Mps3, Ndj1, Csm4) in Sa. cerevisiae and of related structures and pathways in other organisms is only the beginning to understanding how these transnuclear envelope tethers are constructed and regulated. Understanding how these connections and the movements they foster contribute to the faithful segregation of chromosomes in meiosis will be challenging and rewarding, like the middle school dance.

A selfish DNA element engages a meiosis-specific motor and telomeres for germ-line propagation

The yeast 2 micron plasmid engages a meiosis-specific motor that orchestrates telomere-led chromosome movements for its telomere-associated segregation during meiosis I.

Slk19p of Saccharomyces cerevisiae Regulates Anaphase Spindle Dynamics Through Two Independent Mechanisms

Slk19p is a member of the Cdc-14 early anaphase release (FEAR) pathway, a signaling network that is responsible for activation of the cell-cycle regulator Cdc14p in Saccharomyces cerevisiae. Disruption of the FEAR pathway results in defects in anaphase, including alterations in the assembly and behavior of the anaphase spindle. Many phenotypes of slk19Δ mutants are consistent with a loss of FEAR signaling, but other phenotypes suggest that Slk19p may have FEAR-independent roles in modulating the behavior of microtubules in anaphase. Here, a series of SLK19 in-frame deletion mutations were used to test whether Slk19p has distinct roles in anaphase that can be ascribed to specific regions of the protein. Separation-of-function alleles were identified that are defective for either FEAR signaling or aspects of anaphase spindle function. The data suggest that in early anaphase one region of Slk19p is essential for FEAR signaling, while later in anaphase another region is critical for maintaining the coordination between spindle elongation and the growth of interpolar microtubules.

Time-Lapse Fluorescence Microscopy of Saccharomyces cerevisiae in Meiosis

Movements are implicit in the chromosome behaviors of bouquet formation, pairing and synapsis during meiotic prophase. In S. cerevisiae, the positions of chromosomes, specific structures, and individual chromosomal loci marked by fluorescent fusion proteins are easily visualized in living cells. Time-lapse analyses have revealed rapid and varied chromosome movements throughout meiotic prophase. To facilitate the analysis of these movements, we have developed a simple, inexpensive, and efficient method to prepare sporulating cells for fluorescence microscopy. This method produces a monolayer of cells that progress from meiosis through spore formation, allows visualization of hundreds of cells in a single high-resolution frame and is suitable for most methods of fluorescence microscopy.

Sister chromatid cohesion remodeling and meiotic recombination

Proper control of cohesion along the chromosome arms is essential for segregation of homologous chromosomes in meiosis. In a recent study we reported that Tid1p, a protein previously implicated in recombination, is required for resolution of Mcd1p-dependent cohesion in meiosis. Here we demonstrate that Pds5p and Dmc1p promote this cohesion. Pds5p is known to be required for maintenance of cohesion while Dmc1p is recognized as essential for meiotic recombination. Finding that the same defect in separation of sister chromatids could be suppressed by disrupting the functions of these proteins supports the emerging recognition that cohesion is remodeled during recombination and further indicates that cohesion is modified specifically to regulate meiotic recombination. We also find that overexpression of the regulatory subunit of Cdc7p kinase, Dbf4p, suppresses the tid1Δ sporulation defect, suggesting a role for Cdc7p/Dbf4p in regulating cohesion.