Cell Cycle Regulation In C. elegans

我们的实验室对染色体分离的过程以及这一过程中的缺陷如何影响多细胞有机体的发育感兴趣。在过去的几年里，我们一直专注于产生单倍体配子的减数分裂。我们一直在研究一类来自线虫的温度敏感(Ts)胚胎致死突变体，它们在减数分裂I中期停滞。在野生型动物中，减数分裂I前期的卵母细胞通过精子受精。受精后，卵母细胞的染色体经历了两次减数分裂，丢弃了极体中的额外染色体。这些第一次减数分裂是重要的，因为在这个阶段染色体分离的任何错误都可能导致胚胎的染色体数量异常，这可能会导致致命性。在我们的突变体中，卵母细胞的染色体停滞在减数分裂I的中期，并且永远不会分离它们的染色体同源物，也不会伸出极体。我们的减数分裂突变体定义了五个基因；它们编码后期促进复合体或环体(APC/C)的亚单位。这个复合体作为E3泛素连接酶，在细胞周期的中期到后期转变过程中，针对蛋白质进行破坏(由26S蛋白酶体)。我们将这些突变体命名为MAT，因为它们在减数分裂I的中期到后期过渡中存在缺陷。 为了确定这些APC/C亚基的基因外调节因子或底物，我们使用MAT-3突变体进行了遗传抑制筛选。我们的27个抑制子突变中的大多数是显性的。这些抑制基因已经用单核苷酸多态(SNP)技术进行了定位，并定义了至少9个互补组。大量等位基因代表着三个纺锤体检查点组件的突变。这些是线虫MAD1、MAD2和MAD3的同源物。当染色体没有正确地附着在有丝分裂的纺锤体上时，纺锤体检查点阻止中期到后期的过渡。我们的结果表明，这个检查点也在减数分裂过程中起作用。我们在MDF-1(秀丽线虫MAD1同源基因)中鉴定出1个等位基因，在MDF-3基因中鉴定出2个等位基因(MAD3同源基因)，在MDF-2基因中鉴定出12个等位基因(MAD2同源基因)。我们认为，我们的MAT突变体并没有触发检查点，而是检查点在减数分裂过程中作为APC/C的负调节因子正常工作。我们还鉴定了三个显性抑制基因，它们是APC/C正调控基因的突变，该基因被称为fzy-1，是CDc20/Fzy的直系同源基因。这三个突变聚集在蛋白质的一个小区域，被认为对其与MDF-2的相互作用非常重要。这些突变可能破坏了与MDF-2的相互作用，从而阻止了MDF-2对APC/C的抑制。 在过去的一年里，我们鉴定了另一个含有APC亚单位突变的抑制等位基因，如-1。我们之前测试了该基因在减数分裂中的作用(使用RNAi)，但未能发现早期胚胎表型。温度敏感的功能降低等位基因确实表明该基因在减数分裂过程中起作用；减数分裂的1-细胞胚胎在不允许的温度下观察到，但频率很低。在被抑制的菌株中，这种基因的RNAi使该菌株恢复到减数分裂1细胞停滞表型。这一发现有力地表明，我们的抑制等位基因是这样-1的功能获得等位基因。对该突变背景下的That-1基因进行测序证实，That-1的编码序列存在突变。我们的抑制子筛选有助于识别这种罕见的功能获得等位基因，它向我们揭示了这个APC亚单位可以在减数分裂过程中发挥作用。此类-1基因编码一个APC-5同源基因，有趣的是，线虫中有两个类似APC-5的基因。我们最近已经证明，另一个APC-5基因GFI-3在RNAi治疗的基础上并不是必需的。除了一个转座子插入等位基因外，GFI-3没有其他的突变。我们正在努力确定该等位基因是否有相关的表型。GFI-3的RNAi不增强其他APC突变体，而此类-1的RNAi则增强。上述这种-1功能缺失等位基因也增强了其他APC功能缺失表型。这些结果表明，这是一个减数分裂的APC-5亚基。该基因的缺失增强了弱的APC突变，而功能获得的等位基因则抑制了弱的APC突变。这一抑制数据表明，这种蛋白的功能增益突变版本可能维持多亚基APC的功能。我们对GFI-3亚基的假设是，这个APC-5亚基在其他组织中或在发育过程中的其他时间起作用。我们正试图使用GFP记者构造来证明这一预测。

Our lab is interested in the process of chromosome segregation and how defects in this process can affect the development of a multicellular organism. Over the past few years we have focused on the meiotic divisions that produce haploid gametes. We have been studying a class of temperature-sensitive (ts) embryonic lethal mutants from C. elegans that arrest in metaphase of meiosis I. In wildtype animals, oocytes in prophase of meiosis I are fertilized by sperm. Following fertilization, the oocyte chromosomes undergo two meiotic divisions, discarding the extra chromosomes in the polar bodies. These first meiotic divisions are important as any errors in chromosome segregation at this stage can lead to embryos with an abnormal number of chromosomes, which would likely lead to lethality. In our mutants, the oocyte chromosomes arrest in metaphase of meiosis I and never separate their chromosome homologs and never extrude polar bodies. Our meiotic mutants define five genes; they encode subunits of the Anaphase Promoting Complex or Cyclosome (APC/C). This complex serves as an E3 ubiquitin ligase that targets proteins for destruction (by the 26S proteasome) during the metaphase to anaphase transition of the cell cycle. We have named these mutants mat for their defects in the metaphase to anaphase transition during meiosis I. To identify extragenic regulators or substrates of these APC/C subunits, we have carried out a genetic suppression screen using a mat-3 mutant. The majority of our 27 suppressor mutations are dominant. These suppressors have been mapped using single nucleotide polymorphism (SNP) technology and define at least 9 complementation groups. A large number of alleles represent mutations in three spindle checkpoint components. These are the C. elegans orthologs of MAD1, MAD2, and MAD3. The spindle checkpoint prevents the metaphase to anaphase transition when chromosomes are not properly attached to the mitotic spindle. Our results suggest that this checkpoint also operates during meiosis. We identified one allele in the mdf-1 (the C. elegans Mad1 ortholog), two alleles in the mdf-3 gene (the Mad3 ortholog), and 12 alleles in the mdf-2 gene (the Mad2 ortholog). We believe that our mat mutants are not triggering the checkpoint, but rather that the checkpoint normally operates during meiosis as a negative regulator of the APC/C. Perhaps the checkpoint functions to regulate the proper timing of the meiotic divisions. We also identified three dominant suppressors that were mutations in a positive regulator of the APC/C. This gene is called fzy-1 and is the Cdc20/Fzy ortholog. These three mutations cluster in a small region of the protein thought to be important for its interaction with MDF-2. These mutations presumably disrupt the interaction with MDF-2 and thus prevent MDF-2 inhibition of the APC/C. In the past year, we have characterized another suppressor allele that harbors a mutation in an APC subunit, such-1. We had previously tested this gene for a role in the meiotic divisions (using RNAi) yet failed to find an early embryonic phenotype. A temperature-sensitive reduction-of-function allele does suggest that this gene functions during meiosis; meiotic 1-cell embryos are observed at the non-permissive temperature, but at a low frequency. RNAi of the such-1 gene in the suppressed strain reverts the strain back to the meiotic 1-cell arrest phenotype. This finding strongly suggests that our suppressor allele is a gain-of-function allele in such-1. Sequencing of the such-1 gene in this mutant background confirmed that such-1 harbored a mutation in its coding sequence. Our suppressor screen was instrumental in identifying this rare gain-of-function allele that revealed to us that this APC subunit could function during the meiotic divisions. The such-1 gene encodes an APC-5 ortholog and interestingly, there are two apc-5-like genes in C. elegans. We have recently shown that the other apc-5 gene, gfi-3, is not essential based on RNAi treatment. There are no existing mutations in gfi-3 other than a transposon insertion allele. We are working to determine if this allele has an associated phenotype. RNAi of gfi-3 does not enhance other APC mutants, while RNAi of such-1 does. The such-1 reduction-of-function allele mentioned above also does enhance other APC loss-of-function phenotypes. These results suggest that such-1 is a meiotic APC-5 subunit. Depletion of this gene enhances weak APC mutants while a gain-of-function allele suppresses weak APC mutants. This suppression data suggests that a mutant gain-of-function version of the SUCH-1 protein might maintain the function of the multisubunit APC. Our hypothesis for the GFI-3 subunit is that this APC-5 subunit acts in other tissues or at other times during development. We are trying to prove this prediction using GFP reporter constructs.