Molecular analysis of protein complexes involved in pairing of mammalian chromosomes during meiosis

Sammanfattning: How is the accuracy of chromosomal pairing and segregation processes in dividing meiotic cells assured? This is an important question as premature or erratic segregation of chromosomes can give rise to aneuploidy, which is the leading genetic cause of pregnancy loss in humans. Meiosis is a cell division process that produces genetically unique haploid gametes from diploid cells. An evolutionarily conserved and meiosis-specific protein complex, the synaptonemal complex (SC), is important for pairing (synapsis), recombination and segregation of homologous chromosomes. The SC is a proteinaceous structure that connects the homologous chromosomes during meiotic prophase 1. It consists of two axial elements (AEs) that hold the sister chromatids of each homologous chromosome together and transversal filaments (TF) that connect the AEs along the entire length of the chromosomes. Three genes encoding SC proteins in the mammalian system have so far been cloned. These are SCP1, which encodes a component of the TFs, and SCP2 and SCP3, which encode components of the AE. A second protein complex associated with meiotic chromosomes, the cohesin complex, has shown to be essential for sister chromatid cohesion during meiosis and mitosis. The meiotic cohesin complex subunits, REC8, STAG3, SMC1§ and SMC3, are part of an axial core that colocalises with the AEs of the SC in meiotic cells. The main focus of this thesis is to understand how mammalian homologous chromosomes synapse and segregate during meiosis. We have investigated how individual components of the SC and the cohesin complex are temporally organised and assembled into axial cores of homologous chromosomes. Using an SCP3-deficient mouse model system, it has previously been shown that SCP3 is required for AE assembly. We have used this model system to investigate the role of the AE and the function of SCP3 in male meiotic cells. We find that the cohesin core subunits are able to form an axial core in SCP3deficient cells, demonstrating that assembly of the axial core is independent of SCP3. We also find that lack of SCP3 transiently delays assembly of the cohesin core subunits, REC8, STAG3, SMC3 and SMC1§ early in prophase I and that the integrity of the mature axial core structures are affected by the absence of SCP3. We also show that SCP3 is not required for recombination or synapsis, since markers for these two processes are either not attached or are affected to a lesser extent, despite the absence of SCP3 and the AEs. Taken together, we conclude that neither the AEs nor SCP3 are required for axial core formation, recombination or synapsis. Rather, the axial core formed by cohesin subunits is likely to be essential for these processes. We have further characterised the function of SCP3 in vivo and in vitro, as well as the role of SCP3 in SCP2 and SCP1 function. We have studied the fiber-forming properties of all three SC proteins by overexpression in cultured somatic cells. We find that SCP3 self-assembles into thick, multistranded, cross-striated fibers that resemble the AEs of some organisms, whereas SCP2 and SCP1 form cellular foci. Strikingly, cotransfection of SCP3 and SCP2 in cultured cells generates novel fibrillar structures, which form short overlapping fibers. No colocalisation of SCP3 and SCS1 was observed, however, in parallel cotransfection. experiments. Notably, no SCP2 structures are detected in SCP3-null spermatocytes, whereas SCS1 forms TF-like fibers that contain striking axial gaps. We conclude that SCP2 depends on SCP3 for its assembly into AEs and that they interact in vivo. SCP1, however, does not physically interact with either SCP3 or SCP2. We propose that SCP3 constitutes the core of the AEs and that the AE functions as a molecular framework supporting axial core formation and synapsis.