The Role of the Nuclear Envelope in meiotic Prophase I events in C. elegans

During meiosis, homologous chromosomes from each parent must pair, synapse, and recombine before being assorted to the gametes. Any failure in this process leads to chromosome mis-segregation and aneuploidy. In C. elegans, to find the correct pairing partner, telomere-led chromosome movement occurs in a restricted subvolume of the nucleus. This feature is comparable to the widely conserved meiotic bouquet, a configuration where telomeres cluster in a limited area at the nuclear periphery. Chromosomes are moved by cytoskeletal forces transmitted via the SUN/KASH bridge across the nuclear envelope, and abrogation of movement leads to precocious non-homologous synapsis. The C. elegans inner nuclear envelope protein matefin/SUN-1 plays a conserved, pivotal role in chromosome movement. Matefin/SUN-1 is specifically phosphorylated at its nuclear N- terminus at multiple serines (on 6-7 residues) in leptotene/zygotene. SUN-1 phosphorylations coincide with the presence of clustered chromatin, the concentration of SUN-1 into aggregates at chromosome ends and chromosome movement. Substitution lines mimicking constitutive phosphorylation fail to dissolve chromosomal attachment plaques. Therefore SUN-1 phosphorylations define the time window of chromosome movement and regulate progression of recombination. Recent work has revealed that the individual SUN-1 modifications differ with respect to subcellular localization and phenotypes when mutated. Nevertheless all are under the control of the CHK-2 kinase. Besides other defects, chk-2 mutants fail to form SUN-1 aggregates. However, a substitution mimicking constitutive phosphorylation (S12E) cannot rescue the chk-2 phenotype, suggesting additional targets other than SUN-1. In the present project we would like to extend our understanding of the SUN-1 phosphorylation target sites. We plan to study their functional differences and hierarchical relationships and how they might alter the properties of the nuclear envelope. We want to examine how SUN-1 connects to chromosome ends, and if or how SUN-1 dimerization impacts on SUN-1 aggregate formation. One SUN-1 phospho-site (S12) stands out. A certain synapsis dependent recombination intermediate on each chromosome seems to regulate its nucleus wide dephosphorylation thereby possibly feeding into a mechanism to control that each chromosome pair receives a cross-over. The unpaired X chromosome in males must have evolved a strategy to escape this control, a feature we would like to investigate with this study.

Meiosis is defined a series of two specialist cell divisions, needed to produce haploid gametes. During the first meiotic division, faithful chromosome segregation is facilitated by the formation of a physical tether called crossover between the parental homologs. Crossover formation is accompanied by chromosome movement mediated by the conserved SUN/KASH bridge, which spans the nuclear envelope and connects chromosomes in the nucleus to cytoplasmic forces. These forces stir chromosomes, helping to bring homologs together and to inhibit undesired interactions. Unfaithful chromosome segregation leads to infertility, pregnancy loss, and conditions linked to mental retardation. In the frame of this project we conducted a study, where we present evidence that SUN-1 (as a component of the SUN/KASH bridge) in C. elegans is part of a meiotic surveillance mechanism that monitors events leading to crossing over and thus coordinates meiotic progression and chromosome movement with establishment of a crossover. SUN and KASH proteins are constituents of the inner and outer nuclear membranes. They interact in the perinuclear space to form the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex thereby bridging the nuclear envelope. LINC complexes mediate numerous biological processes by connecting chromatin with the cytoplasmic force generating machinery. In the course of this grant we also showed that the coiled-coil domains of SUN-1 are required for oligomerization and retention of the protein in the nuclear envelope, especially at later stages of female gametogenesis. Consistently, deletion of the coiled-coil domain makes SUN-1 sensitive to unilateral force exposure across the nuclear membrane. Premature loss of SUN-1 from the nuclear envelope leads to embryonic death due to loss of centrosome-nuclear envelope attachments. However, in contrast to previous notions we could show that the coiled-coil domain is dispensable for functional LINC complex formation, exemplified by successful crossover formation in meiotic prophase I in its absence.