Mammalian Sperm Head Formation Involves Different Polarization of Two Novel LINC Complexes

Background: LINC complexes are nuclear envelope bridging protein structures formed by interaction of SUN and KASH proteins. They physically connect the nucleus with the peripheral cytoskeleton and are critically involved in a variety of dynamic processes, such as nuclear anchorage, movement and positioning and meiotic chromosome dynamics. Moreover, they are shown to be essential for maintaining nuclear shape. Findings: Based on detailed expression analysis and biochemical approaches, we show here that during mouse sperm development, a terminal cell differentiation process characterized by profound morphogenic restructuring, two novel distinctive LINC complexes are established. They consist either of spermiogenesis-specific Sun3 and Nesprin1 or Sun1g, a novel non-nuclear Sun1 isoform, and Nesprin3. We could find that these two LINC complexes specifically polarize to opposite spermatid poles likely linking to sperm-specific cytoskeletal structures. Although, as shown in co-transfection/ immunoprecipitation experiments, SUN proteins appear to arbitrarily interact with various KASH partners, our study demonstrates that they actually are able to confine their binding to form distinct LINC complexes. Conclusions: Formation of the mammalian sperm head involves assembly and different polarization of two novel spermiogenesis-specific LINC complexes. Together, our findings suggest that theses LINC complexes connect the differentiating spermatid nucleus to surrounding cytoskeletal structures to enable its well-directed shaping and elongation

First molecular-cytogenetic characterization of Fanconi anemia fragile sites in primary lymphocytes of FA-D2 patients in different stages of the disease

Background: Fanconi anemia (FA) is a chromosomal instability syndrome characterized by increased frequency of chromosomal breakages, chromosomal radial figures and accelerated telomere shortening. In this work we performed detailed molecular-cytogenetic characterization of breakpoints in primary lymphocytes of FA-D2 patients in different stages of the disease using fluorescent in situ hybridization. Results: We found that chromosomal breakpoints co-localize on the molecular level with common fragile sites, whereas their distribution pattern depends on the severity of the disease. Telomere quantitative fluorescent in situ hybridization revealed that telomere fusions and radial figures, especially radials which involve telomere sequences are the consequence of critically shortened telomeres that increase with the disease progression and could be considered as a predictive parameter during the course of the disease. Sex chromosomes in FA cells are also involved in radial formation indicating that specific X chromosome regions share homology with autosomes and also could serve as repair templates in resolving DNA damage. Conclusions: FA-D2 chromosomal breakpoints co-localize with common fragile sites, but their distribution pattern depends on the disease stage. Telomere fusions and radials figures which involve telomere sequences are the consequence of shortened telomeres, increase with disease progression and could be of predictive value

Live cell imaging of meiotic chromosome dynamics in yeast

Recombination in first meiotic prophase is initiated by endogenous breaks in double-stranded DNA (DSBs) which occurs during a time when chromosomes are remodeled and proteinaceous cores (axes) are assembled along their length. DSBs are instrumental in homologue recognition and underlie the crossovers that form between parental chromosomes to ensure genome haploidization during the following two successive meiotic divisions. Advances in fluorescence microscopy and genetic engineering of GFP-tagged fusion proteins have made it possible to observe the behavior of entire chromosomes and specific subregions in live cells of the yeast Saccharomyces cerevisiae. In meiosis we observed that telomeres are dynamic and move about the entire nuclear periphery, only interrupted by their fleeting clustering at the spindle pole body (the centrosome equivalent), known as bouquet formation. This mobility translates to whole chromosomes and nuclei during the entire prophase I. Here we describe a simple setup for live cell microscopy that we used to observe chromosome movements during a time when DSBs are formed and transform into crossover and non-crossover products

Cell Biol Int.

Mutations in the lamin A gene have been shown, among other defects, to give rise to Hutchinson-Gilford progeria syndrome (HGPS) and to atypical Werner syndrome (WS), both of which are progeroid disorders. Here, we have investigated well-characterized WS patient cell strains that are compound heterozygous for mutations in the WRN gene. As in HGPS and in atypical WS, we found nuclear deformations to be characteristic of all cell strains studied. In WS cells centrosome number, assembly of the nuclear lamina and nuclear pore distribution occurred normally. Furthermore, nuclear deformations were not associated with a defect in lamin A expression. We propose that nuclear deformation is a universal characteristic of progeroid cells and may result from slow cell cycle progression

Biol. Chem.

Electrophoretic mobility shift assays (EMSA) revealed that under standard cell culture conditions NF- B was induced in Fanconi anemia fibroblasts in contrast to control cells. Dithiothreitol, a potent synthetic redox potential-delivering compound, when added to growing cells, prevented this induction of NF- B and, simultaneously, chromosomal instability was reduced. Fanconi anemia cells possess low endogenous levels of the naturally occurring antioxidant thioredoxin. Transfection of Fanconi anemia cells with thioredoxin cDNA containing a nuclear localization signal prevented both spontaneous as well as mitomycin C-induced chromosomal instability. A promotor construct with two NF- B binding sites in front of the CAT gene induced little CAT expression in cells with low thioredoxin content in spite of induced NF-[kappa]B. In cells with higher thioredoxin content CAT expression was increased. Cotransfection of the NF-[kappa]B-dependent CAT plasmid with the Trx/nuc-plasmid into FA fibroblasts increased the CAT expression to almost that of control cells, indicating that in this model system with diminished thioredoxin content NF-[kappa]B requires thioredoxin for binding to its specific promotor. Since Fanconi anemia cells have low thioredoxin contents, NF-[kappa]B-dependent genes are expressed insufficiently. This explains part of the pathophysiological processes observed in Fanconi anemia

FEBS Lett.

Werner’s syndrome is a rare disease of premature ageing. The WRN gene product defective in this disorder belongs to the RecQ helicase family and is thought to be involved in DNA metabolism. Another protein, which plays an important role in both DNA replication and repair, is the poly-ADP ribosyl transferase. Here we demonstrate an interaction of these two proteins resulting in ADP-ribosylation of the WRN protein. These results imply that WRN is involved in DNA replication and in DNA repair

Synthèse des analogues hétérocycliques des alcaloïdes, Camptothécine, Rutaecarpine, et Luotonine (agents à fort potentiel cytotoxique)

LE HAVRE-BU Centrale (763512101) / SudocSudocFranceF

Chromosome mobility during meiotic prophase in Saccharomyces cerevisiae

In many organisms, a synaptonemal complex (SC) intimately connects each pair of homologous chromosomes during much of the first meiotic prophase and is thought to play a role in regulating recombination. In the yeast Saccharomyces cerevisiae, the central element of each SC contains Zip1, a protein orthologous to mammalian SYCP1. To study the dynamics of SCs in living meiotic cells, a functional ZIP1::GFP fusion was introduced into yeast and analyzed by fluorescence video microscopy. During pachytene, SCs exhibited dramatic and continuous movement throughout the nucleus, traversing relatively large distances while twisting, folding, and unfolding. Chromosomal movements were accompanied by changes in the shape of the nucleus, and all movements were reversibly inhibited by the actin antagonist Latrunculin B. Normal movement required the NDJ1 gene, which encodes a meiosis-specific telomere protein needed for the attachment of telomeres to the nuclear periphery and for normal kinetics of recombination and meiosis. These results show that SC movements involve telomere attachment to the nuclear periphery and are actin-dependent and suggest these movements could facilitate completion of meiotic recombination