Spermatocytes have the capacity to segregate chromosomes despite centriole duplication failure.

Centrosomes are the canonical microtubule organizing centers (MTOCs) of most mammalian cells, including spermatocytes. Centrosomes comprise a centriole pair within a structurally ordered and dynamic pericentriolar matrix (PCM). Unlike in mitosis, where centrioles duplicate once per cycle, centrioles undergo two rounds of duplication during spermatogenesis. The first duplication is during early meiotic prophase I, and the second is during interkinesis. Using mouse mutants and chemical inhibition, we have blocked centriole duplication during spermatogenesis and determined that non-centrosomal MTOCs (ncMTOCs) can mediate chromosome segregation. This mechanism is different from the acentriolar MTOCs that form bipolar spindles in oocytes, which require PCM components, including gamma-tubulin and CEP192. From an in-depth analysis, we identified six microtubule-associated proteins, TPX2, KIF11, NuMA, and CAMSAP1-3, that localized to the non-centrosomal MTOC. These factors contribute to a mechanism that ensures bipolar MTOC formation and chromosome segregation during spermatogenesis when centriole duplication fails. However, despite the successful completion of meiosis and round spermatid formation, centriole inheritance and PLK4 function are required for normal spermiogenesis and flagella assembly, which are critical to ensure fertility.

BLM helicase ortholog Sgs1 is a central regulator of meiotic recombination intermediate metabolism.

The BLM helicase has been shown to maintain genome stability by preventing accumulation of aberrant recombination intermediates. We show here that the Saccharomyces cerevisiae BLM ortholog, Sgs1, plays an integral role in normal meiotic recombination, beyond its documented activity limiting aberrant recombination intermediates. In wild-type meiosis, temporally and mechanistically distinct pathways produce crossover and noncrossover recombinants. Crossovers form late in meiosis I prophase, by polo kinase-triggered resolution of Holliday junction (HJ) intermediates. Noncrossovers form earlier, via processes that do not involve stable HJ intermediates. In contrast, sgs1 mutants abolish early noncrossover formation. Instead, both noncrossovers and crossovers form by late HJ intermediate resolution, using an alternate pathway requiring the overlapping activities of Mus81-Mms4, Yen1, and Slx1-Slx4, nucleases with minor roles in wild-type meiosis. We conclude that Sgs1 is a primary regulator of recombination pathway choice during meiosis and suggest a similar function in the mitotic cell cycle.

Meiotic recombination intermediates are resolved with minimal crossover formation during return-to-growth, an analogue of the mitotic cell cycle.

Accurate segregation of homologous chromosomes of different parental origin (homologs) during the first division of meiosis (meiosis I) requires inter-homolog crossovers (COs). These are produced at the end of meiosis I prophase, when recombination intermediates that contain Holliday junctions (joint molecules, JMs) are resolved, predominantly as COs. JM resolution during the mitotic cell cycle is less well understood, mainly due to low levels of inter-homolog JMs. To compare JM resolution during meiosis and the mitotic cell cycle, we used a unique feature of Saccharomyces cerevisiae, return to growth (RTG), where cells undergoing meiosis can be returned to the mitotic cell cycle by a nutritional shift. By performing RTG with ndt80 mutants, which arrest in meiosis I prophase with high levels of interhomolog JMs, we could readily monitor JM resolution during the first cell division of RTG genetically and, for the first time, at the molecular level. In contrast to meiosis, where most JMs resolve as COs, most JMs were resolved during the first 1.5-2 hr after RTG without producing COs. Subsequent resolution of the remaining JMs produced COs, and this CO production required the Mus81/Mms4 structure-selective endonuclease. RTG in sgs1-ΔC795 mutants, which lack the helicase and Holliday junction-binding domains of this BLM homolog, led to a substantial delay in JM resolution; and subsequent JM resolution produced both COs and NCOs. Based on these findings, we suggest that most JMs are resolved during the mitotic cell cycle by dissolution, an Sgs1 helicase-dependent process that produces only NCOs. JMs that escape dissolution are mostly resolved by Mus81/Mms4-dependent cleavage that produces both COs and NCOs in a relatively unbiased manner. Thus, in contrast to meiosis, where JM resolution is heavily biased towards COs, JM resolution during RTG minimizes CO formation, thus maintaining genome integrity and minimizing loss of heterozygosity.

Amyloids and yeast prion biology.

The prions (infectious proteins) of Saccharomyces cerevisiae are proteins acting as genes, by templating their conformation from one molecule to another in analogy to DNA templating its sequence. Most yeast prions are amyloid forms of normally soluble proteins, and a single protein sequence can have any of several self-propagating forms (called prion strains or variants), analogous to the different possible alleles of a DNA gene. A central issue in prion biology is the structural basis of this conformational templating process. The in-register parallel ß sheet structure found for several infectious yeast prion amyloids naturally suggests an explanation for this conformational templating. While most prions are plainly diseases, the [Het-s] prion of Podospora anserina may be a functional amyloid, with important structural implications. Yeast prions are important models for human amyloid diseases in general, particularly because new evidence is showing infectious aspects of several human amyloidoses not previously classified as prions. We also review studies of the roles of chaperones, aggregate-collecting proteins, and other cellular components using yeast that have led the way in improving the understanding of similar processes that must be operating in many human amyloidoses.

Functional conservation of RecQ helicase BLM between humans and Drosophila melanogaster.

RecQ helicases are a family of proteins involved in maintaining genome integrity with functions in DNA repair, recombination, and replication. The human RecQ helicase family consists of five helicases: BLM, WRN, RECQL, RECQL4, and RECQL5. Inherited mutations in RecQ helicases result in Bloom Syndrome (BLM mutation), Werner Syndrome (WRN mutation), Rothmund-Thomson Syndrome (RECQL4 mutation), and other genetic diseases, including cancer. The RecQ helicase family is evolutionarily conserved, as Drosophila melanogaster have three family members: DmBlm, DmRecQL4, and DmRecQL5 and DmWRNexo, which contains a conserved exonuclease domain. DmBlm has functional similarities to human BLM (hBLM) as mutants demonstrate increased sensitivity to ionizing radiation (IR) and a decrease in DNA double-strand break (DSB) repair. To determine the extent of functional conservation of RecQ helicases, hBLM was expressed in Drosophila using the GAL4 > UASp system to determine if GAL4 > UASp::hBLM can rescue DmBlm mutant sensitivity to IR. hBLM was able to rescue female DmBlm mutant sensitivity to IR, supporting functional conservation. This functional conservation is specific to BLM, as human GAL4 > UASp::RECQL was not able to rescue DmBlm mutant sensitivity to IR. These results demonstrate the conserved role of BLM in maintaining the genome while reinforcing the applicability of using Drosophila as a model system to study Bloom Syndrome.

Hypobaria during aeromedical evacuation exacerbates histopathological injury and modifies inflammatory response in rats exposed to blast overpressure injury.

BACKGROUND: Aeromedical evacuation (AE) is often used as a rapid and effective way to evacuate patients. However, little is known about the possible effects of AE on patients with blast and traumatic brain injury. In the current study, we used blast overpressure (BOP) as a method to introduce traumatic brain injury in rats and investigated the effects of hypobaria during AE on histology and inflammatory response. METHODS: Animals were exposed to a 12-hour flight 2 days after BOP and euthanized 48 hours after flight. Control animals were kept at normobaria. RESULTS: Overall, BOP animals exposed to flight demonstrated higher histopathologic injury scores as compared to control animals in lungs, brain, kidney, heart, and intestine. The BOP animals exposed to normobaria exhibited a proinflammatory response compared to those that were not blasted, an observation that was not seen in BOP animals exposed to hypobaria. CONCLUSION: These data suggest that AE 48 hours post blast may lead to impairment in the inflammatory process and worsening of long-term outcomes. LEVEL OF EVIDENCE: Animal research, level II.

Analogs of the novel phytohormone, strigolactone, trigger apoptosis and synergize with PARP inhibitors by inducing DNA damage and inhibiting DNA repair.

Strigolactones are a novel class of plant hormones produced in roots that regulate shoot and root development. We previously reported that strigolactone analogs (SLs) induce G2/M cell cycle arrest and apoptosis in a variety of human cancer cells and inhibit tumor growth of human breast cancer xenografts in mice. SLs had no significant influences on non-transformed cells. Here we report for the first time that SLs induce DNA damage in the form of DNA double-strand breaks (DSBs) and activate the DNA damage response signaling by inducing phosphorylation of ATM, ATR and DNA-PKcs and co-localization of the DNA damage signaling protein, 53BP1, with γH2AX nuclear foci. We further report that in addition to DSBs induction, SLs simultaneously impair DSBs repair, mostly homology-directed repair (HDR) and to a lesser extent non-homologous end joining (NHEJ). In response to SLs, RAD51, the homologous DSB repair protein, is ubiquitinated and targeted for proteasomal degradation and it fails to co-localize with γH2AX foci. Interestingly, SLs synergize with DNA damaging agents-based therapeutics. The combination of PARP inhibitors and SLs showed an especially potent synergy, but only in BRCA1-proficient cells. No synergy was observed between SLs and PARP inhibitors in BRCA1-deficient cells, supporting a role for SLs in HDR impairment. Together, our data suggest that SLs increase genome instability and cell death by a unique mechanism of inducing DNA damage and inhibiting DNA repair.

The nuclear lamina and its functions in the nucleus.

The nuclear lamina is a structure near the inner nuclear membrane and the peripheral chromatin. It is composed of lamins, which are also present in the nuclear interior, and lamin-associated proteins. The increasing number of proteins that interact with lamins and the compound interactions between these proteins and chromatin-associated proteins make the nuclear lamina a highly complex but also a very exciting structure. The nuclear lamina is an essential component of metazoan cells. It is involved in most nuclear activities including DNA replication, RNA transcription, nuclear and chromatin organization, cell cycle regulation, cell development and differentiation, nuclear migration, and apoptosis. Specific mutations in nuclear lamina genes cause a wide range of heritable human diseases. These diseases include Emery-Dreifuss muscular dystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy (DCM) with conduction system disease, familial partial lipodystrophy (FPLD), autosomal recessive axonal neuropathy (Charcot-Marie-Tooth disorder type 2, CMT2), mandibuloacral dysplasia (MAD), Hutchison Gilford Progeria syndrome (HGS), Greenberg Skeletal Dysplasia, and Pelger-Huet anomaly (PHA). Genetic analyses in Caenorhabditis elegans, Drosophila, and mice show new insights into the functions of the nuclear lamina, and recent structural analyses have begun to unravel the molecular structure and assembly of lamins and their associated proteins.

Yeast prions: structure, biology, and prion-handling systems.

A prion is an infectious protein horizontally transmitting a disease or trait without a required nucleic acid. Yeast and fungal prions are nonchromosomal genes composed of protein, generally an altered form of a protein that catalyzes the same alteration of the protein. Yeast prions are thus transmitted both vertically (as genes composed of protein) and horizontally (as infectious proteins, or prions). Formation of amyloids (linear ordered ß-sheet-rich protein aggregates with ß-strands perpendicular to the long axis of the filament) underlies most yeast and fungal prions, and a single prion protein can have any of several distinct self-propagating amyloid forms with different biological properties (prion variants). Here we review the mechanism of faithful templating of protein conformation, the biological roles of these prions, and their interactions with cellular chaperones, the Btn2 and Cur1 aggregate-handling systems, and other cellular factors governing prion generation and propagation. Human amyloidoses include the PrP-based prion conditions and many other, more common amyloid-based diseases, several of which show prion-like features. Yeast prions increasingly are serving as models for the understanding and treatment of many mammalian amyloidoses. Patients with different clinical pictures of the same amyloidosis may be the equivalent of yeasts with different prion variants.

Amyloid diseases of yeast: prions are proteins acting as genes.

The unusual genetic properties of the non-chromosomal genetic elements [URE3] and [PSI+] led to them being identified as prions (infectious proteins) of Ure2p and Sup35p respectively. Ure2p and Sup35p, and now several other proteins, can form amyloid, a linear ordered polymer of protein monomers, with a part of each molecule, the prion domain, forming the core of this ß-sheet structure. Amyloid filaments passed to a new cell seed the conversion of the normal form of the protein into the same amyloid form. The cell's phenotype is affected, usually from the deficiency of the normal form of the protein. Solid-state NMR studies indicate that the yeast prion amyloids are in-register parallel ß-sheet structures, in which each residue (e.g. Asn35) forms a row along the filament long axis. The favourable interactions possible for aligned identical hydrophilic and hydrophobic residues are believed to be the mechanism for propagation of amyloid conformation. Thus, just as DNA mediates inheritance by templating its own sequence, these proteins act as genes by templating their conformation. Distinct isolates of a given prion have different biological properties, presumably determined by differences between the amyloid structures. Many lines of evidence indicate that the Saccharomyces cerevisiae prions are pathological disease agents, although the example of the [Het-s] prion of Podospora anserina shows that a prion can have beneficial aspects.

Age-related changes of nuclear architecture in Caenorhabditis elegans.

Mutations in lamins cause premature aging syndromes in humans, including the Hutchinson-Gilford Progeria Syndrome (HGPS) and Atypical Werner Syndrome. It has been shown that HGPS cells in culture undergo age-dependent progressive changes in nuclear architecture. However, it is unknown whether similar changes in nuclear architecture occur during the normal aging process. We have observed that major changes of nuclear architecture accompany Caenorhabditis elegans aging. We found that the nuclear architecture in most nonneuronal cell types undergoes progressive and stochastic age-dependent alterations, such as changes of nuclear shape and loss of peripheral heterochromatin. Furthermore, we show that the rate of these alterations is influenced by the insulin/IGF-1 like signaling pathway and that reducing the level of lamin and lamin-associated LEM domain proteins leads to shortening of lifespan. Our work not only provides evidence for changes of nuclear architecture during the normal aging process of a multicellular organism, but also suggests that HGPS is likely a result of acceleration of the normal aging process. Because the nucleus is vital for many cellular functions, our studies raise the possibility that the nucleus is a prominent focal point for regulating aging.