Transverse Filaments Research Articles

SIMULTANEOUS TRANSVERSE OSCILLATIONS OF A PROMINENCE AND A FILAMENT AND LONGITUDINAL OSCILLATION OF ANOTHER FILAMENT INDUCED BY A SINGLE SHOCK WAVE

We present the first stereoscopic and Doppler observations of simultaneous transverse oscillations of a prominence and a filament and longitudinal oscillation of another filament launched by a single shock wave. Using H-alpha Doppler observations, we derive the three-dimensional oscillation velocities at different heights along the prominence axis. The results indicate that the prominence has a larger oscillation amplitude and damping time at higher altitude, but the periods at different heights are the same (i.e., 13.5 minutes). This suggests that the prominence oscillates like a linear vertical rigid body with one end anchored on the Sun. One of the filaments shows weak transverse oscillation after the passing of the shock, which is possibly due to the low altitude of the filament and the weakening (due to reflection) of the shock wave before the interaction. Large amplitude longitudinal oscillation is observed in the other filament after the passing of the shock wave. The velocity amplitude and period are about 26.8 km/s and 80.3 minutes, respectively. We propose that the orientation of a filament or prominence relative to the normal vector of the incoming shock should be an important factor for launching transverse or longitudinal filament oscillations. In addition, the restoring forces of the transverse prominence are most likely due to the coupling of gravity and magnetic tension of the supporting magnetic field, while that for the longitudinal filament oscillation is probably the resultant force of gravity and magnetic pressure.

Non-filamentated ultra-intense and ultra-short pulse fronts in three-dimensional Raman seed amplification

Ultra-intense and ultra-short laser pulses may be generated up to the exawatt-zetawatt regime due to parametric processes in plasmas. The minimization of unwanted plasma processes leads to operational limits which are discussed here with respect to filamentation. Transverse filamentation, which originally was derived for plane waves, is being investigated for seed pulse propagation in the so called π-pulse limit. A three-dimensional (3D) three-wave-interaction model is the basis of the present investigation. To demonstrate the applicability of the three-wave-interaction model, the 1D pulse forms are compared with those obtained from 1D particle in cell and Vlasov simulations. Although wave-breaking may occur, the kinetic simulations show that the leading pumped pulse develops a form similar to that obtained from the three-wave-interaction model. In the main part, 2D and 3D filamentation processes of (localized) pulses are investigated with the three-wave-interaction model. It is shown that the leading pulse front can stay filamentation-free, whereas the rear parts show transverse modulations.

Phylogenies of Central Element Proteins Reveal the Dynamic Evolutionary History of the Mammalian Synaptonemal Complex: Ancient and Recent Components

During meiosis, the stable pairing of the homologous chromosomes is mediated by the assembly of the synaptonemal complex (SC). Its tripartite structure is well conserved in Metazoa and consists of two lateral elements (LEs) and a central region (CR) that in turn is formed by several transverse filaments (TFs) and a central element (CE). In a previous article, we have shown that not only the structure, but also the major structural proteins SYCP1 (TFs) and SYCP3 (LEs) of the mammalian SC are conserved in metazoan evolution. In continuation of this work, we now investigated the evolution of the mammalian CE-specific proteins using phylogenetic and biochemical/cytological approaches. In analogy to the observations made for SYCP1 and SYCP3, we did not detect homologs of the mammalian CE proteins in insects or nematodes, but in several other metazoan clades. We were able to identify homologs of three mammalian CE proteins in several vertebrate and invertebrate species, for two of these proteins down to the basal-branching phylum of Cnidaria. Our approaches indicate that the SC arose only once, but evolved dynamically during diversification of Metazoa. Certain proteins appear to be ancient in animals, but successive addition of further components as well as protein loss and/or replacements have also taken place in some lineages.

CENTRAL REGION COMPONENT1, a Novel Synaptonemal Complex Component, Is Essential for Meiotic Recombination Initiation in Rice

In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated central region component1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID receptor-interacting protein13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with homologous pairing aberration in rice meiosis1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13.

The Cohesion Protein SOLO Associates with SMC1 and Is Required for Synapsis, Recombination, Homolog Bias and Cohesion and Pairing of Centromeres in Drosophila Meiosis

Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome cores.

Multidimensional modeling of biofilm development and fluid dynamics in a hydrogen-based, membrane biofilm reactor (MBfR)

A two-dimensional, particle-based biofilm model coupled with mass transport and computational fluid dynamics was developed to simulate autotrophic denitrification in a spiral-wound membrane biofilm reactor (MBfR), where hydrogen is supplied via hollow-fiber membrane fabric. The spiral-wound configuration consists of alternating layers of plastic spacer net and membrane fabric that create rows of flow channels, with the top and bottom walls comprised of membranes. The transversal filaments of the spacer partially obstruct the channel flow, producing complex mixing and shear patterns that require multidimensional representation. This study investigated the effect of hydrogen and nitrate concentrations, as well as spacer configuration, on biofilm development and denitrification fluxes. The model results indicate that the cavity spacer filaments, which rest on the bottom membranes, cause uneven biofilm growth. Most biofilm resided on the bottom membranes, only in the wake of the filaments where low shear zones formed. In this way, filament configuration may help achieve a desired biofilm thickness. For the conditions tested in this study, the highest nitrate fluxes were attained by minimizing the filament diameter and maximizing the filament spacing. This lowered the shear stress at the top membranes, allowing for more biofilm growth. For the scenarios studied, biomass limitation at the top membranes hindered performance more significantly than diffusion limitation in the thick biofilms at the bottom membranes. The results also highlighted the importance of two-dimensional modeling to capture uneven biofilm growth on a substratum with geometrical complexity.

Quantitative high resolution mapping of HvMLH3 foci in barley pachytene nuclei reveals a strong distal bias and weak interference.

In barley (Hordeum vulgare L.), chiasmata (the physical sites of genetic crossovers) are skewed towards the distal ends of chromosomes, effectively consigning a large proportion of genes to recombination coldspots. This has the effect of limiting potential genetic variability, and of reducing the efficiency of map-based cloning and breeding approaches for this crop. Shifting the sites of recombination to more proximal chromosome regions by forward and reverse genetic means may be profitable in terms of realizing the genetic potential of the species, but is predicated upon a better understanding of the mechanisms governing the sites of these events, and upon the ability to recognize real changes in recombination patterns. The barley MutL Homologue (HvMLH3), a marker for class I interfering crossovers, has been isolated and a specific antibody has been raised. Immunolocalization of HvMLH3 along with the synaptonemal complex transverse filament protein ZYP1, used in conjunction with fluorescence in situ hybridization (FISH) tagging of specific barley chromosomes, has enabled access to the physical recombination landscape of the barley cultivars Morex and Bowman. Consistent distal localization of HvMLH3 foci throughout the genome, and similar patterns of HvMLH3 foci within bivalents 2H and 3H have been observed. A difference in total numbers of HvMLH3 foci between these two cultivars has been quantified, which is interpreted as representing genotypic variation in class I crossover frequency. Discrepancies between the frequencies of HvMLH3 foci and crossover frequencies derived from linkage analysis point to the existence of at least two crossover pathways in barley. It is also shown that interference of HvMLH3 foci is relatively weak compared with other plant species.

The Ecm11-Gmc2 Complex Promotes Synaptonemal Complex Formation through Assembly of Transverse Filaments in Budding Yeast

During meiosis, homologous chromosomes pair at close proximity to form the synaptonemal complex (SC). This association is mediated by transverse filament proteins that hold the axes of homologous chromosomes together along their entire length. Transverse filament proteins are highly aggregative and can form an aberrant aggregate called the polycomplex that is unassociated with chromosomes. Here, we show that the Ecm11-Gmc2 complex is a novel SC component, functioning to facilitate assembly of the yeast transverse filament protein, Zip1. Ecm11 and Gmc2 initially localize to the synapsis initiation sites, then throughout the synapsed regions of paired homologous chromosomes. The absence of either Ecm11 or Gmc2 substantially compromises the chromosomal assembly of Zip1 as well as polycomplex formation, indicating that the complex is required for extensive Zip1 polymerization. We also show that Ecm11 is SUMOylated in a Gmc2-dependent manner. Remarkably, in the unSUMOylatable ecm11 mutant, assembly of chromosomal Zip1 remained compromised while polycomplex formation became frequent. We propose that the Ecm11-Gmc2 complex facilitates the assembly of Zip1 and that SUMOylation of Ecm11 is critical for ensuring chromosomal assembly of Zip1, thus suppressing polycomplex formation.

Experimental Study of Current Filamentation Instability

Current filamentation instability is observed and studied in a laboratory environment with a 60 MeV electron beam and a plasma capillary discharge. Multiple filaments are observed and imaged transversely at the plasma exit with optical transition radiation. By varying the plasma density the transition between single and multiple filaments is found to be k(p)σ(r)~2.2. Scaling of the transverse filament size with the plasma skin depth is predicted in theory and observed over a range of plasma densities. Lowering the bunch charge, and thus the bunch density, suppresses the instability.

Rumors of Its Disassembly Have Been Greatly Exaggerated: The Secret Life of the Synaptonemal Complex at the Centromeres

Rumors of Its Disassembly Have Been Greatly Exaggerated: The Secret Life of the Synaptonemal Complex at the Centromeres

Synaptonemal Complex Components Persist at Centromeres and Are Required for Homologous Centromere Pairing in Mouse Spermatocytes

Recent studies in simple model organisms have shown that centromere pairing is important for ensuring high-fidelity meiotic chromosome segregation. However, this process and the mechanisms regulating it in higher eukaryotes are unknown. Here we present the first detailed study of meiotic centromere pairing in mouse spermatogenesis and link it with key events of the G2/metaphase I transition. In mouse we observed no evidence of the persistent coupling of centromeres that has been observed in several model organisms. We do however find that telomeres associate in non-homologous pairs or small groups in B type spermatogonia and pre-leptotene spermatocytes, and this association is disrupted by deletion of the synaptonemal complex component SYCP3. Intriguingly, we found that, in mid prophase, chromosome synapsis is not initiated at centromeres, and centromeric regions are the last to pair in the zygotene-pachytene transition. In late prophase, we first identified the proteins that reside at paired centromeres. We found that components of the central and lateral element and transverse filaments of the synaptonemal complex are retained at paired centromeres after disassembly of the synaptonemal complex along diplotene chromosome arms. The absence of SYCP1 prevents centromere pairing in knockout mouse spermatocytes. The localization dynamics of SYCP1 and SYCP3 suggest that they play different roles in promoting homologous centromere pairing. SYCP1 remains only at paired centromeres coincident with the time at which some kinetochore proteins begin loading at centromeres, consistent with a role in assembly of meiosis-specific kinetochores. After removal of SYCP1 from centromeres, SYCP3 then accumulates at paired centromeres where it may promote bi-orientation of homologous centromeres. We propose that, in addition to their roles as synaptonemal complex components, SYCP1 and SYCP3 act at the centromeres to promote the establishment and/or maintenance of centromere pairing and, by doing so, improve the segregation fidelity of mammalian meiotic chromosomes.

Mass-transfer entrance effects in narrow rectangular channels with ribbed walls or mesh-type spacers

Entrance effects on the Sherwood number are investigated in a narrow rectangular channel, both with ribbed walls and filled with different mesh-type spacers, and for Reynolds numbers, based on the channel height, varying between 5 and 500. Mass transfer coefficients were measured by the limiting current technique, using the ferri–ferro cyanide redox couple with 0.5M potassium carbonate in a test cell with a narrow rectangular channel (15mm×2mm×170mm) and 8 consecutive segmented nickel electrodes (11.2mm long and 10mm wide) fitted in the bottom wall. Five mesh-type spacers made of two layers of parallel filaments (1mm diameter) and having different flow attack angles were tested. The ribbed walls tested had transverse square ribs (1mm×1mm) uniformly spaced, with inter-ribs distances of 3.8, 7.6 and 11.35mm. Average Sherwood number for each electrode was measured for both cases of the upstream electrodes being active (under limiting current conditions) or inactive (without current). Results show that for stable laminar flow regime strong entrance effects can prevail over the entire channel whenever transverse or oblique filaments are not in contact with the electrodes. Diamond-shape spacers presented no mass-transfer entrance effects even at a lowest Reynolds number of 5. In the transitional flow regime, i.e., at Re above the critical value, the Sherwood number decreases sharply in the first periodic channel segments with posterior stabilization for all the spacer configurations tested. This work gives guidelines for using periodic boundary conditions in the CFD simulation of flow and mass-transfer inside spacer-filled narrow rectangular channels with relevance for membrane modules design.

The isolation and characterisation of the wheat molecular ZIPper I homologue, TaZYP1.

BackgroundThe synaptonemal complex (SC) is a proteinaceous tripartite structure used to hold homologous chromosomes together during the early stages of meiosis. The yeast ZIP1 and its homologues in other species have previously been characterised as the transverse filament protein of the synaptonemal complex. Proper installation of ZYP1 along chromosomes has been shown to be dependent on the axial element-associated protein, ASY1 in Arabidopsis.ResultsHere we report the isolation of the wheat (Triticum aestivum) ZYP1 (TaZYP1) and its expression profile (during and post-meiosis) in wild-type, the ph1b deletion mutant as well as in Taasy1 RNAi knock-down mutants. TaZYP1 has a putative DNA-binding S/TPXX motif in its C-terminal region and we provide evidence that TaZYP1 interacts non-preferentially with both single- and double-stranded DNA in vitro. 3-dimensional dual immunofluorescence localisation assays conducted with an antibody raised against TaZYP1 show that TaZYP1 interacts with chromatin during meiosis but does not co-localise to regions of chromatin where TaASY1 is present. The TaZYP1 signal lengthens into regions of chromatin where TaASY1 has been removed in wild-type but this appears delayed in the ph1b mutant. The localisation profile of TaZYP1 in four Taasy1 knock-down mutants is similar to wild-type but TaZYP1 signal intensity appears weaker and more diffused.ConclusionsIn contrast to previous studies performed on plant species where ZYP1 signal is sandwiched by ASY1 signal located on both axial elements of the SC, data from the 3-dimensional dual immunofluorescence localisation assays conducted in this study show that TaZYP1 signal only lengthens into regions of chromatin after TaASY1 signal is being unloaded. However, the observation that TaZYP1 loading appears delayed in both the ph1b and Taasy1 mutants suggests that TaASY1 may still be essential for TaZYP1 to play a role in SC formation during meiosis. These data further suggest that the temporal installation of ZYP1 onto pairing homologous chromosomes in wheat is different to that of other plant species and highlights the need to study this synaptonemal complex protein on a species to species basis.

Inter-Homolog Crossing-Over and Synapsis in Arabidopsis Meiosis Are Dependent on the Chromosome Axis Protein AtASY3

In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs). Although the frequency of DNA double-strand breaks (DSBs) appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs. Immunolocalization studies in wild-type meiocytes indicate that the HORMA protein AtASY1, which is related to Hop1 in budding yeast, forms hyper-abundant domains along the chromosomes that are spatially associated with DSBs and early recombination pathway proteins. Loss of AtASY3 disrupts the axial organization of AtASY1. Furthermore we show that the AtASY3 and AtASY1 homologs BoASY3 and BoASY1, from the closely related species Brassica oleracea, are co-immunoprecipitated from meiocyte extracts and that AtASY3 interacts with AtASY1 via residues in its predicted coiled-coil domain. Together our results suggest that AtASY3 is a functional homolog of Red1. Since studies in budding yeast indicate that Red1 and Hop1 play a key role in establishing a bias to favor inter-homolog recombination (IHR), we propose that AtASY3 and AtASY1 may have a similar role in Arabidopsis. Loss of AtASY3 also disrupts synaptonemal complex (SC) formation. In Atasy3-1 the transverse filament protein AtZYP1 forms small patches rather than a continuous SC. The few AtMLH1 foci that remain in Atasy3-1 are found in association with the AtZYP1 patches. This is sufficient to prevent the ectopic recombination observed in the absence of AtZYP1, thus emphasizing that in addition to its structural role the protein is important for CO formation.

Together yes, but not coupled: new insights into the roles of RAD51 and DMC1 in plant meiotic recombination

The eukaryotic recombinases RAD51 and DMC1 are essential for DNA strand-exchange between homologous chromosomes during meiosis. RAD51 is also expressed during mitosis, and mediates homologous recombination (HR) between sister chromatids. It has been suggested that DMC1 might be involved in the switch from intersister chromatid recombination in somatic cells to interhomolog meiotic recombination. At meiosis, the Arabidopsis Atrad51 null mutant fails to synapse and has extensive chromosome fragmentation. The Atdmc1 null mutant is also asynaptic, but in this case chromosome fragmentation is absent. Thus in plants, AtDMC1 appears to be indispensable for interhomolog homologous recombination, whereas AtRAD51 seems to be more involved in intersister recombination. In this work, we have studied a new AtRAD51 knock-down mutant, Atrad51-2, which expresses only a small quantity of RAD51 protein. Atrad51-2 mutant plants are sterile and hypersensitive to DNA double-strand break induction, but their vegetative development is apparently normal. The meiotic phenotype of the mutant consists of partial synapsis, an elevated frequency of univalents, a low incidence of chromosome fragmentation and multivalent chromosome associations. Surprisingly, non-homologous chromosomes are involved in 51% of bivalents. The depletion of AtDMC1 in the Atrad51-2 background results in the loss of bivalents and in an increase of chromosome fragmentation. Our results suggest that a critical level of AtRAD51 is required to ensure the fidelity of HR during interchromosomal exchanges. Assuming the existence of asymmetrical DNA strand invasion during the initial steps of recombination, we have developed a working model in which the initial step of strand invasion is mediated by AtDMC1, with AtRAD51 required to check the fidelity of this process.

On the investigation of fast electron beam filamentation in laser-irradiated solid targets using multi-MeV proton emission

The transverse filamentation of beams of fast electrons transported in solid targets irradiated by ultraintense (5 × 1020 W cm−2), picosecond laser pulses is investigated experimentally. Filamentation is diagnosed by measuring the uniformity of a beam of multi-MeV protons accelerated by the sheath field formed by the arrival of the fast electrons at the rear of the target, and is investigated for metallic and insulator targets ranging in thickness from 50 to 1200 µm. By developing an analytical model, the effects of lateral expansion of electron beam filaments in the sheath during the proton acceleration process is shown to account for measured increases in proton beam nonuniformity with target thickness for the insulating targets.

Synaptonemal Complex-Dependent Centromeric Clustering and the Initiation of Synapsis in Drosophila Oocytes

The pairing of homologous chromosomes and the intimate synapsis of the paired homologs by the synaptonemal complex (SC) are essential for subsequent meiotic processes including recombination and chromosome segregation. Here we show that the centromere clustering plays an important role in initiating homolog synapsis during meiosis in Drosophila females. Although centromeres are not clustered prior to the onset of meiosis, all four pairs of centromeres are actively clustered into one or two masses during early meiotic prophase. Within the 16-cell cyst, centromeric clustering appears to define the first step in the initiation of synapsis. Clustering is restricted to the nuclei that form the SC and is dependent on all known SC proteins. Surprisingly, both centromeric clusters and the SC components associated with them persist long after the disassembly of the euchromatic SC at the end of pachytene. The initiation of homologous recombination through the formation of programmed double-strand breaks (DSBs) is not required for either the formation or the maintenance of the centromeric clusters. Our data support a view in which the SC-mediated clustering at the centromeres is the initiating event for meiotic synapsis.

Solving a Meiotic LEGO® Puzzle: Transverse Filaments and the Assembly of the Synaptonemal Complex in Caenorhabditis elegans

The structure of the meiosis-specific synaptonemal complex, which is perhaps the central visible characteristic of meiotic prophase, has been a matter of intense interest for decades. Although a general picture of the interactions between the transverse filament proteins that create this structure has emerged from studies in a variety of organisms, a recent analysis of synaptonemal complex structure in Caenorhabditis elegans by Schild-Prüfert et al. (2011) has provided the clearest picture of the structure of the architecture of a synaptonemal complex to date. Although the transverse filaments of the worm synaptonemal complex are assembled differently then those observed in yeast, mammalian, and Drosophila synaptonemal complexes, a comparison of the four assemblies shows that achieving the overall basic structure of the synaptonemal complex is far more crucial than conserving the structures of the individual transverse filaments.

HORMAD1 Modulates Double Stranded Break Repair During Female Meiosis.

Meiosis is unique to germ cells and essential for reproduction. During the first meiotic division, homologous chromosomes pair, recombine and form chiasmata. The homologues connect via axial elements and numerous transverse filaments to form the synaptonemal complex. The synaptonemal complex is a critical component for chromosome pairing, segregation and recombination. We recently reported that HORMAD1 is a critical component of the synaptonemal complex that affects synapsis, and recombination. But unlike Sycp1, Sycp2 and Sycp3 deficient mice, no significant differences were observed in the number of primordial, primary, secondary and developing follicles between wild type and Hormad1-/- newborn, 8 day and 80 day ovaries. Therefore Hormad1-/- ovaries have seemingly normal ovarian folliculogenesis, with no evidence of accelerated oocyte loss. We investigated molecular anatomy of the HORMAD1 female meiocytes to understand Hormad1-/- oocytes ability to resist loss. Meiosis in mouse ovaries commences circa E13.5 and arrests in the diplotene stage prior to birth. By E16.5, most oocytes show zygotene and pachytene stages of meiosis while at E18.5, pachytene and diplotene stages predominate. We examined the E16.5 and E18.5 day wild type and Hormad1-/- embryonic ovaries using CREST and SYCP2 or SYCP3 antibodies to examine defects in female meiocyte synapsis formation. We didn't find a significant difference in the number of the zygotene stage cells between wild type and Hormad1-/- ovaries. At E16.5, 16% less pachytene-like oocytes and 18% more diplotene stage oocytes were observed in the Hormad1-/- ovaries when compared to the wild-type ovaries. In E18.5 day 30% less Pachytene-like stage and 28% more diplotene stage oocytes were observed in the Hormad1-/- ovaries. These results show that HORMAD1 plays an important role in synapsis formation in the pachytene stage oocytes, and may act as a pachytene checkpoint protein. We hypothesized that HORMAD1 modulates double stranded break (DSB) formation, since unrepaired DSBs lead to accelerated oocyte loss. We showed previously that zygotene stage Hormad1-/- oocytes have drastically reduced γH2AX signal, a marker for DSB formation. We therefore irradiated E16.5 day wild-type and Hormad1-/- ovaries to determine effects on DSB repair and formation. Our results indicate that 82% of γH2AX signal was still present in Hormad1+/- zygotene oocytes after 8h exposure of gamma rays as compared to 1h exposure of Hormad1+/- zygotene oocytes, but only 18% of measured γH2AX signal intensity remained in the Hormad1-/- zygotene oocytes after 8h exposure to gamma rays as compared to the 1hr exposure of Hormad1-/- zygotene oocytes. Our results indicate that HORMAD1 deficiency protects against DSB break formation, which may in part be responsible for the ability of Hormad1-/- oocytes to resist apoptotic loss. HORMAD1 is the mammalian homologue to yeast Hop1. Similar to Hop1 mutants in yeast, lack of HORMAD1 may de-repress DMC1-independent inter-sister repair pathway, resulting in efficient DNA break repairs. The reduced DSBs in Hormad1-/- meiocytes, and de-repression of DMC1-independent inter-sister repair pathways, may not affect regular apoptotic pathways that eliminate substantial number of oocytes during germ cell clusters breakdown to form primordial follicles at the time of birth, resulting in no decrease in Hormad1-/- primordial oocyte numbers. (poster)

Murine Missing in Metastasis (MIM) Mediates Cell Polarity and Regulates the Motility Response to Growth Factors

BackgroundMissing in metastasis (MIM) is a member of the inverse BAR-domain protein family, and in vitro studies have implied MIM plays a role in deforming membrane curvature into filopodia-like protrusions and cell dynamics. Yet, the physiological role of the endogenous MIM in mammalian cells remains undefined.Principal FindingsWe have examined mouse embryonic fibroblasts (MEFs) derived from mice in which the MIM locus was targeted by a gene trapping vector. MIM−/− MEFs showed a less polarized architecture characterized by smooth edges and fewer cell protrusions as compared to wild type cells, although the formation of filopodia-like microprotrusions appeared to be normal. Immunofluorescent staining further revealed that MIM−/− cells were partially impaired in the assembly of stress fibers and focal adhesions but were enriched with transverse actin filaments at the periphery. Poor assembly of stress fibers was apparently correlated with attenuation of the activity of Rho GTPases and partially relieved upon overexpressing of Myc-RhoAQ63L, a constitutively activated RhoA mutant. MIM−/− cells were also spread less effectively than wild type cells during attachment to dishes and substratum. Upon treatment with PDGF MIM−/− cells developed more prominent dorsal ruffles along with increased Rac1 activity. Compared to wild type cells, MIM−/− cells had a slower motility in the presence of a low percentage of serum-containing medium but migrated normally upon adding growth factors such as 10% serum, PDGF or EGF. MIM−/− cells were also partially impaired in the internalization of transferrin, fluorescent dyes, foreign DNAs and PDGF receptor alpha. On the other hand, the level of tyrosine phosphorylation of PDGF receptors was more elevated in MIM depleted cells than wild type cells upon PDGF treatment.ConclusionsOur data suggests that endogenous MIM protein regulates globally the cell architecture and endocytosis that ultimately influence a variety of cellular behaviors, including cell polarity, motility, receptor signaling and membrane ruffling.