HOP1 and HOP2 are involved in salt tolerance by facilitating the brassinosteroid-related nucleo-cytoplasmic partitioning of the HSP90-BIN2 complex.

The co-chaperone heat shock protein (HSP)70-HSP90 organizing protein (HOP) is involved in plant thermotolerance. However, its function in plant salinity tolerance was not yet studied. We found that Arabidopsis HOP1 and HOP2 play critical roles in salt tolerance by affecting the nucleo-cytoplasmic partitioning of HSP90 and brassinosteroid-insensitive 2 (BIN2). A hop1/2 double mutant was hypersensitive to salt-stress. Interestingly, this sensitivity was remedied by exogenous brassinolide application, while the application of brassinazole impeded growth of both wild-type (WT) and hop1/2 plants under normal and salt stress conditions. This suggested that the insufficient brassinosteroid (BR) content was responsible for the salt-sensitivity of hop1/2. After WT was transferred to salt stress conditions, HOP1/2, BIN2 and HSP90 accumulated in the nucleus, brassinazole-resistant 1 (BZR1) was phosphorylated and accumulated in the cytoplasm, and BR content significantly increased. This initial response resulted in dephosphorylation of BZR1 and BR response. This dynamic regulation of BR content was impeded in salt-stressed hop1/2. Thus, we propose that HOP1 and HOP2 are involved in salt tolerance by affecting BR signalling.

Genetic interactions between the chromosome axis-associated protein Hop1 and homologous recombination determinants in Schizosaccharomyces pombe.

Hop1 is a component of the meiosis-specific chromosome axis and belongs to the evolutionarily conserved family of HORMA domain proteins. Hop1 and its orthologs in higher eukaryotes are a major factor in promoting double-strand DNA break formation and inter-homolog recombination. In budding yeast and mammals, they are also involved in a meiotic checkpoint kinase cascade monitoring the completion of double-strand DNA break repair. We used the fission yeast, Schizosaccharomyces pombe, which lacks a canonical synaptonemal complex to test whether Hop1 has a role beyond supporting the generation of double-strand DNA breaks and facilitating inter-homolog recombination events. We determined how mutants of homologous recombination factors genetically interact with hop1, studied the role(s) of the HORMA domain of Hop1, and characterized a bio-informatically predicted interactor of Hop1, Aho1 (SPAC688.03c). Our observations indicate that in fission yeast, Hop1 does require its HORMA domain to support wild-type levels of meiotic recombination and localization to meiotic chromatin. Furthermore, we show that hop1∆ only weakly interacts genetically with mutants of homologous recombination factors, and in fission yeast likely has no major role beyond break formation and promoting inter-homolog events. We speculate that after the evolutionary loss of the synaptonemal complex, Hop1 likely has become less important for modulating recombination outcome during meiosis in fission yeast, and that this led to a concurrent rewiring of genetic pathways controlling meiotic recombination.

Temperature regulates negative supercoils to modulate meiotic crossovers and chromosome organization.

Crossover recombination is a hallmark of meiosis that holds the paternal and maternal chromosomes (homologs) together for their faithful segregation, while promoting genetic diversity of the progeny. The pattern of crossover is mainly controlled by the architecture of the meiotic chromosomes. Environmental factors, especially temperature, also play an important role in modulating crossovers. However, it is unclear how temperature affects crossovers. Here, we examined the distribution of budding yeast axis components (Red1, Hop1, and Rec8) and the crossover-associated Zip3 foci in detail at different temperatures, and found that both increased and decreased temperatures result in shorter meiotic chromosome axes and more crossovers. Further investigations showed that temperature changes coordinately enhanced the hyperabundant accumulation of Hop1 and Red1 on chromosomes and the number of Zip3 foci. Most importantly, temperature-induced changes in the distribution of axis proteins and Zip3 foci depend on changes in DNA negative supercoils. These results suggest that yeast meiosis senses temperature changes by increasing the level of negative supercoils to increase crossovers and modulate chromosome organization. These findings provide a new perspective on understanding the effect and mechanism of temperature on meiotic recombination and chromosome organization, with important implications for evolution and breeding.

Notch signaling without the APH-2/nicastrin subunit of gamma secretase in Caenorhabditis elegans germline stem cells.

The final step in Notch signaling activation is the transmembrane cleavage of Notch receptor by γ secretase. Thus far, genetic and biochemical evidence indicates that four subunits are essential for γ secretase activity in vivo: presenilin (the catalytic core), APH-1, PEN-2, and APH-2/nicastrin. Although some γ secretase activity has been detected in APH-2/nicastrin-deficient mammalian cell lines, the lack of biological relevance for this activity has left the quaternary γ secretase model unchallenged. Here, we provide the first example of in vivo Notch signal transduction without APH-2/nicastrin. The surprising dispensability of APH-2/nicastrin is observed in Caenorhabditis elegans germline stem cells (GSCs) and contrasts with its essential role in previously described C. elegans Notch signaling events. Depletion of GLP-1/Notch, presenilin, APH-1, or PEN-2 causes a striking loss of GSCs. In contrast, aph-2/nicastrin mutants maintain GSCs and exhibit robust and localized expression of the downstream Notch target sygl-1. Interestingly, APH-2/nicastrin is normally expressed in GSCs and becomes essential under conditions of compromised Notch function. Further insight is provided by reconstituting the C. elegans γ secretase complex in yeast, where we find that APH-2/nicastrin increases but is not essential for γ secretase activity. Together, our results are most consistent with a revised model of γ secretase in which the APH-2/nicastrin subunit has a modulatory, rather than obligatory role. We propose that a trimeric presenilin-APH-1-PEN-2 γ secretase complex can provide a low level of γ secretase activity, and that cellular context determines whether or not APH-2/nicastrin is essential for effective Notch signal transduction.

A molecular cell biology toolkit for the study of meiosis in the silkworm Bombyx mori.

Meiosis is usually described as 4 essential and sequential processes: (1) homolog pairing; (2) synapsis, mediated by the synaptonemal complex; (3) crossing over; and (4) segregation. In this canonical model, the maturation of crossovers into chiasmata plays a vital role in holding homologs together and ensuring their segregation at the first meiotic division. However, Lepidoptera (moths and butterflies) undergo 3 distinct meiotic processes, only one of which is canonical. Lepidoptera males utilize 2 meiotic processes: canonical meiosis that produces nucleated fertile sperm, and a noncanonical meiosis that produces anucleated nonfertile sperm which are nonetheless essential for reproduction. Lepidoptera females, which carry heteromorphic sex chromosomes, undergo a completely achiasmate (lacking crossovers) meiosis, thereby requiring an alternative mechanism to ensure proper homolog segregation. Here, we report that the development of a molecular cell biology toolkit designed to properly analyze features of meiosis, including the synaptonemal complex structure and function, in the silkworm Bombyx mori. In addition to standard homology searches to identify Bombyx orthologs of known synaptonemal complex encoding genes, we developed an ortholog discovery app (Shinyapp) to identify Bombyx orthologs of proteins involved in several meiotic processes. We used this information to clone genes expressed in the testes and then created antibodies against their protein products. We used the antibodies to confirm the localization of these proteins in normal male spermatocytes, as well as using in vitro assays to confirm orthologous interactions. The development of this toolkit will facilitate further study of the unique meiotic processes that characterize meiosis in Lepidoptera.

The joint effects of nanoplastics and TBBPA on neurodevelopmental toxicity in Caenorhabditis elegans.

Both of nanoplastics (NPs) and Tetrabromobisphenol A (TBBPA) are organic pollutants widely detected in the environment and organisms. The large specific surface area of NPs makes them ideal vectors for carrying various toxicants, such as organic pollutants, metals, or other nanomaterials, posing potential threats to human health. This study used Caenorhabditis elegans (C. elegans) to investigate the neurodevelopmental toxicity induced by combined exposure of TBBPA and polystyrene NPs. Our results showed that combined exposure caused synergistic inhibitory effects on the survival rate, body length/width, and locomotor ability. Furthermore, the overproduction of reactive oxygen species (ROS), lipofuscin accumulation, and dopaminergic neuronal loss suggested that oxidative stress was involved in induction of neurodevelopmental toxicity in C. elegans. The expressions of Parkinson's disease related gene (pink-1) and Alzheimer's disease related gene (hop-1) were significantly increased after combined exposure of TBBPA and polystyrene NPs. Knock out of pink-1 and hop-1 genes alleviated the adverse effects such as growth retardation, locomotion deficits, dopaminergic loss, and oxidative stress induction, indicating that pink-1 and hop-1 genes play an important role in neurodevelopmental toxicity induced by TBBPA and polystyrene NPs. In conclusion, TBBPA and polystyrene NPs had synergistic effect on oxidative stress induction and neurodevelopmental toxicity in C. elegans, which was mediated through increased expressions of pink-1 and hop-1.

Turning coldspots into hotspots: targeted recruitment of axis protein Hop1 stimulates meiotic recombination in Saccharomyces cerevisiae.

The DNA double-strand breaks that initiate meiotic recombination are formed in the context of the meiotic chromosome axis, which in Saccharomyces cerevisiae contains a meiosis-specific cohesin isoform and the meiosis-specific proteins Hop1 and Red1. Hop1 and Red1 are important for double-strand break formation; double-strand break levels are reduced in their absence and their levels, which vary along the lengths of chromosomes, are positively correlated with double-strand break levels. How axis protein levels influence double-strand break formation and recombination remains unclear. To address this question, we developed a novel approach that uses a bacterial ParB-parS partition system to recruit axis proteins at high levels to inserts at recombination coldspots where Hop1 and Red1 levels are normally low. Recruiting Hop1 markedly increased double-strand breaks and homologous recombination at target loci, to levels equivalent to those observed at endogenous recombination hotspots. This local increase in double-strand breaks did not require Red1 or the meiosis-specific cohesin component Rec8, indicating that, of the axis proteins, Hop1 is sufficient to promote double-strand break formation. However, while most crossovers at endogenous recombination hotspots are formed by the meiosis-specific MutLγ resolvase, crossovers that formed at an insert locus were only modestly reduced in the absence of MutLγ, regardless of whether or not Hop1 was recruited to that locus. Thus, while local Hop1 levels determine local double-strand break levels, the recombination pathways that repair these breaks can be determined by other factors, raising the intriguing possibility that different recombination pathways operate in different parts of the genome.

Topoisomerases Modulate the Timing of Meiotic DNA Breakage and Chromosome Morphogenesis in Saccharomyces cerevisiae.

During meiotic prophase, concurrent transcription, recombination, and chromosome synapsis place substantial topological strain on chromosomal DNA, but the role of topoisomerases in this context remains poorly defined. Here, we analyzed the roles of topoisomerases I and II (Top1 and Top2) during meiotic prophase in Saccharomyces cerevisiae We show that both topoisomerases accumulate primarily in promoter-containing intergenic regions of actively transcribing genes, including many meiotic double-strand break (DSB) hotspots. Despite the comparable binding patterns, top1 and top2 mutations have different effects on meiotic recombination. TOP1 disruption delays DSB induction and shortens the window of DSB accumulation by an unknown mechanism. By contrast, temperature-sensitive top2-1 mutants exhibit a marked delay in meiotic chromosome remodeling and elevated DSB signals on synapsed chromosomes. The problems in chromosome remodeling were linked to altered Top2 binding patterns rather than a loss of Top2 catalytic activity, and stemmed from a defect in recruiting the chromosome remodeler Pch2/TRIP13 to synapsed chromosomes. No chromosomal defects were observed in the absence of TOP1 Our results imply independent roles for Top1 and Top2 in modulating meiotic chromosome structure and recombination.

Structural insights into the recognition of phosphorylated Hop1 by Mek1.

The FHA domain-containing protein Mek1 is a meiosis-specific kinase that is involved in the regulation of interhomolog recombination in meiosis in Saccharomyces cerevisiae. The recruitment and activation of Mek1 require the phosphorylation of the chromosome axis protein Hop1 at Thr318 (pT318), which is necessary for recognition by the Mek1 FHA domain. Here, crystal structures of the Mek1 FHA domain in the apo state and in complex with the Hop1 pT318 peptide are presented, demonstrating that the hydrophobic residues Phe320 and Val321 at the pT+2 and pT+3 positions in the ligand contribute to the preferential recognition. It was further found that in Schizosaccharomyces pombe Mek1 FHA binds both pT15 in its N-terminal SQ/TQ cluster domain (SCD) and pT270 in the Hop1 SCD. The results revealed the structural basis for the preferential recognition of phosphorylated Hop1 by Mek1 in S. cerevisiae and facilitate the understanding of the interaction between the S. pombe Mek1 FHA domain and its binding targets.

Mutation of hop-1 and pink-1 attenuates vulnerability of neurotoxicity in C. elegans: the role of mitochondria-associated membrane proteins in Parkinsonism.

Mitochondrial dysfunction is considered as a critical mechanism in the pathogenesis of Parkinson's disease (PD). Increasing evidence supports the notion of mitochondria-associated membranes (MAMs) in mitochondrial dysfunction; yet little is known about the role of MAMs-related proteins in the pathogenesis of PD. Herein we exposed the nematode Caenorhabditis elegans to 0.5-10.0 µM rotenone (RO) or 0.2-1.6 mM paraquat (PQ) for 3 days. Our results showed that both RO and PQ induced similar Parkinsonism including motor deficits and dopaminergic degeneration. RO/PQ caused mitochondrial damages characterized by the increase of vacuole areas and autophagy vesicles, but the decrease of mitochondrial cristae. RO/PQ-impacted mitochondrial function was also demonstrated by the decrease of ATP level and mitochondrial membrane potential. Additionally, the attachment or surrounding of endoplasmic reticulum to the damaged mitochondria indicates ultrastructural alterations in MAMs. Using fluorescently labeled transgenic nematodes, we further found that the expression of tomm-7 and genes of Complex I, II and III was reduced, whereas the expression of pink-1 was increased in the exposed animals. To determine MAMs in toxicity toward PD, we investigated the mutants of hop-1 and pink-1, encoding presenilin and PTEN-induced putative kinase 1 (PINK1) in mitochondria-associated membranes, respectively. Results demonstrated that the mutation of both hop-1 and pink-1 reduced the vulnerability of lethal, behavioral, and mitochondrial toxicity induced by RO/PQ. These findings suggest that presenilin and PINK1 play important roles in the RO/PQ-induced neurotoxicity through the mechanisms involved in mitochondria-associated membranes.

Ethanol production from sweet sorghum bagasse through process optimization using response surface methodology.

In this study, comparative evaluation of acid- and alkali pretreatment of sweet sorghum bagasse (SSB) was carried out for sugar production after enzymatic hydrolysis. Results indicated that enzymatic hydrolysis of alkali-pretreated SSB resulted in higher production of glucose, xylose and arabinose, compared to the other alkali concentrations and also acid-pretreated biomass. Response Surface Methodology (RSM) was, therefore, used to optimize parameters, such as alkali concentration, temperature and time of pretreatment prior to enzymatic hydrolysis to maximize the production of sugars. The independent variables used during RSM included alkali concentration (1.5-4%), pretreatment temperature (125-140 °C) and pretreatment time (10-30 min) were investigated. Process optimization resulted in glucose and xylose concentration of 57.24 and 10.14 g/L, respectively. Subsequently, second stage optimization was conducted using RSM for optimizing parameters for enzymatic hydrolysis, which included substrate concentration (10-15%), incubation time (24-60 h), incubation temperature (40-60 °C) and Celluclast concentration (10-20 IU/g-dwt). Substrate concentration 15%, (w/v) temperature of 60 °C, Celluclast concentration of 20 IU/g-dwt and incubation time of 58 h led to a glucose concentration of 68.58 g/l. Finally, simultaneous saccharification fermentation (SSF) as well as separated hydrolysis and fermentation (SHF) was evaluated using Pichia kudriavzevii HOP-1 for production of ethanol. Significant difference in ethanol concentration was not found using either SSF or SHF; however, ethanol productivity was higher in case of SSF, compared to SHF. This study has established a platform for conducting scale-up studies using the optimized process parameters.

Transcription dynamically patterns the meiotic chromosome-axis interface.

Meiotic chromosomes are highly compacted yet remain transcriptionally active. To understand how chromosome folding accommodates transcription, we investigated the assembly of the axial element, the proteinaceous structure that compacts meiotic chromosomes and promotes recombination and fertility. We found that the axial element proteins of budding yeast are flexibly anchored to chromatin by the ring-like cohesin complex. The ubiquitous presence of cohesin at sites of convergent transcription provides well-dispersed points for axis attachment and thus chromosome compaction. Axis protein enrichment at these sites directly correlates with the propensity for recombination initiation nearby. A separate modulating mechanism that requires the conserved axial-element component Hop1 biases axis protein binding towards small chromosomes. Importantly, axis anchoring by cohesin is adjustable and readily displaced in the direction of transcription by the transcriptional machinery. We propose that such robust but flexible tethering allows the axial element to promote recombination while easily adapting to changes in chromosome activity.

Transcription of meiotic-like-pathway genes in Giardia intestinalis

The reproductive mechanism of Giardia intestinalis, considered one of the earliest divergent eukaryotes, has not been fully defined yet. Some evidence supports the hypothesis that Giardia is an exclusively asexual organism with a clonal population structure. However, the high genetic variability, the variation in ploidy during its life cycle, the low heterozygosity and the existence of genes involved in the meiotic-like recombination pathway in the parasite's genome cast doubt on exclusively asexual nature of Giardia. In this work, semiquantitative RT-PCR analysis was used to assess the transcription pattern of three meiosis-like-specific genes involved in homologues recombination: dmc1, hop1 and spo11. The mRNAs were amplified during the parasite's differentiation processes, encystation and excystation, and expression was found at each stage of its life cycle. A semiquantitative assessment also suggests that expression of some of the genes is regulated during encystation process.