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Shinohara, M.

Publications and source records attributed to Shinohara, M..

18 recordsLinked to original sources

Enhancing Visuomotor Reaction during Dark Transition via Model-Optimized Transcutaneous Vagus Nerve Stimulation

Sudden reductions in ambient illuminance temporarily impair visuomotor performance due to the inherent latency of dark adaptation, posing significant risks in operational environments such as tunnel entry. To mitigate this degradation, we propose a model-based neuromodulation framework using transcutaneous vagus nerve stimulation (tVNS), which is known to accelerate and augment pupil dilation and potentially enhance sensorimotor processing by activating the locus coeruleus-norepinephrine (LC-NE) system. First, we characterized the temporal dynamics of the tVNS-induced pupillary response and developed a predictive model using a long short-term memory (LSTM) network in standard lighting. The model demonstrated efficacy in reproducing nonlinear physiological characteristics even under sudden dark exposure. Second, we derived optimized stimulation sequences using a genetic algorithm to maximize pupil dilation during immediate adaptation to darkness. Pupillary evaluations during bright-to-dark transitions revealed that the optimized tVNS additively increased pupil diameter. The improvement in visuomotor choice-reaction performance was highly correlated with the amount of tVNS-induced increase in pupil diameter during dark transition across healthy young adults. These findings suggest that the model-optimized tVNS approach may effectively mitigate the degradation of visuomotor performance during dark transitions via LC-NE activation as well as subsequent sensorimotor enhancement. Furthermore, the experimental results demonstrate the potential of the transient pupil response to tVNS to serve as a robust physiological indicator for predicting behavioral improvement.

bioengineering↗

ALDH3A2 acts as a metabolic safeguard that regulates sphingolipid metabolism to suppress DNA damage and cell death

Highly reactive aldehydes are generated during metabolic processes in the body, and their detoxification is essential for maintaining cellular homeostasis. Hexadecenal, a long-chain fatty aldehyde, is formed during the sphingolipid degradation pathway from the lipid mediator sphingosine-1-phosphate (S1P). However, the cytotoxicity resulting from dysregulation of hexadecenal metabolism is still unclear. To elucidate the effects of impaired hexadecenal metabolism, we analyzed the function of ALDH3A2, an aldehyde dehydrogenase in humans. Our results revealed that ALDH3A2 enzymatic activity is crucial for the suppression of DNA damage, particularly interstrand DNA crosslinks, upon S1P exposure. Furthermore, we demonstrated that hexadecenal accumulation promotes cell death accompanied by the activation of cellular stress responses and morphological abnormalities in the endoplasmic reticulum. These findings suggest that ALDH3A2 functions as a metabolic safeguard to suppress DNA damage and cell death in response to the enhanced metabolic flux of hexadecenal.

molecular biology↗

Transposon insertion sequencing identifies genetic determinants of intrinsic rifamycin resistance in Mycobacterium abscessus

Mycobacterium abscessus exhibits intrinsic resistance to many antimicrobial agents, including rifampicin, a frontline anti-tuberculosis drug, severely limiting treatment options. Here, we used transposon insertion sequencing (Tn-Seq) to perform a genomewide screen to identify genes required for intrinsic rifampicin resistance in M. abscessus. We uncovered candidate genes that confer intrinsic resistance to the rifamycin-class antibiotics, rifampicin and rifabutin. The genes we identified included previously reported genes such as arr, helR, and MAB_2807. By comparing our results with the rifampicin intrinsic resistance gene in Mycobacterium tuberculosis, we found that the mechanisms underlying rifampicin intrinsic resistance were distinct between the two species. The contribution of seven representative candidate genes to rifampicin resistance was confirmed by characterizing targeted gene deletion or transposon insertion mutants. Among these determinants, MAB_2807 was identified as a major efflux-based contributor to rifamycin resistance, and its disruption increased intracellular rifampicin accumulation. In addition to known resistance determinants, Tn-Seq revealed contributions from many genes involved in cell envelope processes to rifamycin resistance. Guided by this genetic signature, we evaluated the combined effects of cell wall-targeting antimicrobial agents with rifamycins. We found that rifamycins displayed selective synergistic interactions with specific {beta}-lactam antibiotics. Interestingly, we found that rifamycin exposure altered cell envelope ultrastructure and increased the accumulation of the peptidoglycan precursor UDP-N-acetylglucosamine, suggesting that rifampicin perturbs envelope-associated metabolic homeostasis. These findings highlight rifamycin-induced vulnerable cellular processes that may inform rational combination strategies. ImportanceRifamycins are cornerstone antibiotics for the treatment of mycobacterial infections, yet they are ineffective against Mycobacterium abscessus. Although several key resistance mechanisms have been described, the full genetic basis of intrinsic rifamycin resistance remains to be elucidated. Here, we used transposon insertion sequencing to systematically identify genes that contribute to intrinsic rifamycin resistance in M. abscessus. Because recent studies have shown that chemical modification of rifamycins can overcome specific intrinsic resistance mechanisms, a comprehensive understanding of these determinants could facilitate the development of next-generation rifamycins with improved activity against M. abscessus. In addition, we unexpectedly found that rifamycin exposure alters cell envelope structure, the intracellular levels of a key metabolic intermediate in peptidoglycan biosynthesis, and susceptibility to certain {beta}-lactams in M. abscessus. These findings may help guide the rational development of combination therapeutic strategies with next-generation rifamycins.

microbiology↗

Integrated analysis of Tn-Seq and pangenome reveals core and lineage-specific essential genes in Mycobacterium avium subsp. hominissuis.

Pulmonary disease caused by nontuberculous mycobacteria (NTM-PD) is an emerging global health concern. Among NTM, Mycobacterium avium subsp. hominissuis (MAH) is the major causative agent of NTM-PD. Similar to Mycobacterium tuberculosis (Mtb), MAH exhibits lineage-specific geographical distributions and host adaptations. Here, we characterized three MAH strains from the residential bathrooms of MAH-PD patients in Japan. A genetic population clustering analysis revealed that the three strains belong to the East Asia (EA) lineages that are predominant in Japan and Korea. Pan-genome analysis using the publicly available complete genome sequences of MAH and the newly sequenced MAH strains identified 3,313 core genes that are conserved among distinct MAH lineages. Identification of essential genes in the three strains was conducted using transposon insertion sequencing (Tn-Seq), and their gene essentiality profiles were compared to those of a previously studied SC3 lineage strain, MAC109. Despite their genetic diversity, nearly all essential genes were derived from the core gene set. In addition, we identified a set of common essential genes for the EA and SC3 lineages, as well as lineage-specific essential genes. Our results highlight the evolutionary and clinical importance of lineage-specific adaptations in MAH. ImportanceBy integrating transposon insertion sequencing with pan-genome analysis, we provide the first systematic comparison of essential genes across multiple Mycobacterium avium subsp. hominissuis (MAH) strains. Although MAH strains exhibit remarkable genetic diversity, we found that MAH essential genes are primarily confined to the core genome of MAH. This essential plasticity highlights the evolutionary strategies that underpin MAH survival across diverse environments and patient populations. Recognizing this interplay provides a foundation for identifying robust drug targets and developing lineage-informed therapies for MAH infection.

microbiology↗

Dynamics of Fanconi anemia protein D2 in association with nuclear lipid droplet formation

Fanconi anemia (FA) is a rare genetic disease caused by the loss of function of one of the 22 associated genes and is characterized by bone marrow failure, cancer predisposition, and developmental defects. The proteins encoded by these genes (FA proteins) mainly function in DNA damage response and repair. Although FA deficiency has multiple effects on the regulation of lipid metabolism, the molecular function of FA proteins in the context of FA pathology remains unclear. In the present study, we demonstrated that FANCD2, a key component of FA proteins, interacts with lipid metabolism-related factors and that FANCD2 deficiency downregulates the cellular levels of fatty acids. Moreover, a portion of FANCD2 is localized to nuclear lipid droplets in response to oleic acid treatment. These subcellular dynamics are independent of FANCD2 monoubiquitination, which is essential for the DNA damage response. Collectively, these findings demonstrate that FANCD2 responds to not only DNA damage but also oleic acid exposure, providing insights into the pathogenesis of lipid dysregulation in FA.

cell biology↗

Enhancing radiotherapy response via intratumoral injection of the TLR9 agonist CpG to stimulate CD8 T cells in an autochthonous mouse model of sarcoma

Radiation therapy is frequently used to treat cancers including soft tissue sarcomas. Prior studies established that the toll-like receptor 9 (TLR9) agonist cytosine-phosphate-guanine oligodeoxynucleotide (CpG) enhances the response to radiation therapy (RT) in transplanted tumors, but the mechanism(s) remain unclear. Here, we used CRISPR/Cas9 and the chemical carcinogen 3-methylcholanthrene (MCA) to generate autochthonous soft tissue sarcomas with high tumor mutation burden. Treatment with a single fraction of 20 Gy RT and two doses of CpG significantly enhanced tumor response, which was abrogated by genetic or immunodepletion of CD8+ T cells. To characterize the immune response to RT + CpG, we performed bulk RNA-seq, single-cell RNA-seq, and mass cytometry. Sarcomas treated with 20 Gy and CpG demonstrated increased CD8 T cells expressing markers associated with activation and proliferation, such as Granzyme B, Ki-67, and interferon-{gamma}. CpG + RT also upregulated antigen presentation pathways on myeloid cells. Furthermore, in sarcomas treated with CpG + RT, TCR clonality analysis suggests an increase in clonal T-cell dominance. Collectively, these findings demonstrate that RT + CpG significantly delays tumor growth in a CD8 T cell-dependent manner. These results provide a strong rationale for clinical trials evaluating CpG or other TLR9 agonists with RT in patients with soft tissue sarcoma.

cancer biology↗

Stress-induced vascular remodeling: novel insight into the role of omega-3 fatty acid metabolite, 4-oxoDHA

BackgroundStress has garnered significant attention as a prominent risk factor for inflammation-related diseases, particularly cardiovascular diseases (CVDs). However, the precise mechanisms underlying stress-driven CVDs remain elusive, thereby impeding the development of effective preventive and therapeutic strategies. MethodsTo explore the correlation between plasma lipid metabolites and depressive states, we conducted a study involving healthy volunteers (n=408). Liquid chromatography (LC)/mass spectrometry (MS)/MS-based lipidomics and the self-rating depression (SDS) scale questionnaire were employed for data collection. In addition, we utilized a mouse model by subjecting mice to restraint stress and investigating the impact of stress on plasma lipid metabolites and vascular remodeling following carotid ligation. In vitro functional and mechanistic studies were performed using macrophages, endothelial cells, and neutrophil cells. ResultsOur findings revealed a significant association between depressive state and reduced plasma levels of 4-oxoDHA, a specific omega-3 fatty acid metabolite regulated by 5-lipoxygenase (LO) in neutrophils in healthy volunteers. In mice, restraint stress led to decreased plasma 4-oxoDHA levels and exacerbated vascular remodeling. Moreover, 4-oxoDHA demonstrated the ability to enhance Nrf2-HO-1 pathways, exerting anti-inflammatory effects on endothelial cells and macrophages. Mechanistically, stress-induced noradrenaline triggered the degradation of 5-LO in neutrophils through the proteasome system, facilitated by dopamine D2-like receptor activation. The reduction in circulating 4-oxoDHA resulted in the downregulation of the Nrf2-HO-1 anti-inflammatory axis and an increase in ICAM-1 expression, vascular permeability, and remodeling. ConclusionsOur study unveiled a novel stress-induced pathway of vascular inflammation, mediated through the regulation of omega-3 fatty acid metabolites. Reduced levels of circulating 4-oxoDHA under stress conditions may serve as a promising biomarker for stress. This understanding of the interplay between neurobiology and lipid metabolism provides a potential avenue for the development of treatments aimed at preventing stress-induced systemic neuroinflammation. Highlights- Our study reveals that stress-induced reduction in circulating levels of a specific omega-3 fatty acid metabolite, 4-oxoDHA, contributes to vascular inflammation. - We have identified a novel pathway that explains how stress promotes systemic vascular inflammation by regulating omega-3 fatty acid metabolites in the circulation. - Our findings provide new evidence for the role of 4-oxoDHA in maintaining Nrf2-ARE-related anti-inflammatory functions in endothelial cells and macrophages.

biochemistry↗

Novel monoclonal antibodies showing broad neutralizing activity for SARS-CoV-2 variants including Omicrons BA.5 and BA.2.75

We identified novel neutralizing monoclonal antibodies against SARS-CoV-2 variants (including Omicron) from individuals received two doses of mRNA vaccination after they had been infected with wildtype. We named them MO1, MO2 and MO3. MO1 shows high neutralizing activity against authentic variants: D614G, Delta, BA.1, BA.1.1, BA.2, and BA.2.75 and BA.5. Our findings confirm that the wildtype-derived vaccination can induce neutralizing antibodies that recognize the epitopes conserved among the SARS-CoV-2 variants (including BA.5 and BA.2.75). The monoclonal antibodies obtained herein could serve as novel prophylaxis and therapeutics against not only current SARS-CoV-2 viruses but also future variants that may arise.

microbiology↗

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed passive head motion (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents heads. SUMMARYThe present protocol describes a custom-designed passive head motion system, which reproduces mechanical accelerations at rodents heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

bioengineering↗

Mec1/Tel1-independent role of protein phosphatase 4 in Hop1 assembly to promote meiotic chromosome axis formation in budding yeast

Dynamic changes in chromosomal structure that occur during meiotic prophase play an important role in the progression of meiosis. Among these structures, meiosis-specific chromosomal axis-loop structures are important as a scaffold for integrated control between the meiotic recombination reaction and the associated checkpoint system to ensure accurate chromosome segregation. However, the molecular mechanism of the initial step of chromosome axis-loop construction is not well understood. Here, we showed that in budding yeast, protein phosphatase 4 (PP4) that primarily counteracts Mec1/Tel1 phosphorylation is required to promote the assembly of chromosomal axis components Hop1 and Red1 onto meiotic chromatin via interaction with Hop1. PP4, in contrast, did not affect Rec8 assembly. Notably, unlike the previously known function of PP4, this novel function of PP4 was independent of meiotic DSB-dependent Tel1/Mec1 kinase activities. The defect in Hop1/Red1 assembly in the absence of PP4 function was not suppressed by dysfunction of Pch2, which removes Hop1 protein from the chromosome axis. This suggests that PP4 is required for the initial step of chromatin loading of Hop1 rather than stabilization of Hop1 on axes. These results indicate that phosphorylation/dephosphorylation-mediated regulation of Hop1 recruitment onto chromatin occurs during chromosome axis construction before meiotic double-strand break formation.

molecular biology↗

Mechanical impact on the head has an antihypertensive effect

Physical exercise is known to be beneficial for various brain functions. However, the mechanisms behind the positive effects of exercise on the brain remain to be elucidated. Here we show that passive head motion in hypertensive rats, which reproduces the mechanical accelerations generated in their heads during moderate-velocity treadmill running, decreases the expression of angiotensin II type 1 receptor (AT1R) in astrocytes in the rostral ventrolateral medulla (RVLM), thereby lowering blood pressure. Passive head motion generates interstitial fluid movement that is estimated to exert shear stress with an average magnitude of <1 Pa on the cells in the rat medulla. Fluid shear stress of a sub-Pa magnitude decreases AT1R expression in cultured astrocytes. In hypertensive rats, inhibition of interstitial fluid movement following hydrogel introduction to the RVLM eliminates the antihypertensive effects of passive head motion and treadmill running. Furthermore, vertically oscillating chair riding by hypertensive adult humans, which reproduces the mechanical accelerations generated in their heads during light jogging or fast walking, lowers their blood pressure. Our findings indicate that moderate mechanical intervention can have antihypertensive effects by modulating the function of RVLM astrocytes through interstitial fluid shear stress. We anticipate that mechanical regulation is responsible for a variety of the positive effects of physical exercise on human health, particularly those related to brain functions.

bioengineering↗

Phosphorylation of luminal region of the SUN-domain protein Mps3 promotes nuclear envelope localization during meiosis

During meiosis, protein ensembles in the nuclear envelope (NE) containing SUN- and KASH-domain proteins, called linker nucleocytoskeleton and cytoskeleton (LINC) complex, promote the chromosome motion. Yeast SUN-domain protein, Mps3, forms multiple meiosis-specific ensembles on NE, which show dynamic localisation for chromosome motion; however, the mechanism by which these Mps3 ensembles are formed during meiosis remains largely unknown. Here, we showed that the cyclin-dependent protein kinase (CDK) and Dbf4-dependent Cdc7 protein kinase (DDK) regulate meiosis-specific dynamics of Mps3 on NE, particularly by mediating the resolution of Mps3 clusters and telomere clustering. We also found that the luminal region of Mps3 juxtaposed to the inner nuclear membrane is required for meiosis-specific localisation of Mps3 on NE. Negative charges introduced by meiosis-specific phosphorylation in the luminal region of Mps3 alter its interaction with negatively charged lipids by electric repulsion in reconstituted liposomes. Phospho-mimetic substitution in the luminal region suppresses the localisation of Mps3 via the inactivation of CDK or DDK. Our study revealed multi-layered phosphorylation-dependent regulation of the localisation of Mps3 on NE for meiotic chromosome motion and NE remodelling.

molecular biology↗

APOE2 promotes longevity independent of Alzheimer's disease

ObjectiveAlthough apolipoprotein E (APOE) allele associates with longevity, its mechanism is not understood. The protective effects of APOE2 and the deleterious effects of APOE4 on Alzheimers disease (AD) risk may confound APOE effects on longevity. MethodsWe analyzed a large number of subjects from the National Alzheimers Coordinating Center (NACC), and animal models expressing human apoE isoforms in the absence of AD. ResultsClinically, the APOE2 allele was associated with longer lifespan, while APOE4 associated with shorter lifespan, compared to the common APOE3 allele. This effect was also seen irrespective of clinical AD status, and in subjects with little amyloid pathology or after adjustment for AD-related pathologies. In animal studies, apoE2-TR mice also exhibited longer lifespan, while apoE4 showed some trends of shorter lifespan. Notably, old apoE2-TR mice kept activity measured by open field assay, associated with longer lifespan. Evidence of preserved activity in APOE2 carrier was also obtained in clinical records. In animal studies, higher levels of apoE2 in brain and plasma were correlated with activity. Moreover, lower levels of total cholesterol in the brain and higher levels of high-density lipoprotein cholesterol and triglycerides in the plasma of apoE2-TR mice were associated with apoE levels and more activity. InterpretationAPOE2 can contribute to longevity independent of AD. Preserved activity would be an early-observable feature of apoE2-mediated longevity, where higher levels of apoE2 and its-associated lipid metabolism might be involved.

neuroscience↗

SCFCdc4 ubiquitin ligase regulates synaptonemal complex formation during meiosis

Homologous chromosomes pair with each other during meiosis, culminating in the formation of the synaptonemal complex (SC), which is coupled with meiotic recombination. In this study, we showed that a meiosis-specific depletion mutant of a cullin (Cdc53) of the SCF (Skp-Cullin-F-box) ubiquitin ligase, which plays a critical role in cell cycle regulation during mitosis, is deficient in SC formation, but is proficient in the formation of crossovers, indicating uncoupling of meiotic recombination with SC formation in the mutant. Furthermore, the deletion of the PCH2 gene encoding a meiosis-specific AAA+ ATPase suppresses SC-assembly defect induced by CDC53 depletion. On the other hand, the pch2 cdc53 double mutant is defective in meiotic crossover formation, suggesting the SC assembly with unrepaired DSBs. A temperature-sensitive mutant of the CDC4, which encodes a F-box protein of the SCF, shows similar meiotic defects to the CDC53 depletion mutant. These suggest that SCFCdc4, probably SCFCdc4-dependnet protein ubiquitylation, regulates and collaborates with Pch2 in SC assembly and meiotic recombination. SummaryDuring meiosis, homologous chromosomes pair with each other and form the synaptonemal complex (SC). In this study, components of the SCF (Skp-Cullin-F-box) ubiquitin ligase, Cdc53 and Cdc4, are required for SC formation. A meiosis-specific AAA+ ATPase Pch2 antagonize the functions of Cdc53 and Cdc4 for proper SC assembly.

molecular biology↗

The bakerss yeast Msh4-Msh5 associates with double-strand break hotspots and chromosome axis during meiosis to promote crossovers

Segregation of homologous chromosomes during the first meiotic division requires at least one obligate crossover/exchange event between the homolog pairs. In the bakers yeast Saccharomyces cerevisiae and mammals, the mismatch repair-related factors, Msh4-Msh5 and Mlh1-Mlh3 generate the majority of the meiotic crossovers from programmed double-strand breaks (DSBs). To understand the mechanistic role of Msh4-Msh5 in meiotic crossing over, we performed genome-wide ChIP-sequencing and cytological analysis of the Msh5 protein in cells synchronized for meiosis. We observe that Msh5 associates with DSB hotspots, chromosome axis, and centromeres. We found that the initial recruitment of Msh4-Msh5 occurs following DSB resection. A two-step Msh5 binding pattern was observed: an early weak binding at DSB hotspots followed by enhanced late binding upon the formation of double Holliday junction structures. Msh5 association with the chromosome axis is Red1 dependent, while Msh5 association with the DSB hotspots and axis is dependent on DSB formation by Spo11. Msh5 binding was enhanced at strong DSB hotspots consistent with a role for DSB frequency in promoting Msh5 binding. These data on the in vivo localization of Msh5 during meiosis have implications for how Msh4-Msh5 may work with other crossover and synapsis promoting factors to ensure Holliday junction resolution at the chromosome axis. AUTHOR SUMMARYDuring meiosis, crossovers facilitate physical linkages between homologous chromosomes that ensure their accurate segregation. Meiotic crossovers are initiated from programmed DNA double-strand breaks (DSBs). In the bakers yeast and mammals, DSBs are repaired into crossovers primarily through a pathway involving the highly conserved mismatch repair related Msh4-Msh5 complex along with other crossover promoting factors. In vitro and physical studies suggest that the Msh4-Msh5 heterodimer facilitates meiotic crossover formation by stabilizing Holliday junctions. We investigated the genome-wide in vivo binding sites of Msh5 during meiotic progression. Msh5 was enriched at DSB hotspots, chromosome axis, and centromere sites. Our results suggest Msh5 associates with both DSB sites on the chromosomal loops and with the chromosome axis to promote crossover formation. These results on the in vivo dynamic localization of the Msh5 protein provide novel insights into how the Msh4-Msh5 complex may work with other crossover and synapsis promoting factors to facilitate crossover formation.

genetics↗

DNA damage response clamp loader Rad24(Rad17) and Mec1(ATR) kinase have distinct functions in regulating meiotic crossovers

Crossover (CO) recombination is essential for chromosome segregation during meiosis I. The number and distribution of COs are tightly regulated during meiosis. CO control includes CO assurance and CO interference, which guarantee at least one CO per a bivalent and evenly-spaced CO distribution, respectively. Previous studies showed the role of DNA damage response (DDR) clamp and its loader in efficient formation of meiotic COs by promoting the recruitment of a pro-CO protein Zip3 and interhomolog recombination, and also by suppressing ectopic recombination. In this study, by classical tetrad analysis of Saccharomyces cerevisiae, we showed that a mutant defective in the RAD24 gene (RAD17 in other organisms), which encodes the DDR clamp loader, displayed reduced CO frequencies on two shorter chromosomes (III and V) but not on a long chromosome (chromosome VII). The residual COs in the rad24 mutant do not show interference. In contrast to the rad24 mutant, mutants defective in the ATR kinase homolog Mec1/Esr1, including a mec1 null and a mec1 kinase-dead mutant, show little or no defect in CO frequency. On the other hand, mec1 COs show defects in interference, similar to the rad24 mutant. Moreover, CO formation and its control are implemented in a chromosome-specific manner, which may reflect a role for chromosome size in regulation.

genetics↗

Srs2 helicase prevents the formation of toxic DNA damage during late prophase I of yeast meiosis

Proper repair of double-strand breaks (DSBs) is key to ensure proper chromosome segregation. In this study, we found that the deletion of the SRS2 gene, which encodes a DNA helicase necessary for the control of homologous recombination, induces aberrant chromosome segregation during budding yeast meiosis. This abnormal chromosome segregation in srs2 cells accompanies the formation of a novel DNA damage induced during late meiotic prophase-I. The damage may contain long stretches of single-stranded DNAs (ssDNAs), which lead to aggregate formation of a ssDNA binding protein, RPA, and a RecA homolog, Rad51, as well as other recombination proteins inside of the nuclei. The Rad51 aggregate formation in the srs2 mutant depends on the initiation of meiotic recombination and occurs in the absence of chromosome segregation. Importantly, as an early recombination intermediate, we detected a thin bridge of Rad51 between two Rad51 foci or among the foci in the srs2 mutant, which is rarely seen in wild type. These might be cytological manifestation of the connection of two DSB ends and multi-invasion. The DNA damage with Rad51 aggregates in the srs2 mutant is passed through anaphase-I and -II, suggesting the absence of DNA damage-induced cell-cycle arrest after the pachytene stage. We propose that Srs2 helicase resolves early protein-DNA recombination intermediates to suppress the formation of aberrant lethal DNA damage during late prophase-I.

genetics↗

Unexpected erosion of chromosome cohesion by a mitotic prophase-like pathway in yeast meiosis

Sister chromatid cohesion on chromosome arms is essential for the segregation of homologous chromosomes during meiosis I while it is dispensable for sister chromatid separation during mitosis. It was assumed that, unlike the situation in mitosis, chromosome arms retain cohesion prior to onset of anaphase-I. Paradoxically, reduced immunostaining signals of meiosis-specific cohesin, including the kleisin Rec8, from the chromosomes were observed during late prophase-I of budding yeast. This decrease is seen in the absence of Rec8 cleavage and depends on condensin-mediated recruitment of Polo-like kinase (PLK/Cdc5). In this study, we confirmed that this release indeed accompanies the dissociation of acetylated Smc3 as well as Rec8 from meiotic chromosomes during late prophase-I. This release requires, in addition to PLK, the cohesin regulator, Wapl (Rad61/Wpl1 in yeast), and Dbf4-dependent Cdc7 kinase (DDK). Meiosis-specific phosphorylation of Rad61/Wpl1 and Rec8 by PLK and DDK collaboratively promote this release. This process is similar to the vertebrate \"prophase\" pathway for cohesin release during G2 phase and pro-metaphase. In yeast, meiotic cohesin release coincides with PLK-dependent compaction of chromosomes in late meiotic prophase-I. We suggest that yeast uses this highly regulated cleavage-independent pathway to remove cohesin during late prophase-I to facilitate morphogenesis of condensed metaphase-I chromosomes.\n\nAuthor SummaryIn meiosis the life and health of future generations is decided upon. Any failure in chromosome segregation has a detrimental impact. Therefore, it is currently believed that the physical connections between homologous chromosomes are maintained by meiotic cohesin with exceptional stability. Indeed, it was shown that cohesive cohesin does not show an appreciable turnover during long periods in oocyte development. In this context, it was long assumed but not properly investigated, that the prophase pathway for cohesin release would be specific to mitotic cells and will be safely suppressed during meiosis so as not to endanger the valuable chromosome connections. However, a previous study on budding yeast meiosis suggests the presence of cleavage-independent pathway of cohesin release during late prophase-I. In the work presented here we confirmed that the prophase pathway is not suppressed during meiosis, at least in budding yeast and showed that this cleavage-independent release is regulated by meiosis-specific phosphorylation of two cohesin subunits, Rec8 and Rad61(Wapl) by two cell-cycle regulators, PLK and DDK. Our results suggest that late meiotic prophase-I actively controls cohesin dynamics on meiotic chromosomes for chromosome segregation.

cell biology↗