The apple transcription factor MdbHLH4 regulates plant morphology and fruit development by promoting cell enlargement.

The bHLH family, the second largest transcription factor (TF) family in plants, plays a crucial role in regulating plant growth and development processes. However, the biological functions and mechanisms of most bHLH proteins remain unknown, particularly in apples. In this study, we found that MdbHLH4 positively modulates plant growth and development by enhancing cell expansion. Overexpression (OE) of MdbHLH4 resulted in increased biomass, stem and root length, leaf area, and larger areas of pith, xylem, and cortex with greater cell size compared with wild-type apple plants. Conversely, RNA interference (RNAi)-mediated silencing of MdbHLH4 led to reduced xylem and phloem as well as smaller cell size compared to wild-type apple plants. Ectopic expression of MdbHLH4 in tomatoes resulted in enlarged fruits with impaired color appearance, decreased accumulation of soluble solids, and decreased flesh firmness along with larger seeds. Subsequent investigations have shown that MdbHLH4 directly binds to the promoters of MdARF6b and MdPIF4b, enhancing their expression levels. These findings suggest that MdbHLH4 potentially regulates plant cell expansion through auxin and light signaling pathways. These study results not only provide new insights into the roles of bHLH transcription factors in regulating plant growth and development but also contribute to a deeper understanding of their underlying mechanisms.

Phenotypic Analysis and Molecular Characterization of Enlarged Cell Size Mutant in Nannochloropsis oceanica.

The cell cycle is the fundamental cellular process of eukaryotes. Although cell-cycle-related genes have been identified in microalgae, their cell cycle progression differs from species to species. Cell enlargement in microalgae is an essential biological trait. At the same time, there are various causes of cell enlargement, such as environmental factors, especially gene mutations. In this study, we first determined the phenotypic and biochemical characteristics of a previously obtained enlarged-cell-size mutant of Nannochloropsis oceanica, which was designated ECS. Whole-genome sequencing analysis of the insertion sites of ECS indicated that the insertion fragment is integrated inside the 5'-UTR of U/P-type cyclin CYCU;1 and significantly decreases the gene expression of this cyclin. In addition, the transcriptome showed that CYCU;1 is a highly expressed cyclin. Furthermore, cell cycle analysis and RT-qPCR of cell-cycle-related genes showed that ECS maintains a high proportion of 4C cells and a low proportion of 1C cells, and the expression level of CYCU;1 in wild-type (WT) cells is significantly increased at the end of the light phase and the beginning of the dark phase. This means that CYCU;1 is involved in cell division in the dark phase. Our results explain the reason for the larger ECS size. Mutation of CYCU;1 leads to the failure of ECS to fully complete cell division in the dark phase, resulting in an enlargement of the cell size and a decrease in cell density, which is helpful to understand the function of CYCU;1 in the Nannochloropsis cell cycle.

Elongation enhances encounter rates between phytoplankton in turbulence.

Phytoplankton come in a stunning variety of shapes but elongated morphologies dominate-typically 50% of species have aspect ratio above 5, and bloom-forming species often form chains whose aspect ratios can exceed 100. How elongation affects encounter rates between phytoplankton in turbulence has remained unknown, yet encounters control the formation of marine snow in the ocean. Here, we present simulations of encounters among elongated phytoplankton in turbulence, showing that encounter rates between neutrally buoyant elongated cells are up to 10-fold higher than for spherical cells and even higher when cells sink. Consequently, we predict that elongation can significantly speed up the formation of marine snow compared to spherical cells. This unexpectedly large effect of morphology in driving encounter rates among plankton provides a potential mechanistic explanation for the rapid clearance of many phytoplankton blooms.

Mouse oocytes develop in cysts with the help of nurse cells.

Mouse germline cysts, on average, develop into six oocytes supported by 24 nurse cells that transfer cytoplasm and organelles to generate a Balbiani body. We showed that between E14.5 and P5, cysts periodically activate some nurse cells to begin cytoplasmic transfer, which causes them to shrink and turnover within 2 days. Nurse cells die by a programmed cell death (PCD) pathway involving acidification, similar to Drosophila nurse cells, and only infrequently by apoptosis. Prior to initiating transfer, nurse cells co-cluster by scRNA-seq with their pro-oocyte sisters, but during their final 2 days, they cluster separately. The genes promoting oocyte development and nurse cell PCD are upregulated, whereas the genes that repress transfer, such as Tex14, and oocyte factors, such as Nobox and Lhx8, are under-expressed. The transferred nurse cell centrosomes build a cytocentrum that establishes a large microtubule aster in the primordial oocyte that organizes the Balbiani body, defining the earliest oocyte polarity.

Transcriptional regulation of neural stem cell expansion in the adult hippocampus.

Experience governs neurogenesis from radial-glial neural stem cells (RGLs) in the adult hippocampus to support memory. Transcription factors (TFs) in RGLs integrate physiological signals to dictate self-renewal division mode. Whereas asymmetric RGL divisions drive neurogenesis during favorable conditions, symmetric divisions prevent premature neurogenesis while amplifying RGLs to anticipate future neurogenic demands. The identities of TFs regulating RGL symmetric self-renewal, unlike those that regulate RGL asymmetric self-renewal, are not known. Here, we show in mice that the TF Kruppel-like factor 9 (Klf9) is elevated in quiescent RGLs and inducible, deletion of Klf9 promotes RGL activation state. Clonal analysis and longitudinal intravital two-photon imaging directly demonstrate that Klf9 functions as a brake on RGL symmetric self-renewal. In vivo translational profiling of RGLs lacking Klf9 generated a molecular blueprint for RGL symmetric self-renewal that was characterized by upregulation of genetic programs underlying Notch and mitogen signaling, cell cycle, fatty acid oxidation, and lipogenesis. Together, these observations identify Klf9 as a transcriptional regulator of neural stem cell expansion in the adult hippocampus.

In humans and other mammals, a region of the brain known as the hippocampus plays important roles in memory. New experiences guide cells in the hippocampus known as radial-glial neural stem cells (RGLs) to divide to make new neurons and other types of cells involved in forming memories. Each time an RGL divides, it can choose to divide asymmetrically to maintain a copy of itself and make a new cell of another type, or divide symmetrically (a process known as symmetric self-renewal) to produce two RGLs. Symmetric self-renewal helps to restore and replenish the pool of stem cells in the hippocampus that are lost due to injury or age, allowing us to continue making new neurons. Proteins known as transcription factors are believed to control how RGLs divide. Previous studies have identified several transcription factors that regulate the RGLs splitting asymmetrically to make neurons and other cells. But the identities of the transcription factors that regulate symmetric self-renewal in the adult hippocampus have remained elusive. Here, Guo et al. searched for transcription factors that regulate symmetric self-renewal of RGLs in mice. The experiments found that RGLs that are resting and not dividing (referred to as 'quiescent') have higher levels of a transcription factor called Klf9 than RGLs that are actively dividing. Loss of the gene encoding Klf9 triggered quiescent RGLs to start dividing, and further experiments showed that Klf9 directly inhibited symmetric self-renewal. Guo et al. then used an approach called in vivo translational profiling to generate a blueprint that revealed new insights into the molecular processes involved in this symmetric division. These findings pave the way for researchers to develop strategies that may expand the numbers of stem cells in the hippocampus. This could eventually be used to help replenish brain circuits with neurons and improve the memory of individuals with Alzheimer's disease or other conditions that cause memory loss.

Volume growth in animal cells is cell cycle dependent and shows additive fluctuations.

The way proliferating animal cells coordinate the growth of their mass, volume, and other relevant size parameters is a long-standing question in biology. Studies focusing on cell mass have identified patterns of mass growth as a function of time and cell cycle phase, but little is known about volume growth. To address this question, we improved our fluorescence exclusion method of volume measurement (FXm) and obtained 1700 single-cell volume growth trajectories of HeLa cells. We find that, during most of the cell cycle, volume growth is close to exponential and proceeds at a higher rate in S-G2 than in G1. Comparing the data with a mathematical model, we establish that the cell-to-cell variability in volume growth arises from constant-amplitude fluctuations in volume steps rather than fluctuations of the underlying specific growth rate. We hypothesize that such 'additive noise' could emerge from the processes that regulate volume adaptation to biophysical cues, such as tension or osmotic pressure.

Effect of Ghrelin Intervention on the Ras/ERK Pathway in the Regulation of Heart Failure by PTEN.

OBJECTIVE: To study the possible mechanism of ghrelin in heart failure and how it works. METHOD: In vitro results demonstrated that ghrelin alleviates cardiac function and reduces myocardial fibrosis in rats with heart failure. Moreover, ghrelin intervention increased PTEN expression level and reduced ERK, c-jun, and c-Fos expression level; in vivo experiments demonstrated that ghrelin intervention reduces mast memory expression and increases cardiomyocyte surface area, PTEN expression level, ERK, c-jun, c-Fos expression level, and cell surface area, while ERK blockade suppresses mast gene expression and reduces cell surface area. RESULTS: In vitro experimental results prove that we have successfully constructed a rat model related to heart failure, and ghrelin can alleviate the heart function of heart failure rats and reduce myocardial fibrosis. In addition, ghrelin is closely related to the decrease of the expression levels of ERK, c-jun, and c-Fos, but it can also increase the expression of PTEN in the rat model; in vivo experiments proved that we successfully constructed an in vitro cardiac hypertrophy model, and the intervention of ghrelin would reduce the expression of hypertrophic memory and increase the surface area of cardiomyocytes, increase the expression level of PTEN, and reduce the expression levels of ERK, c-jun, and c-Fos, while the blockade of PTEN will increase the expression of hypertrophy genes and increase the cell surface area, while the blockade of ERK will increase the expression of hypertrophic genes, which in turn will make the cell surface area reducing. CONCLUSION: Ghrelin inhibits the phosphorylation and nuclear entry of ERK by activating PTEN, thereby controlling the transcription of hypertrophic genes, improving myocardial hypertrophy, and enhancing cardiac function.

The bryophytes Physcomitrium patens and Marchantia polymorpha as model systems for studying evolutionary cell and developmental biology in plants.

Bryophytes are nonvascular spore-forming plants. Unlike in flowering plants, the gametophyte (haploid) generation of bryophytes dominates the sporophyte (diploid) generation. A comparison of bryophytes with flowering plants allows us to answer some fundamental questions raised in evolutionary cell and developmental biology. The moss Physcomitrium patens was the first bryophyte with a sequenced genome. Many cell and developmental studies have been conducted in this species using gene targeting by homologous recombination. The liverwort Marchantia polymorpha has recently emerged as an excellent model system with low genomic redundancy in most of its regulatory pathways. With the development of molecular genetic tools such as efficient genome editing, both P. patens and M. polymorpha have provided many valuable insights. Here, we review these advances with a special focus on polarity formation at the cell and tissue levels. We examine current knowledge regarding the cellular mechanisms of polarized cell elongation and cell division, including symmetric and asymmetric cell division. We also examine the role of polar auxin transport in mosses and liverworts. Finally, we discuss the future of evolutionary cell and developmental biological studies in plants.

Distinguishing different modes of growth using single-cell data.

Collection of high-throughput data has become prevalent in biology. Large datasets allow the use of statistical constructs such as binning and linear regression to quantify relationships between variables and hypothesize underlying biological mechanisms based on it. We discuss several such examples in relation to single-cell data and cellular growth. In particular, we show instances where what appears to be ordinary use of these statistical methods leads to incorrect conclusions such as growth being non-exponential as opposed to exponential and vice versa. We propose that the data analysis and its interpretation should be done in the context of a generative model, if possible. In this way, the statistical methods can be validated either analytically or against synthetic data generated via the use of the model, leading to a consistent method for inferring biological mechanisms from data. On applying the validated methods of data analysis to infer cellular growth on our experimental data, we find the growth of length in E. coli to be non-exponential. Our analysis shows that in the later stages of the cell cycle the growth rate is faster than exponential.

All cells ­ from bacteria to humans ­ tightly control their size as they grow and divide. Cells can also change the speed at which they grow, and the pattern of how fast a cell grows with time is called 'mode of growth'. Mode of growth can be 'linear', when cells increase their size at a constant rate, or 'exponential', when cells increase their size at a rate proportional to their current size. A cell's mode of growth influences its inner workings, so identifying how a cell grows can reveal information about how a cell will behave. Scientists can measure the size of cells as they age and identify their mode of growth using single cell imaging techniques. Unfortunately, the statistical methods available to analyze the large amounts of data generated in these experiments can lead to incorrect conclusions. Specifically, Kar et al. found that scientists had been using specific types of plots to analyze growth data that were prone to these errors, and may lead to misinterpreting exponential growth as linear and vice versa. This discrepancy can be resolved by ensuring that the plots used to determine the mode of growth are adequate for this analysis. But how can the adequacy of a plot be tested? One way to do this is to generate synthetic data from a known model, which can have a specific and known mode of growth, and using this data to test the different plots. Kar et al. developed such a 'generative model' to produce synthetic data similar to the experimental data, and used these data to determine which plots are best suited to determine growth mode. Once they had validated the best statistical methods for studying mode of growth, Kar et al. applied these methods to growth data from the bacterium Escherichia coli. This showed that these cells have a form of growth called 'super-exponential growth'. These findings identify a strategy to validate statistical methods used to analyze cell growth data. Furthermore, this strategy ­ the use of generative models to produce synthetic data to test the accuracy of statistical methods ­ could be used in other areas of biology to validate statistical approaches.

Pear xyloglucan endotransglucosylase/hydrolases PcBRU1 promotes stem growth through regulating cell wall elongation.

Brassinosteroids (BRs) play numerous important roles in plant growth and development. Previous studies reported that BRs could promote stem growth by regulating the expression of xyloglucan endotransglucosylase/hydrolases (XTHs). However, the mechanism of XTHs involved in stem growth remains unclear. In this study, PcBRU1, which belonged to the XTH family, was upregulated by exogenous BL treatment in Pyrus communis. The expression of PcBRU1 was highest in stems and lowest in leaves. Subcellular localization analysis indicated that PcBRU1 was located in the plasma membrane. Furthermore, overexpressing PcBRU1 in tobaccos promoted the plant height and internode length. Electron microscopy and anatomical structure analysis showed that the cell wall was significantly thinner and the cells were slenderer in transgenic tobacco lines overexpressing PcBRU1 than in wild-type tobaccos. PcBRU1 promoted stem growth as it loosened the cell wall, leading to the change in cell morphology. In addition, overexpressing PcBRU1 altered the root development and leaf shape of transgenic tobaccos. Taken together, the results could provide a theoretical basis for the XTH family in regulating cell-wall elongation and stem growth.

bHLH transcription factors LP1 and LP2 regulate longitudinal cell elongation.

Basic helix-loop-helix/helix-loop-helix (bHLH/HLH) transcription factors play substantial roles in plant cell elongation. In this study, two bHLH/HLH homologous proteins leaf related protein 1 and leaf-related protein 2 (AtLP1 and AtLP2) were identified in Arabidopsis thaliana. LP1 and LP2 play similar positive roles in longitudinal cell elongation. Both LP1 and LP2 overexpression plants exhibited long hypocotyls, elongated cotyledons, and particularly long leaf blades. The elongated leaves resulted from increased longitudinal cell elongation. lp1 and lp2 loss-of-function single mutants did not display distinct phenotypes, but the lp1lp2 double mutant showed decreased leaf length associated with less longitudinal polar cell elongation. Furthermore, the phenotype of lp1lp2 could be rescued by the expression of LP1 or LP2. Expression of genes related to cell elongation was upregulated in LP1 and LP2 overexpression plants but downregulated in lp1lp2 double mutant plants compared with that of wild type. LP1 and LP2 proteins could directly bind to the promoters of Longifolia1 (LNG1) and LNG2 to activate the expression of these cell elongation related genes. Both LP1 and LP2 could interact with two other bHLH/HLH proteins, IBH1 (ILI1 binding BHLH Protein1) and IBL1 (IBH1-like1), thereby suppressing the transcriptional activation of LP1 and LP2 to the target genes LNG1 and LNG2. Thus, our data suggested that LP1 and LP2 act as positive regulators to promote longitudinal cell elongation by activating the expression of LNG1 and LNG2 genes in Arabidopsis. Moreover, homodimerization of LP1 and LP2 may be essential for their function, and interaction between LP1/LP2 and other bHLH/HLH proteins may obstruct transcriptional regulation of target genes by LP1 and LP2.

Salinity Affects Saxitoxins (STXs) Toxicity in the Dinoflagellate Alexandrium pacificum, with Low Transcription of SXT-Biosynthesis Genes sxtA4 and sxtG.

Salinity is an important factor for regulating metabolic processes in aquatic organisms; however, its effects on toxicity and STX biosynthesis gene responses in dinoflagellates require further elucidation. Herein, we evaluated the physiological responses, toxin production, and expression levels of two STX synthesis core genes, sxtA4 and sxtG, in the dinoflagellate Alexandrium pacificum Alex05 under different salinities (20, 25, 30, 35, and 40 psu). Optimal growth was observed at 30 psu (0.12 cell division/d), but cell growth significantly decreased at 20 psu and was irregular at 25 and 40 psu. The cell size increased at lower salinities, with the highest size of 31.5 µm at 20 psu. STXs eq was highest (35.8 fmol/cell) in the exponential phase at 30 psu. GTX4 and C2 were predominant at that time but were replaced by GTX1 and NeoSTX in the stationary phase. However, sxtA4 and sxtG mRNAs were induced, and their patterns were similar in all tested conditions. PCA showed that gene transcriptional levels were not correlated with toxin contents and salinity. These results suggest that A. pacificum may produce the highest amount of toxins at optimal salinity, but sxtA4 and sxtG may be only minimally affected by salinity, even under high salinity stress.

Tissue-resident M2 macrophages directly contact primary sensory neurons in the sensory ganglia after nerve injury.

BACKGROUND: Macrophages in the peripheral nervous system are key players in the repair of nerve tissue and the development of neuropathic pain due to peripheral nerve injury. However, there is a lack of information on the origin and morphological features of macrophages in sensory ganglia after peripheral nerve injury, unlike those in the brain and spinal cord. We analyzed the origin and morphological features of sensory ganglionic macrophages after nerve ligation or transection using wild-type mice and mice with bone-marrow cell transplants. METHODS: After protecting the head of C57BL/6J mice with lead caps, they were irradiated and transplanted with bone-marrow-derived cells from GFP transgenic mice. The infraorbital nerve of a branch of the trigeminal nerve of wild-type mice was ligated or the infraorbital nerve of GFP-positive bone-marrow-cell-transplanted mice was transected. After immunostaining the trigeminal ganglion, the structures of the ganglionic macrophages, neurons, and satellite glial cells were analyzed using two-dimensional or three-dimensional images. RESULTS: The number of damaged neurons in the trigeminal ganglion increased from day 1 after infraorbital nerve ligation. Ganglionic macrophages proliferated from days 3 to 5. Furthermore, the numbers of macrophages increased from days 3 to 15. Bone-marrow-derived macrophages increased on day 7 after the infraorbital nerve was transected in the trigeminal ganglion of GFP-positive bone-marrow-cell-transplanted mice but most of the ganglionic macrophages were composed of tissue-resident cells. On day 7 after infraorbital nerve ligation, ganglionic macrophages increased in volume, extended their processes between the neurons and satellite glial cells, and contacted these neurons. Most of the ganglionic macrophages showed an M2 phenotype when contact was observed, and little neuronal cell death occurred. CONCLUSION: Most of the macrophages that appear after a nerve injury are tissue-resident, and these make direct contact with damaged neurons that act in a tissue-protective manner in the M2 phenotype. These results imply that tissue-resident macrophages signal to neurons directly through physical contact.

Network pharmacology and experimental investigation of Rhizoma polygonati extract targeted kinase with herbzyme activity for potent drug delivery.

Rhizoma polygonati (Huangjing, RP) has been used for a long history with many chemical components in inducing anti-cancer, anti-aging, anti-diabetes, anti-fatigue, and more prevention of diseases or acts as nutrition sources in food. Here we investigated RP extract combination with kinase inhibitors in anti-cell growth and blockade in pathways targeting kinases. Experimental investigation and network pharmacology analysis were applied to test the potent kinase-mediated signaling. Herbzyme activity was determined by substrate with optical density measurement. Extract of processed RP inhibits cell growth in a much greater manner than alone when applied in combination with inhibitors of mTOR or EGFR. Moreover, processing methods of RP from Mount Tai (RP-Mount Tai) play essential roles in herbzyme activity of phosphatase suggesting the interface is also essential, in addition to the chemical component. The network pharmacology analysis showed the chemical component and target networks involving AKT and mTOR, which is consistent with experimental validation. Finally, EGFR inhibitor could be associated with nano-extract of RP-Mount Tai but not significantly affects the phosphatase herbzyme activity in vitro. Thus the processed extract of RP-Mount Tai may play a dual role in the inhibition of cell proliferation signaling by both chemical component and nanoscale herbzyme of phosphatase activity to inhibit kinases including mTOR/AKT in potent drug delivery of kinase inhibitors.

A prion accelerates proliferation at the expense of lifespan.

In fluctuating environments, switching between different growth strategies, such as those affecting cell size and proliferation, can be advantageous to an organism. Trade-offs arise, however. Mechanisms that aberrantly increase cell size or proliferation-such as mutations or chemicals that interfere with growth regulatory pathways-can also shorten lifespan. Here we report a natural example of how the interplay between growth and lifespan can be epigenetically controlled. We find that a highly conserved RNA-modifying enzyme, the pseudouridine synthase Pus4/TruB, can act as a prion, endowing yeast with greater proliferation rates at the cost of a shortened lifespan. Cells harboring the prion grow larger and exhibit altered protein synthesis. This epigenetic state, [BIG+] (better in growth), allows cells to heritably yet reversibly alter their translational program, leading to the differential synthesis of dozens of proteins, including many that regulate proliferation and aging. Our data reveal a new role for prion-based control of an RNA-modifying enzyme in driving heritable epigenetic states that transform cell growth and survival.

Cells make different proteins to perform different tasks. Each protein is a long chain of building blocks called amino acids that must fold into a particular shape before it can be useful. Some proteins can fold in more than one way, a normal form and a 'prion' form. Prions are unusual in that they can force normally folded proteins with the same amino acid sequence as them to refold into new prions. This means that a single prion can make many more that are inherited when cells divide. Some prions can cause disease, but others may be beneficial. Pus4 is a yeast protein that is typically involved in modifying ribonucleic acids, molecules that help translate genetic information into new proteins. Sometimes Pus4 can adopt a beneficial prion conformation called [BIG⁺]. When yeast cells have access to plenty of nutrients, [BIG⁺] helps them grow faster and larger, but this comes at the cost of a shorter lifespan. Garcia, Campbell et al. combined computational modeling and experiments in baker's yeast (Saccharomyces cerevisiae) to investigate the role of [BIG⁺]. They found that the prion accelerated protein production, leading to both faster growth and a shorter lifespan in these cells, even without any changes in gene sequence. Garcia, Campbell et al.'s findings explain the beneficial activity of prion proteins in baker's yeast cells. The results also describe how cells balance a tradeoff between growth and lifespan without any changes in the genome. This helps to highlight that genetics do not always explain the behaviors of cells, and further methods are needed to better understand cell biology.

An auxin signaling network translates low-sugar-state input into compensated cell enlargement in the fugu5 cotyledon.

In plants, the effective mobilization of seed nutrient reserves is crucial during germination and for seedling establishment. The Arabidopsis H+-PPase-loss-of-function fugu5 mutants exhibit a reduced number of cells in the cotyledons. This leads to enhanced post-mitotic cell expansion, also known as compensated cell enlargement (CCE). While decreased cell numbers have been ascribed to reduced gluconeogenesis from triacylglycerol, the molecular mechanisms underlying CCE remain ill-known. Given the role of indole 3-butyric acid (IBA) in cotyledon development, and because CCE in fugu5 is specifically and completely cancelled by ech2, which shows defective IBA-to-indoleacetic acid (IAA) conversion, IBA has emerged as a potential regulator of CCE. Here, to further illuminate the regulatory role of IBA in CCE, we used a series of high-order mutants that harbored a specific defect in IBA-to-IAA conversion, IBA efflux, IAA signaling, or vacuolar type H+-ATPase (V-ATPase) activity and analyzed the genetic interaction with fugu5-1. We found that while CCE in fugu5 was promoted by IBA, defects in IBA-to-IAA conversion, IAA response, or the V-ATPase activity alone cancelled CCE. Consistently, endogenous IAA in fugu5 reached a level 2.2-fold higher than the WT in 1-week-old seedlings. Finally, the above findings were validated in icl-2, mls-2, pck1-2 and ibr10 mutants, in which CCE was triggered by low sugar contents. This provides a scenario in which following seed germination, the low-sugar-state triggers IAA synthesis, leading to CCE through the activation of the V-ATPase. These findings illustrate how fine-tuning cell and organ size regulation depend on interplays between metabolism and IAA levels in plants.

UBC19 is a new interacting protein of ORANGE for its nuclear localization in Arabidopsis thaliana.

ORANGE (OR) is a member of the DnaJ-like zinc finger domain-containing protein family, of which all orthologs share a highly conserved quadruple repeat of the CxxCxxxG signatures at their C-termini. Dual subcellular localization and different interacting partner proteins have been reported for OR. In plastids, OR interacts with phytoene synthase, the entry enzyme for carotenoid biosynthesis, to promote chromoplast biogenesis and carotenoid accumulation in non-pigmented tissues. In the nucleus, OR interacts with the eukaryotic release factor eRF1-2 to regulate cell elongation in the petiole, and with the transcription factor TCP14 to repress the expression of Early Light-Induced Proteins (ELIPs) and chloroplast biogenesis in de-etiolating cotyledons. In this study, we demonstrated the E2 ubiquitin-conjugating enzyme UBC19 as a new interacting partner of OR. The lysine58 of OR was found to be ubiquitinated, and OR lost its nuclear localization and the capability in repressing ELIPs when lysine58 was substituted by alanine. Our findings raised the possibility that the ubiquitination by UBC19 is essential for the nuclear localization of OR.

Genetic interaction between F-box motif encoding YDR131C and retrograde signaling-related RTG1 regulates the stress response and apoptosis in Saccharomyces cerevisiae.

The retrograde signaling pathway is well conserved from yeast to humans, which regulates cell adaptation during stress conditions and prevents cell death. One of its components, RTG1 encoded Rtg1p in association with Rtg3p communicates between mitochondria, nucleus, and peroxisome during stress for adaptation, by regulation of transcription. The F-box motif protein encoded by YDR131C  constitutes a part of SCF Ydr131c -E3 ligase complex, with unknown function; however, it is known that retrograde signaling is modulated by the E3 ligase complex. This study reports epistasis interaction between YDR131C and RTG1, which regulates cell growth, response to genotoxic stress, decreased apoptosis, resistance to petite mutation, and cell wall integrity. The cells of ydr131cΔrtg1Δ genetic background exhibits growth rate improvement however, sensitivity to hydroxyurea, itraconazole antifungal agent and synthetic indoloquinazoline-based alkaloid (8-fluorotryptanthrin, RK64), which disrupts the cell wall integrity in Saccharomyces cerevisiae. The epistatic interaction between YDR131C and RTG1 indicates a link between protein degradation and retrograde signaling pathways.

Novel cell-based analysis reveals region-dependent changes in microglial dynamics in grey matter in a cuprizone model of demyelination.

Microglia are key players in Multiple Sclerosis (MS), expressing many susceptibility genes for this disease. They constantly survey the brain microenvironment, but the precise functional relationships between microglia and pathological processes remain unknown. We performed a detailed assessment of microglial dynamics in three distinct grey matter regions in a cuprizone-induced demyelination model. We found that microglial activation preceded detectable demyelination and showed regional specificities, such as prominent phagocytic activity in cortical layer 5 and early hypertrophic morphology in hippocampal CA1. Demyelination happened earliest in cortical layer 5, although was more complete in CA1. In cortical layer 2/3, microglial activation and demyelination were less pronounced but microglia became hyper-ramified with slower process movement during remyelination, thereby maintaining local brain surveillance. Profiling of microglia using specific morphological and motility parameters revealed region-specific heterogeneity of microglial responses in the grey matter that might serve as sensitive indicators of progression in CNS demyelinating diseases.

Generation of microRNA-378a-deficient hiPSC as a novel tool to study its role in human cardiomyocytes.

microRNA-378a (miR-378a) is one of the most highly expressed microRNAs in the heart. However, its role in the human cardiac tissue has not been fully understood. It was observed that miR-378a protects cardiomyocytes from hypertrophic growth by regulation of IGF1R and the expression of downstream kinases. Increased levels of miR-378a were reported in the serum of Duchenne muscular dystrophy (DMD) patients and female carriers of DMD gene-associated mutations with developed cardiomyopathy. In order to shed more light on the role of miR-378a in human cardiomyocytes and its potential involvement in DMD-related cardiomyopathy, we generated two human induced pluripotent stem cell (hiPSC) models; one with deletion of miR-378a and the second one with deletion of DMD exon 50 leading to the DMD phenotype. Our results indicate that lack of miR-378a does not influence the pluripotency of hiPSC and their ability to differentiate into cardiomyocytes (hiPSC-CM). miR-378a-deficient hiPSC-CM exhibited, however, significantly bigger size compared to the isogenic control cells, indicating the role of this miRNA in the hypertrophic growth of human cardiomyocytes. In accordance, the level of NFATc3, phosphoAKT, phosphoERK and ERK was higher in these cells compared to the control counterparts. A similar effect was achieved by silencing miR-378a with antagomirs. Of note, the percentage of cells with nuclear localization of NFATc3 was higher in miR-378a-deficient hiPSC-CM. Analysis of electrophysiological properties and Ca2+ oscillations revealed the decrease in the spike slope velocity and lower frequency of calcium spikes in miR-378a-deficient hiPSC-CM. Interestingly, the level of miR-378a increased gradually during cardiac differentiation of hiPSC. Of note, it was low until day 15 in differentiating DMD-deficient hiPSC-CM and then rose to a similar level as in the isogenic control counterparts. In summary, our findings confirmed the utility of hiPSC-based models for deciphering the role of miR-378a in the control and diseased human cardiomyocytes.