Biology subjects

Guo, A.

Publications and source records attributed to Guo, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

A Plant Flavonol Rescues a Pathogenic Mutation Associated with Kinesin in Neurons

KIF1A, a microtubule-based motor protein responsible for axonal transport, is linked to a group of neurological disorders known as KIF1A-associated neurological disorder (KAND). Current therapeutic options for KAND are limited. Here, we introduced the clinically relevant KIF1A(R11Q) variant into the C. elegans homolog UNC-104, resulting in uncoordinated animal behaviors. Through genetic suppressor screens, we identified intragenic mutations in UNC-104s motor domain that rescued synaptic vesicle localization and coordinated movement. We showed that two suppressor mutations partially recovered motor activity in vitro by counteracting the structural defect caused by R11Q at KIF1As nucleotide-binding pocket. We found that supplementation with fisetin, a plant flavonol, improved KIF1A(R11Q) worms movement and morphology. Notably, our biochemical and single-molecule assays revealed that fisetin directly restored the ATPase activity and processive movement of human KIF1A(R11Q) without affecting wild-type KIF1A. These findings suggest fisetin as a potential intervention for enhancing KIF1A(R11Q) activity and alleviating associated defects in KAND.

cell biology↗

bioRxiv2023

Tracking the Metabolic Fate of Exogenous Arachidonic Acid in Ferroptosis Using Dual-Isotope Labeling Lipidomics

Lipid metabolism is implicated in a variety of diseases, including cancer, cell death, and inflammation, but lipidomics has proven to be challenging due to the vast structural diversity over a narrow range of mass and polarity of lipids. Isotope labeling is often used in metabolomics studies to follow the metabolism of exogenously added labeled compounds because they can be differentiated from endogenous compounds by the mass shift associated with the label. The application of isotope labeling to lipidomics has also been explored as a method to track the metabolism of lipids in various disease states. However, it can be difficult to differentiate a single isotopically labeled lipid from the rest of the lipidome due to the variety of endogenous lipids present over the same mass range. Here we report the development of a dual-isotope deuterium labeling method to track the metabolic fate of exogenous polyunsaturated fatty acids, e.g, arachidonic acid (AA), in the context of ferroptosis using hydrophilic interaction-ion mobility-mass spectrometry (HILIC-IM-MS). Ferroptosis is a type of cell death that is dependent on lipid peroxidation. The use of two isotope labels rather than one enables the identification of labeled species by a signature doublet peak in the resulting mass spectra. A Python-based software, D-Tracer, was developed to efficiently extract metabolites with dual-isotope labels. The labeled species were then identified with Lipydomics based on their retention times, collision cross section, and m/z values. Changes in exogenous AA incorporation in the absence and presence of a ferroptosis inducer were elucidated. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/542640v1_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@6bbc67org.highwire.dtl.DTLVardef@11e3a84org.highwire.dtl.DTLVardef@193ce6eorg.highwire.dtl.DTLVardef@d5b757_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

TPR domain assigns versatility of BcTir/Tpr system against viral infection

NAD+-derived signal produced by TIR domain triggered the host immune responses. The ubiquitous TPR domain involved in signal recognition and effector activation were found widely assembled with the TIR domain. However, the immune roles of these assemblies remain elusive. Here, a two-gene operon, one containing a TIR domain, designated as BcTir, and the other, BcTpr, from Bacillus cereus, exhibited anti-phage immunity. BcTpr, but not BcTir, exhibited NADase activity to produce the cyclic ADPR (cADPR) isomer and mediate NAD+ depletion. Noticeably, the truncated N terminus of BcTpr only depleted NAD+ unless at the presence of TPR domain to generate cADPR isomer unveiling its role played for glycosite selection. In addition, the BcTir/Tpr system significantly repressed viral proliferation and increased oxidation resistance by scavenging excessive reactive oxygen species (ROS) upon phage infection. These findings unraveled a multifunctional role of the BcTir/Tpr system during immune responses. In BriefThe bacterial BcTir/Tpr system was identified with the ability to protect against phage infection via NAD+ depletion, viral replication repression, and ROS homeostasis, in which BcTpr played a dual role in NAD+-derived signal production and NAD+ depletion. HighlightsO_LIThe BcTir/Tpr system works as a BcTir-BcTpr complex against phage infection. C_LIO_LIBcTpr, instead of BcTir, generates the NAD+-derived cADPR isomer through its glycosidase domain at N terminus. C_LIO_LIThe amount of cADPR isomer production is regulated by the helix numbers of the TPR domain at C terminus of BcTpr. C_LIO_LIThe BcTir/Tpr system can depress phage proliferation and decrease ROS production upon phage infection. C_LI

microbiology↗

bioRxiv2022

JASPER: a fast genome polishing tool that improves accuracy and creates population-specific reference genomes

Advances in long-read sequencing technologies have dramatically improved the contiguity and completeness of genome assemblies. Using the latest nanopore-based sequencers, we can generate enough data for the assembly of a human genome from a single flow cell. With the long-read data from these sequences, we can now routinely produce de novo genome assemblies in which half or more of a genome is contained in megabase-scale contigs. Assemblies produced from nanopore data alone, though, have relatively high error rates and can benefit from a process called polishing, in which more-accurate reads are used to correct errors in the consensus sequence. In this manuscript, we present a novel tool for genome polishing called JASPER (Jellyfish-based Assembly Sequence Polisher for Error Reduction). In contrast to other polishing methods, JASPER gains efficiency by avoiding the alignment of reads to the assembly. Instead, JASPER uses a database of k-mer counts that it creates from the reads to detect and correct errors in the consensus. In addition to its use for polishing genomes, JASPER can also create population-specific genomes using an existing reference genome along with sequencing reads from multiple individuals from the population of interest. In this mode, JASPER alters the reference genome so that it contains variants that are common in the target population. In our experiments, we show that after creating a Japanese-specific reference genome, we observed a 27% reduction in homozygous variant calls using whole-genome sequencing reads from an individual from Tokyo.

bioinformatics↗

bioRxiv2022

Genetic Impairment of Succinate Metabolism Disrupts Bioenergetic Sensing in Adrenal Neuroendocrine Tumor

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of mitochondrial TCA cycle enzyme, succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect in PC tumors, triggers dysregulation of Ca2+ homeostasis, and aberrantly activates calpain and the protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a cascade of phospho- signaling where GSK3 inhibition inactivates energy sensing by AMP-kinase through dephosphorylation of the AMP-kinase {gamma} subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC tumor formation. A novel Cdk5 inhibitor, MRT3-007, reversed this phospho-cascade, invoking an anti- Warburg effect, cell cycle arrest, and senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important novel mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers. HighlightsO_LILoss of SDHB function in pheochromocytoma causes Ca2+ dysregulation, calpain activation, and aberrant activation of the protein kinase Cdk5. C_LIO_LIHyperactive Cdk5 deregulates a GSK3/PRKAG2/AMPK signaling cascade. C_LIO_LIp25 overexpression and consequent aberrant Cdk5 activity in chromaffin cells causes pheochromocytoma. C_LIO_LIInhibition of Cdk5 activates the PRKAG2/AMPK/p53 signaling to rescue cell senescence and block PC tumor progression. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/475410v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@16f0a48org.highwire.dtl.DTLVardef@10c1c7aorg.highwire.dtl.DTLVardef@21b100org.highwire.dtl.DTLVardef@1eb8674_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

Small RNA profiling in Mycobacterium insights into stress adapt ability

Mycobacteria would encounter a number of environment changes during infection, and respond to it using different mechanisms. sRNA is a posttranscriptionally regulatory system for the function of genes and has been investigated in many other bacteria. Here, we used Mycobacterium tuberculosis and Mycobacterium bovis BCG infection models and sequenced the whole bacterial RNAs before and after host cells infection. Comparison of differential expressed sRNAs, by using GO and KEGG, and target predication, was carried out. Six pathogenically relevant stresses, drug resistance test, growth rate and morphology were used for screening and identify sRNAs. From these data, we identified a subset of sRNAs that are differentially expressed in multiple infection groups and stress conditions. We found that many of them were associated with lipid metabolism. Among them, ncBCG427, was significantly down-regulated when BCG entered into macrophages, and was associated with increase of biofilm formation and changed in drug susceptibility. Then, reduction of virulence possibility depends on regulating lipid metabolism.

zoology↗

bioRxiv2021

Sequence Determinants of Human Junctophilin-2 Protein Nuclear Localization and Phase Separation

Junctophilin-2 (JPH2) was conventionally considered as a structural membrane binding protein. Recently, it was shown that proteolytically truncated mouse JPH2 variants are imported into nucleus to exert alternative functions. However, the intranuclear behaviors of human JPH2 (hJPH2) and underlying molecular determinants have not been explored. Here, we demonstrate that full-length hJPH2 is imported into nucleus in human cells by two nuclear localization signals (NLSs), including a newly discovered one at the C-terminus. Importantly, unlike the JPH2 N-terminal truncation which diffuses throughout the nucleus, full-length hJPH2 forms nuclear bodies behaving like liquid-liquid phase separated droplets that are separated from chromatin. The C-terminal transmembrane domain is required for the formation of hJPH2 droplets. Oxidation mimicking substitution of residues C678 and M679 augments the formation of hJPH2 nuclear droplets, suggesting nuclear hJPH2 liquid-liquid phase separation could be modulated by oxidative stress. Mutation A405D, which introduces a negatively charged residue into an intrinsic disordered region (IDR) of hJPH2, turns liquid-like droplets into amyloid-like aggregates. Depletion of an Alanine Rich Region in the IDR recapitulates the liquid-amyloid phase transition. The MORN repeat regions of hJPH2 encodes intrinsic tendency to form amyloid-like structure. Together, these data for the first time revealed the novel intrinsic properties of hJPH2 to form nuclear liquid droplets, and identified critical functional domains and residues encoding these properties. We propose that hJPH2 droplets could function as membrane-less organelles participating in nuclear regulatory processes.

biochemistry↗

bioRxiv2021

Ultrastructural distribution of presynaptic active zones and dense core vesicles in olfactory projection neurons of Drosophila

In Drosophila melanogaster, olfactory projection neurons (PNs) convey odor information from the peripheral olfactory center to higher brain regions. The anatomical and physiological properties of PNs have been well characterized at the cellular and circuit level. The ultrastructural features of PNs remain unknown however, particularly with respect to presynaptic active zones (PAZs) and dense core vesicles (DCVs). In the current study, membrane-labeled electron microscopy was used to volume-reconstruct 89 PN axonal boutons and identify the internal PAZs and DCVs. Based on ultrastructural parameters, these PN boutons could be classified into three morphological distinct subtypes. Interestingly, the distributions of PAZs and DCVs were distinct within these three subtypes. DCVs were enriched in membrane labeled GH146-positive boutons, suggesting that GH146-positive PNs release both neurotransmitters and neuromodulators. The study identified the detailed distributions of PAZs and DCVs in PN boutons and indicates that neuromodulators mediated by DCVs may play an important role in PNs for olfactory processing.

neuroscience↗

bioRxiv2020

Automation of Leaf Counting in Maize and Sorghum Using Deep Learning

Leaf number and leaf emergence rate are phenotypes of interest to plant breeders, plant geneticists, and crop modelers. Counting the extant leaves of an individual plant is straightforward even for an untrained individual, but manually tracking changes in leaf numbers for hundreds of individuals across multiple time points is logistically challenging. This study generated a dataset including over 150,000 maize and sorghum images for leaf counting projects. A subset of 17,783 images also includes annotations of the positions of individual leaf tips. With these annotated images, we evaluate two deep learning-based approaches for automated leaf counting: the first based on counting-by-regression from whole image analysis and a second based on counting-by-detection. Both approaches can achieve RMSE (root of mean square error) smaller than one leaf, only moderately inferior to the RMSE between human annotators of between 0.57 and 0.73 leaves. The counting-by-regression approach based on CNNs (convolutional neural networks) exhibited lower accuracy and increased bias for plants with extreme leaf numbers which are underrepresented in this dataset. The counting-by-detection approach based on Faster R-CNN object detection models achieve near human performance for plants where all leaf tips are visible. The annotated image data and model performance metrics generated as part of this study provide large scale resources for the comparison and improvement of algorithms for leaf counting from image data in grain crops.

plant biology↗

bioRxiv2020

The deacylase SIRT5 supports melanoma viability by regulating chromatin dynamics

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieves a durable remission. SIRT5 is a member of the sirtuin family of protein deacylases that regulate metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we show that SIRT5 is required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 is required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf;Pten-driven melanoma model. Via metabolite and transcriptomic analyses, we find that SIRT5 is required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably include MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a novel, druggable genotype-independent addiction in melanoma.

cancer biology↗

bioRxiv2020

Towards More Reliable Unsupervised Tissue Segmentation Via Integrating Mass Spectrometry Imaging and Hematoxylin-Erosin Stained Histopathological Image

Mass Spectrometry Imaging (MSI) provides a useful tool to divide a tissue section into sub-regions with similar molecular profiles, namely tissue segmentation. However, owing to the lack of ground truth, there is no reliable evaluation approach to assess the validity of unsupervised segmentation outcomes of MSI. We propose a novel solution grounded on a presumption that a segmentation is reliable if it can be reproduced using distinct bio-information extracted from independent sources. Specifically, besides molecular information from MSI data, we also obtain morphological information over a tissue section from its Hematoxylin-Erosin (H&E) stained histopathological image. MSI has high molecular specificity but low spatial resolving power, the H&E image has no molecular specificity but it can capture microscopic details of the tissue with a spatial resolution two magnitudes higher than MSI. The whole H&E image is split into an array of small patches, which correspond to the spatial pixels of MSI. A spectrum of informative morphological features is computed iteratively for each patch and spatial segmentation can be generated by clustering the patches based on their morphological similarities. Adjusted Mutual Information (AMI) score measures the degree of agreement between MSI-based and H&E image-based segmentation outcomes, which is defined by us as an objective and quantitative evaluation metric of segmentation validity. We investigated various candidate morphological features: a combination of Deep Convolution Neural Network (DCNN) features and handcrafted Threshold Adjacency Statistics (TAS) features finally stood out. The most appropriate number of tissue segments was also determined according to AMI score. Moreover, we introduced Co-Clustering algorithm to MSI data to simultaneously group m/z variables and spatial pixels, so potential biomarkers associated to each sub-region were discovered without the need of further analysis. Eventually, by integrating the segmentation outcomes based on MSI and H&E image data, the confidence level of the segment assignment was displayed for each pixel, which offered a much more informative and compelling way to present the segmentation results.

bioinformatics↗

bioRxiv2020

Reciprocal regulation of vacuolar calcium transport and V-ATPase activity, and the effects of Phosphatidylinositol 3,5-bisphosphate

Yeast vacuoles are acidified by the V-ATPase, a protein complex comprised of the membrane embedded VO complex and the soluble cytoplasmic V1 complex. The assembly of the V1-VO holoenzyme is required for the transfer of H+ into the vacuole lumen for acidification. The assembly of the V1-VO holoenzyme is stabilized by the lipid phosphatidylinositol 3,5-bisphospate (PI(3,5)P2) made by the PI3P 5-kinase Fab1/PIKfyve. The absence of PI(3,5)P2 leads to the dissociation of the V1 complex from the membrane. Separately, PI(3,5)P2 has been shown to modulate Ca2+ transport across the vacuole membrane during fission and fusion. Here we examined whether the regulation of H+ and Ca2+ by PI(3,5)P2 are interdependent. We show that modulating extraluminal Ca2+ concentrations inhibit V-ATPase activity. As extraluminal CaCl2 levels are raised, the activity of H+ pumping is reduced. Conversely, chelating free Ca2+ with EGTA stimulated vacuole acidification. Not only did Ca2+ levels affect H+ translocation, we also show that blocking V-ATPase activity inhibited Ca2+ transport into the vacuole lumen. Together, these data illustrate that Ca2+ transport and V-ATPase regulation are interconnected through the modulation of vacuolar lipid profiles. Summary StatementHere we show that Ca2+ and H+ transport across the vacuole membrane is reciprocally regulated and that it is linked to the production of Phosphatidylinositol 3,5-bisphoshpate.

biochemistry↗

bioRxiv2019

NADPH diaphorase neuronal dystrophy in gracile nucleus, cuneatus nucleus and spinal trigeminal nucleus in aged rat

NADPH-diaphorase (N-d) activity is commonly used to identify NOS-ergic neurons. In our previous study, N-d positive neuritic dystrophy and spheroid termed aging-related N-d Body is discovered in the lumbosacral spinal cord in the normal aging rats. Histological studies also reveal that N-d positive neurodegenerative changes occur in the gracile nucleus. We re-examined N-d activity in gracile nucleus in aged rat. We found N-d positive neuritic dystrophy and spheroid also occurred in the cuneatus nucleus and spinal trigeminal nucleus. Besides regular coronal section, longitudinal oriented dystrophic neurites were detected in the sagittal and horizontal section in gracile nucleus and dorsal column. We fziurther examined the medullary oblongata with regular classical histology including Golgi staining, immunocytochemistry of NOS and phosphorylated tau protein, neuronal tracing method with wheat germ agglutinin conjugated alexa-fluor-488 through sciatic nerve, and spinal cord transection at thoracic level. Most of N-d positive neuritic dystrophy and spheroid did not showed colocalization with NOS or phosphorylated tau protein. Neuronal tracing and spinal cord transection revealed that N-d dystrophic neurites in gracile nucleus originated from terminal of sensory projection from spinal cord and peripheral somatic input. The results suggested that aging-related N-d dystrophy in the gracile nucleus was unique morphological feature. In conclusion, it was postulated that the N-d dystrophy as a morphological marker of aging degenerative damage in normal aged organisms.

neuroscience↗

bioRxiv2019

ImmuCellAI: a unique method for comprehensive T-cell subsets abundance prediction and its application in cancer immunotherapy

The distribution and abundance of immune cells, particularly T-cell subsets, play pivotal roles in cancer immunology and therapy. There are many T-cell subsets with specific function, however current methods are limited in estimating them, thus, a method for predicting comprehensive T-cell subsets is urgently needed in cancer immunology research. Here we introduce Immune Cell Abundance Identifier (ImmuCellAI), a novel gene set signature-based method, for precisely estimating the abundance of 24 immune cell types including 18 T-cell subsets, from gene expression data. Performance evaluation on both our sequencing data with flow cytometry results and public expression data indicated that ImmuCellAI can estimate immune cells with superior accuracy than other methods especially on many T-cell subsets. Application of ImmuCellAI to immunotherapy datasets revealed that the abundance of dendritic cells (DC), cytotoxic T, and gamma delta T cells was significantly higher both in comparisons of on-treatment vs. pre-treatment and responders vs. non-responders. Meanwhile, we built an ImmuCellAI result-based model for predicting the immunotherapy response with high accuracy (AUC 0.80~0.91). These results demonstrated the powerful and unique function of ImmuCellAI in tumor immune infiltration estimation and immunotherapy response prediction. The ImmuCellAI online server is freely available at http://bioinfo.life.hust.edu.cn/web/ImmuCellAI/.

bioinformatics↗

bioRxiv2019

Phosphoprotein-based Biomarkers as Predictors for Cancer Therapy

Disparities in cancer patient responses have prompted widespread searches to identify differences in sensitive vs. non-sensitive populations and form the basis of personalized medicine. This customized approach is dependent upon the development of pathway-specific therapeutics in conjunction with biomarkers that predict patient responses. Here, we show that Cdk5 drives growth in subgroups of human patients with multiple types of neuroendocrine neoplasms. Phosphoproteomics and high throughput screening identified novel phosphorylation sites downstream of Cdk5. These phosphorylation events serve as biomarkers of Cdk5 activity and effectively pinpoint Cdk5-driven tumors. Toward achieving targeted therapy, we demonstrate that mouse models of neuroendocrine cancer are responsive to selective Cdk5 inhibitors and biomimetic nanoparticles are effective vehicles for enhanced tumor targeting and reduction of drug toxicity. Finally, we show that biomarkers of Cdk5-dependent tumors effectively predict response to anti-Cdk5 therapy in patient-derived xenografts. Thus, a phosphoprotein-based diagnostic assay combined with Cdk5-targeted therapy is a rational treatment approach for neuroendocrine malignancies.

cancer biology↗

bioRxiv2019

A comparative assessment of aging-related NADPH diaphorase positivity in the spinal cord and medullary oblongata between pigeon and murine

NADPH diaphorase (N-d) positive neurons has been examined in many animals. N-d neurodegenerative neurites were detected in some animal models. However, detailed information of N-d positivity and aging related changes was still lack in the spinal cord and medulla oblongata of pigeons. In this study, we evaluated the N-d positivity and aging alterations in the spinal cord and medullary oblongata of the pigeon compared with rat and mouse. In pigeons, N-d neurons were more numerous in the dorsal horn, around the central canal and in the column of Terni in the thoracic and lumbar segments and scattered neurons occurred in the ventral horn of spinal segments. N-d neurons also occurred in the white matter of spinal cord. Morphometrical analysis demonstrated in the lumbosacral, cervical and thoracic regions. Compared with young pigeons, the size of N-d soma was significantly altered in aged pigeons. Meanwhile, the dramatic morphological changes occurred in the lumbar to sacral segments. The most important findings of this study were aging-related N-d positive bodies (ANB) in aged pigeons, mainly in the nucleus cuneatus externus (CuE), occasionally in the nuclei gracilis et cuneatus. ANBs were identified in the gracile nuclei in spinal cord in the aged rats and mice. ANBs were also detected in the CuE spinal nucleus in the aged rats. Immunohistochemistry also showed that the aging changes occurred in the cell types and neuropeptides in aged animals. The results suggested the weak inflammation and neuronal dysfunction in the spinal cord in aged pigeons. Our results suggested that the ANB could be considered as aging marker in the central nervous system.

neuroscience↗

bioRxiv2019

Copper Blocks V-ATPase Activity and SNARE Complex Formation to Inhibit Yeast Vacuole Fusion

The accumulation of Copper in organisms can lead to altered functions of various pathways, and become cytotoxic through the generation of reactive oxygen species. In yeast, cytotoxic metals such as Hg+, Cd2+, and Cu2+ are transported into the lumen of the vacuole through various pumps. Copper ions are initially transported into the cell by the copper transporter Ctr1 at the plasma membrane and sequestered by chaperones and other factors to prevent cellular damage by free cations. Excess copper ions can subsequently be transported into the vacuole lumen by an unknown mechanism. Transport across membranes requires the reduction of Cu2+ to Cu+. Labile copper ions can interact with membranes to alter fluidity, lateral phase separation and fusion. Here we found that CuCl2 potently inhibited vacuole fusion by blocking SNARE pairing. This was accompanied by the inhibition of V-ATPase H+ pumping. Deletion of the vacuolar reductase Fre6 had no effect on the inhibition of fusion by copper. This suggests that that Cu2+ is responsible for the inhibition of vacuole fusion and V-ATPase function. This notion is supported by the differential effects chelators. The Cu2+-specific chelator TETA rescued fusion, whereas the Cu+-specific chelator BCS had no effect on the inhibited fusion.

biochemistry↗

bioRxiv2019

Phosphatidylinositol 3,5-Bisphosphate regulates Ca2+ Transport During Yeast Vacuolar Fusion through Activation of the Ca2+ ATPase Pmc1

The transport of Ca2+ across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles during hyperosmotic shock become fragmented through fission events that require Ca2+ efflux of their luminal stores through the TRP channel Yvc1. This requires the production of the lipid PI(3,5)P2 by Fab1. Ca2+ is also released during vacuole fusion upon trans-SNARE complex assembly, however, the role of PI(3,5)P2 remains unclear. Here we demonstrate that elevated PI(3,5)P2 levels abolish Ca2+ efflux during fusion, indicating that PI(3,5)P2 has opposing effects on Ca2+ transport in fission versus fusion. Notably, Ca2+ efflux was enhanced when PI(3,5)P2 levels were reduced. Importantly, the effect of PI(3,5)P2 on Ca2+ flux was independent of Yvc1. Rather, the effect was dependent on the Ca2+ pump Pmc1. Vacuoles lacking Pmc1 were resistant to the effects of PI(3,5)P2, while those lacking Yvc1 remained sensitive. Furthermore altering PI(3,5)P2 levels affects the interactions of Pmc1 with the Vo component Vph1 and the R-SNARE Nyv1. We now propose a model in which elevated PI(3,5)P2 activates continued Pmc1 function to prevent the accumulation of released extraluminal Ca2+. SummaryDuring osmotic stress PI(3,5)P2 triggers Ca2+ release from vacuoles. Here we show PI(3,5)P2 stimulates Ca2+ uptake by vacuoles during fusion, illustrating that it has a dual role in Ca2+ transport.

biochemistry↗