Protein Subcellular Localization Research Articles

Further delineation of EBF3-related syndromic neurodevelopmental disorder in twelve Chinese patients.

Neurodevelopmental disorders (NDDs) have heterogeneity in both clinical characteristics and genetic factors. EBF3 is a recently discovered gene associated with a syndromic form of NDDs characterized by hypotonia, ataxia and facial features. In this study, we report twelve unrelated individuals with EBF3 variants using next-generation sequencing. Five missense variants (four novel variants and one known variant) and seven copy number variations (CNVs) of EBF3 gene were identified. All of these patients exhibited developmental delay/intellectual disability. Ataxia was observed in 33% (6/9) of the patients, and abnormal muscle tone was observed in 55% (6/11) of the patients. Aberrant MRI reports were noted in 64% (7/11) of the patients. Four novel missense variants were all located in the DNA-binding domain. The pathogenicity of these variants was validated by in vitro experiments. We found that the subcellular protein localization of the R152C and F211L mutants was changed, and the distribution pattern of the R163G mutant was changed from even to granular. Luciferase assay results showed that the four EBF3 mutants' transcriptional activities were all significantly decreased (p < 0.01). Our study further expanded the gene mutation spectrum of EBF3-related NDD.

Abstract P2-16-06: Protein expression and subcellular localization of a clathrin adaptor AP-1 associate with tumor growth activity in breast cancer

Abstract [Introduction] Breast cancer is the fifth leading cause of cancer-related death among women, and its prevalence is the highest in all cancers of Japanese women. To improve the prognosis of breast cancer patients, new diagnostic and therapeutic tools need to be developed. Adaptor protein complex-1 (AP-1) is one of the clathrin adaptor molecules regulating intracellular transport of specific cargos between the trans-Golgi network (TGN) and endosomes. Although the detailed mechanisms have been poorly elucidated, previous reports suggest that AP-1 expression is related with cell proliferation and cancer metastasis. The purpose of this study is to investigate the association between AP-1 expression in tumor tissue measured with immunohistological analyses and surrogate markers of cancer aggressiveness, such as Ki-67 labeling index and/or breast cancer subtypes. [Materials and methods] The records of 207 primary breast cancer patients surgically treated at Fukushima Medical University Hospital from 2011 to 2014 were reviewed. Paraffin embedded sections of these patients were processed for immunohistochemistry examination using antibody against γ-adaptin, a subunit of AP-1, and then its immunoreactivity was quantified. The staining patterns were also graded into 4 grade: Negative (Neg) = 1, Perinuclear (PN) = 2, Scattered (Sc) = 3, and Diffuse (Dif) = 4. The association of the stain characteristics, such as intensity and patterns, and clinicopathological factors, including Ki-67 labelling index and tumor subtypes, were assessed. In addition, the detailed intracellular localization of AP-1 was examined with double immunofluorescence microscopy using markers for the TGN and endosome. [Results] AP-1 intensity was associated with Ki-67 index (p &amp;lt; 0.005) and subtypes (p &amp;lt; 0.0001). Patients with high Ki-67 index showed significantly higher AP-1 intensity than those with low Ki-67 index (p &amp;lt; 0.05). Regarding the tumor subtypes, HER2 type (p &amp;lt; 0.001) and TNBC (p &amp;lt; 0.05) showed significantly higher AP-1 intensity than Luminal A. HER2 type also showed significantly higher AP-1 intensity than Luminal B (p &amp;lt; 0.01). In terms of the staining pattern, AP-1 distribution was also significantly associated with Ki-67 index (p &amp;lt; 0.0001) and subtype (p &amp;lt; 0.0001). The staining pattern grade of high Ki-67 index patients was significantly higher (p &amp;lt; 0.0001) than that of low Ki-67 index patients. Regarding the association with subtypes, the grades in Luminal B (p &amp;lt; 0.0001), HER2 type (p &amp;lt; 0.0001), and TNBC (p &amp;lt; 0.005) were significantly higher than that of Luminal A, and HER2 type also showed higher score than Luminal B (p &amp;lt; 0.01). In tumor cells with Sc pattern, AP-1 was mainly localized in EEA1-positive endosomes, but the localization in TGN was not apparent. [Conclusion] Although further confirmative studies are needed, these results suggest that the protein expression and the endosomal localization of AP-1 are likely related to the cellular proliferation activity in breast cancer tissues. Aberrant AP-1 distribution could be a predictive marker of the tumor growth potential. Citation Format: Nobuhiro Hoshi, Kazunoshin Tachibana, Takefumi Uemura, Yuko Nishimagi, Masaru Noda, Maiko Okano, Sadahiko Abe, Tohru Otake, Satoshi Waguri. Protein expression and subcellular localization of a clathrin adaptor AP-1 associate with tumor growth activity in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-06.

MED13L and its disease-associated variants influence the dendritic development of cerebral cortical neurons in the mammalian brain.

The mediator complex comprises multiple subcellular subunits that collectively function as a molecular interface between RNA polymerase II and gene-specific transcription factors. Recently, genetic variants to one subunit of the complex, known as MED13L (mediator complex subunit 13 like), have been implicated in syndromic intellectual disability and distinct facial features, frequently accompanied by congenital heart defects. We investigated the impact of five disease-associated MED13L variants on the subcellular localization and biochemical stability of MED13L protein in vitro and in vivo. In overexpression assays using cortical neurons from embryonic mouse cerebral cortices transduced by in utero electroporation-mediated gene transfer, we found that mouse orthologues of human MED13L-p.P866L and -p.T2162M missense variants accumulated in the nucleus, while the p.S2163L and p.S2177Y variants were diffusely distributed in the cytoplasm. In contrast, we found that the p.Q1922* truncation variant was barely detectable in transduced cells, a phenotype reminiscent of this variant that results in MED13L haploinsufficiency in humans. Next, we analyzed these variants for their effects on neuronal migration, dendritic growth, spine morphology, and axon elongation of cortical neurons in vivo. There, we found that overexpression of the p.P866L variant resulted in reduced number and length of dendrites of cortical layer II/III pyramidal neurons. Furthermore, we show that mMED13L-knockdown abrogated dendritic growth in vivo, and this effect was significantly rescued by co-electroporation of an RNAi-resistant mMED13L, but weakly by the p.T2162M variant, and not at all by the p.S2163L variant. However, overexpression of the p.S2163L variant inhibited mature dendritic spine formation in vivo. Expression of each of the 5 variants did not affect neuronal cell migration and callosal axon elongation in vivo. Taken together, our results demonstrate that MED13L expression is relevant to corticogenesis and influences the dendritic branching characteristics of cortical excitatory neurons. Our study also suggests that disease-associated MED13L variants may directly cause morphological and functional defects in cortical neurons in different ways.

Prediction of apoptosis protein subcellular location based on amphiphilic pseudo amino acid composition.

Introduction: Apoptosis proteins play an important role in the process of cell apoptosis, which makes the rate of cell proliferation and death reach a relative balance. The function of apoptosis protein is closely related to its subcellular location, it is of great significance to study the subcellular locations of apoptosis proteins. Many efforts in bioinformatics research have been aimed at predicting their subcellular location. However, the subcellular localization of apoptotic proteins needs to be carefully studied. Methods: In this paper, based on amphiphilic pseudo amino acid composition and support vector machine algorithm, a new method was proposed for the prediction of apoptosis proteins\x{2019} subcellular location. Results and Discussion: The method achieved good performance on three data sets. The Jackknife test accuracy of the three data sets reached 90.5%, 93.9% and 84.0%, respectively. Compared with previous methods, the prediction accuracies of APACC_SVM were improved.

Loss of Extrasynaptic Inhibitory Glycine Receptors in the Hippocampus of an AD Mouse Model Is Restored by Treatment with Artesunate.

Alzheimer's disease (AD) is characterized by synaptic failure and neuronal loss. Recently, we demonstrated that artemisinins restored the levels of key proteins of inhibitory GABAergic synapses in the hippocampus of APP/PS1 mice, a model of cerebral amyloidosis. In the present study, we analyzed the protein levels and subcellular localization of α2 and α3 subunits of GlyRs, indicated as the most abundant receptor subtypes in the mature hippocampus, in early and late stages of AD pathogenesis, and upon treatment with two different doses of artesunate (ARS). Immunofluorescence microscopy and Western blot analysis demonstrated that the protein levels of both α2 and α3 GlyRs are considerably reduced in the CA1 and the dentate gyrus of 12-month-old APP/PS1 mice when compared to WT mice. Notably, treatment with low-dose ARS affected GlyR expression in a subunit-specific way; the protein levels of α3 GlyR subunits were rescued to about WT levels, whereas that of α2 GlyRs were not affected significantly. Moreover, double labeling with a presynaptic marker indicated that the changes in GlyR α3 expression levels primarily involve extracellular GlyRs. Correspondingly, low concentrations of artesunate (≤1 µM) also increased the extrasynaptic GlyR cluster density in hAPPswe-transfected primary hippocampal neurons, whereas the number of GlyR clusters overlapping presynaptic VIAAT immunoreactivities remained unchanged. Thus, here we provide evidence that the protein levels and subcellular localization of α2 and α3 subunits of GlyRs show regional and temporal alterations in the hippocampus of APP/PS1 mice that can be modulated by the application of artesunate.

Engineering of bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s.

Optogenetic tools for controlling protein-protein interactions (PPIs) have been developed from a small number of photosensory modules that respond to a limited selection of wavelengths. Cyanobacteriochrome (CBCR) GAF domain variants respond to an unmatched array of colors; however, their natural molecular mechanisms of action cannot easily be exploited for optogenetic control of PPIs. Here we developed bidirectional, cyanobacteriochrome-based light-inducible dimers (BICYCL)s by engineering synthetic light-dependent interactors for a red/green GAF domain. The systematic approach enables the future engineering of the broad chromatic palette of CBCRs for optogenetics use. BICYCLs are among the smallest optogenetic tools for controlling PPIs and enable either green-ON/red-OFF (BICYCL-Red) or red-ON/green-OFF (BICYCL-Green) control with up to 800-fold state selectivity. The access to green wavelengths creates new opportunities for multiplexing with existing tools. We demonstrate the utility of BICYCLs for controlling protein subcellular localization and transcriptional processes in mammalian cells and for multiplexing with existing blue-light tools.

Extended preconditioning on soft matrices directs human mesenchymal stem cell fate via YAP transcriptional activity and chromatin organization.

Dynamic extracellular matrix (ECM) mechanics plays a crucial role in tissue development and disease progression through regulation of stem cell behavior, differentiation, and fate determination. Periodontitis is a typical case characterized by decreased ECM stiffness within diseased periodontal tissues as well as with irreversible loss of osteogenesis capacity of periodontal tissue-derived human periodontal tissue-derived MSCs (hMSCs) even returning back to a physiological mechanical microenvironment. We hypothesized that the hMSCs extendedly residing in the soft ECM of diseased periodontal tissues may memorize the mechanical information and have further effect on ultimate cell fate besides the current mechanical microenvironment. Using a soft priming and subsequent stiff culture system based on collagen-modified polydimethylsiloxane substrates, we were able to discover that extended preconditioning on soft matrices (e.g., 7 days of exposure) led to approximately one-third decrease in cell spreading, two-third decrease in osteogenic markers (e.g., RUNX2 and OPN) of hMSCs, and one-thirteenth decrease in the production of mineralized nodules. The significant loss of osteogenic ability may attribute to the long-term residing of hMSCs in diseased periodontal tissue featured with reduced stiffness. This is associated with the regulation of transcriptional activity through alterations of subcellular localization of yes-associated protein and nuclear feature-mediated chromatin organization. Collectively, we reconstructed phenomena of irreversible loss of hMSC osteogenesis capacity in diseased periodontal tissues in our system and revealed the critical effect of preconditioning duration on soft matrices as well as the potential mechanisms in determining ultimate hMSC fate.

An assessment of the penile squamous cell carcinoma surfaceome for therapeutic target discovery.

13 Background: There are limited options for relapsed penile squamous cell carcinoma (PSCC) patients after definitive therapy or chemo-refractory disease. Novel target discovery methods are needed to identify potential treatment options and the cell surface represents an actionable target for molecular and cell-based therapies. We evaluated the cell surface molecular catalogue (e.g., surfaceome) in PSCC to identify underexplored targets. Methods: To evaluate proteins enriched on the surface of PSCC cells, we screened published translatomics data from 5 PSCC cell lines (HPV-negative). Ribosome-bound RNA expression values were then analyzed using a validated surfaceome gene list (n=2,886 proteins) to infer surface presence, which were grouped by consensus protein classes. This was complemented by RNA-Seq (n=37) on resected PSCC tumors (HPV+=16, HPV- = 16, unknown HPV status = 5). We used immunohistochemistry (IHC) to assess protein expression and subcellular localization in PSCC tumors; stain intensity was assessed by a semi-quantitative H-score. Non-parametric statistics compared distributions and Kaplan-Meier analysis estimated overall survival (OS; defined from surgery to death or last follow-up) and groups were compared by log-rank testing. The open-source DRPPM-PATH-SURVEIOR app and R statistical software were used to perform analyses. Results: We identified 604 unique surfaceome proteins which were represented in the top 75% of expression in the PSCC cell line translatome. Half of these proteins have N-glycosylations and 49.5% classified as high-confidence surface targets. The largest proportions of surfaceome proteins included: Receptors (25.6%), Transporters (18.5%), and proteins with unclassified function (32.2%). Of note, 16.2% of this surfaceome linked to at least one compound in the DrugBank database.There was a moderate correlation between cell line and tumor surfaceome RNAs (top 75%; r =0.58, p &lt; 2.2x10-16). We selected targets in the 99% (BSG; CD147; regulates MMPs and lactate transport), 90% (FGFR1; receptor tyrosine kinase) and 75% (SLC16A1; metabolite transporter) of the surfaceome expression. Tumor RNA levels for BSG, FGFR1 and SLC16A1 were elevated, but without differences based on HPV. IHC demonstrated robust expression in tumors with plasma membrane scoring being enriched compared to non-plasma membrane compartments (CD147, p =2.9x10-6; SLC16A1, p = 5.1x10-6; FGFR1, p = 0.007). Of note, patients with elevated BSG RNA (based on median log2 value) had worse OS (p = 0.018), though this lost significance after adjusting for HPV. No differences in OS were seen with FGFR1 or SLC16A1 expression. Conclusions: Our analysis of the surfaceome based on RNA expression was associated with increased protein levels in tumor tissues. Evaluation of the PSCC surfaceome may provide opportunity to investigate novel therapeutic targets, which may be actionable regardless of HPV status.

CBL-Interacting Protein Kinases 18 (CIPK18) Gene Positively Regulates Drought Resistance in Potato.

Sensor-responder complexes comprising calcineurin B-like (CBL) proteins and CBL-interacting protein kinases (CIPKs) are plant-specific Ca2+ receptors, and the CBL-CIPK module is widely involved in plant growth and development and a large number of abiotic stress response signaling pathways. In this study, the potato cv. "Atlantic" was subjected to a water deficiency treatment and the expression of StCIPK18 gene was detected by qRT-PCR. The subcellular localization of StCIPK18 protein was observed by a confocal laser scanning microscope. The StCIPK18 interacting protein was identified and verified by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). StCIPK18 overexpression and StCIPK18 knockout plants were constructed. The phenotypic changes under drought stress were indicated by water loss rate, relative water content, MDA and proline contents, and CAT, SOD and POD activities. The results showed that StCIPK18 expression was upregulated under drought stress. StCIPK18 is localized in the cell membrane and cytoplasm. Y2H shows the interaction between StCIPK18 and StCBL1, StCBL4, StCBL6 and StCBL8. BiFC further verifies the reliability of the interaction between StCIPK18 and StCBL4. Under drought stress, StCIPK18 overexpression decreased the water loss rate and MDA, and increased RWC, proline contents and CAT, SOD and POD activities; however, StCIPK18 knockout showed opposite results, compared with the wild type, in response to drought stress. The results can provide information for the molecular mechanism of the StCIPK18 regulating potato response to drought stress.

Genome-wide identification of Mg2+ transporters and functional characteristics of DlMGT1 in Dimocarpus longan.

Longan (Dimocarpus Longan) is one of the most important fruit crops in Southern China. Lack of available Mg in acidic soil conditions is a limitation to further increasing longan yield. Magnesium transporter (MGT/MRS2) mediates the uptake, transport, and redistribution of Mg2+ in higher plants. To understand the role of MGTs family members in longan Mg deficiency. We identified and analyzed the protein characteristics, phylogeny, expression changes, subcellular localization, and transcriptional regulation of DlMGTs members. The results showed that, twelve DlMGTs are localized in the cell membrane, chloroplast, and nucleus. The evolutionary differences in MGTs between herbaceous and woody species in different plants. The DlMGTs promoters contained many cis-acting elements and transcription factor binding sites related to the hormone, environmental, and stress response. Subcellular localization assays showed that DlMGT1 localizes in the cell membrane of Arabidopsis protoplasts. The candidate transcription factor DlGATA16, which may regulate the expression of DlMGT1, was localized in the nucleus of tobacco leaves. Dual luciferase analysis demonstrated that DlGATA16 is a potential factor regulating the transcriptional activity of DlMGT1. In this study, we identified and analyzed DlMGTs on a genome-wide scale and the subcellular localization and interaction of DlMGT1 and DlGATA16, which has important implications for further functional analysis studies of MGTs and the use of MGT for longan genetic improvement.

GliaMorph: a modular image analysis toolkit to quantify Müller glial cell morphology.

Cell morphology is crucial for all cell functions. This is particularly true for glial cells as they rely on complex shape to contact and support neurons. However, methods to quantify complex glial cell shape accurately and reproducibly are lacking. To address this, we developed the image analysis pipeline 'GliaMorph'. GliaMorph is a modular analysis toolkit developed to perform (1) image pre-processing, (2) semi-automatic region-of-interest selection, (3) apicobasal texture analysis, (4) glia segmentation, and (5) cell feature quantification. Müller glia (MG) have a stereotypic shape linked to their maturation and physiological status. Here, we characterized MG on three levels: (1) global image-level, (2) apicobasal texture, and (3) regional apicobasal vertical-to-horizontal alignment. Using GliaMorph, we quantified MG development on a global and single-cell level, showing increased feature elaboration and subcellular morphological rearrangement in the zebrafish retina. As proof of principle, we analysed expression changes in a mouse glaucoma model, identifying subcellular protein localization changes in MG. Together, these data demonstrate that GliaMorph enables an in-depth understanding of MG morphology in the developing and diseased retina.

Identification and In silico analysis of proline-glutamate/proline-proline-glutamate proteins of Mycobacterium tuberculosis complex: A comparison of computational web-based tools.

Understanding the protein's subcellular localization and secretory nature can greatly improve the target identification for diagnostic assays and drug discovery, although their identification in laboratory experiments is a time-consuming and labor-intensive process. In order to identify proteins that could be targeted for therapeutic intervention or the development of diagnostic assays, we used a variety of computational tools to predict the subcellular localization or secretory nature of mycobacterial proline-glutamate/proline-proline-glutamate (PE/PPE) proteins. PSORTb version 3.0.3, TBpred, and Gpos-mPLoc analyses were performed on 30 selected PE/PPE protein sequences, while, SignalP 6.0, SignalP 5.0, Phobius, PSORTb version 3.0.3 and TBpred were used for signal sequence predictions. Gpos-mPLoc and TBpred had the highest concordance for extracellular prediction, while PSORTb and TBpred had the highest concordance for prediction of membrane localization. The tools for predicting the secretory nature of proteins had little agreement. Multiple computational tools must be considered to provide an indication of the subcellular localization of PE/PPE proteins. Laboratory experiments should be used to confirm the findings of the tools.

Atlas of interactions between SARS-CoV-2 macromolecules and host proteins.

The proteins and RNAs of viruses extensively interact with host proteins after infection. We collected and reanalyzed all available datasets of protein-protein and RNA-protein interactions related to SARS-CoV-2. We investigated the reproducibility of those interactions and made strict filters to identify highly confident interactions. We systematically analyzed the interaction network and identified preferred subcellular localizations of viral proteins, some of which such as ORF8 in ER and ORF7A/B in ER membrane were validated using dual fluorescence imaging. Moreover, we showed that viral proteins frequently interact with host machinery related to protein processing in ER and vesicle-associated processes. Integrating the protein- and RNA-interactomes, we found that SARS-CoV-2 RNA and its N protein closely interacted with stress granules including 40 core factors, of which we specifically validated G3BP1, IGF2BP1, and MOV10 using RIP and Co-IP assays. Combining CRISPR screening results, we further identified 86 antiviral and 62 proviral factors and associated drugs. Using network diffusion, we found additional 44 interacting proteins including two proviral factors previously validated. Furthermore, we showed that this atlas could be applied to identify the complications associated with COVID-19. All data are available in the AIMaP database (https://mvip.whu.edu.cn/aimap/) for users to easily explore the interaction map.

Domain organization, expression, subcellular localization, and biological roles of ARGONAUTE proteins in Arabidopsis.

ARGONAUTE (AGO) proteins are the final effectors of small RNA-mediated transcriptional and post-transcriptional silencing pathways. Plant AGO proteins are essential for preserving genome integrity, regulating developmental processes, and in stress responses and pathogen defense. Since the discovery of the first eukaryotic AGO in Arabidopsis, our understanding of these proteins has grown exponentially throughout all the eukaryotes. However, many aspects of AGO proteins' modes of action and how they are influenced by their subcellular localization are still to be elucidated. Here, we provide an updated and comprehensive view of the evolution, domain architecture and roles, expression pattern, subcellular localization, and biological functions of the 10 AGO proteins in Arabidopsis.

Subcellular protein localisation of Trypanosoma brucei bloodstream form-upregulated proteins maps stage-specific adaptations

Background: Genome-wide subcellular protein localisation in Trypanosoma brucei, through our TrypTag project, has comprehensively dissected the molecular organisation of this important pathogen. Powerful as this resource is, T. brucei has multiple developmental forms and we previously only analysed the procyclic form. This is an insect life cycle stage, leaving the mammalian bloodstream form unanalysed. The expectation is that between life stages protein localisation would not change dramatically (completely unchanged or shifting to analogous stage-specific structures). However, this has not been specifically tested. Similarly, which organelles tend to contain proteins with stage-specific expression can be predicted from known stage specific adaptations but has not been comprehensively tested. Methods: We used endogenous tagging with mNG to determine the sub-cellular localisation of the majority of proteins encoded by transcripts significantly upregulated in the bloodstream form, and performed comparison to the existing localisation data in procyclic forms. Results: We have confirmed the localisation of known and identified the localisation of novel stage-specific proteins. This gave a map of which organelles tend to contain stage specific proteins: the mitochondrion for the procyclic form, and the endoplasmic reticulum, endocytic system and cell surface in the bloodstream form. Conclusions: This represents the first genome-wide map of life cycle stage-specific adaptation of organelle molecular machinery in T. brucei.

Characterization of the soybean KRP gene family reveals a key role for GmKRP2a in root development.

Kip-related proteins (KRPs), as inhibitory proteins of cyclin-dependent kinases, are involved in the growth and development of plants by regulating the activity of the CYC-CDK complex to control cell cycle progression. The KRP gene family has been identified in several plants, and several KRP proteins from Arabidopsis thaliana have been functionally characterized. However, there is little research on KRP genes in soybean, which is an economically important crop. In this study, we identified nine GmKRP genes in the Glycine max genome using HMM modeling and BLASTP searches. Protein subcellular localization and conserved motif analysis showed soybean KRP proteins located in the nucleus, and the C-terminal protein sequence was highly conserved. By investigating the expression patterns in various tissues, we found that all GmKRPs exhibited transcript abundance, while several showed tissue-specific expression patterns. By analyzing the promoter region, we found that light, low temperature, an anaerobic environment, and hormones-related cis-elements were abundant. In addition, we performed a co-expression analysis of the GmKRP gene family, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) set enrichment analysis. The co-expressing genes were mainly involved in RNA synthesis and modification and energy metabolism. Furthermore, the GmKRP2a gene, a member of the soybean KRP family, was cloned for further functional analysis. GmKRP2a is located in the nucleus and participates in root development by regulating cell cycle progression. RNA-seq results indicated that GmKRP2a is involved in cell cycle regulation through ribosome regulation, cell expansion, hormone response, stress response, and plant pathogen response pathways. To our knowledge, this is the first study to identify and characterize the KRP gene family in soybean.

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons.

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal neurons allows the study of individual neurons or subcellular components. We have shown subcellular protein localization within a neuron by immunocytochemistry, neuronal polarity, synaptic morphology, and its developmental change using a low-density primary hippocampal culture. Recently, ready-to-use frozen stocks of neurons have become commercially available. These frozen stocks of neurons reduce the time needed to prepare animal experiments and also contribute to the reduction of the number of animals used. Here, we introduce a reproducible low-density primary culture method using a 96-well plate. We used a commercially available frozen stock of neurons from the rat embryonic hippocampus. The neurons can be stably cultured long-term without media changes by reducing the growth of glial cells at particular timepoints. This high-throughput assay using low-density culture allows reproducible imaging-based evaluations of synaptic plasticity.

S-Palmitoylation of Synaptic Proteins in Neuronal Plasticity in Normal and Pathological Brains.

Protein lipidation is a common post-translational modification of proteins that plays an important role in human physiology and pathology. One form of protein lipidation, S-palmitoylation, involves the addition of a 16-carbon fatty acid (palmitate) onto proteins. This reversible modification may affect the regulation of protein trafficking and stability in membranes. From multiple recent experimental studies, a picture emerges whereby protein S-palmitoylation is a ubiquitous yet discrete molecular switch enabling the expansion of protein functions and subcellular localization in minutes to hours. Neural tissue is particularly rich in proteins that are regulated by S-palmitoylation. A surge of novel methods of detection of protein lipidation at high resolution allowed us to get better insights into the roles of protein palmitoylation in brain physiology and pathophysiology. In this review, we specifically discuss experimental work devoted to understanding the impact of protein palmitoylation on functional changes in the excitatory and inhibitory synapses associated with neuronal activity and neuronal plasticity. The accumulated evidence also implies a crucial role of S-palmitoylation in learning and memory, and brain disorders associated with impaired cognitive functions.

Establishment of an efficient cotton root protoplast isolation protocol suitable for single-cell RNA sequencing and transient gene expression analysis

BackgroundCotton has tremendous economic value worldwide; however, its allopolyploid nature and time-consuming transformation methods have hampered the development of cotton functional genomics. The protoplast system has proven to be an important and versatile tool for functional genomics, tissue-specific marker gene identification, tracking developmental trajectories, and genome editing in plants. Nevertheless, the isolation of abundant viable protoplasts suitable for single-cell RNA sequencing (scRNA-seq) and genome editing remains a challenge in cotton.ResultsWe established an efficient transient gene expression system using protoplasts isolated from cotton taproots. The system enables the isolation of large numbers of viable protoplasts and uses an optimized PEG-mediated transfection protocol. The highest yield (3.55 × 105/g) and viability (93.3%) of protoplasts were obtained from cotton roots grown in hydroponics for 72 h. The protoplasts isolated were suitable for scRNA-seq. The highest transfection efficiency (80%) was achieved when protoplasts were isolated as described above and transfected with 20 μg of plasmid for 20 min in a solution containing 200 mM Ca2+. Our protoplast-based transient expression system is suitable for various applications, including validation the efficiency of CRISPR vectors, protein subcellular localization analysis, and protein–protein interaction studies.ConclusionsThe protoplast isolation and transfection protocol developed in this study is stable, versatile, and time-saving. It will accelerate functional genomics and molecular breeding in cotton.

An Artificial Intelligence-Based Stacked Ensemble Approach for Prediction of Protein Subcellular Localization in Confocal Microscopy Images

Predicting subcellular protein localization has become a popular topic due to its utility in understanding disease mechanisms and developing innovative drugs. With the rapid advancement of automated microscopic imaging technology, approaches using bio-images for protein subcellular localization have gained a lot of interest. The Human Protein Atlas (HPA) project is a macro-initiative that aims to map the human proteome utilizing antibody-based proteomics and related c. Millions of images have been tagged with single or multiple labels in the HPA database. However, fewer techniques for predicting the location of proteins have been devised, with the majority of them relying on automatic single-label classification. As a result, there is a need for an automatic and sustainable system capable of multi-label classification of the HPA database. Deep learning presents a potential option for automatic labeling of protein’s subcellular localization, given the vast image number generated by high-content microscopy and the fact that manual labeling is both time-consuming and error-prone. Hence, this research aims to use an ensemble technique for the improvement in the performance of existing state-of-art convolutional neural networks and pretrained models were applied; finally, a stacked ensemble-based deep learning model was presented, which delivers a more reliable and robust classifier. The F1-score, precision, and recall have been used for the evaluation of the proposed model’s efficiency. In addition, a comparison of existing deep learning approaches has been conducted with respect to the proposed method. The results show the proposed ensemble strategy performed exponentially well on the multi-label classification of Human Protein Atlas images, with recall, precision, and F1-score of 0.70, 0.72, and 0.71, respectively.