Protein Localization Patterns Research Articles

Cell-type-specific Labeling of Endogenous Proteins Using the Split GFP System in Drosophila.

i. Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise cell-type-specific labeling of proteins has been challenging in vivo . A notable solution to this challenge is the self-complementing split green fluorescent protein (GFP 1-10/11 ) system. In this paper, we present a detailed protocol for labeling endogenous proteins in a cell-type-specific manner using the GFP 1-10/11 system in fruit flies. This approach depends on the reconstitution of the GFP 1-10 and GFP 11 fragments, creating a fluorescence signal. We insert the GFP 11 fragment into a specific genomic locus while expressing its counterpart, GFP 1-10 , through an available Gal4 driver line. The unique aspect of this system is that neither GFP 1-10 nor GFP 11 alone emits fluorescence, enabling the precise detection of protein localization only in Gal4-positive cells expressing the GFP 11 tagged endogenous protein. We illustrate this technique using the adhesion molecule gene teneurin-m ( Ten-m ) as a model, highlighting the generation and validation of GFP 11 protein trap lines via Minos-mediated integration cassette (MiMIC) insertion. Furthermore, we demonstrate the cell-type-specific labeling of Ten-m proteins in the larval brains of fruit flies. This method significantly enhances our ability to image endogenous protein localization patterns in a cell-type-specific manner and is adaptable to various model organisms beyond fruit flies.

PIFiA: self-supervised approach for protein functional annotation from single-cell imaging data

Fluorescence microscopy data describe protein localization patterns at single-cell resolution and have the potential to reveal whole-proteome functional information with remarkable precision. Yet, extracting biologically meaningful representations from cell micrographs remains a major challenge. Existing approaches often fail to learn robust and noise-invariant features or rely on supervised labels for accurate annotations. We developed PIFiA (Protein Image-based Functional Annotation), a self-supervised approach for protein functional annotation from single-cell imaging data. We imaged the global yeast ORF-GFP collection and applied PIFiA to generate protein feature profiles from single-cell images of fluorescently tagged proteins. We show that PIFiA outperforms existing approaches for molecular representation learning and describe a range of downstream analysis tasks to explore the information content of the feature profiles. Specifically, we cluster extracted features into a hierarchy of functional organization, study cell population heterogeneity, and develop techniques to distinguish multi-localizing proteins and identify functional modules. Finally, we confirm new PIFiA predictions using a colocalization assay, suggesting previously unappreciated biological roles for several proteins. Paired with a fully interactive website (https://thecellvision.org/pifia/), PIFiA is a resource for the quantitative analysis of protein organization within the cell.

Claudin-3 in the non-neural ectoderm is essential for neural fold fusion in chicken embryos

The neural tube, the embryonic precursor to the brain and spinal cord, begins as a flat sheet of epithelial cells, divided into non-neural and neural ectoderm. Proper neural tube closure requires that the edges of the neural ectoderm, the neural folds, to elevate upwards and fuse along the dorsal midline of the embryo. We have previously shown that members of the claudin protein family are required for the early phases of chick neural tube closure. Claudins are transmembrane proteins, localized in apical tight junctions within epithelial cells where they are essential for regulation of paracellular permeability, strongly involved in apical-basal polarity, cell-cell adhesion, and bridging the tight junction to cytoplasmic proteins. Here we explored the role of Claudin-3 (Cldn3), which is specifically expressed in the non-neural ectoderm. We discovered that depletion of Cldn3 causes folic acid-insensitive primarily spinal neural tube defects due to a failure in neural fold fusion. Apical cell surface morphology of Cldn3-depleted non-neural ectodermal cells exhibited increased membrane blebbing and smaller apical surfaces. Although apical-basal polarity was retained, we observed altered Par3 and Pals1 protein localization patterns within the apical domain of the non-neural ectodermal cells in Cldn3-depleted embryos. Furthermore, F-actin signal was reduced at apical junctions. Our data presents a model of spina bifida, and the role that Cldn3 is playing in regulating essential apical cell processes in the non-neural ectoderm required for neural fold fusion.

Assessment of Expression Levels and Localization Patterns of Phospholipase C zeta in Different Grades of HOST in Human Sperm.

Phospholipase C zeta (PLC-ζ) deficiency in sperm can underlie oocyte activation failure after intracytoplasmic sperm injection (ICSI). The aim of this study was to determine PLC-ζ expression and location in individual spermatozoa in each host score so that a hypo-osmotic swelling test (HOST) may be used to help routine sperm selection for ICSI. In this experimental study, fresh semen samples were randomly obtained from 30 men who were referred to the Andrology Unit of the Infertility Center. Samples were processed by density gradient centrifugation (DGC) and exposed to hypotonic conditions. Seven different tail patterns, classified from 'a' to 'g' can be detected according to World Health Organization (WHO) criteria. Then, the PLC-ζ protein localization pattern was assessed by quantitative Immunofluorescence in individual sperm Host grades. Moreover, the sperm content of PLC-ζ protein was evaluated by flow cytometry correlated with semen analysis parameters. In the present study, quantitive immunofluorescence analysis indicated that sperm from different host grades exhibited seven localization patterns of PLC-ζ of acrosomal (A); equatorial (EQ), and postacrosomal (PA) patterns. A+EQ=acrosomal and equatorial, A+PA=acrosomal and post-acrosomal, EQ+PA=equatorial and post-crosomal, and A+EQ+PA. The sperm from HOST grade 'd' exhibited significantly higher PLC-ζ (A+PA) and (A+EQ+PA) staining compared to sperm from other grades (P=0.006). The sperm from grade 'd' exhibited higher PLC-ζ (EQ+PA) compared with other grades (P=0.001). However, grade 'd' was not significantly different from 'c' (P=0.087). Analysis of the combined results confirmed that there was a clear reduction in PLC-ζ immunofluorescence in Host grades 'a', 'f' and 'g' sperms. Our data suggest that HOST may represent a useful diagnostic tool for the selection of sperms exhibiting a higher level of PLC-ζ expression.

Mutations in SLC45A2 lead to loss of melanin in parrot feathers.

Bird plumage coloration is a complex and multifactorial process that involves both genetic and environmental factors. Diverse pigment groups contribute to plumage variation in different birds. In parrots, the predominant green color results from the combination of 2 different primary colors: yellow and blue. Psittacofulvin, a pigment uniquely found in parrots, is responsible for the yellow coloration, while blue is suggested to be the result of light scattering by feather nanostructures and melanin granules. So far, genetic control of melanin-mediated blue coloration has been elusive. In this study, we demonstrated that feather from the yellow mutant rose-ringed parakeet displays loss of melanosome granules in spongy layer of feather barb. Using whole genome sequencing, we found that mutation in SLC45A2, an important solute carrier protein in melanin synthetic pathway, is responsible for the sex-linked yellow phenotype in rose-ringed parakeet. Intriguingly, one of the mutations, P53L found in yellow Psittacula krameri is already reported as P58A/S in the human albinism database, known to be associated with human OCA4. We further showed that mutations in SLC45A2 gene affect melanin production also in other members of Psittaculidae family such as alexandrine and plum-headed parakeets. Additionally, we demonstrate that the mutations associated with the sex-linked yellow phenotype, localized within the transmembrane domains of the SLC45A2 protein, affect the protein localization pattern. This is the first evidence of plumage color variation involving SLC45A2 in parrots and confirmation of associated mutations in the transmembrane domains of the protein that affects its localization.

Palmitoylation Regulates Human Serotonin Transporter Activity, Trafficking, and Expression and Is Modulated by Escitalopram.

In the central nervous system, serotonergic signaling modulates sleep, mood, and cognitive control. During serotonergic transmission, the synaptic concentration of serotonin is tightly controlled in a spatial and temporal manner by the serotonin transporter (SERT). Dysregulation of this process is implicated in the pathogenesis of major-depressive, obsessive-compulsive, and autism-spectrum disorders, which makes SERT a primary target for prescription therapeutics, most notably selective serotonin reuptake inhibitors (SSRIs). S-Palmitoylation, the reversible addition of a 16-carbon fatty acid to proteins, is an increasingly recognized dynamic post-translational modification responsible for modulating protein kinetics, trafficking, and localization patterns in response to physiologic/cellular stimuli. In this study, we reveal that human SERTs are a target for palmitoylation, and using the irreversible palmitoyl acyltransferase inhibitor 2-bromopalmitate (2BP), we have identified several associated functions. Using a lower dose of 2BP in shorter time frames, inhibition of palmitoylation was associated with reductions in SERT Vmax, without changes in Km or surface expression. With higher doses of 2BP for longer time intervals, inhibition of palmitoylation was consistent with the loss of cell surface and total SERT protein, suggesting palmitoylation is an important mechanism in regulating SERT trafficking and maintenance of SERT protein through biogenic or anti-degradative processes. Additionally, we have identified that treatment with the SSRI escitalopram decreases SERT palmitoylation analogous to 2BP, reducing SERT surface expression and transport capacity. Ultimately, these results reveal that palmitoylation is a major regulatory mechanism for SERT kinetics and trafficking and may be the mechanism responsible for escitalopram-induced internalization and ultimately decreased cellular SERT protein levels.

Presence of sodefrin precursor-like factor pheromone candidates in mental and dorsal tail base glands in the plethodontid salamander, Karsenia koreana.

Plethodontid salamanders are well known for their distinct courtship rituals and the associated pheromonal signaling. However, little is known about pheromones produced in the lone Asian plethodontid species Karsenia koreana. Here, we examined the localization patterns of proteins of the sodefrin precursor-like factor (SPF) pheromone system in K. koreana. Using an antibody generated against SPF proteins from another plethodontid, Desmognathus ocoee, we tested three types of skin glands in K. koreana males via immunohistochemistry: the mental gland and two types of dorsal tail base glands-caudal courtship glands and dorsal granular glands. SPF immunoreactivity was detected in the known courtship gland, the mental gland, as well as granular glands, but not in caudal courtship glands. Due to immunoreaction specificity, we hypothesize the proteins of the SPF system in K. koreana and D. ocoee are structurally and functionally related and are used as courtship pheromones in K. koreana. Also, we hypothesize that K. koreana males transmit SPF to the female during the tail-straddling walk via dorsal granular glands. Finally, K. koreana male caudal courtship glands may be producing SPF proteins that are not recognized by our SPF antibody or these glands may play a different role in courtship than anticipated.

Single-cell subcellular protein localisation using novel ensembles of diverse deep architectures

Unravelling protein distributions within individual cells is vital to understanding their function and state and indispensable to developing new treatments. Here we present the Hybrid subCellular Protein Localiser (HCPL), which learns from weakly labelled data to robustly localise single-cell subcellular protein patterns. It comprises innovative DNN architectures exploiting wavelet filters and learnt parametric activations that successfully tackle drastic cell variability. HCPL features correlation-based ensembling of novel architectures that boosts performance and aids generalisation. Large-scale data annotation is made feasible by our AI-trains-AI approach, which determines the visual integrity of cells and emphasises reliable labels for efficient training. In the Human Protein Atlas context, we demonstrate that HCPL is best performing in the single-cell classification of protein localisation patterns. To better understand the inner workings of HCPL and assess its biological relevance, we analyse the contributions of each system component and dissect the emergent features from which the localisation predictions are derived.

Molecular characterization suggests kinetic modulation of expression of accessory viral protein, W, in Newcastle disease virus infected DF1 cells.

The online version contains supplementary material available at 10.1007/s13337-023-00813-2.

Sleep loss impairs intestinal stem cell function and gut homeostasis through the modulation of the GABA signalling pathway in Drosophila.

Sleep is essential for maintaining health. Indeed, sleep loss is closely associated with multiple health problems, including gastrointestinal disorders. However, it is not yet clear whether sleep loss affects the function of intestinal stem cells (ISCs). Mechanical sleep deprivation and sss mutant flies were used to generate the sleep loss model. qRT-PCR was used to measure the relative mRNA expression. Gene knock-in flies were used to observe protein localization and expression patterns. Immunofluorescence staining was used to determine the intestinal phenotype. The shift in gut microbiota was observed using 16S rRNA sequencing and analysis. Sleep loss caused by mechanical sleep deprivation and sss mutants disturbs ISC proliferation and intestinal epithelial repair through the brain-gut axis. In addition, disruption of SSS causes gut microbiota dysbiosis in Drosophila. As regards the mechanism, gut microbiota and the GABA signalling pathway both partially played a role in the sss regulation of ISC proliferation and gut function. The research shows that sleep loss disturbed ISC proliferation, gut microbiota, and gut function. Therefore, our results offer a stem cell perspective on brain-gut communication, with details on the effect of the environment on ISCs.

PIN-FORMED1 polarity in the plant shoot epidermis is insensitive to the polarity of neighboring cells

SummaryAt the Arabidopsis shoot apex, epidermal cells are planar-polarized along an axis marked by the asymmetric localization patterns of several proteins including PIN-FORMED1 (PIN1), which facilitates the directional efflux of the plant hormone auxin to pattern phyllotaxis. While PIN1 polarity is known to be regulated non-cell autonomously via the MONOPTEROS (MP) transcription factor, how this occurs has not been determined. Here, we use mosaic expression of the serine threonine kinase PINOID (PID) to test whether PIN1 polarizes according to the polarity of neighboring cells. Our findings reveal that PIN1 is insensitive to the polarity of PIN1 in neighboring cells arguing against auxin flux or extracellular auxin concentrations acting as a polarity cue, in contrast to previous model proposals.

Mechanical conflict caused by a cell-wall-loosening enzyme activates de novo shoot regeneration.

Cellular heterogeneity is a hallmark of multicellular organisms. During shoot regeneration from undifferentiated callus, only a select few cells, called progenitors, develop into shoot. How these cells are selected and what governs their subsequent progression to a patterned organ system is unknown. Using Arabidopsis thaliana, we show that it is not just the abundance of stem cell regulators but rather the localization pattern of polarity proteins that predicts the progenitor's fate. A shoot-promoting factor, CUC2, activated the expression of the cell-wall-loosening enzyme, XTH9, solely in a shell of cells surrounding the progenitor, causing different mechanical stresses in these cells. This mechanical conflict then activates cell polarity in progenitors to promote meristem formation. Interestingly,genetic or physical perturbationsto cells surrounding the progenitor impaired the progenitor and vice versa. These suggest a feedback loop between progenitors and their neighbors for shoot regeneration in the absence of tissue-patterning cues.

A synthetic biology approach reveals diverse and dynamic CDK response profiles via multisite phosphorylation of NLS-NES modules.

The complexity of multisite phosphorylation mechanisms in regulating nuclear localization signals (NLSs) and nuclear export signals (NESs) is not understood, and its potential has not been used in synthetic biology. The nucleocytoplasmic shuttling of many proteins is regulated by cyclin-dependent kinases (CDKs) that rely on multisite phosphorylation patterns and short linear motifs (SLiMs) to dynamically control proteins in the cell cycle. We studied the role of motif patterns in nucleocytoplasmic shuttling using sensors based on the CDK targets Dna2, Psy4, and Mcm2/3 of Saccharomyces cerevisiae. We designed multisite phosphorylation modules by rearranging phosphorylation sites, cyclin-specific SLiMs, phospho-priming, phosphatase specificity, and NLS/NES phospho-regulation and obtained very different substrate localization dynamics. These included ultrasensitive responses with and without a delay, graded responses, and different homeostatic plateaus. Thus, CDK can do much more than trigger sequential switches during the cell cycle as it can drive complex patterns of protein localization and activity by using multisite phosphorylation networks.

A review on pilus assembly mechanisms in Gram-positive and Gram-negative bacteria

The surface of Gram-positive and Gram-negative bacteria contains long hair-like proteinaceous protrusion known as pili or fimbriae. Historically, pilin proteins were considered to play a major role in the transfer of genetic material during bacterial conjugation. Recent findings however elucidate their importance in virulence, biofilm formation, phage transduction, and motility. Therefore, it is crucial to gain mechanistic insights on the subcellular assembly of pili and the localization patterns of their subunit proteins (major and minor pilins) that aid the macromolecular pilus assembly at the bacterial surface. In this article, we review the current knowledge of pilus assembly mechanisms in a wide range of Gram-positive and Gram-negative bacteria, including subcellular localization patterns of a few pilin subunit proteins and their role in virulence and pathogenesis.

NEDD4L inhibits glycolysis and proliferation of cancer cells in oral squamous cell carcinoma by inducing ENO1 ubiquitination and degradation

ABSTRACT Glycolysis contributes to cell metabolism and facilitates cell proliferation of oral squamous cell carcinoma (OSCC), the most common type of oral cancer. Understanding the regulatory mechanisms involved in the glycolysis of OSCC cells may provide important therapeutic inspirations. Immunohistochemistry was used to examine protein localization patterns in human OSCC tissues and Western blot was conducted to gauge protein level. Lentivirus transduction was used to overexpress or silence genes of interest. Cell proliferation was assessed by Cell Counting Kit (CCK)-8 assay while glycolysis was examined via measurement of extracellular acidification rate, oxygen consumption rate, and lactate and ATP production. In vivo cancer development was evaluated with a mouse tumor growth model. OSCC tissues displayed reduced expression of NEDD4L compared with normal tissues. NEDD4L expression positively correlated with 5-year patient survival rate, indicating that NEDD4L may be a prognosis marker for OSCC. NEDD4L overexpression suppressed proliferation, cell cycle transition, and glycolysis in OSCC cells, and inhibited in vivo tumor growth. UbiBrowser identified ENO1, an enzyme that catalyzes glycolysis, as a substrate of NEDD4L. Overexpression of NEDD4L resulted in the ubiquitination and subsequent degradation of ENO1 whereas overexpression of ENO1 reversed the functional effects of NEDD4L overexpression, restoring proliferation, cell cycle transition, and glycolysis in OSCC cells. NEDD4L elicits tumor-suppressive functions via inhibition of OSCC cell proliferation, cell cycle transition, and glycolysis by stimulating ENO1 ubiquitination and degradation. Our results unraveled a signaling axis important for OSCC cell survival and metabolism, which can serve as a potential therapeutic target.

OpenCell: Endogenous tagging for the cartography of human cellular organization.

Elucidating the wiring diagram of the human cell is a central goal of the postgenomic era. We combined genome engineering, confocal live-cell imaging, mass spectrometry, and data science to systematically map the localization and interactions of human proteins. Our approach provides a data-driven description of the molecular and spatial networks that organize the proteome. Unsupervised clustering of these networks delineates functional communities that facilitate biological discovery. We found that remarkably precise functional information can be derived from protein localization patterns, which often contain enough information to identify molecular interactions, and that RNA binding proteins form a specific subgroup defined by unique interaction and localization properties. Paired with a fully interactive website (opencell.czbiohub.org), our work constitutes a resource for the quantitative cartography of human cellular organization.

Assembly of Bacillus subtilis Dynamin into Membrane-Protective Structures in Response to Environmental Stress Is Mediated by Moderate Changes in Dynamics at a Single Molecule Level

Dynamin-like proteins are membrane-associated GTPases, conserved in bacteria and in eukaryotes, that can mediate nucleotide-driven membrane deformation or membrane fusion reactions. Bacillus subtilis’ DynA has been shown to play an important role in protecting cells against chemicals that induce membrane leakage, and to form an increased number of membrane-associated structures after induction of membrane stress. We have studied the dynamics of DynA at a single molecule level in real time, to investigate how assembly of stress-induced structures is accompanied by changes in molecule dynamics. We show that DynA molecule displacements are best described by the existence of three distinct populations, a static mode, a low-mobility, and a fast-mobile state. Thus, DynA is most likely freely diffusive within the cytosol, moves along the cell membrane with a low mobility, and arrests at division sites or at stress-induced lesions at the membrane. In response to stress-inducing membrane leakage, but not to general stress, DynA molecules become slightly more static, but largely retain their mobility, suggesting that only few molecules are involved in the repair of membrane lesions, while most molecules remain in a dynamic mode scanning for lesions. Our data suggest that even moderate changes in single molecule dynamics can lead to visible changes in protein localization patterns.

Single-cell RNA-sequencing analysis and characterisation of testicular cells in giant panda (Ailuropoda melanoleuca).

The giant panda (Ailuropoda melanoleuca ) is a rare and endangered species to be preserved in China. The giant panda has a low reproductive capacity, and due to the scarcity of samples, studies on testes from giant panda are very limited, with little knowledge about the process of spermatogenesis in this species. To establish the gene expression profiles in cells from the testis of a giant panda. The 10×Genomics single-cell RNA-sequencing platform was applied to cells from the testis of an adult giant panda. We identified eight testicular cell types including six somatic and two germ cell types from our single-cell RNA-sequencing datasets. We also identified the differentially expressed genes (DEGs) in each cell type, and performed functional enrichment analysis for the identified testicular cell types. Furthermore, by immunohistochemistry we explored the protein localisation patterns of several marker genes in testes from giant panda. Our study has for the first time established the gene expression profiles in cells from the testis of a giant panda. Our data provide a reference catalogue for spermatogenesis and testicular cells in the giant panda, laying the foundation for future breeding and preservation of this endangered species.

ClC-c regulates the proliferation of intestinal stem cells via the EGFR signalling pathway in Drosophila.

ObjectivesAdult stem cells uphold a delicate balance between quiescent and active states, which is crucial for tissue homeostasis. Whereas many signalling pathways that regulate epithelial stem cells have been reported, many regulators remain unidentified.Materials and MethodsFlies were used to generate tissue‐specific gene knockdown and gene knockout. qRT‐PCR was used to assess the relative mRNA levels. Immunofluorescence was used to determine protein localization and expression patterns. Clonal analyses were used to observe the phenotype. RNA‐seq was used to screen downstream mechanisms.ResultsHere, we report a member of the chloride channel family, ClC‐c, which is specifically expressed in Drosophila intestinal stem/progenitor cells and regulates intestinal stem cell (ISC) proliferation under physiological conditions and upon tissue damage. Mechanistically, we found that the ISC loss induced by the depletion of ClC‐c in intestinal stem/progenitor cells is due to inhibition of the EGFR signalling pathway.ConclusionOur findings reveal an ISC‐specific function of ClC‐c in regulating stem cell maintenance and proliferation, thereby providing new insights into the functional links among the chloride channel family, ISC proliferation and tissue homeostasis.

Rapid Androgen-Responsive Proteome Is Involved in Prostate Cancer Progression.

Androgen exerts its functions by binding with an androgen receptor (AR). It can activate many signaling pathways that are important to the progression of castration-resistant prostate cancer (CRPC). Here, we characterized the rapid proteomic changes seen at 5, 15, 30, and 60 min after the androgen treatment of VCaP cells via the tandem mass tag (TMT) labeling strategy. A total of 5529 proteins were successfully identified and quantified. Dynamic time profiling of protein expression patterns allowed us to identify five protein clusters involved in various stages of androgen-initiated signal transmission and processing. More details of protein functions and localization patterns, and our elucidation of an AR-interacting protein network, were obtained. Finally, we validated the expression level of AR-regulated proteins known to be significantly regulated in CRPC patients using the mouse xenograft model and patient samples. Our work offers a systematic analysis of the rapid proteomic changes induced by androgen and provides a global view of the molecular mechanisms underlying CRPC progression.