Differential Subcellular Localization Research Articles

Subfunctionalisation and self-repression of duplicated E1 homologues finetunes soybean flowering and adaptation

Soybean is a photoperiod-sensitive staple crop. Its photoperiodic flowering has major consequences for latitudinal adaptation and grain yield. Here, we identify and characterise a flowering locus named Time of flower 4b (Tof4b), which encodes E1-Like b (E1Lb), a homologue of the key soybean floral repressor E1. Tof4b protein physically associates with the promoters of two FLOWERING LOCUS T (FT) genes to repress their transcription and delay flowering to impart soybean adaptation to high latitudes. Three E1 homologues undergo subfunctionalisation and show differential subcellular localisation. Moreover, they all possess self-repression capability and each suppresses the two homologous counterparts. Subfunctionalisation and the transcriptional regulation of E1 genes collectively finetune flowering time and high-latitude adaptation in soybean. We propose a model for the functional fate of the three E1 genes after the soybean whole-genome duplication events, refine the molecular mechanisms underlying high-latitude adaption, and provide a potential molecular-breeding resource.

Post-translational Regulation of BRI1-EMS Suppressor 1 and Brassinazole-resistant 1.

BRI1-EMS Suppressor 1 (BES1) and Brassinazole resistant 1 (BZR1) are two highly similar master transcription factors of the brassinosteroid (BR) signaling pathway that regulate a variety of plant growth and development processes as well as stress responses. Previous genetic and biochemical analyses have established a complex regulatory network to control the two transcription factors. This network includes coordination with other transcription factors and interactors, multiple post-translational modifications (PTMs), and differential subcellular localizations. In this review, we systematically detail the functions and regulatory mechanisms of various PTMs: phosphorylation/dephosphorylation, ubiquitination/deubiquitination, SUMOylation/deSUMOylation, oxidation/reduction, in regulating the subcellular localization, protein stability, and the transcriptional activity of BES1/BZR1. We also discuss the current knowledge about the BES1/BZR1-interactors mediating the dynamic nucleocytoplasmic shuttling of BES1 and BZR1.

Construction of a TP53 mutation-associated ceRNA network as prognostic biomarkers in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) continues to endanger human health worldwide. Regulatory networks of competing endogenous RNAs (ceRNAs) play important roles in HCC. TP53 is the second most often altered gene in HCC and has a significant role in regulating target genes such as miRNAs and lncRNAs. MethodsData from patients with TP53 mutation were collected through the cBioPortal database and differential analysis was performed to screen RNAs related to TP53 mutation. The lncRNA-miRNA–mRNA relationship was predicted by the miRcode, miRDB, and TargetScan databases. The ceRNA networks were screened and visualized by Cytoscape. Core ceRNA networks were generated by differential analysis, coexpression analysis, prognostic analysis and subcellular localization. Finally, methylation, mutation, PPI, GSEA, immunity and drug sensitivity analyses of MEX3A were performed to determine the role of MEX3A in HCC. ResultsWe identified 1508 DEmRNAs, 85 DEmiRNAs and 931 DElncRNAs and obtained a ceRNA network including 28 lncRNAs, 4 miRNAs and 31 mRNAs. Twenty hub DERNAs in the TP53-altered-related ceRNA network were screened out by Cytoscape and the core ceRNA network (LINC00491/TCL6-hsa-miR-139-5p-MEX3A) was obtained by multiple analyses. In addition, we discovered that the methylation level of MEX3A was decreased and the mutation frequency was raised in HCC. Furthermore, elevated MEX3A expression was associated with alterations in the HCC immunological microenvironment. ConclusionWe successfully constructed a reciprocal ceRNA network, which could provide new ideas for exploring HCC mechanisms and therapeutic approaches.

Differential subcellular localization of ASPM RNA and protein.

RNAs encoding some centrosomal components are trafficked to the organelle during mitosis. Some RNAs, including ASPM , localize to the centrosome co-translationally. However, the relative position of these RNAs and their protein after trafficking to centrosomes remained unclear. We find that mislocalization of ASPM RNA from the centrosome does not affect the localization of ASPM protein. Further, ASPM RNA and ASPM protein reside in two physically close yet distinct subcellular spaces, with ASPM RNA on the astral side of the centrosome and ASPM protein on the spindle side. This suggests subtly distinct locations of ASPM RNA translation and ASPM protein function.

The stem region of group A transferase is crucial for its specificity, and its alteration promotes heterologous Forssman synthase activity

Some stem region mutants of human blood group A transferase (hAT) possess Forssman synthase (FS) activity, but very little is known about the mechanisms responsible for this enzymatic crosstalk. We performed confocal microscopy and image analysis to determine whether different intra-Golgi localization was accountable for this acquired activity. We also performed structural modeling and mutational and normal mode analyses. We introduced new mutations in the stem region and tested its FS and AT activities. No differences in subcellular localization were found between hAT and FS-positive mutants. AlphaFold models of hAT and mFS (mouse Forssman synthase) showed that the hAT stem region has a tether-like stem region, while in mFS, it encircles its catalytic domain. In silico analysis of FS-positive mutants indicated that stem region mutations induced structural changes, decreasing interatomic interactions and mobility of hAT that correlated with FS activity. Several additional mutations introduced in that region also bestowed FS activity without altering the AT activity: hAT 37–55 aa substitution by mFS 34–52, 37–55 aa deletion, and missense mutations: S46P, Q278Y, and Q286M. Stem region structure, mobility, and interactions are crucial for hAT specificity. Moreover, stem region mutations can lead to heterologous Forssman activity without changes in the catalytic machinery.

Sirtuin family in autoimmune diseases.

In recent years, epigenetic modifications have been widely researched. As humans age, environmental and genetic factors may drive inflammation and immune responses by influencing the epigenome, which can lead to abnormal autoimmune responses in the body. Currently, an increasing number of studies have emphasized the important role of epigenetic modification in the progression of autoimmune diseases. Sirtuins (SIRTs) are class III nicotinamide adenine dinucleotide (NAD)-dependent histone deacetylases and SIRT-mediated deacetylation is an important epigenetic alteration. The SIRT family comprises seven protein members (namely, SIRT1-7). While the catalytic core domain contains amino acid residues that have remained stable throughout the entire evolutionary process, the N- and C-terminal regions are structurally divergent and contribute to differences in subcellular localization, enzymatic activity and substrate specificity. SIRT1 and SIRT2 are localized in the nucleus and cytoplasm. SIRT3, SIRT4, and SIRT5 are mitochondrial, and SIRT6 and SIRT7 are predominantly found in the nucleus. SIRTs are key regulators of various physiological processes such as cellular differentiation, apoptosis, metabolism, ageing, immune response, oxidative stress, and mitochondrial function. We discuss the association between SIRTs and common autoimmune diseases to facilitate the development of more effective therapeutic strategies.

TRNATyr has an unusually short half-life in Trypanosoma brucei.

Following transcription, tRNAs undergo a series of processing and modification events to become functional adaptors in protein synthesis. Eukaryotes have also evolved intracellular transport systems whereby nucleus-encoded tRNAs may travel out and into the nucleus. In trypanosomes, nearly all tRNAs are also imported from the cytoplasm into the mitochondrion, which lacks tRNA genes. Differential subcellular localization of the cytoplasmic splicing machinery and a nuclear enzyme responsible for queuosine modification at the anticodon "wobble" position appear to be important quality control mechanisms for tRNATyr, the only intron-containing tRNA in T. brucei Since tRNA-guanine transglycosylase (TGT), the enzyme responsible for Q formation, cannot act on an intron-containing tRNA, retrograde nuclear transport is an essential step in maturation. Unlike maturation/processing pathways, the general mechanisms of tRNA stabilization and degradation in T. brucei are poorly understood. Using a combination of cellular and molecular approaches, we show that tRNATyr has an unusually short half-life. tRNATyr, and in addition tRNAAsp, also show the presence of slow-migrating bands during electrophoresis; we term these conformers: alt-tRNATyr and alt-tRNAAsp, respectively. Although we do not know the chemical or structural nature of these conformers, alt-tRNATyr has a short half-life resembling that of tRNATyr; the same is not true for alt-tRNAAsp We also show that RRP44, which is usually an exosome subunit in other organisms, is involved in tRNA degradation of the only intron-containing tRNA in T. brucei and is partly responsible for its unusually short half-life.

Genome-wide analysis of calcium-dependent protein kinase (CDPK) family and functional characterization of TaCDPK25-U in response to drought stress in wheat

Calcium (Ca2+)-dependent protein kinase (CDPK) is one kind of biomacromolecule that can not only act as the unique Ca2+ sensors for the increase and decrease of calcium concentrations, but also as an effector for calcium signaling. It plays a vital role in regulating plant growth and development as well as stress responses. Although it has been extensively characterized in many plants, the importance of CDPK family in wheat is not well studied to date. In this study, a total of 85, 57, 27, and 27 CDPKs were identified in Triticum aestivum (T. aestivum), Triticum dicoccoides (T. dicoccoides), Triticum urartu (T. urartu) and Aegilops tauschii (A. tauschii), respectively, and grouped them into four phylogenetic subclades. Then, the gene structure, protein domains, conserved motifs and cis-acting elements of them were comprehensively investigated. Synteny analysis demonstrated that segmental duplication events and polyploidization contributed to the expansion of TaCDPK family. Furthermore, the tissue-specific as well as stress-responsive candidates were detected based on RNA-seq analysis. Results showed that under drought stress, TaCDPK25-U was induced to transcribe two alternative spliced isoforms (TaCDPK25-U-AS1 and TaCDPK25-U-AS2), which displayed differential expression levels and different subcellular localization. Finally, over-expression of TaCDPK25-U-AS1 or TaCDPK25-U-AS2 enhanced the drought tolerance in Arabidopsis thaliana (A. thaliana). Additionally, the expression of TaCDPK25-U-AS2 was demonstrated to be positively regulated by TaDREB3 to improve drought tolerance in vivo and in vitro. This study laid the foundation to better understand the evolution and function of CDPK family in wheat.

CCNB2 as a potential biomarker of bladder cancer via the high throughput technology.

Bladder cancer and oral squamous cell carcinoma (OSCC) seriously affect people's health. However, the relationship between bladder cancer and OSCC remains unclear. Got GSE138206, GSE146483, GSE184616, and bladder cancer datasets GSE65635, GSE100926 from Gene Expression Omnibus database. Weighted gene co-expression network analysis was used to identify the significant module. Functional enrichment analysis was performed via the Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes. Furthermore, the Gene Set Enrichment Analysis was also used to complete the enrichment analysis. Comparative Toxicogenomics Database found most relevant diseases to core genes. TargetScan is used to forecast analysis of microRNA and target genes. In Gene Ontology analysis, differentially expressed genes were mostly concentrated in cell differentiation, extrallular region, structural molecule activity, and actin binding. In Kyoto Encyclopedia of Genes and Genomes analysis, the differentially expressed genes were mainly enriched in PI3K-Akt signaling pathway, pathway in cancer, and extracellular matrix-receptor interaction. Seven hub genes (cyclin B2 [CCNB2], TK1, CDC20, PCNA, CKS1B, CDCA5, MCM4) were obtained. Hub genes (CCNB2, CDC20) are highly expressed in OSCC and bladder cancer samples. CCNB2 was one common oncogene of bladder cancer and OSCC.

Distinctly localized lipid phosphate phosphatases mediate endoplasmic reticulum glycerolipid metabolism in Arabidopsis.

Inter-organelle communication is an integral subcellular process in cellular homeostasis. In plants, cellular membrane lipids are synthesized in the plastids and endoplasmic reticulum (ER). However, the crosstalk between these organelles in lipid biosynthesis remains largely unknown. Here, we show that a pair of lipid phosphate phosphatases (LPPs) with differential subcellular localizations is required for ER glycerolipid metabolism in Arabidopsis (Arabidopsis thaliana). LPPα2 and LPPε1, which function as phosphatidic acid phosphatases and thus catalyze the core reaction in glycerolipid metabolism, were differentially localized at ER and chloroplast outer envelopes despite their similar tissue expression pattern. No mutant phenotype was observed in single knockout mutants; however, genetic suppression of these LPPs affected pollen growth and ER phospholipid biosynthesis in mature siliques and seeds with compromised triacylglycerol biosynthesis. Although chloroplast-localized, LPPε1 was localized close to the ER and ER-localized LPPα2. This proximal localization is functionally relevant, because overexpression of chloroplastic LPPε1 enhanced ER phospholipid and triacylglycerol biosynthesis similar to the effect of LPPα2 overexpression in mature siliques and seeds. Thus, ER glycerolipid metabolism requires a chloroplast-localized enzyme in Arabidopsis, representing the importance of inter-organelle communication in membrane lipid homeostasis.

Protein engineering reveals that gasdermin A preferentially targets mitochondrial membranes over the plasma membrane during pyroptosis

When activated, gasdermin family members are thought to be pore-forming proteins that cause lytic cell death. Despite this, numerous studies have suggested that the threshold for lytic cell death is dependent on which gasdermin family member is activated. Determination of the propensity of various gasdermin family members to cause pyroptosis has been handicapped by the fact that for many of them, the mechanisms and timing of their activation are uncertain. In this article, we exploit the recently discovered exosite-mediated recognition of gasdermin D (GSDMD) by the inflammatory caspases to develop a system that activates gasdermin family members in an efficient and equivalent manner. We leverage this system to show that upon activation, GSDMD and gasdermin A (GSDMA) exhibit differential subcellular localization, differential plasma membrane permeabilization, and differential lytic cell death. While GSDMD localizes rapidly to both the plasma membrane and organelle membranes, GSDMA preferentially localizes to the mitochondria with delayed and diminished accumulation at the plasma membrane. As a consequence of this differential kinetics of subcellular localization, N-terminal GSDMA results in early mitochondrial dysfunction relative to plasma membrane permeabilization. This study thus challenges the assumption that gasdermin family members effect cell death through identical mechanisms and establishes that their activation in their respective tissues of expression likely results in different immunological outcomes.

Inferring differential subcellular localisation in comparative spatial proteomics using BANDLE

The steady-state localisation of proteins provides vital insight into their function. These localisations are context specific with proteins translocating between different subcellular niches upon perturbation of the subcellular environment. Differential localisation, that is a change in the steady-state subcellular location of a protein, provides a step towards mechanistic insight of subcellular protein dynamics. High-accuracy high-throughput mass spectrometry-based methods now exist to map the steady-state localisation and re-localisation of proteins. Here, we describe a principled Bayesian approach, BANDLE, that uses these data to compute the probability that a protein differentially localises upon cellular perturbation. Extensive simulation studies demonstrate that BANDLE reduces the number of both type I and type II errors compared to existing approaches. Application of BANDLE to several datasets recovers well-studied translocations. In an application to cytomegalovirus infection, we obtain insights into the rewiring of the host proteome. Integration of other high-throughput datasets allows us to provide the functional context of these data.

Fluorescent Auxin Analogs Report Two Auxin Binding Sites with Different Subcellular Distribution and Affinities: A Cue for Non-Transcriptional Auxin Signaling.

The complexity of auxin signaling is partially due to multiple auxin receptors that trigger differential signaling. To obtain insight into the subcellular localization of auxin-binding sites, we used fluorescent auxin analogs that can undergo transport but do not deploy auxin signaling. Using fluorescent probes for different subcellular compartments, we can show that the fluorescent analog of 1-naphthaleneacetic acid (NAA) associates with the endoplasmic reticulum (ER) and tonoplast, while the fluorescent analog of indole acetic acid (IAA) binds to the ER. The binding of the fluorescent NAA analog to the ER can be outcompeted by unlabeled NAA, which allows us to estimate the affinity of NAA for this binding site to be around 1 μM. The non-transportable auxin 2,4-dichlorophenoxyacetic acid (2,4-D) interferes with the binding site for the fluorescent NAA analog at the tonoplast but not with the binding site for the fluorescent IAA analog at the ER. We integrate these data into a working model, where the tonoplast hosts a binding site with a high affinity for 2,4-D, while the ER hosts a binding site with high affinity for NAA. Thus, the differential subcellular localization of binding sites reflects the differential signaling in response to these artificial auxins.

Regulation of Cardiac Contractility by the Alpha 2 Subunit of the Na+/K+-ATPase.

Cytosolic Na + concentrations regulate cardiac excitation-contraction coupling and contractility. Inhibition of the Na+/K+-ATPase (NKA) activity increases cardiac contractility by increasing cytosolic Ca2+ levels, as increased cytosolic Na+ levels are coupled to less Ca2+ extrusion and/or increased Ca2+ influx from the Na+/Ca2+-exchanger. NKA consists of one α subunit and one β subunit, with α1 and α2 being the main α isoforms in cardiomyocytes. Substantial evidence suggests that NKAα2 is the primary regulator of cardiac contractility despite being outnumbered by NKAα1 in cardiomyocytes. This review will mainly focus on differential regulation and subcellular localization of the NKAα1 and NKAα2 isoforms, and their relation to the proposed concept of subcellular gradients of Na+ in cardiomyocytes. We will also discuss the potential roles of NKAα2 in mediating cardiac hypertrophy and ventricular arrhythmias.

Differential Trafficking and Expression of PIR Proteins in Acute and Chronic Plasmodium Infections.

Plasmodium multigene families are thought to play important roles in the pathogenesis of malaria. Plasmodium interspersed repeat (pir) genes comprise the largest multigene family in many Plasmodium species. However, their expression pattern and localisation remain to be elucidated. Understanding protein subcellular localisation is fundamental to reveal the functional importance and cell-cell interactions of the PIR proteins. Here, we use the rodent malaria parasite, Plasmodium chabaudi chabaudi, as a model to investigate the localisation pattern of this gene family. We found that most PIR proteins are co-expressed in clusters during acute and chronic infection; members of the S7 clade are predominantly expressed during the acute-phase, whereas members of the L1 clade dominate the chronic-phase of infection. Using peptide antisera specific for S7 or L1 PIRS, we show that these PIRs have different localisations within the infected red blood cells. S7 PIRs are exported into the infected red blood cell cytoplasm where they are co-localised with parasite-induced host cell modifications termed Maurer’s clefts, whereas L1 PIRs are localised on or close to the parasitophorous vacuolar membrane. This localisation pattern changes following mosquito transmission and during progression from acute- to chronic-phase of infection. The presence of PIRs in Maurer’s clefts, as seen for Plasmodium falciparum RIFIN and STEVOR proteins, might suggest trafficking of the PIRs on the surface of the infected erythrocytes. However, neither S7 nor L1 PIR proteins detected by the peptide antisera are localised on the surface of infected red blood cells, suggesting that they are unlikely to be targets of surface variant-specific antibodies or to be directly involved in adhesion of infected red blood cells to host cells, as described for Plasmodium falciparum VAR proteins. The differences in subcellular localisation of the two major clades of Plasmodium chabaudi PIRs across the blood cycle, and the apparent lack of expression on the red cell surface strongly suggest that the function(s) of this gene family may differ from those of other multigene families of Plasmodium, such as the var genes of Plasmodium falciparum.

Nucleo-cytoplasmic RNA distribution responsible for maintaining neuroinflammatory microenvironment

ABSTRACT Subcellular localization of transcripts is highly associated with regulation of gene expression, synthesis of protein, and also the development of the human brain cortex. Although many mechanisms are prevalent in the occurrence of neuroinflammation, the mechanisms based on differences in subcellular localization of transcripts have not been explored. To characterize the dynamic profile of nuclear and cytoplasmic transcripts during the progress of haemorrhage-induced neuroinflammation, we isolated nucleo-cytoplasmic RNA fractions of oxyhaemoglobin (oxy-Hb) treated microglia cells and sequenced both fractions. We discovered that cytoplasmic retained genes were the major forces to maintain the neuroinflammatory microenvironment with 10 hub genes and 40 conserved genes were identified. Moreover, antisense RNA Gm44096 and lincRNA Gm47270, which co-expressed with a crowd of inflammatory genes in the cytoplasm, were discovered as regulatory strategies for sustaining the neuroinflammatory microenvironment. Thus, our study provides a new perspective on understanding haemorrhage-induced neuroinflammation and also reveals a mechanism of lncRNA responsible for maintaining the neuroinflammatory microenvironment.

Artificial Self-assembling Nanocompartment for Organizing Metabolic Pathways in Yeast.

Metabolic pathways are commonly organized by sequestration into discrete cellular compartments. Compartments prevent unfavorable interactions with other pathways and provide local environments conducive to the activity of encapsulated enzymes. Such compartments are also useful synthetic biology tools for examining enzyme/pathway behavior and for metabolic engineering. Here, we expand the intracellular compartmentalization toolbox for budding yeast (Saccharomyces cerevisiae) with Murine polyomavirus virus-like particles (MPyV VLPs). The MPyV system has two components: VP1 which self-assembles into the compartment shell and a short anchor, VP2C, which mediates cargo protein encapsulation via binding to the inner surface of the VP1 shell. Destabilized green fluorescent protein (GFP) fused to VP2C was specifically sorted into VLPs and thereby protected from host-mediated degradation. An engineered VP1 variant displayed improved cargo capture properties and differential subcellular localization compared to wild-type VP1. To demonstrate their ability to function as a metabolic compartment, MPyV VLPs were used to encapsulate myo-inositol oxygenase (MIOX), an unstable and rate-limiting enzyme in d-glucaric acid biosynthesis. Strains with encapsulated MIOX produced ∼20% more d-glucaric acid compared to controls expressing “free” MIOX—despite accumulating dramatically less expressed protein—and also grew to higher cell densities. This is the first demonstration in yeast of an artificial biocatalytic compartment that can participate in a metabolic pathway and establishes the MPyV platform as a promising synthetic biology tool for yeast engineering.

RNA-Binding Proteins in the Post-transcriptional Control of Skeletal Muscle Development, Regeneration and Disease.

Embryonic myogenesis is a temporally and spatially regulated process that generates skeletal muscle of the trunk and limbs. During this process, mononucleated myoblasts derived from myogenic progenitor cells within the somites undergo proliferation, migration and differentiation to elongate and fuse into multinucleated functional myofibers. Skeletal muscle is the most abundant tissue of the body and has the remarkable ability to self-repair by re-activating the myogenic program in muscle stem cells, known as satellite cells. Post-transcriptional regulation of gene expression mediated by RNA-binding proteins is critically required for muscle development during embryogenesis and for muscle homeostasis in the adult. Differential subcellular localization and activity of RNA-binding proteins orchestrates target gene expression at multiple levels to regulate different steps of myogenesis. Dysfunctions of these post-transcriptional regulators impair muscle development and homeostasis, but also cause defects in motor neurons or the neuromuscular junction, resulting in muscle degeneration and neuromuscular disease. Many RNA-binding proteins, such as members of the muscle blind-like (MBNL) and CUG-BP and ETR-3-like factors (CELF) families, display both overlapping and distinct targets in muscle cells. Thus they function either cooperatively or antagonistically to coordinate myoblast proliferation and differentiation. Evidence is accumulating that the dynamic interplay of their regulatory activity may control the progression of myogenic program as well as stem cell quiescence and activation. Moreover, the role of RNA-binding proteins that regulate post-transcriptional modification in the myogenic program is far less understood as compared with transcription factors involved in myogenic specification and differentiation. Here we review past achievements and recent advances in understanding the functions of RNA-binding proteins during skeletal muscle development, regeneration and disease, with the aim to identify the fundamental questions that are still open for further investigations.

A differential subcellular localization of two copper transporters from the COPT family suggests distinct roles in copper homeostasis in Physcomitrium patens

The moss Physcomitrium (Physcomitrella) patens is a bryophyte that provides genetic information about the adaptation to the life on land by early Embryophytes and is a reference organism for comparative evolutionary studies in plants. Copper is an essential micronutrient for every living organism, its transport across the plasma membrane is achieved by the copper transport protein family COPT/CTR. Two genes related to the COPT family were identified in Physcomitrella patens, PpaCOPT1 and PpaCOPT2. Homology modelling of both proteins showed the presence of three putative transmembrane domains (TMD) and the Mx3M motif, constituting a potential Cu + selectivity filter present in other members of this family. Functional characterization of PpaCOPT1 and PpaCOPT2 in the yeast mutant ctr1Δctr3Δ restored its growth on medium with non-fermentable carbon sources at micromolar Cu concentrations, providing support that these two moss proteins function as high affinity Cu + transporters. Localization of PpaCOPT1 and PpaCOPT2 in yeast cells was observed at the tonoplast and plasma membrane, respectively. The heterologous expression of PpaCOPT2 in tobacco epidermal cells co-localized with the plasma membrane marker. Finally, only PpaCOPT1 was expressed in seven-day old protonema and was influenced by extracellular copper levels. This evidence suggests different roles of PpaCOPT1 and PpaCOPT2 in copper homeostasis in Physcomitrella patens.

Cellular Diversity and Differential Subcellular Localization of the G-Protein Gαo Subunit in the Mouse Cerebellum.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) transduce signals from G protein-coupled receptors (GPCRs) to effector ion channels and enzymes Gαo, a member of the pertussis toxin-sensitive Gi/o family, is widely expressed in the brain, although its role within a neuronal context remains largely unknown. Using immunohistochemical and quantitative immunoelectron microscopy techniques, we have investigated the expression, cellular and subcellular localization of Gαo in the cerebellar cortex. Histoblot revealed that Gαo is expressed in many brain regions, including the cerebellum. At the cellular level, Gαo protein was distributed in Purkinje cells, basket cells, stellate cells, granule cells and Golgi cells. At the subcellular level, pre-embedding immunoelectron microscopy revealed mainly a postsynaptic localization of Gαo along the extrasynaptic plasma membrane of Purkinje cell dendritic shafts and spines, and dendrites of basket, stellate and granule cells. To a lesser extent, immunolabeling for Gαo was localized in different types of axon terminals establishing excitatory synapses. Moreover, post-embedding immunoelectron microscopy revealed the synaptic localization of Gαo on PSDs of glutamatergic synapses between Purkinje cell spines and parallel fiber terminals and its co-localization with GABAB1 in the same spines. Quantitative analysis of Gαo immunoparticles revealed they preferentially localized on the cytoplasmic face of the plasma membrane. Furthermore, the analysis revealed a high concentration of Gαo around excitatory synapses on Purkinje cell dendritic spines, but a uniform distribution in granule cell dendrites. These molecular-anatomical findings suggest that Gαo is a major signal transducer of specific GPCRs in different neuronal populations in the cerebellum.