Golgi Vesicles Research Articles

In Vitro Methods to Study the Golgi Apparatus Role in Proteoglycan and Glycosaminoglycan Synthesis.

Subcellular fractionation is an introductory step in a variety of experimental approaches designed to study intracellular components, like membranes and organelle systems. Subcellular fractions enriched in membranes of the Golgi apparatus of mammalian cells have been isolated to address localization and activity of proteins, including enzymes, to study intracellular membrane transport mechanisms, and to reconstitute in vitro cellular processes associated with the Golgi apparatus. Here, I describe methods to purify Golgi membranes by subcellular fractionation, to assay nucleotide sulfate (PAPS) uptake into Golgi vesicles, and to measure sulfate incorporation into in vitro synthesized glycosaminoglycans.

ArfGAP3 regulates vesicle transport and glucose uptake in myoblasts

Skeletal muscle injuries are common, and damaged myofibers are repaired through proliferation and differentiation of muscle satellite cells. GLUT4 is enriched in GLUT4 storage vesicles (GSVs) and plays a crucial role in the maintenance of muscle function. ArfGAP3 regulates the vesicle transport especially for COPI coat assembly, but its effects on GSVs and the repair process after skeletal muscle injury remains unclear. In this study, datasets related to skeletal muscle injury and myoblast differentiation GSE469, GSE5413, GSE45577 and GSE108040 were retrieved through the GEO database and the expression of heptameric coat protein complex I (COPI) and Golgi vesicle transport-related genes in various datasets, as well as the expression correlation between ArfGAP2, ArfGAP3 and COPI-related genes COPA, COPB1, COPB2, COPE, COPZ1, COPZ2 were analyzed. The results suggested that ArfGAP3 was expressed in the process of repair after skeletal muscle injury and myoblast differentiation and that ArfGAP3 was positively correlated with COPI-related genes. In vitro experimental results showed that ArfGAP3 was colocalized with COPA, COPB, COPG protein, and GLUT4 in C2C12 myoblasts. After the downregulation of ArfGAP3 expression, intracellular vesicle transport, and glucose uptake were blocked, the proliferation of myoblasts under low glucose culture conditions was impaired, the proportion of apoptosis increased, and myotube differentiation was impaired. In summary, ArfGAP3 regulates COPI-associated vesicle and GSVs transport and affects the proliferation and differentiation ability of myoblasts by influencing glucose uptake, thereby modulating the repair process after skeletal muscle injury.

Pathway-level mutation analysis in primary high-grade serous ovarian cancer and matched brain metastases

Brain metastases (BMs) in ovarian cancer (OC) are a rare event. BMs occur most frequently in high-grade serous (HGS) OC. The molecular features of BMs in HGSOC are poorly understood. We performed a whole-exome sequencing analysis of ten matched pairs of formalin-fixed paraffin-embedded samples from primary HGSOC and corresponding BMs. Enrichment significance (p value; false discovery rate) was computed using the Reactome, the Kyoto Encyclopedia of Genes and Genomes pathway collections, and the Gene Ontology Biological Processes. Germline DNA damage repair variants were found in seven cases (70%) and involved the BRCA1, BRCA2, ATM, RAD50, ERCC4, RPA1, MLHI, and ATR genes. Somatic mutations of TP53 were found in nine cases (90%) and were the only stable mutations between the primary tumor and BMs. Disturbed pathways in BMs versus primary HGSOC constituted a complex network and included the cell cycle, the degradation of the extracellular matrix, cell junction organization, nucleotide metabolism, lipid metabolism, the immune system, G-protein-coupled receptors, intracellular vesicular transport, and reaction to chemical stimuli (Golgi vesicle transport and olfactory signaling). Pathway analysis approaches allow for a more intuitive interpretation of the data as compared to considering single-gene aberrations and provide an opportunity to identify clinically informative alterations in HGSOC BM.

An electron microscopic study of neocortex of Syrian hamsters (<i>Mesocricetus auratus</i>) infected with SARS-CoV-2 (Coronaviridae: <i>Coronavirinae: Betacoronavirus: Sarbecovirus</i>)

Convalescent COVID-19 patients have various signs of central nervous system damage, including those directly associated with SARS-CoV-2. Hence, studies of SARS-COV-2 related morphological changes in neocortex are particularly relevant for understanding the mechanisms of their formation and development of approaches to preclinical evaluation of the effectiveness of antiviral drugs. The purpose of the research is a longitudinal study of the ultrastructural alterations in Syrian hamsters neocortex after experimental SARS-CoV-2 infection. Male Syrian hamsters weighing 80100 g, aged 4 to 6 weeks, were infected with 26 l SARS-CoV-2 intranasally with 4104 TCD50/ml of viral particles. The animals were euthanized on days 3, 7 or 28 post-infection, the brain was extracted with the cortex excision. The material analysis was performed using transmission electron microscopy. On day 3 post-infection, the number of moderately hyperchromic neurons in neocortex increased, while by the day 7 the number of apoptotic cells significantly increased. Simultaneously, an increased signs of neuronophagy and representation of atypical glia were observed. Increased number of altered oligodendrocytes was observed on day 28 post-infection. Viral invasion was accompanied by changes in neocortical cells since day 3 post-infection, such as transformation of their nucleus, the rough endoplasmic reticulum and the Golgi vesicles as well as microvascular spasm with perivascular edema. As a result of electron microscopic study, the ultrastructural alterations in neocortex were described in an experimental model of SARS-CoV-2 infection. The findings can be used to identify the mechanisms of infection pathogenesis and to search for the new directions in development of medicines.

Dominant ARF3 variants disrupt Golgi integrity and cause a neurodevelopmental disorder recapitulated in zebrafish

Vesicle biogenesis, trafficking and signaling via Endoplasmic reticulum-Golgi network support essential developmental processes and their disruption lead to neurodevelopmental disorders and neurodegeneration. We report that de novo missense variants in ARF3, encoding a small GTPase regulating Golgi dynamics, cause a developmental disease in humans impairing nervous system and skeletal formation. Microcephaly-associated ARF3 variants affect residues within the guanine nucleotide binding pocket and variably perturb protein stability and GTP/GDP binding. Functional analysis demonstrates variably disruptive consequences of ARF3 variants on Golgi morphology, vesicles assembly and trafficking. Disease modeling in zebrafish validates further the dominant behavior of the mutants and their differential impact on brain and body plan formation, recapitulating the variable disease expression. In-depth in vivo analyses traces back impaired neural precursors’ proliferation and planar cell polarity-dependent cell movements as the earliest detectable effects. Our findings document a key role of ARF3 in Golgi function and demonstrate its pleiotropic impact on development.

Co-expression network analysis for identification of novel biomarkers of bronchopulmonary dysplasia model.

Bronchopulmonary dysplasia (BPD) is the most common neonatal chronic lung disease. However, its exact molecular pathogenesis is not understood. We aimed to identify relevant gene modules that may play crucial roles in the occurrence and development of BPD by weighted gene co-expression network analysis (WGCNA). We used RNA-Seq data of BPD and healthy control rats from our previous studies, wherein data from 30 samples was collected at days 1, 3, 7, 10, and 14. Data for preprocessing analysis included 17,613 differentially expressed genes (DEGs) with false discovery rate <0.05. We grouped the highly correlated genes into 13 modules, and constructed a network of mRNA gene associations, including the 150 most associated mRNA genes in each module. Lgals8, Srpra, Prtfdc1, and Thap11 were identified as the key hub genes. Enrichment analyses revealed Golgi vesicle transport, coated vesicle, actin-dependent ATPase activity and endoplasmic reticulum pathways associated with these genes involved in the pathological process of BPD in module. This is a study to analyze data obtained from BPD animal model at different time-points using WGCNA, to elucidate BPD-related susceptibility modules and disease-related genes.

Golgi Apparatus fragmentation in dilated cardiomyopathy and its relationship with the alteration of vesicular transport

Abstract Background The Golgi Apparatus (GA) is an important membrane-bound organelle implied in many physiological events, as trafficking, processing and sorting of newly membrane and secretory proteins and lipids. Nevertheless, these functions can be dysregulated in a diversity of pathologies due to the alteration of the GA morphology, such as cardiovascular diseases. Previously, we described in dilated cardiomyopathy (DCM) alterations in shape and size of the secretion vesicles of GA that was related to a worse functional situation. The patients had smaller, more ellipsoidal and more numerous vesicles with a higher content of natriuretic peptides (1). This situation can be related with Golgi fragmentation, a status characterized by the presence of a dispersed GA both in physiological and pathophysiological conditions (2). However, a greater understanding of Golgi fragmentation in DCM is necessary. Purpose We attempted to analyse the components of the vesicular Golgi transport and the Golgi fragmentation in cardiac tissue samples from DCM patients, paying special attention to GM130, as one of the main molecules related with the Golgi fragmentation. Methods We analysed vesicular Golgi transport genes in human hearts by RNA-sequencing in 13 samples from DCM patients and 10 control hearts (CNT), and the miRNAs that regulate their expression by ncRNA sequencing (DCM, n=20; and CNT, n=8). Finally, we investigated changes in the protein levels of GM130 and its phosphorylated state (pGM130) by western blot with 22 DCM and 6 CNT samples in total. Results We found 134 differentially expressed genes related to the GA, and 15 of them were involved in the secretion of Golgi vesicles. Among these molecules, VAMP4, a v-SNARE related with the Golgi fragmentation, is found to be downregulated (−1.27-Fold; P&amp;lt;0.05). Moreover, we have observed a downregulation in the expression of GOLGA2, the gene that encodes GM130 (−1.24; P&amp;lt;0.05). Furthermore, its regulator, hsa-miR-17-5p (−1.42; P&amp;lt;0.01) was also downregulated. In this sense, we have found no changes in GM130 protein levels between DCM patients and CNT group. In contrast, pGM130, molecule that it has been used as a biomarker of Golgi fragmentation, was overexpressed in DCM patients (1.19; P&amp;lt;0.05). Conclusion We found a deregulation of genes related to the vesicle secretion in the GA, which together with the changes in vesicular morphology previously observed, suggests an alteration in the rate of secretion in response to the needs of the DCM patients. In addition, the increase in the phosphorylated protein GM130 suggests the presence of fragmentation in DCM patients that could be related to the alteration in vesicular transport. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): National Institute of Health Carlos III European Regional Development Fund (ERDF)

Localization of CgVPE1 in secondary cell wall formation during tracheary element differentiation in the pericarp of Citrus grandis 'Tomentosa' fruits.

CgVPE1 is important in the differentiation of TE cells in C. grandis 'Tomentosa' fruits as it may directly affects secondary cell wall construction while participating in PCD. The vacuolar processing enzyme (VPE) plays an important role in both developmental and environmentally inducible programmed cell death (PCD); it was originally identified as a cysteine protease localized in the vacuole to activate and mature vacuolar proteins in plants. Interestingly, we found a VPE called CgVPE1 to be associated with deposition of the secondary cell wall in tracheary element (TE) cells in the pericarp of Citrus grandis 'Tomentosa' fruits. We then used ultrathin sections and the TUNEL assay to verify that PCD is involved in TE development. Furthermore, CgVPE1 was found to be mainly expressed in secretory cavities and TEs in the pericarp of Citrus grandis 'Tomentosa' fruits. Immunolocalization of CgVPE1 in the pericarp indicated that CgVPE1 is mainly distributed in the central large vacuole, endoplasmic reticulum, Golgi vesicles, cytosol, and secondary wall before TE maturation. CgVPE1 appeared earlier in the endoplasmic reticulum and Golgi vesicles of TEs cells. The vesicles containing CgVPE1 near the large central vacuole and secondary wall were observed, respectively. CgVPE1 proteins content in the cytoplasm decreased sharply, while the CgVPE1 content in the secondary cell wall did not change significantly after vacuole rupture. CgVPE1 protein contents in the secondary cell wall were significantly reduced until the TE cells developed into hollow thick-walled cells. Furthermore, labeling of VPE homologues in Arabidopsis thaliana using immunoelectron microscopy with anti-CgVPE1 antibody revealed that VPE homologues were specifically distributed in the secondary cell wall of stem TEs. Overall, these results suggested that CgVPE1 is not only involved PCD during TE cell development; furthermore, it may directly participate in the construction of plant secondary cell walls.

Neurotransmitter release cycle-related genes predict the prognosis of lung adenocarcinoma.

Because of the limitations of therapeutic approaches, patients suffering from lung adenocarcinoma (LUAD) have unsatisfactory prognoses. Studies have shown that neurotransmitters participated in tumorigenesis and development. In LUAD, the expression of neurotransmitter release cycle-related genes (NRCRGs) has been reported to be disordered. This study aimed to study the correlation between NRCRGs and LUAD. In this study, based on the Cancer Genome Atlas cohort, consensus clustering analyses were performed on ten neurotransmitter release cycle-related (NRCR) differentially expressed genes. Neurotransmitter release cycle (NRC) scores were derived by the Least Absolute Shrinkage and Selection Operator-Cox regression model constituted by 3 NRCRGs. Univariate and multivariate Cox regression analyses were performed to evaluate the prognosis value of the NRC score. In addition, single-Sample Gene Set Enrichment Analysis and CIBERSORT were conducted in the Cancer Genome Atlas cohort. Finally, gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses were also performed. As a result, the NRC-low group showed a good prognosis instead of the NRC-high group. NRC score was identified to be an independent prognosis factor for LUAD. In general, the NRC score based on the prognostic model was found to be closely correlated with immunotherapy-related anti-cancer immunity and inflamed tumor microenvironment. Functional enrichment results demonstrated that differentially expressed genes between 2 NRC groups were closely correlated with DNA replication, cell-substrate adhesion, Golgi vesicle transport, MAPK signal pathway, and many others. Novel biomarkers were offered for predicting the prognoses of LUAD patients. The NRC score might contribute to guiding LUAD patients with immunotherapy selection.

MTORC1 controls Golgi architecture and vesicle secretion by phosphorylation of SCYL1

The protein kinase mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation, supporting anabolic reactions and inhibiting catabolic pathways like autophagy. Its hyperactivation is a frequent event in cancer promoting tumor cell proliferation. Several intracellular membrane-associated mTORC1 pools have been identified, linking its function to distinct subcellular localizations. Here, we characterize the N-terminal kinase-like protein SCYL1 as a Golgi-localized target through which mTORC1 controls organelle distribution and extracellular vesicle secretion in breast cancer cells. Under growth conditions, SCYL1 is phosphorylated by mTORC1 on Ser754, supporting Golgi localization. Upon mTORC1 inhibition, Ser754 dephosphorylation leads to SCYL1 displacement to endosomes. Peripheral, dephosphorylated SCYL1 causes Golgi enlargement, redistribution of early and late endosomes and increased extracellular vesicle release. Thus, the mTORC1-controlled phosphorylation status of SCYL1 is an important determinant regulating subcellular distribution and function of endolysosomal compartments. It may also explain the pathophysiology underlying human genetic diseases such as CALFAN syndrome, which is caused by loss-of-function of SCYL1.

Integrated Analysis of Microarray, Small RNA, and Degradome Datasets Uncovers the Role of MicroRNAs in Temperature-Sensitive Genic Male Sterility in Wheat.

Temperature-sensitive genic male sterile (TGMS) line Beijing Sterility 366 (BS366) has been utilized in hybrid breeding for a long time, but the molecular mechanism underlying male sterility remains unclear. Expression arrays, small RNA, and degradome sequencing were used in this study to explore the potential role of miRNA in the cold-induced male sterility of BS366. Microspore observation showed defective cell plates in dyads and tetrads and shrunken microspores at the vacuolated stage. Differential regulation of Golgi vesicle transport, phragmoplast formation, sporopollenin biosynthesis, pollen exine formation, and lipid metabolism were observed between cold and control conditions. Pollen development was significantly represented in the 352 antagonistic miRNA-target pairs in the integrated analysis of miRNA and mRNA profiles. The specific cleavage of ARF17 and TIR1 by miR160 and miR393 were found in the cold-treated BS366 degradome, respectively. Thus, the cold-mediated miRNAs impaired cell plate formation through repression of Golgi vesicle transport and phragmoplast formation. The repressed expression of ARF17 and TIR1 impaired pollen exine formation. The results of this study will contribute to our understanding of the roles of miRNAs in male sterility in wheat.

Systematic Identification of Essential Genes Required for Yeast Cell Wall Integrity: Involvement of the RSC Remodelling Complex.

Conditions altering the yeast cell wall lead to the activation of an adaptive transcriptional response mainly governed by the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway. Two high-throughput screenings were developed using the yTHC collection of yeast conditional mutant strains to systematically identify essential genes related to cell wall integrity, and those required for the transcriptional program elicited by cell wall stress. Depleted expression of 52 essential genes resulted in hypersensitivity to the dye Calcofluor white, with chromatin organization, Golgi vesicle transport, rRNA processing, and protein glycosylation processes, as the most highly representative functional groups. Via a flow cytometry-based quantitative assay using a CWI reporter plasmid, 97 strains exhibiting reduced gene-reporter expression levels upon stress were uncovered, highlighting genes associated with RNA metabolism, transcription/translation, protein degradation, and chromatin organization. This screening also led to the discovery of 41 strains displaying a basal increase in CWI-associated gene expression, including mainly putative cell wall-related genes. Interestingly, several members of the RSC chromatin remodelling complex were uncovered in both screenings. Notably, Rsc9 was necessary to regulate the gene expression of CWI-related genes both under stress and non-stress conditions, suggesting distinct requirements of the RSC complex for remodelling particular genes.

Roles of Endomembrane Alkali Cation/Proton Exchangers in Synaptic Function and Neurodevelopmental Disorders.

Endomembrane alkali cation (Na+, K+)/proton (H+) exchangers (eNHEs) are increasingly associated with neurological disorders. These eNHEs play integral roles in regulating the luminal pH, processing, and trafficking of cargo along the secretory (Golgi and post-Golgi vesicles) and endocytic (early, recycling, and late endosomes) pathways, essential regulatory processes vital for neuronal development and plasticity. Given the complex morphology and compartmentalization of multipolar neurons, the contribution of eNHEs in maintaining optimal pH homeostasis and cargo trafficking is especially significant during periods of structural and functional development and remodeling. While the importance of eNHEs has been demonstrated in a variety of non-neuronal cell types, their involvement in neuronal function is less well understood. In this review, we will discuss their emerging roles in excitatory synaptic function, particularly as it pertains to cellular learning and remodeling. We will also explore their connections to neurodevelopmental conditions, including intellectual disability, autism, and attention deficit hyperactivity disorders.

Dynamic Changes in the Proteome of Early Bovine Embryos Developed In Vivo.

Early embryo development is a dynamic process involving important molecular and structural changes leading to the embryonic genome activation (EGA) and early cell lineage differentiation. Our aim was to elucidate proteomic changes in bovine embryos developed in vivo. Eleven females were used as embryo donors and pools of embryos at the 4–6 cell, 8–12 cell, morula, compact morula and blastocyst stages were analyzed by nanoliquid chromatography coupled with label free quantitative mass spectrometry. A total of 2,757 proteins were identified, of which 1,950 were quantitatively analyzed. Principal component analysis of data showed a clear separation of embryo pools according to their developmental stage. The hierarchical clustering of differentially abundant proteins evidenced a first cluster of 626 proteins that increased in abundance during development and a second cluster of 400 proteins that decreased in abundance during development, with most significant changes at the time of EGA and blastocyst formation. The main pathways and processes overrepresented among upregulated proteins were RNA metabolism, protein translation and ribosome biogenesis, whereas Golgi vesicle transport and protein processing in endoplasmic reticulum were overrepresented among downregulated proteins. The pairwise comparison between stages allowed us to identify specific protein interaction networks and metabolic pathways at the time of EGA, morula compaction and blastocyst formation. This is the first comprehensive study of proteome dynamics in non-rodent mammalian embryos developed in vivo. These data provide a number of protein candidates that will be useful for further mechanistic and functional studies.

A GDPase/UDPase bifunctional enzyme from Candida albicans: purification and biochemical characterization.

The most frequently isolated human fungal pathogen is Candida albicans which is responsible for about 50% of all Candida infections. In healthy individuals, this organism resides as a part of the normal microbiota in equilibrium with the host. However, under certain conditions, particularly in immunocompromised patients, this opportunistic pathogen adheres to host cells causing serious systemic infections. Thus, much effort has been dedicated to the study of its physiology with emphasis on factors associated to pathogenicity. A representative analysis deals with the mechanisms of glycoprotein assembly as many cell surface antigens and other macromolecules that modulate the immune system fall within this chemical category. In this regard, studies of the terminal protein glycosylation stage which occurs in Golgi vesicles has led to the identification of nucleotidases that convert glycosyltransferase-generated dinucleotides into the corresponding mononucleotides, thus playing a double function: their activity prevent inhibition of further glycosyl transfer by the accumulation of dinucleotides and the resulting mononucleotides are exchanged by specific membrane transporters for equimolecular amounts of sugar donors from the cytosol. Here, using a simple protocol for protein separation we isolated a bifunctional nucleotidase from C. albicans active on GDP and UDP that was characterized in terms of its molecular mass, response to bivalent ions and other factors, substrate specificity and affinity. Results are discussed in terms of the similarities and differences of this nucleotidase with similar counterparts from other organisms thus contributing to the knowledge of a bifunctional diphosphatase not described before in C. albicans.

E-Cadherin Modulation and Inter-Cellular Trafficking in Tubular Gastric Adenocarcinoma: A High-Resolution Microscopy Pilot Study.

Despite the numerous advances in tumor molecular biology and chemotherapy options, gastric adenocarcinoma is still the most frequent form of gastric cancer. One of the core proteins that regulates inter-cellular adhesion, E-cadherin plays important roles in tumorigenesis as well as in tumor progression; however, the exact expression changes and modulation that occur in gastric cancer are not yet fully understood. In an attempt to estimate if the synthesis/degradation balance matches the final membrane expression of this adhesion molecule in cancer tissue, we assessed the proportion of E-cadherin that is found in the Golgi vesicles as well as in the lysosomal pathway We utilized archived tissue fragments from 18 patients with well and poorly differentiated intestinal types of gastric cancer and 5 samples of normal gastric mucosa, by using high-magnification multispectral microscopy and high-resolution fluorescence deconvolution microscopy. Our data showed that E-cadherin is not only expressed in the membrane, but also in the cytoplasm of normal and tumor gastric epithelia. E-cadherin colocalization with the Golgian vesicles seemed to be increasing with less differentiated tumors, while co-localization with the lysosomal system decreased in tumor tissue; however, the membrane expression of the adhesion molecule clearly dropped from well to poorly differentiated tumors. Thus E-cadherin seems to be more abundantly synthetized than eliminated via lysosomes/exosomes in less differentiated tumors, suggesting that post-translational modifications, such as cleavage, conformational inactivation, or exocytosis, are responsible for the net drop of E-cadherin at the level of the membrane in more anaplastic tumors. This behavior is in perfect accordance with the concept of partial epithelial-to-mesenchymal transition (P-EMT), when the E-cadherin expression of tumor cells is in fact not downregulated but redistributed away from the membrane in recycling vesicles. Moreover, our high-resolution deconvolution microscopy study showed for the first time, at the tissue level, the presence of Lysosome-associated membrane glycoprotein 1 (LAMP1)-positive exosomes/multivesicular bodies being trafficked across the membranes of tumor epithelial cells. Altogether, a myriad of putative modulatory pathways is available as a treatment turning point, even if we are to only consider the metabolism of membrane E-cadherin regulation. Future super-resolution microscopy studies are needed to clarify the extent of lysosome/exosome exchange between tumor cells and with the surrounding stroma, in histopathology samples or even in vivo.

Multi-Omics Analyses Revealed GOLT1B as a Potential Prognostic Gene in Breast Cancer Probably Regulating the Immune Microenvironment.

As recently reported by The International Agency for Research on Cancer (IARC), breast cancer has the highest incidence of all cancers in 2020. Many studies have revealed that golgi apparatus is closely associated with the development of breast cancer. However, the role of golgi apparatus in immune microenvironment is still not clear. In this study, using RNA-Seq datasets of breast cancer patients from the public database (n = 1080), we revealed that GOLT1B, encoding a golgi vesicle transporter protein, was significantly higher expressed in human breast cancer tissues versus normal tissues. Besides, we verified GOLT1B expression in five breast cancer cell line using our original data and found GOLT1B was significantly up-regulated in MDA-MB-231, MCF-7, SKBR3. Subsequently, we identified GOLT1B as a potential independent prognostic factor for breast cancer. After a multi-omics analysis, we uncovered that the higher expression of GOLT1B in breast cancer tissues versus normal tissues might be due to the amplification of GOLT1B and altered phosphorylation of its potential transcriptional factors, including JUN and SIN3A. Subsequently, we discovered that GOLT1B potentially regulated the immune microenvironment basing on the finding that its expression was closely related to the tumor microenvironment score and infiltration of immune cells. Moreover, we revealed that GOLT1B might affect the overall survival rates of breast cancer through regulating the immune cell infiltration. Finally, we disclosed the potential pathways involved in the functions of GOLT1B in breast cancer, including metabolism and ECM-receptor interaction pathways. To sum up, we identified GOLT1B as a potential prognostic gene for breast cancer and disclosed its role in regulating the immune microenvironment.

Linking GOLPH3 and Extracellular Vesicles Content-a Potential New Route in Cancer Physiopathology and a Promising Therapeutic Target is in Sight?

Golgi phosphoprotein 3 (GOLPH3), a highly conserved phosphatidylinositol 4-phosphate effector, is required for maintenance of Golgi architecture, vesicle trafficking, and Golgi glycosylation. GOLPH3 overexpression has been reported in several human solid cancers, including glioblastoma, breast cancer, colorectal cancer, nonsmall cell lung cancer, epithelial ovarian cancer, prostate cancer, gastric cancer, and hepatocellular carcinoma. Although the molecular mechanisms that link GOLPH3 to tumorigenesis require further investigation, it is likely that GOLPH3 may act by controlling the intracellular movement of key oncogenic molecules, between the Golgi compartments and/or between the Golgi and the endoplasmic reticulum. Indeed, numerous evidence indicates that deregulation of intracellular vesicle trafficking contributes to several aspects of cancer phenotypes. However, a direct and clear link between extracellular vesicle movements and GOLPH3 is still missing. In the past years several lines of evidence have implicated GOLPH3 in the regulation of extracellular vesicle content. Specifically, a new role for GOLPH3 has emerged in controlling the internalization of exosomes containing either oncogenic proteins or noncoding RNAs, especially micro-RNA. Although far from being elucidated, growing evidence indicates that GOLPH3 does not increase quantitatively the excretion of exosomes, but rather regulates the exosome content. In particular, recent data support a role for GOLPH3 for loading specific oncogenic molecules into the exosomes, driving both tumor malignancy and metastasis formation. Additionally, the older literature indirectly implicates GOLPH3 in cancerogenesis through its function in controlling hepatitis C virus secretion, which in turn is linked to hepatocellular carcinoma formation. Thus, GOLPH3 might promote tumorigenesis in unexpected ways, involving both direct and indirect routes. If these data are further confirmed, the spectrum of action of GOLPH3 in tumor formation will significantly expand, indicating this protein as a strong candidate for targeted cancer therapy.

Rapid Isolation of Functional Synaptic Vesicles from Tissues Through Cryogrinding, Ultracentrifugation, and Size Exclusion Chromatography.

Many biochemical and biophysical related questions require the isolation of functional synaptic vesicles. Isolated synaptic vesicles can be used for transporter kinetics studies, synaptic vesicle content analysis and immuno-labeling of specific synaptic vesicle proteins, etc. Here I describe a fast and reliable isolation procedure to allow researchers to isolate a large amount, as well as physiologically functional synaptic vesicles, by following the subsequent order of cryogrinding, gradient ultracentrifugation, and size exclusion liquid chromatography. This process enriches over 90% of the synaptic vesicle population, with low contamination of Golgi or endoplasmic reticulum vesicles.

Lighting up Individual Organelles With Fluorescent Carbon Dots.

Cell organelles play crucial roles in the normal functioning of an organism, therefore the disruption of their operation is associated with diseases and in some cases death. Thus, the detection and monitoring of the activities within these organelles are of great importance. Several probes based on graphene oxide, small molecules, and other nanomaterials have been developed for targeting specific organelles. Among these materials, organelle-targeted fluorescent probes based on carbon dots have attracted substantial attention in recent years owing to their superior characteristics, which include facile synthesis, good photostability, low cytotoxicity, and high selectivity. The ability of these probes to target specific organelles enables researchers to obtain valuable information for understanding the processes involved in their functions and/or malfunctions and may also aid in effective targeted drug delivery. This review highlights recently reported organelle-specific fluorescent probes based on carbon dots. The precursors of these carbon dots are also discussed because studies have shown that many of the intrinsic properties of these probes originate from the precursor used. An overview of the functions of the discussed organelles, the types of probes used, and their advantages and limitations are also provided. Organelles such as the mitochondria, nucleus, lysosomes, and endoplasmic reticulum have been the central focus of research to date, whereas the Golgi body, centrosome, vesicles, and others have received comparatively little attention. It is therefore the hope of the authors that further studies will be conducted in an effort to design probes with the ability to localize within these less studied organelles so as to fully elucidate the mechanisms underlying their function.