Intracellular Localization Of Protein Research Articles

Identification and in silico analysis of PHANTASTICA gene in Saintpaulia ionantha H. Wendl (Gesneriaceae)

Saintpaulia ionantha H. Wendl (synonym Streptocarpus ionanthus) has been considered an ornamental plant. The PHANTASTICA (PHAN) gene has a significant role in the formation of plant organs adaxial?abaxial polarity. In the present study the PHAN gene was identified in Saintpaulia for the first time using PCR. To determine the expression pattern of the PHAN gene, gene expression was compared at 3 developmental stages using real time PCR. For in silico analyses, protein characteristics, secondary and 3D structures of protein, intracellular localization, and the phylogenetic tree were investigated using bioinformatics tools. The partial CDS of PHAN gene contained 756 nucleotides, a predicted protein encoded with 252 amino acids with a molecular weight of 28,474 Da. The presence of the HTH MYB-type motif in this sequence indicated that the PHAN protein belonged to the MYB family. The secondary structure of the PHAN protein was composed of 45% ?-helix. The predicted PHAN protein 3D structure revealed that this protein can bind to nucleic acids. The binding site sequence included amino acids 10(R), 12(W), 13(H), 66(W), and 67(K). The intracellular localization of PHAN protein is believed to be in the nucleus. Phylogenetic analysis revealed that species belonging to each family were grouped in same clade. Gene expression revealed that in the later stages of petal development, expression was reduced. The results suggest that the expression pattern of the PHAN gene is that of adaxial?abaxial symmetry initiation during petal development as well as in the leaves of Saintpaulia. It appears that genetic engineering using biotechnology would be beneficial for improving the ornamental value of Saintpaulia.

Rapid Isolation of Rare Isotype-Switched Hybridoma Variants: Application to the Generation of IgG2a and IgG2b MAb to CD63, a Late Endosome and Exosome Marker.

CD63, a member of the tetraspanin superfamily, is used as a marker of late endosomes and lysosome-related organelles, as well as a marker of exosomes. Here, we selected rare isotype variants of TS63 by sorting hybridoma cells on the basis of their high expression of surface immunoglobulins of the IgG2a and IgG2b subclass. Pure populations of cells secreting IgG2a and IgG2b variants of TS63 (referred to as TS63a and TS63b) were obtained using two rounds of cell sorting and one limited dilution cloning step. We validate that these new TS63 variants are suitable for co-labeling with mAb of the IgG1 subclass directed to other molecules, using anti mouse subclass antibodies, and for the labeling of exosomes through direct binding to protein A-coated gold particles. These mAbs will be useful to study the intracellular localization of various proteins and facilitate electron microscopy analysis of CD63 localization.

Autophagy regulates the localization and degradation of p16INK4a.

The tumor suppressor protein p16INK4a (p16) is a well‐established hallmark of aging that induces cellular senescence in response to stress. Previous studies have focused primarily on p16 regulation at the transcriptional level; comparatively little is known about the protein's intracellular localization and degradation. The autophagy–lysosomal pathway has been implicated in the subcellular trafficking and turnover of various stress‐response proteins and has also been shown to attenuate age‐related pathologies, but it is unclear whether p16 is involved in this pathway. Here, we investigate the role of autophagy, vesicular trafficking, and lysosomal degradation on p16 expression and localization in human epithelial cells. Time‐lapse fluorescence microscopy using an endogenous p16‐mCherry reporter revealed that serum starvation, etoposide, and hydrogen peroxide stimulate autophagy and drive p16 recruitment to acidic cytoplasmic vesicles within 4 hr. Blocking lysosomal proteases with leupeptin and ammonium chloride resulted in the accumulation of p16 within lysosomes and increased total p16 levels suggesting that p16 is degraded by this pathway. Furthermore, autophagy blockers chloroquine and bafilomycin A1 caused p16 aggregation within stalled vesicles containing autophagosome marker LC3. Increase of p16 within these vesicles coincided with the accumulation of LC3‐II. Knockdown of autophagosome chaperone p62 attenuated the formation of p16 aggregates in lysosomes, suggesting that p16 is targeted to these vesicles by p62. Taken together, these results implicate the autophagy pathway as a novel regulator of p16 degradation and localization, which could play a role in the etiology of cancer and age‐related diseases.

The nuclear receptor NR4A1 is regulated by SUMO modification to induce autophagic cell death.

NR4A is a nuclear receptor protein family whose members act as sensors of cellular environment and regulate multiple processes such as metabolism, proliferation, migration, apoptosis, and autophagy. Since the ligand binding domains of these receptors have no cavity for ligand interaction, their function is most likely regulated by protein abundance and post-translational modifications. In particular, NR4A1 is regulated by protein abundance, phosphorylation, and subcellular distribution (nuclear-cytoplasmic translocation), and acts both as a transcription factor and as a regulator of other interacting proteins. SUMOylation is a post-translational modification that can affect protein stability, transcriptional activity, alter protein-protein interactions and modify intracellular localization of target proteins. In the present study we evaluated the role of SUMOylation as a posttranslational modification that can regulate the activity of NR4A1 to induce autophagy-dependent cell death. We focused on a model potentially relevant for neuronal cell death and demonstrated that NR4A1 needs to be SUMOylated to induce autophagic cell death. We observed that a triple mutant in SUMOylation sites has reduced SUMOylation, increased transcriptional activity, altered intracellular distribution, and more importantly, its ability to induce autophagic cell death is impaired.

Mitochondrial fission is necessary for mitophagy, development and virulence of the insect pathogenic fungus Beauveria bassiana.

Mitochondrial fission is an essential dynamics that maintains mitochondrial morphology and function. This study seeks to determine the roles of mitochondrial fission in the filamentous entomopathogenic fungus Beauveria bassiana. Three fission-related genes (BbFis1, BbMdv1 and BbDnm1) were functionally characterized via protein intracellular localization and construction of gene disruption mutants. Mitochondrial localization was only observed for BbFis1 which interacts with BbMdv1, but BbMdv1 did not have interaction with BbDnm1. Single disruption mutant of three genes generated the elongated and enlarged mitochondria which could not be eliminated via the mitophagy. Three mutant strains displayed the reduced ATP synthesis and vegetative growth compared with the wild type. Three genes were involved in the early stage of conidiation and unnecessary for the late stage. However, all three genes significantly contribute to blastospore development under submerged condition, and the loss of BbMdv1 had the greatest effects compared with the losses of BbFis1 or BbDnm1. Finally, disruption of three genes significantly attenuated fungal virulence, but their mutations had different influences. In addition to their consistent roles in mitochondrial division and mitophagy, three fission-related genes perform divergent roles in the development and virulence of the entomopathogenic fungus B. bassiana. This study shows that mitochondrial fission is associated with lifecycle of B. bassiana. These findings provide information for the manipulation of fungal physiology and facilitate the application of entomopathogenic fungi.

The Effects of Histone Deacetylase (HDAC) Inhibitors on FASN Intracellular Localization in Cancer Cells

Understanding mechanisms underlying cancer biology is crucial for discovering novel and effective therapies to improve patient outcome. Increased lipid production is a major metabolic feature in cancer. The fatty acid synthase (FASN) is a key enzyme for lipid synthesis and is upregulated in cancer. Although fatty acid synthesis is generally thought to take place in the cytoplasm, it has been reported that this enzyme also localizes to the nucleus in cancer cells. We hypothesize that the intracellular localization FASN could be a potential target to decrease lipid synthesis and ultimately halt cell proliferation. Protein acetylation has been shown to regulate protein intracellular localization. We aim to assess the impact histone deacetylase inhibitors (HDACi) have on the intracellular location of FASN with the goal of reducing de novo lipid production and cancer cell proliferation. We have examined intracellular localization of FASN in cells using immunofluorescence microscopy in cancer cells treated with HDACi and did not detect obvious HDACi-induced changes in FASN localization. FASN is also regulated by other mechanisms such as phosphorylation. Future studies will examine effects of kinase inhibitors on FASN intracellular localization.

SnapShot: Lysosomal Storage Diseases

Lysosomal storage diseases (LSDs) represent a group of monogenic inherited metabolic disorders characterized by the progressive accumulation of undegraded substrates inside lysosomes, resulting in aberrant lysosomal activity and homeostasis. This SnapShot summarizes the intracellular localization and function of proteins implicated in LSDs. Common aspects of LSD pathogenesis and the major current therapeutic approaches are noted. To view this SnapShot, open or download the PDF.

Rab GTPases: Emerging Oncogenes and Tumor Suppressive Regulators for the Editing of Survival Pathways in Cancer.

The Rab GTPase family of proteins are mediators of membrane trafficking, conferring identity to the cell membranes. Recently, Rab and Rab-associated factors have been recognized as major regulators of the intracellular positioning and activity of signaling pathways regulating cell growth, survival and programmed cell death or apoptosis. Membrane trafficking mediated by Rab proteins is controlled by intracellular localization of Rab proteins, Rab-membrane interactions and GTP-activation processes. Aberrant expression of Rab proteins has been reported in multiple cancers such as lung, brain and breast malignancies. Mutations in Rab-coding genes and/or post-translational modifications in their protein products disrupt the cellular vesicle trafficking network modulating tumorigenic potential, cellular migration and metastatic behavior. Conversely, Rabs also act as tumor suppressive factors inducing apoptosis and inhibiting angiogenesis. Deconstructing the signaling mechanisms modulated by Rab proteins during apoptosis could unveil underlying molecular mechanisms that may be exploited therapeutically to selectively target malignant cells.

Septins, a cytoskeletal protein family, with emerging role in striated muscle

Appropriate organization of cytoskeletal components are required for normal distribution and intracellular localization of different ion channels and proteins involved in calcium homeostasis, signal transduction, and contractile function of striated muscle. Proteins of the contractile system are in direct or indirect connection with the extrasarcomeric cytoskeleton. A number of other molecules which have essential role in regulating stretch-, voltage-, and chemical signal transduction from the surface into the cytoplasm or other intracellular compartments are already well characterized. Sarcomere, the basic contractile unit, is comprised of a precisely organized system of thin (actin), and thick (myosin) filaments. Intermediate filaments connect the sarcomeres and other organelles (mitochondria and nucleus), and are responsible for the cellular integrity. Interacting proteins have a very diverse function in coupling of the intracellular assembly components and regulating the normal physiological function. Despite the more and more intense investigations of a new cytoskeletal protein family, the septins, only limited information is available regarding their expression and role in striated, especially in skeletal muscles. In this review we collected basic and specified knowledge regarding this protein group and emphasize the importance of this emerging field in skeletal muscle biology.

Monitoring of intracellular localization of Hepatitis B virus P22 protein using Laser Scanning Confocal Microscopy and Airyscan.

The aim of this study was to assess nucleo-cytoplasmic protein localization to better understand the exact intracellular localization of viral proteins involved with infections. Having determined the general protein localization of hepatitis B virus P22 precore protein, the aim was to more specifically resolve its intracellular organization. This was done using both laser scanning microscopy and Airyscan techniques. Using a 63× objective, the resolution obtained with Airyscan was increased by 1.5-fold as compared to confocal microscopy (p value <.00001).

Protein expression of prenyltransferase subunits in postmortem schizophrenia dorsolateral prefrontal cortex

The pathophysiology of schizophrenia includes altered neurotransmission, dysregulated intracellular signaling pathway activity, and abnormal dendritic morphology that contribute to deficits of synaptic plasticity in the disorder. These processes all require dynamic protein–protein interactions at cell membranes. Lipid modifications target proteins to membranes by increasing substrate hydrophobicity by the addition of a fatty acid or isoprenyl moiety, and recent evidence suggests that dysregulated posttranslational lipid modifications may play a role in multiple neuropsychiatric disorders, including schizophrenia. Consistent with these emerging findings, we have recently reported decreased protein S-palmitoylation in schizophrenia. Protein prenylation is a lipid modification that occurs upstream of S-palmitoylation on many protein substrates, facilitating membrane localization and activity of key intracellular signaling proteins. Accordingly, we hypothesized that, in addition to palmitoylation, protein prenylation may be abnormal in schizophrenia. To test this, we assayed protein expression of the five prenyltransferase subunits (FNTA, FNTB, PGGT1B, RABGGTA, and RABGGTB) in postmortem dorsolateral prefrontal cortex from patients with schizophrenia and paired comparison subjects (n = 13 pairs). We found decreased levels of FNTA (14%), PGGT1B (13%), and RABGGTB (8%) in schizophrenia. To determine whether upstream or downstream factors may be driving these changes, we also assayed protein expression of the isoprenoid synthases FDPS and GGPS1 and prenylation-dependent processing enzymes RCE and ICMT. We found these upstream and downstream enzymes to have normal protein expression. To rule out effects from chronic antipsychotic treatment, we assayed FNTA, PGGT1B, and RABGGTB in the cortex from rats treated long-term with haloperidol decanoate and found no change in the expression of these proteins. Given the role prenylation plays in localization of key signaling proteins found at the synapse, these data offer a potential mechanism underlying abnormal protein–protein interactions and protein localization in schizophrenia.

Different localization of lysosomal-associated membrane protein 1 (LAMP1) in mammalian cultured cell lines.

Lysosomal-associated membrane protein 1 (LAMP1) mainly localizes to lysosomes and late endosomes. We herein investigated the intracellular localization of lysosomal membrane proteins in five mammalian cultured cell lines. Rat LAMP1 fused to enhanced green fluorescent protein (EGFP) mostly accumulated at a particular cytoplasmic area and barely co-localized with LysoTracker® Red DND-99 in golden hamster kidney BHK-21 cells and Chinese hamster ovary CHO-K1 cells. Golden hamster, Chinese hamster, and human LAMP1-EGFP showed a similar intracellular distribution to rat LAMP1-EGFP in BHK-21 cells. Endogenous LAMP1 was also detected in a perinuclear area in BHK-21 cells and CHO-K1 cells, and co-localized with rat CD63-EGFP in BHK-21 cells. Moreover, rat LAMP1-DsRed-Monomer co-localized well with the human trans-Golgi network protein 2-EGFP in BHK-21 cells. These results reveal that LAMP1 predominantly localizes to the trans-Golgi network in BHK-21 cells.

FAM20A is essential for amelogenesis, but is dispensable for dentinogenesis.

Mutations in the gene encoding family with sequence similarity 20, member A (FAM20A) caused amelogenesis imperfecta (AI), in humans. However, the roles of FAM20A in amelogenesis and dentinogenesis are poorly understood. In this study, we generated a Fam20a knockout (Sox2-Cre;Fam20afl/fl) mouse model by crossing Fam20afl/fl mice with Sox2-Cre transgenic mice, in which Fam20a was ablated in both dental epithelium and dental mesenchyme. We found that these mice developed an enamel phenotype that resembles human AI associated with FAM20A mutations, but did not have apparent dentin defects. The secretory stage ameloblasts in the mandibular incisors from the Sox2-Cre;Fam20afl/fl mice were shorter and detached from the enamel matrix, and subsequently lost their polarity, became disorganized and formed numerous spherical extracellular matrices in place of normal enamel. At the molecular level, the Sox2-Cre;Fam20afl/fl mice displayed dramatically reduced expression levels of the genes encoding the enamel matrix proteins, but unaltered levels of the genes encoding the dentin matrix proteins. Moreover, Fam20a ablation resulted in a great decrease in FAM20C protein level, but it did not alter the intracellular localization of FAM20C protein in ameloblasts and odontoblasts. These results indicate that FAM20A is essential for amelogenesis, but is dispensable for dentinogenesis.

Geleophysic dysplasia: novel missense variants and insights into ADAMTSL2 intracellular trafficking

Geleophysic dysplasia (GPHYSD1, MIM231050; GPHYSD2, MIM614185; GPHYSD3, MIM617809) is an autosomal disorder characterized by short-limb dwarfism, brachydactyly, cardiac valvular disease, and laryngotracheal stenosis. Mutations in ADAMTSL2, FBN1, and LTBP3 genes are responsible for this condition. We found that three previously described cases of GPHYSD diagnosed clinically were homozygote or compound heterozygotes for five ADAMTSL2 variants, four of which not being previously reported. By electron microscopy, skin fibroblasts available in one case homozygote for an ADAMTSL2 variant showed a defective intracellular localization of mutant ADAMTSL2 protein that did not accumulate within lysosome-like intra-cytoplasmic inclusions. Moreover, this mutant ADAMTSL2 protein was less secreted in medium and resulted in increased SMAD2 phosphorylation in transfected HEK293 cells.

Observation of intracellular protein localization area in a single neuron using gold nanoparticles with a scanning electron microscope

The localization areas of intracellular proteins in rat cortical neurons were visualized using a scanning electron microscope (SEM) coupled with a focused ion beam (FIB) system. To obtain a clear contrast in the SEM images, gold nanoparticles (GNPs) were bound to specific intracellular proteins by antigen-antibody reactions. By obtaining a cross section of the desired location of the neurons by FIB milling under the SEM imaging condition, it was possible to observe the proteins inside the cells as clear bright spots. When a neuron was stained with anti-tau and anti-histone H1 antibodies, the bright spots were localized in the cross section of the axon and the nucleus, respectively. It was confirmed that targeted proteins in a single neuron on a substrate could be successfully identified. The development of FIB/SEM observation with immunological GNP staining will offer important information for the stable growth of neurons on various substrate structures, since the elongation and turning of axons on the substrates are activated by the redistribution of intracellular proteins.

Mechanical stress alters protein O-GlcNAc in human periodontal ligament cells.

Protein O‐linked N‐acetylglucosamine (O‐GlcNAc) is a post‐translational modification of intracellular proteins that regulates several physiological and pathophysiological process, including response to various stressors. However, O‐GlcNAc's response to mechanical stress has not been investigated yet. As human periodontal ligament (PDL) cells are stimulated by compression force during orthodontic tooth movement that results in structural remodelling, in this study we investigated whether mechanical stress induces any alteration in protein O‐GlcNAc in PDL cells. In this study, PDL cells isolated from premolars extracted for orthodontic indications were exposed to 0, 1.5, 3, 7 and 14 g/cm2 compression forces for 12 hours. Cell viability was measured by flow cytometry, and protein O‐GlcNAc was analysed by Western blot. Cellular structure and intracellular distribution of O‐GlcNAc was studied by immunofluorescence microscopy. We found that between 1.5 and 3 g/cm2 mechanical compression, O‐GlcNAc significantly elevated; however, at higher forces O‐GlcNAc level was not increased. We also found that intracellular localization of O‐GlcNAc proteins became more centralized under 2 g/cm2 compression force. Our results suggest that structural changes stimulated by compression forces have a significant effect on the regulation of O‐GlcNAc; thus, it might play a role in the mechanical stress adaptation of PDL cells.

Alternative splicing induces cytoplasmic localization of RBFOX2 protein in calcific tendinopathy

BackgroundCalcific tendinopathy (CT) is characterized by deposits of calcium, most commonly found in the shoulder tendons. The exact cause and pathogenesis of CT are not fully understood. This study analyzed the expression pattern of RNA-binding protein fox-1 homolog 2 (RBFOX2), a crucial splicing regulator in tissue differentiation. MethodsNormal and calcific tendons were compared for RBFOX2 mRNA level using quantitative reverse-transcription polymerase chain reaction. Intracellular localization of RBFOX2 protein was investigated using immunofluorescence microscopy. Normal and calcific tendon cDNAs were used to clone RBFOX2. Sequencing analysis identified coding sequences of the RBFOX2 isoform. ResultsThe intracellular localization of RBFOX2 protein differed with disease status, with RBFOX2 localized in the cytoplasm in calcific tendons and the nucleus in normal tendons. Analysis of the RBFOX2 protein-coding sequence showed that exon 10, responsible for nuclear localization, was absent in calcific tendons. Splicing of RBFOX2 target genes CHD2 and MBNL1 was significantly affected by cytoplasmic localization of RBFOX2 in calcific tendons. DiscussionGiven the function of RBFOX2 as a splicing regulator in the nucleus, cytoplasmic localization of RBFOX2 protein in calcific tendons may have affected overall splicing events and altered gene expression. These results provide insights for comprehension of CT pathogenesis.

Intracellular localization of vitellogenin receptor mRNA and protein during oogenesis of a parthenogenetic tick, Haemaphysalis longicornis

BackgroundVitellogenin (Vg), a key molecule for oocyte development synthesized in the fat body during blood-feeding, is released into the hemolymph and then taken into the oocytes via Vg receptor (VgR) in ticks. Previously, we showed that VgR mRNA is expressed in the ovary at the adult stage of parthenogenetic Haemaphysalis longicornis ticks and its expression increases after blood-feeding. However, intracellular localization of VgR mRNA and protein at each developmental stage of oocytes during oogenesis remains largely unclear.MethodsmRNA and protein expression profiles of H. longicornis VgR (HlVgR) in the oocytes from the unfed to oviposition periods were analyzed by real-time PCR, in situ hybridization, and immunostaining. To elucidate the timing of the onset of Vg uptake, RNA interference (RNAi)-mediated gene silencing of HlVgR was performed.ResultsIn situ hybridization revealed that HlVgR mRNA was detected in the cytoplasm of stage I-III oocytes, and weaker positive signals for HlVgR mRNA were found in the cell periphery of stage IV and V oocytes. Likewise, HlVgR protein was detected by immunostaining in the cytoplasm of stage I-III oocytes and in the cell periphery of stage IV and V oocytes. Each developmental stage of the oocytes showed distinct patterns of mRNA and protein expression of HlVgR. Moreover, RNAi of HlVgR caused delayed or arrested development in the oocytes. The ovaries of control ticks showed all developmental stages of oocytes, whereas stage I-III oocytes were found in the ovaries of HlVgR-RNAi ticks at 5 days after engorgement.ConclusionsThese results suggest that active uptake of Vg is required for development from stage III to stage IV during oogenesis. Our data clearly revealed an apparent shift in the intracellular localization of VgR for both mRNA and protein level in oocytes during oogenesis.

Expression and intracellular localization of Nanos2-homologue protein in primordial germ cells and spermatogonial stem cells.

SummaryThe decision by germ cells to differentiate and undergo either oogenesis or spermatogenesis takes place during embryonic development and Nanos plays an important role in this process. The present study was designed to investigate the expression patterns in rat of Nanos2-homologue protein in primordial germ cells (PGCs) over different embryonic developmental days as well as in spermatogonial stem cells (SSCs). Embryos from three different embryonic days (E8.5, E10.5, E11.5) and SSCs were isolated and used to detect Nanos2-homologue protein using immunocytochemistry, western blotting, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Interestingly, Nanos2 expression was detected in PGCs at day E11.5 onwards and up to colonization of PGCs in the genital ridge of fetal gonads. No Nanos2 expression was found in PGCs during early embryonic days (E8.5 and 10.5). Furthermore, immunohistochemical and immunofluorescence data revealed that Nanos2 expression was restricted within a subpopulation of undifferentiated spermatogonia (As, single type A SSCs and Apr, paired type A SSCs). The same results were confirmed by our western blot and RT-PCR data, as Nanos2 protein and transcripts were detected only in PGCs from day E11.5 and in undifferentiated spermatogonia (As and Apr). Furthermore, Nanos2-positive cells were also immunodetected and sorted using flow cytometry from the THY1-positive SSCs population, and this strengthened the idea that these cells are stem cells. Our findings suggested that stage-specific expression of Nanos2 occurred on different embryonic developmental days, while during the postnatal period Nanos2 expression is restricted to As and Apr SSCs.

Opto-magnetic Selection and Isolation of Single Cells.

Capturing single cells from large heterogenous populations based solely on observable traits is necessary for many cell biology applications and remains a major technical challenge. The protocol we present allows the isolation of viable and metabolically active cells selected for their shape, migration speed, contact to other cells, or intracellular protein localization. We previously introduced a method termed Cell Labeling via Photobleaching (CLaP) for the efficient tagging of cells chosen for visual criteria. Here we describe a new protocol for capturing such cells using ferromagnetic beads termed single-cell magneto-optical capture (scMOCa). This technology is especially useful when the number of target cells represents an extremely low fraction of the total population (potentially one single cell), a situation in which conventional sorting techniques like fluorescent or magnetic activated cell sorting (F/MACS) cannot provide satisfactory results in terms of capture efficiency and specificity. scMOCa uses the lasers of a confocal microscope to photobleach and crosslink biotin-4-fluorecein molecules to cell membranes. Streptavidin coated magnetic beads then adhere to biotin moieties and a magnet allows the capture of illuminated cells. By precisely controlling liquid volumes and spacing between the different parts of a simple setup, high cell selectivity and capture efficacy can be achieved. scMOCA allows visual selection and isolation of any number of cells in a microscopy field and captured cells remain viable to generate new colonies of chosen phenotypes for downstream analyses.