Intracellular Localization Of Protein Research Articles

Subcellular Localization of Diacylglycerol-responsive Protein Kinase C Isoforms in HeLa Cells

Subcellular localization of protein kinase often plays an important role in determining its activity and specificity. Protein kinase C (PKC), a family of multi-gene protein kinases has long been known to be translocated to the particular cellular compartments in response to DAG or its analog phorbol esters. We used C-terminal green fluorescent protein (GFP) fusion proteins of PKC isoforms to visualize the subcellular distribution of individual PKC isoforms. Intracellular localization of PKC-GFP proteins was monitored by fluorescence microscopy after transient transfection of PKC-GFP expression vectors in the HeLa cells. In unstimulated HeLa cells, all PKC isoforms were found to be distributed throughout the cytoplasm with a few exceptions. PKC<TEX>$\theta$</TEX> was mostly localized to the Golgi, and PKC<TEX>$\gamma$</TEX>, PKC<TEX>$\delta$</TEX> and PKC<TEX>$\eta$</TEX> showed cytoplasmic distribution with Golgi localization. DAG analog TPA induced translocation of PKC-GFP to the plasma membrane. PKC<TEX>$\alpha$</TEX>, PKC<TEX>$\eta$</TEX> and PKC<TEX>$\theta$</TEX> were also localized to the Golgi in response to TPA. Only PKC<TEX>$\delta$</TEX> was found to be associated with the nuclear membrane after transient TPA treatment. These results suggest that specific PKC isoforms are translocated to different intracellular sites and exhibit distinct biological effects.

H3N2 influenza A virus replicates in immortalized human first trimester trophoblast cell lines and induces their rapid apoptosis.

Epidemiological data suggested that pandemic influenza increased the risks of spontaneous abortion and premature labor, while seasonal influenza also increased the risk of schizophrenia in adolescence. However, their pathogenesis is so far unknown. The first trimester trophoblast cell lines, namely, Swan71 and HTR8 cells were challenged with A/Udorn/72 influenza virus (H3N2). At indicated time points, cells were examined for expression of influenza proteins. Viral replication in culture media, apoptosis and the expression of human leukocyte antigen (HLA)-G were also examined. Intracellular localization of viral proteins was observed. Twenty-four hours after inoculation, virus was detected in culture media while most cells fell into apoptosis. During apoptosis, expression of HLA-G was unchanged. We revealed replication of low pathogenic influenza virus in the first trimester trophoblast cell lines. Placental damages are likely to be induced by direct cytopathic effects of influenza virus and subsequent apoptosis rather than down regulation of HLA-G expression and subsequent rejection by maternal immune system.

Overexpression of the downward leaf curling (DLC) gene from melon changes leaf morphology by controlling cell size and shape in Arabidopsis leaves.

A plant-specific gene was cloned from melon fruit. This gene was named downward leaf curling (CmDLC) based on the phenotype of transgenic Arabidopsis plants overexpressing the gene. This expression level of this gene was especially upregulated during melon fruit enlargement. Overexpression of CmDLC in Arabidopsis resulted in dwarfism and narrow, epinastically curled leaves. These phenotypes were found to be caused by a reduction in cell number and cell size on the adaxial and abaxial sides of the epidermis, with a greater reduction on the abaxial side of the leaves. These phenotypic characteristics, combined with the more wavy morphology of epidermal cells in overexpression lines, indicate that CmDLC overexpression affects cell elongation and cell morphology. To investigate intracellular protein localization, a CmDLC-GFP fusion protein was made and expressed in onion epidermal cells. This protein was observed to be preferentially localized close to the cell membrane. Thus, we report here a new plant-specific gene that is localized to the cell membrane and that controls leaf cell number, size and morphology.

ANKRD1, the Gene Encoding Cardiac Ankyrin Repeat Protein, Is a Novel Dilated Cardiomyopathy Gene

We evaluated ankyrin repeat domain 1 (ANKRD1), the gene encoding cardiac ankyrin repeat protein (CARP), as a novel candidate gene for dilated cardiomyopathy (DCM) through mutation analysis of a cohort of familial or idiopathic DCM patients, based on the hypothesis that inherited dysfunction of mechanical stretch-based signaling is present in a subset of DCM patients. CARP, a transcription coinhibitor, is a member of the titin-N2A mechanosensory complex and translocates to the nucleus in response to stretch. It is up-regulated in cardiac failure and hypertrophy and represses expression of sarcomeric proteins. Its overexpression results in contractile dysfunction. In all, 208 DCM patients were screened for mutations/variants in the coding region of ANKRD1 using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct deoxyribonucleic acid sequencing. In vitro functional analyses of the mutation were performed using yeast 2-hybrid assays and investigating the effect on stretch-mediated gene expression in myoblastoid cell lines using quantitative real-time reverse transcription-polymerase chain reaction. Three missense heterozygous ANKRD1 mutations (P105S, V107L, and M184I) were identified in 4 DCM patients. The M184I mutation results in loss of CARP binding with Talin 1 and FHL2, and the P105S mutation in loss of Talin 1 binding. Intracellular localization of mutant CARP proteins is not altered. The mutations result in differential stretch-induced gene expression compared with wild-type CARP. ANKRD1 is a novel DCM gene, with mutations present in 1.9% of DCM patients. The ANKRD1 mutations may cause DCM as a result of disruption of the normal cardiac stretch-based signaling.

Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta

Here, we report on the construction of a novel series of Gateway-compatible plant transformation vectors containing genes encoding autofluorescent proteins, including Cerulean, Dendra2, DRONPA, TagRFP and Venus, for the expression of protein fusions in plant cells. To assist users in the selection of vectors, we have determined the relative in planta photostability and brightness of nine autofluorescent proteins (AFPs), and have compared the use of DRONPA and Dendra2 in photoactivation and photoconversion experiments. Additionally, we have generated transgenic Nicotiana benthamiana lines that express fluorescent protein markers targeted to nuclei, endoplasmic reticulum or actin filaments. We show that conducting bimolecular fluorescence complementation assays in plants that constitutively express cyan fluorescent protein fused to histone 2B provides enhanced data quality and content over assays conducted without the benefit of a subcellular marker. In addition to testing protein interactions, we demonstrate that our transgenic lines that express red fluorescent protein markers offer exceptional support in experiments aimed at defining nuclear or endomembrane localization. Taken together, the new combination of pSITE-BiFC and pSITEII vectors for studying intracellular protein interaction, localization and movement, in conjunction with our transgenic marker lines, constitute powerful tools for the plant biology community.

Molecular Determinants for Subcellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Open Reading Frame 3b Protein

Viruses such as hepatitis C and the severe acute respiratory syndrome coronavirus (SARS-CoV) encode proteins that are distributed between mitochondria and the nucleus, but little is known about the factors that control partitioning between these sites. SARS-CoV encodes a unique accessory gene called open reading frame (ORF) 3b that, like other unique accessory genes in SARS-CoV, likely contributes to viral pathogenicity. The ORF 3b protein is 154 amino acids and is predicted to express from the second ORF in subgenomic RNA3. In this report, we have characterized the molecular components that regulate intracellular localization of the ORF 3b protein. We demonstrate unique shuttling behavior of ORF 3b, whereby the protein initially accumulates in the nucleus and subsequently translocates to mitochondria. Following nuclear localization, ORF 3b traffics to the outer membrane of mitochondria via a predicted amphipathic alpha-helix. Additionally, ORF 3b contains a consensus nuclear export sequence, and we demonstrate that nuclear export and thus mitochondrial translocation are dependent on a leptomycin B-sensitive nuclear export mechanism. We further show that ORF 3b inhibits induction of type I interferon induced by retinoic acid-induced gene 1 and the mitochondrial antiviral signaling protein. Our observations provide insights into the cellular localization of ORF 3b that may enhance our understanding of the mechanisms by which ORF 3b contributes to SARS-CoV pathogenesis. The findings reported here reveal that for multilocalized proteins, consideration of the spatiotemporal distribution may be crucial for understanding viral protein behavior and function.

Intracellular localization of hepatitis delta virus proteins in the presence and absence of viral RNA accumulation.

Hepatitis delta virus (HDV) encodes one protein, hepatitis delta antigen (deltaAg), a 195-amino-acid RNA binding protein essential for the accumulation of HDV RNA-directed RNA transcripts. It has been accepted that deltaAg localizes predominantly to the nucleolus in the absence of HDV genome replication while in the presence of replication, deltaAg facilitates HDV RNA transport to the nucleoplasm and helps redirect host RNA polymerase II (Pol II) to achieve transcription and accumulation of processed HDV RNA species. This study used immunostaining and confocal microscopy to evaluate factors controlling the localization of deltaAg in the presence and absence of replicating and nonreplicating HDV RNAs. When deltaAg was expressed in the absence of full-length HDV RNAs, it colocalized with nucleolin, a predominant nucleolar protein. With time, or more quickly after induced cell stress, there was a redistribution of both deltaAg and nucleolin to the nucleoplasm. Following expression of nonreplicating HDV RNAs, deltaAg moved to the nucleoplasm, but nucleolin was unchanged. When deltaAg was expressed along with replicating HDV RNA, it was found predominantly in the nucleoplasm along with Pol II. This localization was insensitive to inhibitors of HDV replication, suggesting that the majority of deltaAg in the nucleoplasm reflects ribonucleoprotein accumulation rather than ongoing transcription. An additional approach was to reevaluate several forms of deltaAg altered at specific locations considered to be essential for protein function. These studies provide evidence that deltaAg does not interact directly with either Pol II or nucleolin and that forms of deltaAg which support replication are also capable of prior nucleolar transit.

Differential intracellular localization of Sry proteins expressed in Rattus norvegicus

Sry is a transcription factor with a conserved HMG domain that facilitates DNA interactions and nuclear localization, through amino (n) and carboxy (c) terminal nuclear localization sequences (NLS). There are seven Sry loci on the SHR/Akr Y chromosome. Amino acid (aa) differences among Sry proteins are in the nNLS at aa 21 (His vs. Arg), cNLS at aa 76 (Pro vs. Thr), and in the presence/absence of 13 aa in a c‐ terminal activation motif. The objective of this study was to determine if these aa differences between proteins alter nuclear localization. Native, chimeric, mutated and truncated Sry sequences were transfected into CHO cells followed by immunocytochemistry and Western blot analysis of nuclear and cytoplasmic CHO cell fractions. Results showed that Sry2 exhibits both cytoplasmic and nuclear accumulation, while Sry1 and 3 localize only to the nucleus. Sry1 and 3 sequences containing the truncated or deleted activation region exhibit no reduction in nuclear localization, while mutations to the NLS result in cytoplasmic accumulation. Changing His to Arg at aa 21 of Sry1 or Sry3 results in nuclear localization patterns equivalent to native Sry2. Results demonstrate that aa variability in Sry proteins leads to different intracellular localization in vivo and may account for the different phenotypes observed in rats after delivery of exogenous Sry1, Sry2 or Sry3.Support from NIH HL71579 and the University of Akron.

Comparison of intracellular localization of Nubp1 and Nubp2 using GFP fusion proteins

Nubp1 (also known as Nbp35) and Nubp2 (also known as Cfd1) proteins are known to be responsible for regulating centrosome duplication in mouse and ribosome biogenesis in yeast. Nubp proteins contribute to diverse physiological functions. It is thought that Nubp1 and Nubp2 proteins interact with each other and regulate their functions. However, little is known about the intracellular localization of Nubp proteins. In this study, we compared the intracellular localization of human Nubp1 and Nubp2 by fusing these proteins with green fluorescent protein (GFP) in HeLa cells. The nuclear transfer of Nubp1-GFP, where GFP was fused to the C-terminus, was not observed. However, GFP-Nubp1, where GFP was fused to the N-terminus, did accumulate in the nucleus. In addition, GFP-modification at the N-terminal of Nubp2 induced nuclear transformation. Our data suggest that the C-terminal region of Nubp1 is important for nuclear transfer and the N-terminal of Nubp2 contributes to the morphology of the nucleus.

Expression of the melanoma-associated antigen is associated with progression of human thyroid cancer

Thyroid cancer exhibits a spectrum from relatively indolent tumors to tumors that are invasive, metastatic, or progress to poorly differentiated carcinoma. Microarray expression analysis of thyroid cancer cell lines has implicated a member of the melanoma-associated (MAGE) family of cancer-testis antigens in thyroid cancer development and progression. We performed this study to validate the role of MAGE in human thyroid cancers. A tissue microarray (TMA) of samples from 375 patients with thyroid cancer was analyzed with immunohistochemistry (IHC) to localize MAGE. Western blotting of fractionated proteins from MAGE-transfected cells was used to confirm intracellular localization of proteins. Automated analysis of TMA samples was evaluated and subjected to statistical analysis. MAGE immunoreactivity was identified in nuclear and cytoplasmic compartments of normal and malignant tissues. Specificity of staining was proved by fractionation studies that confirmed MAGE expression in nucleus and cytoplasm. Normal thyroid tissue exhibited weak cytoplasmic and strong nuclear MAGE reactivity. Tumors exhibited an increase in cytoplasmic MAGE scores that correlated with clinical behavior: larger tumors had higher MAGE scores, and there was a positive and significant correlation between MAGE cytoplasmic score and the number of histologically proven lymph node metastases. There was a statistically significant negative correlation between cytoplasmic MAGE and the percentage of p53-positive nuclei. Our data confirm gene-profiling evidence that members of the MAGE family play a role in thyroid cancer progression. The use of TMA analyses identifies IHC techniques that are translatable to the clinical setting for prognostic assessment of patients with thyroid cancer.

Genome-wide survey and expression profiling of heat shock proteins and heat shock factors revealed overlapped and stress specific response under abiotic stresses in rice

Heat shock proteins (Hsps) are molecular chaperons, which function in protein folding and assembly, protein intracellular localization and secretion, and degradation of misfolded and truncated proteins. Heat shock factors (Hsfs) are the transcriptional activators of Hsps. It has been reported that Hsps and Hsfs are widely involved in response to various abiotic stresses such as heat, drought, salinity and cold. To elucidate the function and regulation of rice Hsp and Hsf genes, we examined a global expression profiling with heat stressed rice seedling, and then compared our results with the previous rice data under cold, drought and salt stresses. The comparison revealed that, while most Hsfs and Hsps had highly similar and overlapped response and regulation patterns under different stresses, some of those genes showed significantly specific response to distinct stress. We also found that heat-responsive gene profiling differed largely from those under cold/drought/salt stresses, and that drought treatment was more effective to up-regulate Hsf expression in rice than in Arabidopsis. Overall, our data suggests that Hsps and Hsfs might be important elements in cross-talk of different stress signal transduction networks.

Synthesis and intracellular transportation of type I procollagen during functional differentiation of odontoblasts

The expression of type I collagen, the most component of dentin extracellular matrix proteins (ECMs) in odontoblast is correlated with the activity of dentin formation. Since odontoblast possesses a distinct cellular process for protein transport into the dentinal tubule, it is important to examine the intracellular protein localization. However, a study focusing on odontoblast processes has not been performed. Type I collagen is synthesized as procollagen, which is immediately converted to collagen upon secretion. After characterization of antiserum to rat type I procollagen, we investigated the intracellular localization of type I procollagen in odontoblasts during and after dentinogenesis, using immunohistochemistry and in situ hybridization. The level of mRNA expression decreased during dentinogenesis, whereas the intracellular localization of type I procollagen in odontoblast processes become more distinct. The percentage of dentinal tubules with type I procollagen increased significantly with aging. Odontoblasts in pulp horn, in particular, showed moderate expression of type I procollagen after dentinogenesis. Since loss of occlusion also caused a significant decrease in type I procollagen, we concluded that occlusal stimulation activated type I procollagen synthesis in odontoblasts. We also suggest that analysis of intracellular transport of type I procollagen via odontoblast processes may be a new approach to evaluation of odontoblast function.

The modification of quantum dot probes used for the targeted imaging of his-tagged fusion proteins

In molecular biology and protein detection the immobilized metal ion clusters using a NTA-chelator is a powerful technique in identification and isolation of histidine-tagged fusion proteins. The Oligo-histidine tag should serve as a high affinity binding sequence for the purification of any fusion protein via metal chelating adsorbents. We described the preparation and characterization of bioinorganic conjugates made with highly luminescent semiconductor CdTe–CdS core–shell quantum dots (QDs) for biological labeling. A biocompatible surface-functionalized nanoparticle was designed to sense histidine-tagged fusion proteins. This study demonstrates the synthesis of Ni–NTA conjugated QD nanoparticles and the successful application of these nanoparticles to the detection of histidine-tagged fusion proteins. It is believed that this approach will provide a more convenient methodology for the intracellular localization of histidine-tagged protein, as compared with current methods. These Ni–NTA–QD clusters were shown to target the 6 x histidine region of tagged proteins specially.

Characterization of Mce4A protein of Mycobacterium tuberculosis: role in invasion and survival

BackgroundThe mce4 operon is one of the four homologues of mammalian cell entry (mce) operons of Mycobacterium tuberculosis. The mce4A (Rv3499c) gene within this operon is homologous to mce1A (Rv0169), that has a role in host cell invasion by M. tuberculosis. Our earlier reports show that mce4 operon is expressed during the stationary phase of growth of the bacillus in culture and during the course of infection in mammalian hosts. M. tuberculosis carrying mutation in mce4 operon shows growth defect and reduced survival in infected mice. However, the intracellular localization of Mce4A protein and its direct role in cell entry or survival of the bacillus has not been demonstrated so far.ResultsBy transmission electron microscopy we have demonstrated that recombinant Mce4A protein facilitates the invasion of non-pathogenic strain of E. coli into non-phagocytic HeLa cells. We observe that mce4A gene has a role comparable to mce1A in the survival of recombinant E. coli in human macrophages. Using antibodies raised against Mce4A protein, we show that the protein is localized in the cell wall fraction of M. tuberculosis H37Rv stationary phase culture only.ConclusionMce4A protein is expressed during the stationary phase of broth culture and localizes in the cell wall fraction of M. tuberculosis. Mce4A protein expressed in non-pathogenic E. coli enables it to enter and survive within HeLa cells and the macrophages. As Mce4A protein is expressed during later phase of mycobacterial growth, our results raise the possibility of it playing a role in maintenance of persistent tubercular infection.

Formalin can alter the intracellular localization of some transcription factors inSaccharomyces cerevisiae

Indirect immunofluorescence (IF) microscopy is a frequently used method to determine intracellular protein localization. It is especially useful for low abundance proteins, for example the GATA-factors (Gln3, Gat1) which activate nitrogen catabolite repression (NCR)-sensitive transcription. Limiting nitrogen or treating cells with Tor pathway inhibitor, rapamycin, elicits nuclear GATA-factor localization and increased NCR-sensitive transcription, whereas excess nitrogen restricts these proteins to the cytoplasm and decreases transcription. The initial step of the IF procedure is formalin-fixation that quenches cellular activity and fixes protein locations via cross-linking. We find that under some conditions, formalin itself can influence GATA-factor localization. With low formalin (0.8% or 1.6%), Gat1-Myc(13) became more nuclear, and with higher concentrations (5.6%), it became more cytoplasmic. Gln3-Myc(13) localization, on the other hand, did not respond to low formalin, but became more cytoplasmic at the higher concentration. Interestingly, the high concentration of formalin had no demonstrable effect when the GATA factors were completely nuclear, i.e. after rapamycin (Gat1-Myc(13)) or Msx (Gln3-Myc(13)) treatment. These effects are most likely elicited by polyoxymethylene glycols, which significantly increase the osmolarity of the medium (0.5-2). We suggest that varying degrees of osmotic stress and transcription factor movement in response to it can occur after the beginning of fixation but before proteins become immobilized.

Therapeutic Strategies for Long-QT Syndrome

The discovery of genetic defects underlying long-QT syndrome (LQTS) has allowed to identify important genotype-phenotype correlations that are now being used for risk stratification. The next challenge is to exploit the new information on the pathophysiology of the disease derived from molecular genetics to devise more effective therapies. The successful response of LQT1 patients to β-blockers, the QT-shortening action of sodium channel blockers in at least some LQT3 patients, and the importance of maintaining adequate plasma potassium levels in LQT2 patients clearly demonstrate the importance of selecting therapy in the context of the molecular substrate. The hurdles on the road toward the development of novel therapeutic strategies are represented by the variable expressivity of the disease, the high prevalence of “private” mutations, and the role of genetic and nongenetic factors that act as modifiers of the phenotype. Given the large number of mutations identified and their phenotypic complexity, it is clearly impossible to anticipate a scenario in which each mutation will be managed through a specific therapy. However, the evidence that mutations in the LQTS gene may be clustered based on their electrophysiological profile and on their response to specific drugs may provide the rationale for the development of mutation-specific therapies. In this article, we will review the most relevant genotype-phenotype features of LQTS and the strategies explored to develop novel therapeutic approaches. ### Definition and Prevalence The congenital LQTS is an inherited arrhythmogenic disease characterized by abnormally prolonged QT interval leading to life-threatening arrhythmias in the presence of a structurally normal heart.1 Different clinical variants of LQTS have been identified. Romano Ward syndrome, transmitted as an autosomal dominant trait, is the most common form of LQTS and presents only a cardiac phenotype. Less prevalent variants of LQTS, such as Andersen and Timothy syndromes or the recessive Jervell and Lange-Nielsen syndromes, also present …

イネにおける抗酸化タンパク質（ペルオシキレドキシン）の細胞内局在

イネにおける抗酸化タンパク質（ペルオシキレドキシン）の細胞内局在

Mechanisms and molecular adaptation to extreme dehydration in tardigrades: Hsp gene expression in Milnesium tardigradum

The tardigrade Milnesium tardigradum Doyere 1840 is a well-known cosmopolitan species and a typical inhabitant of moist environments which facilitate the animal's gaseous exchange and avoid desiccation. However, such habitats frequently undergo seasonal changes that impact animal life. M. tardigradum is able to survive these periods of adverse conditions due to its ability to enter into a anhydrobiotic state. In adverse environments, all terrestrial and freshwater tardigades arrest their metabolic activity and get dehydrated to form the tun state. In the anhydrobiotic stage, tardigrades showextraordinary tolerance tophysical extremes includinghigh-energy radiation, immersion inorganic solvents, brief exposure to high temperatures and prolonged exposure to indefinitely low temperatures. When environmental conditions are adequate, tuns rehydrate and the animals resume metabolic activity. Drying of cells generally leads to massive damage to cellular membranes and proteins, which eventually results in cell death and, consequently, in the death of the entire organism.Heat shock proteins and theirmolecular partners are known to play diverse roles, even in unstressed cells, in successful folding, assembly, intracellular localization, secretion, regulation, and degradation of other proteins. In this study, a complementary focus is on tardigrades undergoing stress in nature and on the roles of stress genes of the hsp10, hsp20/30, hsp40, hsp60, hsp70 and hsp90 family in the stress physiology ofwhole organisms in different life history stages. This study is part of the project www.FUNCRYPTA.de, funded by the German Federal Ministry of Education and Research, BMBF (0313838).

Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease

The ATP-binding cassette transporter ABCA3 mediates uptake of choline-phospholipids into intracellular vesicles and is essential for surfactant metabolism in lung alveolar type II cells. We have shown previously that ABCA3 mutations in fatal surfactant deficiency impair intracellular localization or ATP hydrolysis of ABCA3 protein. However, the mechanisms underlying the less severe phenotype of patients with ABCA3 mutation are unclear. In this study, we characterized ABCA3 mutant proteins identified in pediatric interstitial lung disease (pILD). E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes as wild-type protein. Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, whereas those of E690K and T1114M mutant proteins are severely impaired. Vanadate-induced nucleotide trapping and photoaffinity labeling of wild-type and mutant proteins using 8-azido-[(32)P]ATP revealed an aberrant catalytic cycle in these mutant proteins. These results demonstrate the importance of a functional catalytic cycle in lipid transport of ABCA3 and suggest a pathophysiological mechanism of pILD due to ABCA3 mutation.

Intracellular localization of the heat shock protein, HSP110, in Xenopus laevis A6 kidney epithelial cells

Heat shock protein 110 (HSP110) is a large molecular mass chaperone that is part of the HSP70/DnaK superfamily. In the present study, we examined the accumulation of HSP110 in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis, using a homologous antibody, detected the presence of HSP110 in A6 cells maintained at 22 °C. The relative levels of HSP110 accumulation increased after heat shock or sodium arsenite treatment. Immunocytochemical analysis revealed that constitutively expressed HSP110 was localized in the cytoplasm in a diffuse granular pattern with enrichment in the nucleus. In A6 cells heat shocked at 33 °C or 35 °C for 2 to 4 h, HSP110 accumulation was enhanced and detected primarily in the cytoplasm as thread- or spindle-like structures. In contrast, HSP30 was not detected constitutively and heat shock treatment of A6 cells induced a relatively uniform punctate pattern primarily in the cytoplasm. Also, treatment of A6 cells at 35 °C for 6 h resulted in the presence of HSP110 and HSP30 enriched in the nucleus of most cells. Finally, A6 cells treated with 25 μM sodium arsenite produced very dense HSP110 structures primarily in the cytoplasm while HSP30 was enriched in the cytoplasm in a granular pattern.