HSF1 granules: a novel stress-induced nuclear compartment of human cells

1997 ◽  
Vol 110 (23) ◽  
pp. 2925-2934 ◽  
Author(s):  
J. Cotto ◽  
S. Fox ◽  
R. Morimoto
Keyword(s):  
Heat Shock ◽  
Spatial Organization ◽  
Fluorescent Protein ◽  
Normal Growth ◽  
Growth Conditions ◽  
Heat Shock Gene ◽  
Heat Shock Factor 1 ◽  
Flag Epitope ◽  
Nuclear Structures ◽  
Stress Dependent

Heat shock factor 1 (HSF1) is the ubiquitous stress-responsive transcriptional activator which is essential for the inducible transcription of genes encoding heat shock proteins and molecular chaperones. HSF1 localizes within the nucleus of cells exposed to heat shock, heavy metals, and amino acid analogues, to form large, irregularly shaped, brightly staining granules which are not detected during attenuation of the heat shock response or when cells are returned to their normal growth conditions. The kinetics of detection of HSF1 granules parallels the transient induction of heat shock gene transcription. HSF1 granules are also detected using an HSF1-Flag epitope tagged protein or a chimeric HSF1-green fluorescent protein which reveals that these nuclear structures are stress-induced and can be detected in living cells. The spatial organization of HSF1 granules in nuclei of stressed cells reveals that they are novel nuclear structures which are stress-dependent and provides evidence that the nucleus undergoes dynamic reorganization in response to stress.

scholarly journals Localization and Expression of MreB in Vibrio parahaemolyticus under Different Stresses

2008 ◽  
Vol 74 (22) ◽  
pp. 7016-7022 ◽  
Author(s):  
Shen-Wen Chiu ◽  
Shau-Yan Chen ◽  
Hin-chung Wong
Keyword(s):  
Stationary Phase ◽  
Vibrio Parahaemolyticus ◽  
Spatial Organization ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Yellow Fluorescent Protein ◽  
Normal Growth ◽  
Growth Conditions ◽  
Cellular Stress Response ◽  
Vbnc State

ABSTRACT MreB, the homolog of eukaryotic actin, may play a vital role when prokaryotes cope with stress by altering their spatial organization, including their morphology, subcellular architecture, and localization of macromolecules. This study investigates the behavior of MreB in Vibrio parahaemolyticus under various stresses. The behavior of MreB was probed using a yellow fluorescent protein-MreB conjugate in merodiploid strain SC9. Under normal growth conditions, MreB formed helical filaments in exponential-phase cells. The shape of starved or stationary-phase cells changed from rods to small spheroids. The cells differentiated into the viable but nonculturable (VBNC) state with small spherical cells via a “swelling-waning” process. In all cases, drastic remodeling of the MreB cytoskeleton was observed. MreB helices typically were loosened and fragmented into short filaments, arcs, and spots in bacteria under these stresses. The disintegrated MreB exhibited a strong tendency to attach to the cytoplasmic membrane. The expression of mreB generally declined in bacteria in the stationary phase and under starvation but was upregulated during the initial periods of cold shock and VBNC state differentiation and decreased afterwards. Our findings demonstrated the behavior of MreB in the morphological changes of V. parahaemolyticus under intrinsic or extrinsic stresses and may have important implications for studying the cellular stress response and aging.

Structure, organization, and expression of the 16-kDa heat shock gene family of Caenorhabditis elegans

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 690-697 ◽  
Author(s):  
E. P. M. Candido ◽  
D. Jones ◽  
D. K. Dixon ◽  
R. W. Graham ◽  
R. H. Russnak ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Normal Growth ◽  
Growth Conditions ◽  
Amino Acid Sequences ◽  
Heat Shock Gene ◽  
Coding Region ◽  
Heat Shock Element ◽  
Hypersensitive Sites ◽  
Intergenic Regions

Exposure of the nematode Caenorhabditis elegans to a heat shock results in the induction of a number of genes not normally expressed in the animals under normal growth conditions. Among these are a family of genes encoding 16 kDa heat shock proteins (hsp16s). The major hsp16 genes have been cloned and characterized, and found to reside at two clusters in the C. elegans genome. One cluster contains two distinct genes, hsp16-1 and hsp16-48, arranged in divergent orientations separated by only 348 base pairs (bp). An identical pair, duplicated and inverted with respect to the first pair, is located 415 bp away. This cluster, located on chromosome V, therefore contains four genes as two identical pairs within less than 4 kilobases of DNA, and the pairs form the arms of a large inverted repeat. A second pair of genes, hsp16-2 and hsp16-41, constitutes a second hsp16 locus with an organization very similar to that of the hsp16-1/48 locus, except that it is not duplicated. Comparisons of the derived amino acid sequences show that hsp16-1 and hsp16-2 form a closely related pair, as do hsp16-41 and hsp16-48. These hsps show extensive sequence identity with the small hsps of Drosophila, as well as with mammalian alpha-crystallins. The coding region of each gene is interrupted by a single intron of approximately 50 bp, in a position homologous to that of the first intron in a mouse alpha-crystallin gene. The compact intergenic regions of both hsp16 loci contain a TATA element and a heat shock element (HSE) for each member of the pair, and are very similar in sequence overall. Expression studies, however, show that the level of transcripts from the hsp16-2/41 pair may be up to 14-fold higher on a per gene basis, as the level of RNA from the hsp16-1/48 pair, depending upon the induction conditions and developmental stage. This difference in message levels seems to be due to differences in the kinetics of inactivation of the genes rather than in transcription rates or rates of mRNA turnover. Distinct DNAseI hypersensitive sites are present upstream of each HSE in chromatin when the genes are inactive; these disappear and the whole intergenic region seems to become DNAse sensitive when the genes are maximally active.Key words: heat shock, 16-kDa polypeptides, gene structure, transcription, DNAseI hypersensitive sites, Caenorhabditis elegans.

scholarly journals Sequence and regulation of a gene encoding a human 89-kilodalton heat shock protein.

1989 ◽  
Vol 9 (6) ◽  
pp. 2615-2626 ◽  
Author(s):  
E Hickey ◽  
S E Brandon ◽  
G Smale ◽  
D Lloyd ◽  
L A Weber
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Normal Growth ◽  
Gene Families ◽  
Growth Conditions ◽  
Complete Sequence ◽  
Start Codon ◽  
Hybridization Probe ◽  
Reading Frame ◽  
Alpha Gene

Vertebrate cells synthesize two forms of the 82- to 90-kilodalton heat shock protein that are encoded by distinct gene families. In HeLa cells, both proteins (hsp89 alpha and hsp89 beta) are abundant under normal growth conditions and are synthesized at increased rates in response to heat stress. Only the larger form, hsp89 alpha, is induced by the adenovirus E1A gene product (M. C. Simon, K. Kitchener, H. T. Kao, E. Hickey, L. Weber, R. Voellmy, N. Heintz, and J. R. Nevins, Mol. Cell. Biol. 7:2884-2890, 1987). We have isolated a human hsp89 alpha gene that shows complete sequence identity with heat- and E1A-inducible cDNA used as a hybridization probe. The 5'-flanking region contained overlapping and inverted consensus heat shock control elements that can confer heat-inducible expression on a beta-globin reporter gene. The gene contained 10 intervening sequences. The first intron was located adjacent to the translation start codon, an arrangement also found in the Drosophila hsp82 gene. The spliced mRNA sequence contained a single open reading frame encoding an 84,564-dalton polypeptide showing high homology with the hsp82 to hsp90 proteins of other organisms. The deduced hsp89 alpha protein sequence differed from the human hsp89 beta sequence reported elsewhere (N. F. Rebbe, J. Ware, R. M. Bertina, P. Modrich, and D. W. Stafford (Gene 53:235-245, 1987) in at least 99 out of the 732 amino acids. Transcription of the hsp89 alpha gene was induced by serum during normal cell growth, but expression did not appear to be restricted to a particular stage of the cell cycle. hsp89 alpha mRNA was considerably more stable than the mRNA encoding hsp70, which can account for the higher constitutive rate of hsp89 synthesis in unstressed cells.

Cdk5 mediates changes in morphology and promotes apoptosis of astrocytoma cells in response to heat shock

2001 ◽  
Vol 114 (6) ◽  
pp. 1145-1153 ◽  
Author(s):  
C. Gao ◽  
S. Negash ◽  
H.S. Wang ◽  
D. Ledee ◽  
H. Guo ◽  
...  
Keyword(s):  
Heat Shock ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Normal Growth ◽  
Dominant Negative ◽  
Specific Expression ◽  
Cell Matrix ◽  
Dominant Negative Mutation ◽  
U373 Cells

The cyclin-dependent kinase member, Cdk5, is expressed in a variety of cell types, but neuron-specific expression of its activator, p35, is thought to limit its activity to neurons. Here we demonstrate that both Cdk5 and p35 are expressed in the human astrocytoma cell line, U373. Cdk5 and p35 are present in the detergent-insoluble cytoskeletal fraction of this cell line and Cdk5 localizes to filopodia and vinculin-rich regions of cell-matrix contact in lamellopodia. When exposed to a 46(o)C heat shock, U373 cells change shape, lose cell-matrix contacts and show increased levels of apoptosis. To test whether Cdk5 activation might play a role in these events, U373 cells were stably transfected with histidine-tagged or green fluorescent protein-tagged constructs of Cdk5 or a dominant negative mutation, Cdk5T33. Under normal growth conditions, growth characteristics of the stably transfected lines were indistinguishable from untransfected U373 cells and Cdk5 localization was not changed. However, when subjected to heat shock, cells stably transfected with Cdk5-T33 remained flattened, showed little loss of cell-matrix adhesion, and exhibited significantly lower levels of apoptosis. In contrast, cells that overexpressed wild-type Cdk5 showed morphological changes similar to those seen in untransfected U373 cells in response to heat shock and had significantly higher levels of apoptosis. Heat-shocked cells showed changes in p35 mobility and stability of the Cdk5/p35 complex consistent with endogenous Cdk5 activity. Together these findings suggest that endogenous Cdk5 activity may play a key role in regulating morphology, attachment, and apoptosis in U373 cells, and raise the possibility that Cdk5 may be a general regulator of cytoskeletal organization and cell adhesion in both neuronal and non-neuronal cells.

Heat shock factor is required for growth at normal temperatures in the fission yeast Schizosaccharomyces pombe

1993 ◽  
Vol 13 (2) ◽  
pp. 749-761
Author(s):  
G J Gallo ◽  
H Prentice ◽  
R E Kingston
Keyword(s):  
Heat Shock ◽  
Schizosaccharomyces Pombe ◽  
Normal Growth ◽  
Growth Conditions ◽  
Heat Shock Factor ◽  
Binding Ability ◽  
Lower Growth ◽  
General Role ◽  
Functional Conservation ◽  
Cellular Processes

Schizosaccharomyces pombe is becoming an increasingly useful organism for the study of cellular processes, since in certain respects, such as the cell cycle and splicing, it is similar to metazoans. Previous biochemical studies have shown that the DNA binding ability of S. pombe heat shock factor (HSF) is fully induced only under stressed conditions, in a manner similar to that of Drosophila melanogaster and humans but differing from the constitutive binding by HSF in the budding yeasts. We report the isolation of the cDNA and gene for the HSF from S. pombe. S. pombe HSF has a domain structure that is more closely related to the structure of human and D. melanogaster HSFs than to the structure of the budding yeast HSFs, further arguing that regulation of HSF in S. pombe is likely to reflect regulation in metazoans. Surprisingly, the S. pombe HSF gene is required for growth at normal temperatures. We show that the S. pombe HSF gene can be replaced by the D. melanogaster HSF gene and that strains containing either of these genes behave similarly to transiently heat-shocked strains with respect to viability and the level of heat-induced transcripts from heat shock promoters. Strains containing the D. melanogaster HSF gene, however, have lower growth rates and show altered morphology at normal growth temperatures. These data demonstrate the functional conservation of domains of HSF that are required for response to heat shock. They further suggest a general role for HSF in growth of eukaryotic cells under normal (nonstressed) growth conditions.

scholarly journals Functional Analysis of the ATG8 Homologue Aoatg8 and Role of Autophagy in Differentiation and Germination in Aspergillus oryzae

2006 ◽  
Vol 5 (8) ◽  
pp. 1328-1336 ◽  
Author(s):  
Takashi Kikuma ◽  
Mamoru Ohneda ◽  
Manabu Arioka ◽  
Katsuhiko Kitamoto
Keyword(s):  
Aspergillus Oryzae ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Normal Growth ◽  
Nitrogen Sources ◽  
Growth Conditions ◽  
Conidial Germination ◽  
Aerial Hyphae ◽  
Green Fluorescent ◽  
Starvation Conditions

ABSTRACT Autophagy is a well-known degradation system, induced by nutrient starvation, in which cytoplasmic components and organelles are digested via vacuoles/lysosomes. Recently, it was reported that autophagy is involved in the turnover of cellular components, development, differentiation, immune responses, protection against pathogens, and cell death. In this study, we isolated the ATG8 gene homologue Aoatg8 from the filamentous fungus Aspergillus oryzae and visualized autophagy by the expression of DsRed2-AoAtg8 and enhanced green fluorescent protein-AoAtg8 fusion proteins in this fungus. While the fusion proteins were localized in dot structures which are preautophagosomal structure-like structures under normal growth conditions, starvation or rapamycin treatment caused their accumulation in vacuoles. DsRed2 expressed in the cytoplasm was also taken up into vacuoles under starvation conditions or during the differentiation of conidiophores and conidial germination. Deletion mutants of Aoatg8 did not form aerial hyphae and conidia, and DsRed2 was not localized in vacuoles under starvation conditions, indicating that Aoatg8 is essential for autophagy. Furthermore, Aoatg8 conditional mutants showed delayed conidial germination in the absence of nitrogen sources. These results suggest that autophagy functions in both the differentiation of aerial hyphae and in conidial germination in A. oryzae.

scholarly journals AIRAP, a New Human Heat Shock Gene Regulated by Heat Shock Factor 1

2010 ◽  
Vol 285 (18) ◽  
pp. 13607-13615 ◽  
Author(s):  
Antonio Rossi ◽  
Edoardo Trotta ◽  
Rossella Brandi ◽  
Ivan Arisi ◽  
Marta Coccia ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Factor ◽  
Heat Shock Gene ◽  
Heat Shock Factor 1 ◽  
Shock Gene

scholarly journals Heat Shock Factor 1 Attenuates 4-Hydroxynonenal-mediated Apoptosis

2007 ◽  
Vol 282 (46) ◽  
pp. 33412-33420 ◽  
Author(s):  
Aaron T. Jacobs ◽  
Lawrence J. Marnett
Keyword(s):  
Lipid Peroxidation ◽  
Heat Shock ◽  
Nuclear Translocation ◽  
Ectopic Expression ◽  
Antioxidant Response ◽  
Heat Shock Factor ◽  
Heat Shock Gene ◽  
Heat Shock Factor 1 ◽  
Inhibitory Interaction ◽  
Heat Shock Responses

Lipid peroxidation is a consequence of both normal physiology and oxidative stress that generates various reactive metabolites, a principal end product being 4-hydroxynonenal (HNE). As a diffusible electrophile, HNE reacts extensively with cellular nucleophiles. Consequently, HNE alters cellular signaling and activates the intrinsic apoptotic cascade. We have previously demonstrated that in addition to promoting apoptosis, HNE activates stress response pathways, including the antioxidant, endoplasmic reticulum stress, DNA damage, and heat shock responses. Here we demonstrate that activation of the heat shock response by HNE is dependent on the expression and nuclear translocation of heat shock factor 1 (HSF1), which promotes the expression of heat shock protein 40 (Hsp40) and Hsp70-1. Ectopic expression and immunoprecipitation of c-Myc-tagged Hsp70-1 indicates that HNE disrupts the inhibitory interaction between Hsp70-1 and HSF1, leading to the activation heat shock gene expression. Using siRNA to silence HSF1 expression, we observe that HSF1 is necessary for the induction of Hsp40 and Hsp70-1 by HNE, and the lack of Hsp expression is correlated with an increase in apoptosis. Nrf2, the transcription factor that mediates the antioxidant response, was also silenced using siRNA. Silencing Nrf2 also enhanced the cytotoxicity of HNE, but not as effectively as HSF1. Silencing HSF1 expression facilitates the activation of JNK pro-apoptotic signaling and selectively decreases expression of the anti-apoptotic Bcl-2 family member Bcl-XL. Overexpression of Bcl-XL attenuates HNE-mediated apoptosis in HSF1-silenced cells. Overall, activation of HSF1 and stabilization of Bcl-XL mediate a protective response that may contribute significantly to the cellular biology of lipid peroxidation.

scholarly journals RNA aptamer capture of macromolecular complexes for mass spectrometry analysis

2020 ◽  
Vol 48 (15) ◽  
pp. e90-e90
Author(s):  
Judhajeet Ray ◽  
Angela Kruse ◽  
Abdullah Ozer ◽  
Takuya Kajitani ◽  
Richard Johnson ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Heat Shock ◽  
High Resolution ◽  
Fluorescent Protein ◽  
Affinity Purification ◽  
Detection Sensitivity ◽  
Mass Spectrometry Analysis ◽  
Heat Shock Factor 1 ◽  
Rna Aptamer ◽  
Macromolecular Complexes

Abstract Specific genomic functions are dictated by macromolecular complexes (MCs) containing multiple proteins. Affinity purification of these complexes, often using antibodies, followed by mass spectrometry (MS) has revolutionized our ability to identify the composition of MCs. However, conventional immunoprecipitations suffer from contaminating antibody/serum-derived peptides that limit the sensitivity of detection for low-abundant interacting partners using MS. Here, we present AptA–MS (aptamer affinity–mass spectrometry), a robust strategy primarily using a specific, high-affinity RNA aptamer against Green Fluorescent Protein (GFP) to identify interactors of a GFP-tagged protein of interest by high-resolution MS. Utilizing this approach, we have identified the known molecular chaperones that interact with human Heat Shock Factor 1 (HSF1), and observed an increased association with several proteins upon heat shock, including translation elongation factors and histones. HSF1 is known to be regulated by multiple post-translational modifications (PTMs), and we observe both known and new sites of modifications on HSF1. We show that AptA–MS provides a dramatic target enrichment and detection sensitivity in evolutionarily diverse organisms and allows identification of PTMs without the need for modification-specific enrichments. In combination with the expanding libraries of GFP-tagged cell lines, this strategy offers a general, inexpensive, and high-resolution alternative to conventional approaches for studying MCs.

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