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.

Epithelial cell spreading induced by hepatocyte growth factor influences paxillin protein synthesis and posttranslational modification

2004 ◽  
Vol 287 (4) ◽  
pp. G886-G898 ◽  
Author(s):  
Ann M. Hopkins ◽  
Matthias Bruewer ◽  
G. Thomas Brown ◽  
A’Drian A. Pineda ◽  
Julie J. Ha ◽  
...  
Keyword(s):  
Growth Factor ◽  
Epithelial Cell ◽  
Hepatocyte Growth Factor ◽  
Fluorescent Protein ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Epithelial Migration ◽  
Cell Matrix ◽  
Hepatocyte Growth

Superficial wounds in the gastrointestinal tract rapidly reseal by coordinated epithelial cell migration facilitated by cytokines such as hepatocyte growth factor (HGF)/scatter factor released in the wound vicinity. However, the mechanisms by which HGF promotes physiological and pathophysiologic epithelial migration are incompletely understood. Using in vitro models of polarized T84 and Caco-2 intestinal epithelia, we report that HGF promoted epithelial spreading and RhoA GTPase activation in a time-dependent manner. Inducible expression of enhanced green fluorescent protein-tagged dominant-negative RhoA significantly attenuated HGF-induced spreading. HGF expanded a zone of partially flattened cells behind the wound edge containing basal F-actin fibers aligned in the direction of spreading. Concomitantly, plaques positive for the focal adhesion protein paxillin were enhanced. HGF induced an increase in the translation of paxillin and, to a lesser extent, β1-integrin. This was independent of cell-matrix adhesion through β1-integrin. Subcellular fractionation revealed increased cosedimentation of paxillin with plasma membrane-containing fractions following HGF stimulation, without corresponding enhancements in paxillin coassociation with β1 integrin or actin. Tyrosine phosphorylation of paxillin was reduced by HGF and was sensitive to the Src kinase inhibitor PP2. With these taken together, we propose that HGF upregulates a free cytosolic pool of paxillin that is unaffiliated with either the cytoskeleton or focal cell-matrix contacts. Thus early spreading responses to HGF may partly relate to increased paxillin availability for incorporation into, and turnover within, dynamic cytoskeletal/membrane complexes whose rapid and transient adhesion to the matrix drives migration.

scholarly journals Selective renal overexpression of human heat shock protein 27 reduces renal ischemia-reperfusion injury in mice

2010 ◽  
Vol 299 (2) ◽  
pp. F347-F358 ◽  
Author(s):  
Minjae Kim ◽  
Sang Won Park ◽  
Mihwa Kim ◽  
Sean W. C. Chen ◽  
William T. Gerthoffer ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Fluorescent Protein ◽  
Ischemia Reperfusion Injury ◽  
Therapeutic Option ◽  
Ischemia Reperfusion ◽  
Heat Shock Protein 27 ◽  
Renal Ischemia ◽  
Specific Expression ◽  
Mrna Induction

We have previously shown that exogenous and endogenous A1 adenosine receptor (A1AR) activation protected against renal ischemia-reperfusion (IR) injury in mice by induction and phosphorylation of heat shock protein 27 (HSP27). With global overexpression of HSP27 in mice, however, there was a paradoxical increase in systemic inflammation with increased renal injury after an ischemic insult due to increased NK1.1 cytotoxicity. In this study, we hypothesized that selective renal expression of HSP27 in mice would improve renal function and reduce injury after IR. Mice were subjected to renal IR injury 2 days after intrarenal injection of saline or a lentiviral construct encoding enhanced green fluorescent protein (EGFP) or human HSP27 coexpressing EGFP (EGFP-huHSP27). Mice with kidney-specific reconstitution of huHSP27 had significantly lower plasma creatinine, renal necrosis, apoptosis, and inflammation as demonstrated by decreased proinflammatory cytokine mRNA induction and neutrophil infiltration. In addition, there was better preservation of the proximal tubule epithelial filamentous (F)-actin cytoskeleton in the huHSP27-reconstituted groups than in the control groups. Furthermore, huHSP27 overexpression led to increased colocalization with F-actin in renal proximal tubules. Taken together, these findings have important clinical implications, as they imply that kidney-specific expression of HSP27 through lentiviral delivery is a viable therapeutic option in attenuating the effects of renal IR.

scholarly journals Dynamics of Nontypical Apoptotic Morphological Changes Visualized by Green Fluorescent Protein in Living Cells with Infectious Pancreatic Necrosis Virus Infection

1999 ◽  
Vol 73 (6) ◽  
pp. 5056-5063 ◽  
Author(s):  
Jiann-Ruey Hong ◽  
Tai-Lang Lin ◽  
Jer-Yen Yang ◽  
Ya-Li Hsu ◽  
Jen-Leih Wu
Keyword(s):  
Cell Line ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Membrane Integrity ◽  
Infectious Pancreatic Necrosis Virus ◽  
Fish Cell Line ◽  
Fish Cell ◽  
Infectious Pancreatic Necrosis ◽  
Pancreatic Necrosis Virus ◽  
Green Fluorescent

ABSTRACT Morphologically, apoptotic cells are characterized by highly condensed membrane blebbing and formation of apoptotic bodies. Recently, we reported that apoptosis precedes necrosis in a fish cell line infected with infectious pancreatic necrosis virus (IPNV). In the present study, we tested the possibility that nontypical apoptosis is a component of IPNV-induced fish cell death. A variant type of green fluorescent protein (EGFP) was expressed in a fish cell line such that EGFP served as a protein marker for visualizing dynamic apoptotic cell morphological changes and for tracing membrane integrity changes during IPNV infection. Direct morphological changes were visualized by fluorescence microscopy by EGFP in living cells infected with IPNV. The nontypical apoptotic morphological change stage occurred during the pre-late stage (6 to 7 h postinfection). Nontypical apoptotic features, including highly condensed membrane blebbing, occurred during the middle apoptotic stage. At the pre-late apoptotic stage, membrane vesicles quickly formed, blebbed, and were finally pinched off from the cell membrane. At the same time, at this pre-late apoptotic stage, apoptotic cells formed unique small holes in their membranes that ranged from 0.39 to 0.78 μm according to examination by scanning electron microscopy and immunoelectron microscopy. Quantitation of the intra- and extracellular release of EGFP by CHSE-214-EGFP cells after IPNV infection was done by Western blotting and fluorometry. Membrane integrity was quickly lost during the late apoptotic stage (after 8 h postinfection), and morphological change and membrane integrity loss could be prevented and blocked by treatment with apoptosis inhibitors such as cycloheximide, genistein, and EDTA before IPNV infection. Together, these findings show the apoptotic features at the onset of pathology in host cells (early and middle apoptotic stages), followed secondarily by nontypical apoptosis (pre-late apoptotic stage) and then by postapoptotic necrosis (late apoptotic stage), of a fish cell line. Our results demonstrate that nontypical apoptosis is a component of IPNV-induced fish cell death.

scholarly journals A Novel, C-Terminal Dominant Negative Mutation of the GR Causes Familial Glucocorticoid Resistance through Abnormal Interactions with p160 Steroid Receptor Coactivators

2002 ◽  
Vol 87 (6) ◽  
pp. 2658-2667 ◽  
Author(s):  
Alessandra Vottero ◽  
Tomoshige Kino ◽  
Herve Combe ◽  
Pierre Lecomte ◽  
George P. Chrousos
Keyword(s):  
Ligand Binding ◽  
Transcriptional Activity ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Glucocorticoid Resistance ◽  
Base Substitution ◽  
Wild Type ◽  
Nuclear Speckles ◽  
Dominant Negative Mutation ◽  
Type Receptor

Primary cortisol resistance is a rare, inherited or sporadic form of generalized end-organ insensitivity to glucocorticoids. Here, we report a kindred in which affected members had a heterozygous T to G base substitution at nucleotide 2373 of exon 9α of the GR gene, causing substitution of Ile by Met at position 747. This mutation was located close to helix 12, at the C terminus of the ligand-binding domain, which has a pivotal role in the formation of activation function (AF)-2, a subdomain that interacts with p160 coactivators. The affinity of the mutant GR for dexamethasone was decreased by about 2-fold, and its transcriptional activity on the glucocorticoid-responsive mouse mammary tumor virus promoter was compromised by 20- to 30-fold. In addition, the mutant GR functioned as a dominant negative inhibitor of wild-type receptor-induced transactivation. The mutant GR through its intact AF-1 domain bound to a p160 coactivator, but failed to do so through its AF-2 domain. Overexpression of a p160 coactivator restored the transcriptional activity and reversed the negative transdominant activity of the mutant GR. Interestingly, green fluorescent protein (GFP)-fused GRαI747M had a slight delay in its translocation from the cytoplasm into the nucleus and formed coarser nuclear speckles than GFP-fused wild-type GRα. Similarly, a GFP-fused p160 coactivator had a distinctly different distribution in the nucleus in the presence of mutant vs. wild-type receptor, presenting also as coarser speckling. We conclude that the mutation at amino acid 747 of the GR causes familial, autosomal dominant glucocorticoid resistance by decreasing ligand binding affinity and transcriptional activity, and by exerting a negative transdominant effect on the wild-type receptor. The mutant receptor has an ineffective AF-2 domain, which leads to an abnormal interaction with p160 coactivators and a distinct nuclear distribution of both.

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.

scholarly journals Regulation of Longevity inCaenorhabditis elegansby Heat Shock Factor and Molecular Chaperones

2004 ◽  
Vol 15 (2) ◽  
pp. 657-664 ◽  
Author(s):  
James F. Morley ◽  
Richard I. Morimoto
Keyword(s):  
Heat Shock ◽  
Molecular Chaperones ◽  
Heat Shock Factor ◽  
Dominant Negative ◽  
Wild Type ◽  
Down Regulation ◽  
Specific Expression ◽  
Genetic Switch ◽  
Sense And Respond ◽  
Dauer Larvae

The correlation between longevity and stress resistance observed in long-lived mutant animals suggests that the ability to sense and respond to environmental challenges could be important for the regulation of life span. We therefore examined the role of heat shock factor (HSF-1), a master transcriptional regulator of stress-inducible gene expression and protein folding homeostasis, in the regulation of longevity. Down-regulation of hsf-1 by RNA interference suppressed longevity of mutants in an insulin-like signaling (ILS) pathway that functions in the nervous system of Caenorhabditis elegans to influence aging. hsf-1 was also required for temperature-induced dauer larvae formation in an ILS mutant. Using tissue-specific expression of wild-type or dominant negative HSF-1, we demonstrated that HSF-1 acts in multiple tissues to regulate longevity. Down-regulation of individual molecular chaperones, transcriptional targets of HSF-1, also decreased longevity of long-lived mutant but not wild-type animals. However, suppression by individual chaperones was to a lesser extent, suggesting an important role for networks of chaperones. The interaction of ILS with HSF-1 could represent an important molecular strategy to couple the regulation of longevity with an ancient genetic switch that governs the ability of cells to sense and respond to stress.

scholarly journals Protein Kinase D Controls the Integrity of Golgi Apparatus and the Maintenance of Dendritic Arborization in Hippocampal Neurons

2009 ◽  
Vol 20 (7) ◽  
pp. 2108-2120 ◽  
Author(s):  
Katalin Czöndör ◽  
Kornelia Ellwanger ◽  
Yannick F. Fuchs ◽  
Sylke Lutz ◽  
Márton Gulyás ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Golgi Complex ◽  
Hippocampal Neurons ◽  
Fluorescent Protein ◽  
Dendritic Tree ◽  
Secretory Vesicle ◽  
Dendritic Arborization ◽  
Dominant Negative ◽  
Protein Kinase D ◽  
Specific Expression

Protein kinase D (PKD) is known to participate in various cellular functions, including secretory vesicle fission from the Golgi and plasma membrane-directed transport. Here, we report on expression and function of PKD in hippocampal neurons. Expression of an enhanced green fluorescent protein (EGFP)-tagged PKD activity reporter in mouse embryonal hippocampal neurons revealed high endogenous PKD activity at the Golgi complex and in the dendrites, whereas PKD activity was excluded from the axon in parallel with axonal maturation. Expression of fluorescently tagged wild-type PKD1 and constitutively active PKD1S738/742E (caPKD1) in neurons revealed that both proteins were slightly enriched at the trans-Golgi network (TGN) and did not interfere with its thread-like morphology. By contrast, expression of dominant-negative kinase inactive PKD1K612W (kdPKD1) led to the disruption of the neuronal Golgi complex, with kdPKD1 strongly localized to the TGN fragments. Similar findings were obtained from transgenic mice with inducible, neuron-specific expression of kdPKD1-EGFP. As a prominent consequence of kdPKD1 expression, the dendritic tree of transfected neurons was reduced, whereas caPKD1 increased dendritic arborization. Our results thus provide direct evidence that PKD activity is selectively involved in the maintenance of dendritic arborization and Golgi structure of hippocampal neurons.

scholarly journals Epidermolysis Bullosa Simplex-Type Mutations Alter the Dynamics of the Keratin Cytoskeleton and Reveal a Contribution of Actin to the Transport of Keratin Subunits

2004 ◽  
Vol 15 (3) ◽  
pp. 990-1002 ◽  
Author(s):  
Nicola Susann Werner ◽  
Reinhard Windoffer ◽  
Pavel Strnad ◽  
Christine Grund ◽  
Rudolf Eberhard Leube ◽  
...  
Keyword(s):  
Epidermolysis Bullosa ◽  
Fluorescent Protein ◽  
Dynamic Equilibrium ◽  
Yellow Fluorescent Protein ◽  
Dominant Negative ◽  
Cell Periphery ◽  
Epidermolysis Bullosa Simplex ◽  
Enhanced Yellow Fluorescent Protein ◽  
Dominant Negative Mutation

Dominant keratin mutations cause epidermolysis bullosa simplex by transforming keratin (K) filaments into aggregates. As a first step toward understanding the properties of mutant keratins in vivo, we stably transfected epithelial cells with an enhanced yellow fluorescent protein-tagged K14R125C mutant. K14R125C became localized as aggregates in the cell periphery and incorporated into perinuclear keratin filaments. Unexpectedly, keratin aggregates were in dynamic equilibrium with soluble subunits at a half-life time of <15 min, whereas filaments were extremely static. Therefore, this dominant-negative mutation acts by altering cytoskeletal dynamics and solubility. Unlike previously postulated, the dominance of mutations is limited and strictly depends on the ratio of mutant to wild-type protein. In support, K14R125C-specific RNA interference experiments resulted in a rapid disintegration of aggregates and restored normal filaments. Most importantly, live cell inhibitor studies revealed that the granules are transported from the cell periphery inwards in an actin-, but not microtubule-based manner. The peripheral granule zone may define a region in which keratin precursors are incorporated into existing filaments. Collectively, our data have uncovered the transient nature of keratin aggregates in cells and offer a rationale for the treatment of epidermolysis bullosa simplex by using short interfering RNAs.

scholarly journals The jaagsiekte sheep retrovirus envelope gene induces transformation of the avian fibroblast cell line DF-1 but does not require a conserved SH2 binding domain

2002 ◽  
Vol 83 (11) ◽  
pp. 2733-2742 ◽  
Author(s):  
Thomas E. Allen ◽  
Kate J. Sherrill ◽  
Sara M. Crispell ◽  
Matthew R. Perrott ◽  
Jonathan O. Carlson ◽  
...  
Keyword(s):  
Cell Line ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Mutational Analysis ◽  
Morphological Changes ◽  
Fibroblast Cell ◽  
Envelope Gene ◽  
Avian Embryo ◽  
Fibroblast Cell Line ◽  
Jaagsiekte Sheep Retrovirus

Ovine pulmonary adenocarcinoma, caused by jaagsiekte sheep retrovirus (JSRV), is a naturally occurring retrovirus-induced pulmonary neoplasm of sheep. We report here that expression of the JSRV env gene is sufficient to transform an avian embryo fibroblast cell line, DF-1. DF-1 cells transfected with an avian sarcoma–leukaemia retroviral expression vector containing the JSRV env gene [pRCASBP(A)-J:env] exhibited changes consistent with transformation, including contraction and rounding of cells with formation of dense foci. Transfection with a reporter construct expressing the green fluorescent protein did not induce morphological changes in DF-1 cells, eliminating the possibility that the vector, the transfection protocol or culturing techniques were responsible for the transformed phenotype. When pRCASBP(A)-J:env-transfected cells were inoculated into nude mice, tumours formed, verifying that the DF-1 cells were tumorigenic. Analysis of the JSRV env gene revealed a conserved tyrosine (597) and methionine (600) residue in the cytoplasmic tail within the transmembrane domain of the envelope, which creates a known binding site of SH2 domains in the p85 subunit of phosphatidylinositol 3-kinase. However, when this tyrosine residue was mutated to serine or alanine, transformation was not affected. Furthermore, mutation of the methionine residue to valine or leucine also failed to eliminate JSRV env-mediated transformation. These results are in contrast to mutational analysis performed in JSRV env-transformed murine NIH-3T3 cells in which both the tyrosine and methionine residues are necessary for transformation. These findings suggest that more than one mechanism may be involved in JSRV env-mediated transformation.

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