RESUMO
The heat shock (HS) response is crucial for cell survival in harmful environments. Nuclear lamin A/C, encoded by the LMNA gene, contributes towards altered gene expression during HS, but the underlying mechanisms are poorly understood. Here, we show that upon HS, lamin A/C was reversibly phosphorylated at serine 22 in concert with HSF1 activation in human cells, mouse cells and Drosophila melanogaster in vivo. Consequently, the phosphorylation facilitated nucleoplasmic localization of lamin A/C and nuclear sphericity in response to HS. Interestingly, lamin A/C knock-out cells showed deformed nuclei after HS and were rescued by ectopic expression of wild-type lamin A, but not by a phosphomimetic (S22D) lamin A mutant. Furthermore, HS triggered concurrent downregulation of lamina-associated protein 2α (Lap2α, encoded by TMPO) in wild-type lamin A/C-expressing cells, but a similar response was perturbed in lamin A/C knock-out cells and in LMNA mutant patient fibroblasts, which showed impaired cell cycle arrest under HS and compromised survival at recovery. Taken together, our results suggest that the altered phosphorylation stoichiometry of lamin A/C provides an evolutionarily conserved mechanism to regulate lamina structure and serve nuclear adaptation and cell survival during HS.
Assuntos
Lamina Tipo A , Serina , Humanos , Camundongos , Animais , Lamina Tipo A/genética , Fosforilação , Serina/metabolismo , Drosophila melanogaster/metabolismo , Núcleo Celular/metabolismoRESUMO
The intestine is divided into functionally distinct regions along the anteroposterior (A/P) axis. How the regional identity influences the function of intestinal stem cells (ISCs) and their offspring remain largely unresolved. We introduce an imaging-based method, "Linear Analysis of Midgut" (LAM), which allows quantitative, regionally defined cellular phenotyping of the whole Drosophila midgut. LAM transforms image-derived cellular data from three-dimensional midguts into a linearized representation, binning it into segments along the A/P axis. Through automated multivariate determination of regional borders, LAM allows mapping and comparison of cellular features and frequencies with subregional resolution. Through the use of LAM, we quantify the distributions of ISCs, enteroblasts, and enteroendocrine cells in a steady-state midgut, and reveal unprecedented regional heterogeneity in the ISC response to a Drosophila model of colitis. Altogether, LAM is a powerful tool for organ-wide quantitative analysis of the regional heterogeneity of midgut cells.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Intestinos , Células Enteroendócrinas , Células-TroncoRESUMO
Energy storage and growth are coordinated in response to nutrient status of animals. How nutrient-regulated signaling pathways control these processes in vivo remains insufficiently understood. Here, we establish an atypical MAP kinase, ERK7, as an inhibitor of adiposity and growth in Drosophila. ERK7 mutant larvae display elevated triacylglycerol (TAG) stores and accelerated growth rate, while overexpressed ERK7 is sufficient to inhibit lipid storage and growth. ERK7 expression is elevated upon fasting and ERK7 mutant larvae display impaired survival during nutrient deprivation. ERK7 acts in the fat body, the insect counterpart of liver and adipose tissue, where it controls the subcellular localization of chromatin-binding protein PWP1, a growth-promoting downstream effector of mTOR. PWP1 maintains the expression of sugarbabe, encoding a lipogenic Gli-similar family transcription factor. Both PWP1 and Sugarbabe are necessary for the increased growth and adiposity phenotypes of ERK7 loss-of-function animals. In conclusion, ERK7 is an anti-anabolic kinase that inhibits lipid storage and growth while promoting survival on fasting conditions.
Assuntos
Adiposidade , MAP Quinases Reguladas por Sinal Extracelular , Animais , Drosophila/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fosforilação , Transdução de SinaisRESUMO
Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure and heart transplantation. A portion of familial DCM is due to mutations in the LMNA gene encoding the nuclear lamina proteins lamin A and C and without adequate treatment these patients have a poor prognosis. To get better insights into pathobiology behind this disease, we focused on modeling LMNA-related DCM using human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM). Primary skin fibroblasts from DCM patients carrying the most prevalent Finnish founder mutation (p.S143P) in LMNA were reprogrammed into hiPSCs and further differentiated into cardiomyocytes (CMs). The cellular structure, functionality as well as gene and protein expression were assessed in detail. While mutant hiPSC-CMs presented virtually normal sarcomere structure under normoxia, dramatic sarcomere damage and an increased sensitivity to cellular stress was observed after hypoxia. A detailed electrophysiological evaluation revealed bradyarrhythmia and increased occurrence of arrhythmias in mutant hiPSC-CMs on ß-adrenergic stimulation. Mutant hiPSC-CMs also showed increased sensitivity to hypoxia on microelectrode array and altered Ca2+ dynamics. Taken together, p.S143P hiPSC-CM model mimics hallmarks of LMNA-related DCM and provides a useful tool to study the underlying cellular mechanisms of accelerated cardiac degeneration in this disease.
Assuntos
Cardiomiopatia Dilatada/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Lamina Tipo A/metabolismo , Modelos Biológicos , Adulto , Animais , Arritmias Cardíacas/complicações , Arritmias Cardíacas/patologia , Cálcio/metabolismo , Cardiomiopatia Dilatada/complicações , Agregação Celular , Diferenciação Celular , Linhagem Celular , Feminino , Humanos , Hipóxia/patologia , Masculino , Camundongos , Microeletrodos , Pessoa de Meia-Idade , Isquemia Miocárdica/complicações , Isquemia Miocárdica/patologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/ultraestrutura , Sarcômeros/metabolismo , Estresse Fisiológico , Adulto JovemRESUMO
Cytoplasmic intermediate filaments (cIFs) are found in all eumetazoans, except arthropods. To investigate the compatibility of cIFs in arthropods, we expressed human vimentin (hVim), a cIF with filament-forming capacity in vertebrate cells and tissues, transgenically in Drosophila Transgenic hVim could be recovered from whole-fly lysates by using a standard procedure for intermediate filament (IF) extraction. When this procedure was used to test for the possible presence of IF-like proteins in flies, only lamins and tropomyosin were observed in IF-enriched extracts, thereby providing biochemical reinforcement to the paradigm that arthropods lack cIFs. In Drosophila, transgenic hVim was unable to form filament networks in S2 cells and mesenchymal tissues; however, cage-like vimentin structures could be observed around the nuclei in internal epithelia, which suggests that Drosophila retains selective competence for filament formation. Taken together, our results imply that although the filament network formation competence is partially lost in Drosophila, a rudimentary filament network formation ability remains in epithelial cells. As a result of the observed selective competence for cIF assembly in Drosophila, we hypothesize that internal epithelial cIFs were the last cIFs to disappear from arthropods.-Gullmets, J., Torvaldson, E., Lindqvist, J., Imanishi, S. Y., Taimen, P., Meinander, A., Eriksson, J. E. Internal epithelia in Drosophila display rudimentary competence to form cytoplasmic networks of transgenic human vimentin.
Assuntos
Citoplasma/metabolismo , Drosophila/metabolismo , Epitélio/metabolismo , Vimentina/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Linhagem Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/enzimologia , Filamentos Intermediários/metabolismo , Laminas/genética , Laminas/metabolismo , Tropomiosina/genética , Tropomiosina/metabolismo , Vimentina/genéticaRESUMO
Nestin, a member of the cytoskeletal family of intermediate filaments, regulates the onset of myogenic differentiation through bidirectional signaling with the kinase Cdk5. Here, we show that these effects are also reflected at the organism level, as there is a loss of skeletal muscle mass in nestin-/- (NesKO) mice, reflected as reduced lean (muscle) mass in the mice. Further examination of muscles in male mice revealed that these effects stemmed from nestin-deficient muscles being more prone to spontaneous regeneration. When the regeneration capacity of the compromised NesKO muscle was tested by muscle injury experiments, a significant healing delay was observed. NesKO satellite cells showed delayed proliferation kinetics in conjunction with an elevation in p35 (encoded by Cdk5r1) levels and Cdk5 activity. These results reveal that nestin deficiency generates a spontaneous regenerative phenotype in skeletal muscle that relates to a disturbed proliferation cycle that is associated with uncontrolled Cdk5 activity.
Assuntos
Homeostase , Músculo Esquelético/fisiologia , Nestina/metabolismo , Regeneração , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Quinase 5 Dependente de Ciclina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares Esqueléticas/fisiologia , Mioblastos/metabolismo , Nestina/deficiência , Tamanho do Órgão , Fenótipo , Células Satélites de Músculo Esquelético/metabolismo , CicatrizaçãoRESUMO
Keratins (K) are intermediate filament proteins important in protection from cellular stress. K8, K18 and K19 are the main components of keratin filaments in colonic epithelia but their role in intestinal diseases remains ambiguous. A function for keratins in intestinal health is supported by the K8-knock-out (K8(-/-)) mouse which manifests an early chronic ulcerative colitis-like inflammatory bowel disease and epithelial hyperproliferation. We tested whether K8(-/-) mice are more susceptible to colorectal cancer (CRC) compared to K8 wild type (K8(+/+)), and K8 heterozygote (K8(+/-)) mice showing increased proliferation but no inflammation. K8(-/-) mice did not develop CRC spontaneously, but had dramatically increased numbers of tumors in the distal colon in the azoxymethane (AOM) and Apc(Min/+) CRC models while neither K8(+/+) nor K8(+/-) mice were susceptible. Upregulation of IL-22 in combination with a complete loss of its negative regulator IL-22BP, and increased downstream STAT3-signaling in K8(-/-) and K8(-/-)Apc(Min/+) colonic epithelia confirmed that the IL-22 pathway, important in inflammation, proliferation and tissue regeneration, was activated. The nearly total loss of IL-22BP correlated with an activated inflammasome leading to increased cleaved caspase-1, and the putative IL-22BP inhibitor, IL-18, as well as a decrease in ALDH1/2. Ablation of K8 in a colorectal cancer cell line similarly resulted in increased IL-18 and decreased ALDH1/2. K8/K18 co-immunoprecipitated with pro-caspase-1, a component of the inflammasome in the colon, which suggests that keratins modulate inflammasome activity and protect the colon from inflammation and tumorigenesis. The K8-null mouse models also provide novel epithelial-derived robust colon-specific CRC models.
Assuntos
Neoplasias Colorretais/genética , Interleucinas/genética , Queratina-8/genética , Neoplasias Experimentais/genética , Família Aldeído Desidrogenase 1 , Animais , Colite/genética , Colite/patologia , Neoplasias Colorretais/patologia , Humanos , Inflamassomos/genética , Inflamação/genética , Inflamação/patologia , Interleucinas/metabolismo , Mucosa Intestinal/patologia , Isoenzimas/genética , Camundongos , Camundongos Knockout , Neoplasias Experimentais/patologia , Retinal Desidrogenase/genética , Deleção de Sequência , Interleucina 22RESUMO
Mutation of the LMNA gene, encoding nuclear lamin A and lamin C (hereafter lamin A/C), is a common cause of familial dilated cardiomyopathy (DCM). Among Finnish DCM patients, the founder mutation c.427T>C (p.S143P) is the most frequently reported genetic variant. Here, we show that p.S143P lamin A/C is more nucleoplasmic and soluble than wild-type lamin A/C and accumulates into large intranuclear aggregates in a fraction of cultured patient fibroblasts as well as in cells ectopically expressing either FLAG- or GFP-tagged p.S143P lamin A. In fluorescence loss in photobleaching (FLIP) experiments, non-aggregated EGFP-tagged p.S143P lamin A was significantly more dynamic. In in vitro association studies, p.S143P lamin A failed to form appropriate filament structures but instead assembled into disorganized aggregates similar to those observed in patient cell nuclei. A whole-genome expression analysis revealed an elevated unfolded protein response (UPR) in cells expressing p.S143P lamin A/C. Additional endoplasmic reticulum (ER) stress induced by tunicamycin reduced the viability of cells expressing mutant lamin further. In summary, p.S143P lamin A/C affects normal lamina structure and influences the cellular stress response, homeostasis and viability.