RESUMEN

Hirano bodies are paracrystalline F-actin-rich structures associated with diverse conditions, including neurodegeneration and aging. Generation of model Hirano bodies using altered forms of Dictyostelium 34-kDa actin-bundling protein allows studies of their physiological function and mechanism of formation. We describe a novel 34-kDa protein mutant, E60K, with a point mutation within the inhibitory domain of the 34-kDa protein. Expression of E60K in Dictyostelium induces the formation of model Hirano bodies. The E60K protein has activated actin binding and is calcium regulated, unlike other forms of the 34-kDa protein that induce Hirano bodies and that have activated actin binding but lack calcium regulation. Actin filaments in the presence of E60K in vitro show enhanced resistance to disassembly induced by latrunculin B. Actin filaments in model Hirano bodies are also protected from latrunculin-induced depolymerization. We used nocodazole and blebbistatin to probe the role of the microtubules and myosin II, respectively, in the formation of model Hirano bodies. In the presence of these inhibitors, model Hirano bodies can form but are smaller than controls at early times of formation. The ultrastructure of model Hirano bodies did not reveal any major difference in structure and organization in the presence of inhibitors. In summary, these results support the conclusion that formation of model Hirano bodies is promoted by gain-of-function actin filament bundling, which enhances actin filament stabilization. Microtubules and myosin II contribute to but are not required for formation of model Hirano bodies.

Asunto(s)

Citoesqueleto de Actina/metabolismo , Dictyostelium/metabolismo , Cuerpos de Inclusión/metabolismo , Proteínas Protozoarias/metabolismo , Citoesqueleto de Actina/genética , Actinas/genética , Actinas/metabolismo , Dictyostelium/genética , Cuerpos de Inclusión/genética , Mutación Missense , Proteínas Protozoarias/genética

RESUMEN

Glycolysis is essential to Trypanosoma brucei, the causative agent of African sleeping sickness, suggesting enzymes in the pathway could be targets for drug development. Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one, EbSe) was identified in a screen as a potent inhibitor of T. brucei hexokinase 1 (TbHK1), the first enzyme in the pathway. EbSe has a history of promiscuity as an enzyme inhibitor, inactivating proteins through seleno-sulfide conjugation with Cys residues. Indeed, dilution of TbHK1 and inhibitor following incubation did not temper inhibition suggesting conjugate formation. Using mass spectrometry to analyze EbSe-based modifications revealed that two Cys residues (C327 and C369) were oxidized after treatment. Site-directed mutagenesis of C327 led to enzyme inactivation indicating that C327 was essential for catalysis. C369 was not essential, suggesting that EbSe inhibition of TbHK1 was the consequence of modification of C327 via thiol oxidation. Additionally, neither EbSe treatment nor mutation of the nine TbHK1 Cys residues appreciably altered enzyme quaternary structure.

RESUMEN

Hirano bodies are cytoplasmic inclusions composed mainly of actin and actin-associated proteins. The formation of Hirano bodies during various neurodegenerative disorders, including Alzheimer's disease and amyotrophic lateral sclerosis, has been reported. Although the underlying molecular mechanisms that lead to the formation of these inclusions in the brain are not known, expression of the C-terminal fragment (CT) (amino acids 124 to 295) from the endogenous 34-kDa actin-binding protein of Dictyostelium discoideum leads to the formation of actin inclusions in vivo. In the current study, we report the development of an inducible expression system to study the early phases of Hirano body formation using an inducible promoter system (rnrB). By fusing the CT to a green fluorescent protein (CT-GFP), we monitored protein expression and localization by fluorescence microscopy, flow cytometry, and Western blot analysis. We observed an increase in the number and size of inclusions formed following induction of the CT-GFP vector system. Time-lapse microscopy studies revealed that the CT-GFP foci associated with the cell cortex and fused to form a single large aggregate. Transmission electron microscopy further demonstrates that these inclusions have a highly ordered ultrastructure, a pathological hallmark of Hirano bodies observed in postmortem brain samples from patients with various neurodegenerative disorders. Collectively, this system provides a method to visualize and characterize the events that surround early actin inclusion formation in a eukaryotic model.

Asunto(s)

Enfermedad de Alzheimer/metabolismo , Dictyostelium/metabolismo , Expresión Génica , Cuerpos de Inclusión/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Protozoarias/metabolismo , Actinas/genética , Actinas/metabolismo , Enfermedad de Alzheimer/genética , Secuencias de Aminoácidos , Animales , Dictyostelium/química , Dictyostelium/genética , Humanos , Cuerpos de Inclusión/química , Cuerpos de Inclusión/genética , Proteínas de Microfilamentos/química , Modelos Biológicos , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética

RESUMEN

Hirano bodies are paracrystalline actin filament-containing structures reported to be associated with a variety of neurodegenerative diseases. However, the biological function of Hirano bodies remains poorly understood, since nearly all prior studies of these structures were done with postmortem samples of tissue. In the present study, we generated a full-length form of a Dictyostelium 34-kDa actin cross-linking protein with point mutations in the first putative EF hand, termed 34-kDa DeltaEF1. The 34-kDa DeltaEF1 protein binds calcium normally but has activated actin binding that is unregulated by calcium. The expression of the 34-kDa DeltaEF1 protein in Dictyostelium induces the formation of Hirano bodies, as assessed by both fluorescence microscopy and transmission electron microscopy. Dictyostelium cells bearing Hirano bodies grow normally, indicating that Hirano bodies are not associated with cell death and are not deleterious to cell growth. Moreover, the expression of the 34-kDa DeltaEF1 protein rescues the phenotypes of cells lacking the 34-kDa protein and cells lacking both the 34-kDa protein and alpha-actinin. Finally, the expression of the 34-kDa DeltaEF1 protein also initiates the formation of Hirano bodies in cultured mouse fibroblasts. These results show that the failure to regulate the activity and/or affinity of an actin cross-linking protein can provide a signal for the formation of Hirano bodies. More generally, the formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton.

Asunto(s)

Actinas/metabolismo , Dictyostelium/metabolismo , Cuerpos de Inclusión/metabolismo , Mutación/genética , Enfermedades Neurodegenerativas/metabolismo , Proteínas Protozoarias/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Diferenciación Celular/genética , División Celular/genética , Células Cultivadas , Dictyostelium/crecimiento & desarrollo , Dictyostelium/ultraestructura , Fibroblastos/metabolismo , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/ultraestructura , Ratones , Microscopía Electrónica , Peso Molecular , Proteínas Protozoarias/genética

RESUMEN

The actin cytoskeleton is sensitive to changes in calcium, which affect contractility, actin-severing proteins, actin-crosslinking proteins and calmodulin-regulated enzymes. To dissect the role of calcium control on the activity of individual proteins from effects of calcium on other processes, calcium-insensitive forms of these proteins were prepared and introduced into living cells to replace a calcium-sensitive form of the same protein. Crosslinking and bundling of actin filaments by the Dictyostelium 34 kDa protein is inhibited in the presence of micromolar free calcium. A modified form of the 34 kDa protein with mutations in the calcium binding EF hand (34 kDa deltaEF2) was prepared using site-directed mutagenesis and expressed in E. coli. Equilibrium dialysis using [(45)Ca]CaCl(2) revealed that the wild-type protein is able to bind one calcium ion with a Kd of 2.4 microM. This calcium binding is absent in the 34 kDa deltaEF2 protein. The actin-binding activity of the 34 kDa deltaEF2 protein was equivalent to wildtype but calcium insensitive in vitro. The wild-type and 34 kDa deltaEF2 proteins were expressed in 34-kDa-null and 34 kDa/alpha-actinin double null mutant Dictyostelium strains to test the hypothesis that calcium regulation of actin crosslinking is important in vivo. The 34 kDa deltaEF2 failed to supply function of the 34 kDa protein important for control of cell size and for normal growth to either of these 34-kDa-null strains. Furthermore, the distribution of the 34 kDa protein and actin were abnormal in cells expressing 34 kDa deltaEF2. Thus, calcium regulation of the formation and/or dissolution of crosslinked actin structures is required for dynamic behavior of the actin cytoskeleton important for cell structure and growth.

Asunto(s)

Citoesqueleto de Actina/metabolismo , Señalización del Calcio/fisiología , Calcio/metabolismo , Citoesqueleto/metabolismo , Dictyostelium/metabolismo , Proteínas de Microfilamentos/metabolismo , Actinina/genética , Actinina/metabolismo , Animales , Sitios de Unión/genética , Células Cultivadas , Proteínas de Microfilamentos/genética , Peso Molecular , Mutación/genética , Unión Proteica/genética

Asunto(s)

Actinas/metabolismo , Citoplasma/metabolismo , Citoplasma/patología , Hipocampo/metabolismo , Hipocampo/patología , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Actinas/ultraestructura , Animales , Técnicas de Cultivo de Célula , Citoplasma/ultraestructura , Dictyostelium , Hipocampo/ultraestructura , Proteínas de Microfilamentos/ultraestructura , Proteínas Asociadas a Microtúbulos/ultraestructura , Unión Proteica , Desnaturalización Proteica

RESUMEN

We report the serendipitous development of the first cultured cell models of Hirano bodies. Myc-epitope-tagged forms of the 34 kDa actin bundling protein (amino acids 1-295) and the CT fragment (amino acids 124-295) of the 34 kDa protein that exhibits activated actin binding and calcium-insensitive actin filament crosslinking activity were expressed in Dictyostelium and mammalian cells to assess the behavior of these modified forms in vivo. Dictyostelium cells expressing the CT-myc fragment: (1) form ellipsoidal regions that contain ordered assemblies of F-actin, CT-myc, myosin II, cofilin and alpha-actinin; (2) grow and develop more slowly than wildtype, but produce normal morphogenetic structures; (3) perform pinocytosis and phagocytosis normally; and (4) produce a level of total actin equivalent to wildtype, but a higher level of F-actin. The paracrystalline inclusions bear a striking resemblance to Hirano bodies, which are associated with a number of pathological conditions. Furthermore, expression of the CT fragment in murine L cells results in F-actin rearrangements characterized by loss of stress fibers, accumulation of numerous punctate foci, and large perinuclear aggregates, the Hirano bodies. Thus, failure to regulate the activity and/or affinity of an actin crosslinking protein can provide a signal for formation of Hirano bodies. More generally, formation of Hirano bodies is a cellular response to or a consequence of aberrant function of the actin cytoskeleton. The results reveal that formation of Hirano bodies is not necessarily related to cell death. These cultured cell models should facilitate studies of the biochemistry, genetics and physiological effects of Hirano bodies.

Asunto(s)

Actinas/metabolismo , Dictyostelium/metabolismo , Cuerpos de Inclusión/metabolismo , Proteínas de Microfilamentos/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Actinas/genética , Actinas/ultraestructura , Animales , Células Cultivadas , Dictyostelium/genética , Dictyostelium/ultraestructura , Modelos Animales de Enfermedad , Endocitosis/genética , Epítopos/genética , Epítopos/metabolismo , Vectores Genéticos/genética , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/ultraestructura , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/ultraestructura , Microscopía Electrónica , Peso Molecular , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Pliegue de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

The kinetics of binding, uptake and degradation of bacteria by vegetative Dictyostelium amoeba using Escherichia coli expressing the recombinant fluorescent protein DsRed have been characterized. There are significant advantages to using DsRed-expressing bacteria for phagocytosis assays. Stable expression of the fluorescent protein, DsRed, provides living bacteria with a bright internal fluorescent signal that is degradable in the phagolysosomal pathway. Unlike assays with chemically labelled bacteria or latex beads, the bacteria are alive and possess a natural, unaltered external surface for receptor interaction. Dictyostelium cells rapidly bind and phagocytose DsRed bacteria. Pulse-chase experiments show that the signal derived from DsRed is degraded with a half-life of approximately 45 min. To distinguish internalized bacteria from those bound to the surface, an assay was developed in which sodium azide was used to release surface-bound particles. Surprisingly, surface particle release appears to be independent of myosin II function. Using this assay it was shown that the uptake of bacteria into cells is extremely rapid. After 1 min incubation, 20% of the signal is derived from internalized bacteria. The proportion of the signal from internalized bacteria increases gradually and reaches 50% at steady state. This assay will be useful in investigations of the molecular machinery of phagocytosis and post-internalization vesicle trafficking.

Asunto(s)

Dictyostelium/microbiología , Dictyostelium/fisiología , Fagocitosis/fisiología , Animales , Adhesión Bacteriana , Escherichia coli/genética , Expresión Génica , Cinética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Miosina Tipo II/fisiología , Fagosomas/microbiología , Fagosomas/fisiología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo