Ruth Furukawa

The Structure, Function, and Assembly of Actin Filament Bundles

The cellular organization, function, and molecular composition of selected biological systems with prominent actin filament bundles are reviewed. An overall picture of the great variety of functions served by actin bundles emerges from this overview. A unifying theme is that the actin cross-linking proteins are conserved throughout the eukaryotic kingdom and yet assembled in a variety of combinations to produce actin bundles of differing functions. Mechanisms of actin bundle formation in vitro are considered illustrating the variety of physical and chemical driving forces in this exceedingly complex process. Our limited knowledge regarding the formation of actin filament bundles in vivo is contrasted with the elegant biophysical studies performed in vitro but nonetheless reveals that interactions with membranes, nucleation sites, and other organizational components must contribute to formation of actin bundles in vivo.

Calcium regulation of actin crosslinking is important for function of the actin cytoskeleton in Dictyostelium

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 ΔEF2) was prepared using site-directed mutagenesis and expressed in E. coli. Equilibrium dialysis using [45Ca]CaCl2 revealed that the wild-type protein is able to bind one calcium ion with a Kd of 2.4 μM. This calcium binding is absent in the 34 kDa ΔEF2 protein. The actin-binding activity of the 34 kDaΔ EF2 protein was equivalent to wildtype but calcium insensitive in vitro. The wild-type and 34 kDa ΔEF2 proteins were expressed in 34-kDa-null and 34 kDa/α-actinin double null mutant Dictyostelium strains to test the hypothesis that calcium regulation of actin crosslinking is important in vivo. The 34 kDa ΔEF2 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 ΔEF2. 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.

Formation of Hirano bodies induced by expression of an actin cross-linking protein with a gain-of-function mutation.

ABSTRACT 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 ΔEF1. The 34-kDa ΔEF1 protein binds calcium normally but has activated actin binding that is unregulated by calcium. The expression of the 34-kDa ΔEF1 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 ΔEF1 protein rescues the phenotypes of cells lacking the 34-kDa protein and cells lacking both the 34-kDa protein and α-actinin. Finally, the expression of the 34-kDa ΔEF1 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.

A cell culture model for investigation of Hirano bodies

Hirano bodies are paracrystalline F-actin-rich aggregations associated with a variety of conditions including aging, and neurodegenerative diseases. The composition and structure of these inclusions have been described by immunohistochemistry and ultrastructure, respectively. However, studies of the physiological function and dynamics of Hirano bodies have been hindered due to lack of a facile in vitro experimental system. We have developed a model for formation of Hirano bodies in mammalian cell cultures by expression of the carboxy-terminal fragment (CT) of a 34-kDa actin-bundling protein. Expression of the CT protein induces F-actin rearrangement in HEK 293, HeLa, Cos7 cells, neuroblastoma and astrocytic cells, and in primary neurons. We have termed these structures model Hirano bodies, since their composition and ultrastructure is quite similar to that reported in vivo. Model Hirano bodies in cell cultures sometimes appeared to be formed of a number of smaller domains, suggesting that small aggregates are intermediates in the formation of Hirano bodies. Stable lines expressing CT and bearing model Hirano bodies exhibit normal growth, morphology, and motility. This model provides a valuable system for the study of the dynamics of Hirano bodies, and their role in disease processes.

Autophagy contributes to degradation of Hirano bodies

Hirano bodies are actin-rich inclusions reported most frequently in the hippocampus in association with a variety of conditions including neurodegenerative diseases, and aging. We have developed a model system for formation of Hirano bodies in Dictyostelium and cultured mammalian cells to permit detailed studies of the dynamics of these structures in living cells. Model Hirano bodies are frequently observed in membrane-enclosed vesicles in mammalian cells consistent with a role of autophagy in the degradation of these structures. Clearance of Hirano bodies by an exocytotic process is supported by images from electron microscopy showing extracellular release of Hirano bodies, and observation of Hirano bodies in the culture medium of Dictyostelium and mammalian cells. An autophagosome marker protein Atg8-GFP, was co-localized with model Hirano bodies in wild type Dictyostelium cells, but not in atg5- or atg1-1 autophagy mutant strains. Induction of model Hirano bodies in Dictyostelium with a high level expression of 34 kDa ΔEF1 from the inducible discoidin promoter resulted in larger Hirano bodies and a cessation of cell doubling. The degradation of model Hirano bodies still occurred rapidly in autophagy mutant (atg5-) Dictyostelium, suggesting that other mechanisms such as the ubiquitin-mediated proteasome pathway could contribute to the degradation of Hirano bodies. Chemical inhibition of the proteasome pathway with lactacystin, significantly decreased the turnover of Hirano bodies in Dictyostelium providing direct evidence that autophagy and the proteasome can both contribute to degradation of Hirano bodies. Short term treatment of mammalian cells with either lactacystin or 3-methyl adenine results in higher levels of Hirano bodies and a lower level of viable cells in the cultures, supporting the conclusion that both autophagy and the proteasome contribute to degradation of Hirano bodies.

Alterations in synaptic plasticity coincide with deficits in spatial working memory in presymptomatic 3xTg-AD mice

Alzheimers disease is a neurodegenerative condition believed to be initiated by production of amyloid-beta peptide, which leads to synaptic dysfunction and progressive memory loss. Using a mouse model of Alzheimers disease (3xTg-AD), an 8-arm radial maze was employed to assess spatial working memory. Unexpectedly, the younger (3month old) 3xTg-AD mice were as impaired in the spatial working memory task as the older (8month old) 3xTg-AD mice when compared with age-matched NonTg control animals. Field potential recordings from the CA1 region of slices prepared from the ventral hippocampus were obtained to assess synaptic transmission and capability for synaptic plasticity. At 3months of age, the NMDA receptor-dependent component of LTP was reduced in 3xTg-AD mice. However, the magnitude of the non-NMDA receptor-dependent component of LTP was concomitantly increased, resulting in a similar amount of total LTP in 3xTg-AD and NonTg mice. At 8months of age, the NMDA receptor-dependent LTP was again reduced in 3xTg-AD mice, but now the non-NMDA receptor-dependent component was decreased as well, resulting in a significantly reduced total amount of LTP in 3xTg-AD compared with NonTg mice. Both 3 and 8month old 3xTg-AD mice exhibited reductions in paired-pulse facilitation and NMDA receptor-dependent LTP that coincided with the deficit in spatial working memory. The early presence of this cognitive impairment and the associated alterations in synaptic plasticity demonstrate that the onset of some behavioral and neurophysiological consequences can occur before the detectable presence of plaques and tangles in the 3xTg-AD mouse model of Alzheimers disease.

Elongation factor 1β is an actin-binding protein

Abstract A 17 kDa polypeptide found in association with actin in cellular extracts of Dictyostelium discoideum was identified as a proteolytic fragment of eEF1β. Antibody elicited against the 17 kDa protein reacted with a single 29 kDa polypeptide in Dictyostelium , indicating that the 17 kDa peptide arises from degradation of a larger precursor. The cDNA isolated from a Dictyostelium library using this antibody as a probe encodes Dictyostelium elongation factor 1β. Amino acid degradation of the 17 kDa protein fragment confirmed the identity of the protein as eEF1β. Direct interaction of eEF1β with actin in vitro was further demonstrated in mixtures of actin with the 17 kDa protein fragment of Dictyostelium eEF1β, recombinant preparations of Dictyostelium eEF1β expressed in Escherichia coli , and the intact eEF1βγ complex purified from wheat germ. Localization of eEF1β in Dictyostelium by immunofluorescence microscopy reveals both diffuse cytoplasmic staining, and some concentration in the cortical and hyaline cytoplasm. The results support the existence of physical and functional interactions of the translation apparatus with the cytoskeleton, and suggest that eEF1β may function in a dual role both to promote the elongation phase of protein synthesis, and to interact with cytoplasmic actin.

Association of AICD and Fe65 with Hirano bodies reduces transcriptional activation and initiation of apoptosis

Hirano bodies are cytoplasmic inclusions predominantly found in the central nervous system associated with various conditions including aging and Alzheimers disease (AD). Since most studies of Hirano bodies have been performed in post-mortem samples, the physiological roles of Hirano bodies have not been investigated. Astrocytoma H4 cells were employed to test the hypothesis that Hirano bodies interact with and modulate signaling by the C-terminal fragment of amyloid-β precursor protein (AICD). We demonstrated by immunofluorescence and immunoprecipitation that model Hirano bodies accumulate AICD. Since stimulation of transcription by AICD is dependent on its interaction with the nuclear adaptor protein Fe65, we examined localization of Fe65, and employed a dual luciferase reporter assay to test the effects of Hirano bodies on AICD- and Fe65-dependent modulation of gene expression. We find that both AICD and Fe65 are co-localized in model Hirano bodies. Model Hirano bodies also down-regulate both AICD-dependent apoptosis and AICD- and Fe65-dependent transcriptional activity. Thus, association of AICD and Fe65 with Hirano bodies impedes their function in promoting apoptosis and modulating transcription.

Model Hirano Bodies Protect against Tau-Independent and Tau-Dependent Cell Death Initiated by the Amyloid Precursor Protein Intracellular Domain

The main pathological hallmarks of Alzheimer’s disease are amyloid-beta plaques and neurofibrillary tangles, which are primarily composed of amyloid precursor protein (APP) and tau, respectively. These proteins and their role in the mechanism of neurodegeneration have been extensively studied. Hirano bodies are a frequently occurring pathology in Alzheimer’s disease as well as other neurodegenerative diseases. However, the physiological role of Hirano bodies in neurodegenerative diseases has yet to be determined. We have established cell culture models to study the role of Hirano bodies in amyloid precursor protein and tau-induced cell death mechanisms. Exogenous expression of APP and either of its c-terminal fragments c31 or Amyloid Precursor Protein Intracellular Domain c58 (AICDc58) enhance cell death. The presence of tau is not required for this enhanced cell death. However, the addition of a hyperphosphorylated tau mimic 352PHPtau significantly increases cell death in the presence of both APP and c31 or AICDc58 alone. The mechanism of cell death induced by APP and its c-terminal fragments and tau was investigated. Fe65, Tip60, p53, and caspases play a role in tau-independent and tau-dependent cell death. In addition, apoptosis was determined to contribute to cell death. The presence of model Hirano bodies protected against cell death, indicating Hirano bodies may play a protective role in neurodegeneration.

Preparation of 30,000-Da actin cross-linking protein from Dictyostelium discoideum

Publisher Summary The 30,000-Da actin cross-linking protein from Dictyostelium discoideum is a monomeric molecule that cross-links actin filaments into bundies or aggregates in which the cross-linked filaments lie parallel to one another in highly organized structures in vitro. This protein is a potential direct target of calcium action in the cytosol, since binding of micromolar calcium in the presence of millimolar magnesium has been demonstrated. This chapter outlines the method for the preparation of this protein from D. discoideum, whose strain (Ax-3) is grown axenically in shaking culture, and used directly after harvesting the cultures. The yield is 5–10 ml (packed wet volume) of cells per 2-liter flask containing 750 ml of HL-5 medium. The method described below has been used with 100 to 350 g of cells. The 30,000-Da protein is purified from Ax-3 lysed by decompression of nitrogen in a Parr bomb (Parr Instruments, Moline, IL). The 30,000-Da protein is greatly enriched in the material sedimented following gelation and syneresis of the soluble extract in vitro. The method has been modified significantly from that originally employed by Fechheimer and Taylor by omission of the actomyosin precipitation step, and changes in the conditions of chromatography on DEAE-cellulose and hydroxylapatite.