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The Dictyostelium discoideum 30 kDa actin-bundling protein cross-links actin filaments into bundles in vitro, and is present in filopodia and pseudopodia in living cells. Monoclonal antibodies reactive with this protein have been isolated, and employed as specific probes for the function of this protein. The monoclonal antibody B2C blocks the interaction of the 30 kDa protein with F-actin in vitro, and decreases phagocytosis of E. coli when introduced into living Dictyostelium cells by controlled sonication. Use of this monoclonal antibody for visualization of the 30 kDa protein by immunofluorescence microscopy reveals striking localization around food particles during the process of phagocytosis. Double staining with rhodamine-labelled phalloidin and the monoclonal antibody documents the co-localization of the 30 kDa protein and actin during formation of phagocytic cups. The dissociation of the 30 kDa protein occurs during the process of maturation to form phagolysosomes. These results support the hypothesis that this actin cross-linking protein participates in dynamic rearrangements of actin filaments accompanying phagocytosis. [ABSTRACT FROM AUTHOR]

Looks at the role of apoptosis, a morphologically and biochemically defined type of programmed cell death initiated in vaculovirus replication in insect cells. Role of programmed cell death in multicellular animals; Block of apoptopic response by the viral gene product p35; Possible identification of molecular pathways involved in programmed cell death; Possible role of apoptosis in invertebrate viral defense systems.

Hirano bodies are eosinophilic, actin-rich inclusions found in autopsied brains in numerous neurodegenerative diseases. The mechanism of Hirano body formation is unknown. Mass spectrometry analysis was performed to identify proteins from partially purified model Hirano bodies from Dictyostelium This analysis identified proteins primarily belonging to ribosomes, proteasomes, mitochondria and cytoskeleton. Profilin, Arp/2/3 and WASH identified by mass spectrometry were found to colocalise with model Hirano bodies. Due to their roles in actin regulation, we selected these proteins for further investigation. Inhibition of the Arp2/3 complex by CK666 prevented formation of model Hirano bodies. Since Arp2/3 activation occurs via the WASH or WAVE complex, we next investigated how these proteins affect Hirano body formation. Whereas model Hirano bodies could form in WASH-deficient cells, they failed to form in cells lacking HSPC300, a member of the WAVE complex. We identified other proteins required for Hirano body formation that include profilin and VASP, an actin nucleation factor. In the case of VASP, both its G- and F-actin binding domains were required for model Hirano body formation. Collectively, our results indicate that de novo actin polymerization is required to form model Hirano bodies., (© 2016. Published by The Company of Biologists Ltd.)

Alzheimer's 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 Alzheimer's 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 Alzheimer's disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: Hirano bodies are actin-rich intracellular inclusions found in the brains of patients with neurodegenerative conditions such as Alzheimer's disease or frontotemporal lobar degeneration-tau. While Hirano body ultrastructure and protein composition have been well studied, little is known about the physiological function of Hirano bodies in an animal model system., Results: Utilizing a Cre/Lox system, we have generated a new mouse model which develops an age-dependent increase in the number of model Hirano bodies present in both the CA1 region of the hippocampus and frontal cortex. These mice develop normally and experience no overt neuron loss. Mice presenting model Hirano bodies have no abnormal anxiety or locomotor activity as measured by the open field test. However, mice with model Hirano bodies develop age-dependent impairments in spatial working memory performance assessed using a delayed win-shift task in an 8-arm radial maze. Synaptic transmission, short-term plasticity, and long-term plasticity was measured in the CA1 region from slices obtained from both the ventral and dorsal hippocampus in the same mice whose spatial working memory was assessed. Baseline synaptic responses, paired pulse stimulation and long-term potentiation measurements in the ventral hippocampus were indistinguishable from control mice. In contrast, in the dorsal hippocampus, synaptic transmission at higher stimulus intensities were suppressed in 3 month old mice with Hirano bodies as compared with control mice. In addition, long-term potentiation was enhanced in the dorsal hippocampus of 8 month old mice with Hirano bodies, concurrent with observed impairment of spatial working memory. Finally, an inflammatory response was observed at 8 months of age in mice with Hirano bodies as assessed by the presence of reactive astrocytes., Conclusion: This study shows that the presence of model Hirano bodies initiates an inflammatory response, alters hippocampal synaptic responses, and impairs spatial working memory in an age-dependent manner. This suggests that Hirano bodies may promote disease progression. This new model mouse provides a tool to investigate how Hirano bodies interact with other pathologies associated with Alzheimer's disease. Hirano bodies likely play a complex and region specific role in the brain during neurodegenerative disease progression.

Background: Hirano bodies are actin-rich paracrystalline inclusions found in brains of patients with Alzheimer's disease (AD), frontotemporal dementia (FTD), and in normal aged individuals. Although studies of post-mortem brain tissue provide clues of etiology, the physiological function of Hirano bodies remains unknown. A cell culture model was utilized to study the interactions of mutant tau proteins, model Hirano bodies, and GSK3β in human astrocytoma cells., Results: Most tau variants showed co-localization with model Hirano bodies. Cosedimentation assays revealed this interaction may be direct, as recombinant purified forms of tau are all capable of binding F-actin. Model Hirano bodies had no effect or enhanced cell death induced by tau in the absence of amyloid precursor protein intracellular domain (AICD). In the presence of AICD and tau, synergistic cell death was observed in most cases, and model Hirano bodies decreased this synergistic cell death, except for forms of tau that caused significant cell death in the presence of Hirano bodies only. A role for the kinase GSK3β is suggested by the finding that a dominant negative form of GSK3β reduces this synergistic cell death. A subset of Hirano bodies in brain tissue of both Alzheimer's disease and normal aged individuals was found to contain tau, with some Hirano bodies in Alzheimer's disease brains containing hyperphosphorylated tau., Conclusion: The results demonstrate a complex interaction between tau and AICD involving activation of GSK3β in promoting cell death, and the ability of Hirano bodies to modulate this process.

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.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

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.

Hirano bodies are cytoplasmic inclusions predominantly found in the central nervous system associated with various conditions including aging and Alzheimer's 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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: Hirano bodies are actin-rich cytoplasmic inclusions found predominantly in the brain in association with a variety of conditions including aging and Alzheimer's disease. The function of Hirano bodies in normal aging and in progression of disease has not been extensively investigated due to a lack of experimental model systems. We have developed a transgenic mouse model by expression of a gain-of-function actin cross-linking protein mutant., Results: We used the Cre/loxP system to permit tissue specific expression of Hirano bodies, and employed the murine Thy 1 promoter to drive expression of Cre recombinase in the brain. Hirano bodies were observed in the cerebral cortex and hippocampus of homozygous double transgenic 6 month old mice containing Cre. The Hirano bodies were eosinophilic rods, and also exhibited the paracrystalline F-actin filament organization that is characteristic of these inclusions. Mice with Hirano bodies appear healthy and fertile, but exhibited some alterations in both short-term and long-term synaptic plasticity, including paired-pulse depression rather than facilitation, and decreased magnitude of early LTP., Conclusions: Hirano bodies are not lethal and appear to have little or no effect on histology and tissue organization. Hirano bodies do modulate synaptic plasticity and exert clearly discernable effects on LTP and paired-pulse paradigms. This model system will allow us to investigate the impact of Hirano bodies in vivo, the pathways for formation and degradation of Hirano bodies, and whether Hirano bodies promote or modulate development of pathology and disease progression.

Assessment of undergraduate research (UR) programs using participant surveys has produced a wealth of information about design, implementation, and perceived benefits of UR programs. However, measurement of student participation university wide, and the potential contribution of research experience to student success, also require the study of extrinsic measures. In this essay, institutional data on student credit-hour generation and grade point average (GPA) from the University of Georgia are used to approach these questions. Institutional data provide a measure of annual enrollment in UR classes in diverse disciplines. This operational definition allows accurate and retrospective analysis, but does not measure all modes of engagement in UR. Cumulative GPA is proposed as a quantitative extrinsic measure of student success. Initial results show that extended participation in research for more than a single semester is correlated with an increase in GPA, even after using SAT to control for the initial ability level of the students. While the authors acknowledge that correlation does not prove causality, continued efforts to measure the impact of UR programs on student outcomes using GPA or an alternate extrinsic measure is needed for development of evidence-based programmatic recommendations.

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 DeltaEF1 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.

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.

Fundamental processes in the life of Dictyostelium, such as locomotion, endocytosis, cytokinesis, and morphogenesis, are mediated by the actin cytoskeleton, which is composed of actin, myosins, and numerous actin-binding proteins. An understanding of these processes at the molecular level will require characterization of the structure, function, and dynamics of the actin and actin-binding proteins both in vivo and in vitro. Dictyostelium has more than a dozen actin cross-linking proteins that can mediate the formation of isotropic actin gels and/or anisotropic actin bundles. We describe the use of transmitted polarized light and polarized light scattering in studies of actin and actin-binding proteins during formation of nematic, gelled, or bundled structures. These methods have allowed quantitative studies of the effects of actin filament length, the concentration of actin, and the concentration of the cross-linking protein on the formation of cross-linked actin structures. Such methods hold great promise for characterization of novel cross-linking proteins, and for interpretation of phenotypes from strains lacking or expressing altered forms of these proteins. These methods are also applicable to studies of other systems such as the interactions of microtubules and microtubule associated proteins.

Actin-bundling proteins organize actin filaments into densely packed bundles. In Dictyostelium discoideum two abundant proteins display calcium-regulated bundling activity, fimbrin and the 34-kDa protein (ABP34). Using a GFP fusion we observed transient localization of fimbrin at the phagocytic cup and macropinosomes. The distribution of truncated constructs encompassing the EF hands and the first actin-binding domain (EA1) or both actin-binding domains devoid of EF hands (A1A2) was indistinguishable from that of the full length protein. The role of fimbrin and a possible functional overlap with ABP34 was investigated in fim- and double 34-/fim- mutants. Except for a moderate cell size defect, fim- mutants did not show defects in growth, endocytosis, exocytosis, and chemotaxis. Double mutants were characterized by a small cell size and a defect in morphogenesis resulting in small fruiting bodies and a low spore yield. The cell size defect could not be overcome by expression of fimbrin fragments EA1 or A1A2, suggesting that both bundling activity and regulation by calcium are important. Induction of filopod formation in 34-/fim- cells was not impaired, indicating that both proteins are dispensable for this process. We searched in the Dictyostelium genome database for fimbrin-like proteins that could compensate for the fimbrin defect and identified three unconventional fimbrins and two more proteins with actin-binding domains of the type present in fimbrins., (Copyright 2005 Wiley-Liss, Inc)

The rickettsial pathogen Anaplasma marginale assembles an actin filament bundle during intracellular infection. Unlike other bacterial pathogens that generate actin filament tails, A. marginale infects mature erythrocytes, and the F-actin appendages are assembled on the cytoplasmic surface of a vacuole containing several organisms. To identify A. marginale molecules associated with these filaments, two complementary approaches were used: matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and tandem mass spectrometry of A. marginale proteins identified with an appendage-specific monoclonal antibody and expression screening of an A. marginale phage library. Amino acid and nucleotide sequences were mapped to a full-length gene in the genome of the St. Maries strain of A. marginale; the correct identification was confirmed by expression of full-length recombinant protein and its reactivity with appendage-specific antibodies. Interestingly, there is marked variation in the abilities of diverse A. marginale strains to assemble the F-actin appendages. Comparison of four strains, the Florida, Illinois, St. Maries, and Virginia strains, revealed substantial polymorphism in the gene encoding the appendage-associated protein, with amino acid sequence identity of as low as 34% among strains. However, this variation does not underlie the differences in expression, as there is no specific polymorphism associated with loss of ability to assemble actin appendages. In contrast, the ability to assemble an actin filament bundle reflected dramatic strain-specific differences in the expression level of the appendage-associated protein. Understanding how this protein influences the cycle of invasion, replication, and egress in the host cell may provide new insights into pathogen-host interactions.

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.

In the past decade the first Arabidopsis genes encoding cytoskeletal proteins were identified. A few dozen genes in the actin and tubulin cytoskeletal systems have been characterized thoroughly, including gene families encoding actins, profilins, actin depolymerizing factors, α-tubulins, and β-tubulins. Conventional molecular genetics have shown these family members to be differentially expressed at the temporal and spatial levels with an ancient split separating those genes expressed in vegetative tissues from those expressed in reproductive tissues. A few members of other cytoskeletal gene families have also been partially characterized, including an actin-related protein, annexins, fimbrins, kinesins, myosins, and villins. In the year 2001 the Arabidopsis genome sequence was completed. Based on sequence homology with well-characterized animal, fungal, and protist sequences, we find candidate cytoskeletal genes in the Arabidopsis database: more than 150 actin-binding proteins (ABPs), including monomer binding, capping, cross-linking, attachment, and motor proteins; more than 200 microtubule-associated proteins (MAPs); and, surprisingly, 10 to 40 potential intermediate filament (IF) proteins. Most of these sequences are uncharacterized and were not identified as related to cytoskeletal proteins. Several Arabidopsis ABPs, MAPs, and IF proteins are represented by individual genes and most were represented as as small gene families. However, several classes of cytoskeletal genes including myosin, eEF1α, CLIP, tea1, and kinesin are part of large gene families with 20 to 70 potential gene members each. This treasure trove of data provides an unprecedented opportunity to make rapid advances in understanding the complex plant cytoskeletal proteome. However, the functional analysis of these proposed cytoskeletal proteins and their mutants will require detailed analysis at the cell biological, molecular genetic, and biochemical levels. New approaches will be needed to move more efficiently and rapidly from this mass of DNA sequence to functional studies on cytoskeletal proteins.

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.