Actin cytoskeletal network in aging and cancer.

The cytoskeleton is being recognized as an important modulator of metabolic functions of the cell. The actin cytoskeletal network, in particular, is involved in events regulating cell proliferation and differentiation. The state of actin in a variety of cell types is regulated by signals arising from the cell surface through a wide spectrum of interactions. In this review, we explore the role of actin cytoskeletal network in a series of events which are known to influence cell proliferation and differentiation. These include interaction of actin network with extracellular matrix proteins, cell surface membranes, second messengers, cytoplasmic enzymes and the nucleus. Because of the involvement of the actin network in such diverse interactions, we propose that alterations in the actin cytoskeletal function may be an important aspect of generalized decrease in cellular functions associated with aging. Preliminary data indicate that alterations in the cytoskeletal network do occur in cells obtained from older individuals. Alterations in actin state are also reported during malignant transformation of cells in culture, and in naturally occurring tumors. Taken together, the existing data seem to suggest that changes in the actin cytoskeletal network may be a part of the aging process as well as malignant transformation. Therefore, the study of the actin cytoskeletal network and its regulation has the potential to yield important information regarding cellular senescence and neoplastic transformation.     

How depolymerization can promote polymerization: the case of actin and profilin

Rapid polymerization and depolymerization of actin filaments in response to extracellular stimuli is required for normal cell motility and development. Profilin is one of the most important actin‐binding proteins; it regulates actin polymerization and interacts with many cytoskeletal proteins that link actin to extracellular membrane. The molecular mechanism of profilin has been extensively considered and debated in the literature for over two decades. Here we discuss several accepted hypotheses regarding the mechanism of profilin function as well as new recently emerged possibilities. Thermal noise is routine in molecular world and unsurprisingly, nature has found a way to utilize it. An increasing amount of theoretical and experimental research suggests that fluctuation‐based processes play important roles in many cell events. Here we show how a fluctuation‐based process of exchange diffusion is involved in the regulation of actin polymerization.     

Actin polymerization and pseudopod extension during amoeboid chemotaxis

Amoebae of the cellular slime mold Dictyostelium discoideum are an excellent model system for the study of amoeboid chemotaxis. These cells can be studied as a homogeneous population whose response to chemotactic stimulation is sufficiently synchronous to permit the correlation of the changes in cell shape and biochemical events during chemotaxis. Having demonstrated this synchrony of response, we show that actin polymerization occurs in two stages during stimulation with chemoattractants. The assembly of F-actin that peaks between 40 and 60 sec after the onset of stimulation is temporally correlated with the growth of new pseudopods. F-actin, which is assembled by 60 sec after stimulation begins, is localized in the new pseudopods that are extended at this time. Both stages of actin polymerization during chemotactic stimulation involve polymerization at the barbed ends of actin filaments based on the cytochalasin sensitivity of this response. We present a hypothesis in which actin polymerization is one of the major driving forces for pseudopod extension during chemotaxis. The predictions of this model, that localized regulation of actin nucleation activity and actin filament cross-linking must occur, are discussed in the context of current models for signal transduction and of recent information regarding the types of actin-binding proteins that are present in the cell cortex.     

Trypanosoma cruzi-cardiomyocytes: new contributions regarding a better understanding of this interaction

The present paper summarizes new approaches regarding the progress done to the understanding of the interaction of Trypanosoma cruzi-cardiomyocytes. Mannose receptors localized at the surface of heart muscle cell are involved in binding and uptake of the parasite. One of the most striking events in the parasite-heart muscle cells interaction is the disruption of the actin cytoskeleton. We have investigated the regulation of the actin mRNA during the cytopathology induced in myocardial cells by the parasite. T. cruzi invasion increases calcium resting levels in cardiomyocytes. We have previously shown that Ca2+ ATPase of the sarcoplasmic reticulum (SERCA) is involved in the invasion of T. cruzi in cardiomyocytes. Treating the cells with thapsigargin, a drug that binds to all SERCA ATPases and causes depletion of intracellular calcium stores, we found a 75% inhibition in the T. cruzi-cardiomyocytes invasion.     

Filamin is essential for shedding of the transmembrane serine protease, epithin

Epithin is a type II transmembrane serine protease that exists in a soluble and membrane-bound form. Shedding is thought to be important in regulating its action, but little is known regarding the intracellular events that trigger such shedding. Here, we show that phorbol myristate acetate (PMA) causes the release of epithin. It also causes accumulation of the protein at the site of cell-cell contacts, and this accumulation is dependent on the formation of cortical actin. In addition, we have identified the actin-binding protein, filamin, as the linker between epithin and the actin cytoskeleton. The interaction of epithin and filamin was enhanced by PMA, and epithin was not released from filamin-deficient M2 cells. We also show that the release of epithin does not require its own activity and is blocked by a metalloprotease inhibitor, GM6001. These results show that filamin has an essential role in shedding by linking epithin to the as yet unidentified metalloprotease-shedding enzyme(s).     

Use of interplasmid recombination to generate stable selectable markers for yeast transformation: application to studies of actin gene control

A plasmid recombination system has been developed that relies upon interplasmid exchanges for yeast cell viability. Two types of plasmids, one carrying the LEU2 allele inserted within yeast actin gene sequences and the other carrying 2-microns plasmid DNA and an intact actin gene, were constructed. Neither plasmid alone yielded transformants in the haploid Leu- strain AH22, but when cotransformed, a number of colonies were obtained. Southern blot analysis revealed that transformants arose because of recombination events within the homologous actin sequences that transferred the LEU2 gene to the actin gene on the 2-microns plasmid. The recombinant plasmids could be recovered, and sequence analysis of one recombination site revealed that the exchange event was faithful at the nucleotide level. The resulting recombinant plasmids carried a defective actin gene and presumably arose because of a double-crossover event. Deletion mutations that prevented actin gene expression on one donor plasmid enabled the recovery at a high frequency of transformants resulting primarily from single-crossover events between the two plasmids. This was presumably because such events no longer generated an intact actin gene on a multicopy plasmid. Infrequently a transformant from a plasmid with an intact gene was recovered, but in these cases the plasmid was not present in multiple copies. These cells exhibited a slower growth rate, and Northern blot analysis revealed an elevated level of actin mRNA.     

Changes in actin organization in the living egg apparatus of Torenia fournieri during fertilization

Changes in actin organization in the living egg apparatus of Torenia fournieri from anthesis to post-fertilization have been investigated using microinjection and confocal microscopy. Our results revealed that the actin cytoskeleton displays dramatic changes in the egg apparatus and appears to coordinate the events of synergid degeneration, pollen tube arrival and gametic fusion during fertilization. Synergid degeneration occurs after anthesis and is accompanied by actin fragmentation and degradation. The actin cytoskeleton becomes organized with numerous aggregates in the chalazal end of the degenerating synergid, and some of the actin infiltrates into the intercellular gap between synergids, egg and central cell, forming a distinct actin band. An actin cap is present near the filiform apparatus after anthesis and disappears after pollen tube arrival. In the egg cell, actin filaments initially organize into a network and after pollination become fragmented into numerous patches in the cortex. These structures, along with the actin in the degenerating synergid and intercellular spaces form two distinct actin coronas during fertilization. The actin coronas vanish after gametic fusion. This is the first report of changes in actin organization in the living egg apparatus. The reorganization of the actin cytoskeleton in the egg apparatus and the presence of the actin coronas during fertilization suggest these events may be a necessary prelude to reception of the pollen tube and fusion of the male and female gametes. Received: 11 November 1999 / Accepted: 31 January 2000     

Unique actin and microtubule arrays co-ordinate the differentiation of microspores to mature pollen in Nicotiana tabacum

The complex cellular events that occur during development of the male gametophyte of higher plants suggest a role for the cytoskeleton. This investigation has revealed that unique microtubule arrays mediate events that occur during microspore development; both actin and microtubule arrays have important roles during the asymmetrical microspore mitosis and unique actin arrays mediate events that occur during early pollen development. Migration of the nucleus to the generative pole during cellular polarization of the microspore is mediated by a microtubule cage that encloses the nucleus. Nuclear position at the generative pole is maintained by an actin net that tethers it to the pole prior to the asymmetrical mitosis. During entry into mitosis, the microtubule cage becomes modified and transforms into the asymmetrical mitotic spindle. Actin is localized within the region of the mitotic spindle and in the phragmoplast. Following mitosis, actin networks enclose first the generative cell and then the vegetative nucleus. These actin networks function during migration of the generative cell and vegetative nucleus toward the centre of the pollen grain. Mature pollen contains a dense cortical actin meshwork and a disc-shaped microtubule array enclosing the generative cell. The functional importance of the unique actin and microtubule arrays is verified by their targeted disruption with specific cytoskeletal inhibitors, which disrupt normal development and cellular morphology. In summary, these data provide evidence that the co-ordinated reorganization of unique actin and microtubule arrays is an essential determinant of microspore and pollen development.     

Actin cytoskeleton regulates calcium dynamics and NFAT nuclear duration

T-cell activation by antigen-presenting cells is accompanied by actin polymerization, T-cell receptor (TCR) capping, and formation of the immunological synapse. However, whether actin-dependent events are required for T-cell function is poorly understood. Herein, we provide evidence for an unexpected negative regulatory role of the actin cytoskeleton on TCR-induced cytokine production. Disruption of actin polymerization resulted in prolonged intracellular calcium elevation in response to anti-CD3, thapsigargin, or phorbol myristate acetate plus ionomycin, leading to persistent NFAT (nuclear factor of activated T cells) nuclear duration. These events were dominant, as the net effect of actin blockade was augmented interleukin 2 promoter activity. Increased surface expression of the plasma membrane Ca(2+) ATPase was observed upon stimulation, which was inhibited by cytochalasin D, suggesting that actin polymerization contributes to calcium export. Our results imply a novel role for the actin cytoskeleton in modulating the duration of Ca(2+)-NFAT signaling and indicate that actin dynamics regulate features of T-cell activation downstream of receptor clustering.     

Actin remodeling to facilitate membrane fusion

Actin and its associated proteins participate in several intracellular trafficking mechanisms. This review assesses recent work that shows how actin participates in the terminal trafficking event of membrane bilayer fusion. A recent flurry of reports defines a role for Rho proteins in membrane fusion and also demonstrates that this role is distinct from any vesicle transport mechanism. Rho proteins are well known to govern actin remodeling, which implicates this process as a condition of membrane fusion. A small but significant body of work examines actin-regulated events of intracellular membrane fusion, exocytosis and endocytosis. In general, actin has been shown to act as a negative regulator of exocytosis. Cortical actin filaments act as a barrier that requires transient removal to allow vesicles to undergo docking at the plasma membrane. However, once docked, F-actin synthesis may act as a positive regulator to give the final stimulus to drive membrane fusion. F-actin synthesis is clearly needed for endocytosis and intracellular membrane fusion events. What may seem like dissimilar results are perhaps snapshots of a single mechanism of membranous actin remodeling (i.e. dynamic disassembly and reassembly) that is universally needed for all membrane fusion events.     

Cellular actin is affected by interaction with Candida albicans

Attachment of Candida albicans, an important opportunistic pathogen, to host tissues is an initial step in the development of the infection. The events occurring in the fungal and in the host cells after interaction are poorly understood. In this study we concentrated on the events occurring in the mammalian cells after the interaction with Candida, with emphasis on the cytoskeleton actin. Human cell line cells (HEp2) were exposed to C. albicans or C. albicans-secreted material (culture filtrate) (actin-rearranging Candida-secreted factor, arcsf). The HEp2 cells were examined for cellular changes using confocal laser microscopy (CLSM), transmission and scanning electron microscopy (TEM and SEM). The CLSM studies, using fluorescein isothiocyanate-labeled C. albicans and rhodamine phalloidin actin staining, revealed yeasts adhering to the HEp2 cells or internalized into the cells, with actin surrounding the fungi. Furthermore, actin rearrangement from filamentous network to actin aggregates was noticed. Interaction between the HEp2 cells and C. albicans could be demonstrated also by SEM and TEM after a 2-4-h exposure of the cells to the fungus. Yeasts and hyphae were found attaching to the surface and within the cells. CLSM studies revealed that exposure of HEp2 cells to arcsf was also followed by cellular actin rearrangement, reduced membrane ruffling and decreased cellular motility. The effect was dose- and time-dependent. All these data indicate that the interaction of Candida with HEp2 cells involves signaling events and affects the cellular actin.     

Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes

The mechanisms responsible for initiating the conversion of globular to filamentous actin (assembly) after stimulation of B lymphocytes and the role of these cytoskeletal changes in cell activation are incompletely understood. We investigated the molecular basis of the signals leading to actin polymerization and concentrated on the involvement of guanosine triphosphate (GTP)-binding regulatory proteins, and protein kinase C (PKC). In addition, we related these early events to later events in B-cell activation, including cell proliferation. Cross-linking the Ag receptor with Staphylococcus aureus Cowan I (SAC) or anti-IgM antibodies, or stimulation of PKC with phorbol ester induced a time- and concentration-dependent increase in the filamentous actin content of B cells. Inhibition or depletion of PKC resulted in decreased actin assembly induced by anti-IgM, SAC, and PMA, suggesting that the signal for polymerization is generated distally to PKC activation. Pertussis toxin pretreatment inhibited the responses to anti-IgM and SAC but not PMA, and direct stimulation of permeabilized cells with GTP gamma S induced microfilament assembly, indicating the involvement of a GTP-binding protein for receptor-mediated events. Disruption of actin polymerization with botulinum C2 toxin or cytochalasin D inhibited the assembly of actin and [3H]TdR incorporation induced by all stimuli. We conclude that human B cell activation by receptor-mediated stimuli results in actin polymerization by signaling pathways coupled to GTP-binding proteins. These changes in the cytoskeleton may be involved in the transduction of messages leading to responses such as proliferation in B lymphocytes.     

Bantam regulates the axonal geometry of <Emphasis Type="Italic">Drosophila</Emphasis> larval brain by modulating actin regulator enabled

During development, axonogenesis, an integral part of neurogenesis, is based on well-concerted events comprising generation, rearrangement, migration, elongation, and adhesion of neurons. Actin, specifically the crosstalk between the guardians of actin polymerization, like enabled, chickadee, capping protein plays an essential role in crafting several events of axonogenesis. Recent evidences reflect multifaceted role of microRNA during axonogenesis. Here, we investigated the role of bantam miRNA, a well-established miRNA in Drosophila, in regulating the actin organization during brain development. Our immunofluorescence studies showed altered arrangement of neurons and actin filaments whereas both qPCR and western blot revealed elevated expression of enabled, one of the actin modulators in bantam mutant background. Collectively, our results clearly demonstrate that bantam plays an instrumental role in shaping the axon architecture regulating the actin geometry through its modulator enabled.     

Identification and purification of actin from the subpellicular network of Toxoplasma gondii tachyzoites

Toxoplasma gondii infects cells through dynamic events dependent on actin. Although the presence of cortical actin has been widely suggested, visualisation and localisation of actin filaments has not been reported. The subpellicular cytoskeleton network is a recently described structure possibly involved in the dynamic events. Using non-ionic detergent extractions, the cortical cytoskeleton network was enriched and used for the isolation and identification of actin. Actin was detected by Western blots in extracts of cytoskeleton networks, and it was localised by gold staining in the network and in both the apical end and the posterior polar ring. Actin was isolated from subpellicular cytoskeleton extracts by binding to DNase I, and it polymerised in vitro as filaments that were gold-decorated by a monoclonal anti-actin antibody. Filaments bound the subfragment 1 of heavy meromyosin, although with atypical arrangements in comparison with the arrowheads observed in muscle actin filaments. Treatment with cytochalasin D and colchicine altered the structural organisation of the subpellicular network indicating the participation of actin filaments and microtubules in the maintenance of its structure. Actin filaments and microtubules, in the subpellicular network, participate reciprocally in the maintaining of the parasite's shape and the gliding motility.     

Actin microfilament dynamics and actin side-binding proteins in plants

Actin microfilaments are highly organized and essential intracellular components of organelle movement and cell morphogenesis in plants. The organization of these microfilaments undergoes dynamic changes during cell division, elongation, and differentiation. Recent live-cell imaging of plant actin microfilaments has revealed their native organization and remarkable dynamics. In addition, characterization of plant actin side-binding proteins has progressed rapidly by genetic, biochemical, and bioinformatic approaches. The gathering and integration of microscopy-based information from actin microfilament dynamics and the molecular identification of actin side-binding proteins have provided considerable insights into actin microfilament-dependent events and actin microfilament organization in plants.     

Cordon-bleu: a new taste in actin nucleation

Polymerization of actin filaments powers many dynamic cellular events and is directed by a small group of actin nucleators. In this issue, Ahuja et al. (2007) identify and characterize a new actin nucleator called cordon-bleu (Cobl) that may drive morphogenesis of the central nervous system during development.     

Participation of Rho, ROCK-2, and GAP activities during actin microfilament rearrangements in Entamoeba histolytica induced by fibronectin signaling

In Entamoeba histolytica little is known about the microfilament rearrangements formed by actin and ABPs. Fibronectin regulates many aspects of cell behavior involving the actin cytoskeleton and members of the Rho family of small GTPases. Using trophozoites interacted with fibronectin and glass, we present evidence related with the formation and regulation of different microfilament rearrangements and their cellular distribution, the effect of actin affecting drugs on these arrangements, and on trophozoites adhesion; we also demonstrate that actin isoforms are induced after adhesion, and also the selective participation of specific actin binding proteins such as ABP-120 and phospho-paxillin, regarding their location in the different actin structures. In addition, we show results that confirm the participation of EhRho, ROCK-2, and GAP activities. We propose that fibronectin induced signaling in E. histolytica trophozoites have important consequences in the actin cytoskeleton that may affect its behavior during the invasive process in the host.     

Fertilization and the cytoskeleton in the red alga Bostrychia moritziana (Rhodomelaceae,Rhodophyta)

Time-lapse videomicroscopy was used to observe the effects of various cytoskeletal inhibitors on three important fertilization events in Bostrychia moritziana: spermatial mitosis, gamete fusion (formation of a fertilization pore) and nuclear migration along the trichogyne. The microtubule inhibitor oryzalin disrupted spermatial mitosis but had no other effect on fertilization. The actin inhibitors, jasplakinolide, cytochalasin B, latrunculin A and B and mycalolide B inhibited gamete fusion while BDM, a myosin-disrupting drug, inhibited all three major fertilization events. FL-Phallacidin was used to stain actin filaments in spermatia and trichogynes while microtubules were labelled with antibodies at appropriate stages of fertilization. Microtubules were only evident during spermatial nuclear division. Actin filaments were present in both trichogynes and spermatia throughout fertilization; they formed a discrete ring around the fertilization pore and ensheathed male nuclei as the latter migrated into and along the trichogyne. These results suggest that the actin/myosin system plays a role in the events of fertilization.