Actin Nuclei Research Articles

Revealing the nanoindentation response of a single cell using a 3D structural finite element model

Changes in the apparent moduli of cells have been reported to correlate with cell abnormalities and disease. Indentation is commonly used to measure these moduli; however, there is evidence to suggest that the indentation protocol employed affects the measured moduli, which can affect our understanding of how physiological conditions regulate cell mechanics. Most studies treat the cell as a homogeneous material or a simple core–shell structure consisting of cytoplasm and a nucleus: both are far from the real structure of cells. To study indentation protocol-dependent cell mechanics, a finite element model of key intracellular components (cortex layer, cytoplasm, actin stress fibres, microtubules, and nucleus) has instead been developed. Results have shown that the apparent moduli obtained with conical indenters decreased with increasing cone angle; however, this change was less significant for spherical indenters of increasing radii. Furthermore, the interplay between indenter geometry and intracellular components has also been studied, which is useful for understanding structure-mechanics-function relationships of cells.

Changes in the Mechanical Interaction Between Actin Cytoskeleton and Nucleus in Osteogenic Differentiation in Human Mesenchymal Stem Cells

Changes in the Mechanical Interaction Between Actin Cytoskeleton and Nucleus in Osteogenic Differentiation in Human Mesenchymal Stem Cells

Measurement of dynamics actin cytoskeleton and nucleus during osteogenic differentiation of mesenchymal stem cells

Measurement of dynamics actin cytoskeleton and nucleus during osteogenic differentiation of mesenchymal stem cells

Label-Free Visualisation of Actin Nucleation and Polymerisation at the Single-Molecule Level using Interferometric Scattering Microscopy

Actin filaments are a major component of the cytoskeleton involved in many basic processes such as cell migration, cell adhesion, cell division and muscle contraction. The most important property of actin is that it forms filaments (F-actin) from globular subunits (G-actin). In the 1960s, Oosawa and co-workers developed a model for the G- to F-actin transition where the rate-limiting step is the formation of a stable actin nucleus, later determined to consist of 2-4 actin monomers. Nuclei are elongated by the addition of monomers to the filament ends. This model is widely accepted, although the molecular details could never be visualised experimentally. Here, we used interferometric scattering microscopy (iSCAT) to monitor nucleation and elongation of actin filaments at the single-molecule level. Building on recent improvements in label-free single molecule sensitivity and mass accuracy, we could reveal the polydispersity of G-actin at concentrations relevant to nucleation and filament growth, showing signatures of small protein oligomers in line with the nuclei sizes discussed above. Using the same approach, we could monitor the nanoscopic arrival of individual subunits dynamically attaching to and detaching from the filament tip during polymerisation. Contrary to the standard model, where actin grows exclusively from monomers, we also found signatures of larger oligomers being added to the filament. Together with our polydispersity measurements, these results point towards a nucleation and growth mechanism for actin filaments based on monomers and small oligomers, rather than exclusively monomers. These results demonstrate the potential of iSCAT for label-free single-molecule imaging and for investigating the mechanisms of mesoscopic dynamics in solution.

Palladin Nucleates Actin Assembly and Regulates Cytoskeleton Architecture

The actin scaffold protein palladin regulates both normal cell migration and invasive cell motility, processes that require the coordinated regulation of actin dynamics. Palladin localizes to actin-rich protrusions and has a well-documented effect on metastasis of invasive cancers. However, its potential effects on actin dynamics have remained elusive. Here, we show that the C-terminal immunoglobulin-like domain of palladin (Ig3) that is directly responsible for actin binding and bundling also potently nucleates the formation of actin filaments in vitro. Palladin eliminated the lag phase that is characteristic of the slow nucleation process of actin polymerization under both G- and F-actin buffer conditions. Furthermore palladin did not alter the critical concentration, and only had a modest effect on the rate of elongation of actin filaments. Therefore the increase in polymerization rate brought about by palladin can be attributed to a direct role for palladin in stabilizing the formation of actin nuclei. We also present evidence that nucleation is likely achieved by a mechanism involving actin-induced dimerization. In addition, we monitored actin polymerization in real-time using TIRF microscopy and found that palladin bundles the actin filaments while promoting polymerization. Finally, we examined whether the Ig3 domain of palladin is required for actin organization in a cellular context. In cells transfected with a full-length palladin construct containing either a deletion of the actin-binding domain or point mutations that disrupt actin-binding we observe dramatically altered cellular distributions of both palladin and actin, which suggests that this direct interaction with actin is critical for regulating cytoskeletal organization and dynamics. These observations define a new function for palladin and support an emerging view of actin-binding proteins that exhibit a dual cellular-nuclear localization and also participate in the regulation of the actin cytoskeleton architecture.

Structural Basis of Actin Filament Nucleation by Tandem W Domains

Spontaneous nucleation of actin is very inefficient in cells. To overcome this barrier, cells have evolved a set of actin filament nucleators to promote rapid nucleation and polymerization in response to specific stimuli. However, the molecular mechanism of actin nucleation remains poorly understood. This is hindered largely by the fact that actin nucleus, once formed, rapidly polymerizes into filament, thus making it impossible to capture stable multisubunit actin nucleus. Here, we report an effective double-mutant strategy to stabilize actin nucleus by preventing further polymerization. Employing this strategy, we solved the crystal structure of AMPPNP-actin in complex with the first two tandem W domains of Cordon-bleu (Cobl), a potent actin filament nucleator. Further sequence comparison and functional studies suggest that the nucleation mechanism of Cobl is probably shared by the p53 cofactor JMY, but not Spire. Moreover, the double-mutant strategy opens the way for atomic mechanistic study of actin nucleation and polymerization.

Comparative analysis of the fluorescent labeling pattern of nuclei of early mouse embryos by using antibodies to various actin molecule domains

New data are presented on the peculiarities of immunofluorescent detection of nuclear actin in mouse two-cell embryos by using antibodies against the C- and N-terminal domains of actin molecule. Redistribution of actin is studied at artificial inhibition of transcription, as well as after enzymatic digestion of DNA. Visual observations are supplemented by comparative analysis of integral fluorescence intensity on confocal images. In both experimental groups, both upon use of transcription inhibitors and after treatment of embryos with DNAse, there has been revealed an increase of fluorescence intensity as compared with control at using antibody to the C-terminal, but not to the N-terminal actin domain. The obtained data allow suggesting that antibody to the C-terminal domain is more efficient upon immunofluorescent detection of actin in nuclei of mouse cleavage embryos.

Molecular architecture of the Spire–actin nucleus and its implication for actin filament assembly

The Spire protein is a multifunctional regulator of actin assembly. We studied the structures and properties of Spire-actin complexes by X-ray scattering, X-ray crystallography, total internal reflection fluorescence microscopy, and actin polymerization assays. We show that Spire-actin complexes in solution assume a unique, longitudinal-like shape, in which Wiskott-Aldrich syndrome protein homology 2 domains (WH2), in an extended configuration, line up actins along the long axis of the core of the Spire-actin particle. In the complex, the kinase noncatalytic C-lobe domain is positioned at the side of the first N-terminal Spire-actin module. In addition, we find that preformed, isolated Spire-actin complexes are very efficient nucleators of polymerization and afterward dissociate from the growing filament. However, under certain conditions, all Spire constructs--even a single WH2 repeat--sequester actin and disrupt existing filaments. This molecular and structural mechanism of actin polymerization by Spire should apply to other actin-binding proteins that contain WH2 domains in tandem.

Mechanism of actin filament nucleation by Vibrio VopL and implications for tandem W domain nucleation

Pathogen proteins targeting the actin cytoskeleton often serve as model systems to understand their more complex eukaryotic analogs. We show that the strong actin filament nucleation activity of Vibrio VopL depends on its three W domains and dimerization through a unique VopL C-terminal domain (VCD). The VCD displays a novel all-helical fold and interacts with the pointed end of the actin nucleus, contributing to the nucleation activity directly and through duplication of the W domain repeat. VopL promotes rapid cycles of filament nucleation and detachment, but generally has no effect on elongation. Profilin inhibits VopL-induced nucleation by competing for actin binding to the W domains. Combined, the results suggest that VopL stabilizes a hexameric double-stranded pointed end nucleus. Analysis of hybrid constructs of VopL and the eukaryotic nucleator Spire suggest that Spire may also function as a dimer in cells.

Assessment of Actin Cytoskeleton and Nuclei in Bovine Blastocysts Developed Under Different Culture Conditions Using a Novel Computer Program

This study was performed to investigate the effects, in terms of nuclear material and actin cytoskeleton quantities (fluorescent pixel counts), of four different bovine blastocyst culturing techniques (in vitro, stepwise in vitro-to-in vivo, or purely in vivo). Cumulus oocyte complexes from abattoir-sourced ovaries were matured in vitro and allocated to four groups: IVP-group embryos developed up to blastocyst stage in vitro. Gamete intra-fallopian transfer (GIFT)-group oocytes were co-incubated with semen for 4 h before transfer to oviducts of heifers. Following in vitro fertilization, cleaved embryos (day 2 of embryo development, day 2-7 group) were transferred into oviducts on day 2. Multiple ovulation embryo transfer (MOET)-group embryos were obtained by superovulating and inseminating heifers; the heifers' genital tracts were flushed at day 7 of blastocyst development. Within each group, ten blastocysts were selected to be differentially dyed (for nuclei and actin cytoskeleton) with fluorescent stains. A novel computer program (ColorAnalyzer) provided differential pixel counts representing organelle quantities. Blastocysts developed only in vivo (MOET group) showed significantly more nuclear material than did blastocysts produced by any other technique. In terms of actin cytoskeleton quantity, blastocysts produced by IVP and by day 2-7 transfer did not differ significantly from each other. Gamete intra-fallopian transfer- and MOET-group embryos showed significantly larger quantities of actin cytoskeleton when compared with any other group and differed significantly from each other. The results of this study indicate that culturing under in vitro conditions, even with part time in vivo techniques, may adversely affect the quantity of blastocyst nuclear material and actin cytoskeleton. The software employed may be useful for culture environment evaluation/developmental competence assessment.

MICRO-CONSTITUENT BASED VISCOELASTIC FINITE ELEMENT ANALYSIS OF BIOLOGICAL CELLS

A viscoelastic analysis of the biological cell considering the microcellular material properties is carried out in this work. Three separate regions of the cell: the actin cortex, cytoplasm and nucleus are considered. The outer cortex and cytoplasm are modeled using standard linear viscoelastic model (SLS) and standard neo-Hookean viscoelastic solid, and a linear elastic material model is considered for the nucleus. The effect of the material properties of cytoplasm and actin cortex on the derivable parameters from three major experimental studies of magnetic twisting cytometry (MTC) and atomic force microscopy (AFM) and micropipette aspiration (MPA) are analyzed using the finite element method. The bead center displacement for the MTC, reaction force for AFM, and aspiration length ratio for the MPA are the major quantities derived from the finite element analysis. A number of parametric studies are also conducted and it is observed that SLS and SnHS models predict nearly identical results for the material constants.

Actin Filament Nucleation: Structure-Function Relationships

The actin cytoskeleton is intimately involved in most cellular functions, including cell motility, endo/exocytosis and intracellular trafficking. These processes are characterized by rapid oscillations of actin polymerization/depolymerization under tight temporal and spatial regulation. Hundreds of G- and F-actin-binding proteins, along with signaling and scaffolding proteins regulate the assembly of actin networks. Among these proteins, filament nucleators play a critical role by determining the time and location for actin polymerization, as well as the specific structures of the actin networks that they generate. Eukaryotic cells and certain pathogens use filament nucleators to stabilize actin nuclei (small oligomers of 2-4 actin subunits), whose formation is rate-limiting. Known filament nucleators include the Arp2/3 complex and its large family of Nucleation Promoting Factors (NPFs), Formins, Spire, Cobl, Lmod, VopL/VopF and TARP. Structural and functional studies are beginning to shed light on the diverse mechanisms used by these molecules to stabilize actin nuclei. Thus, with the exception of Formins known filament nucleators use the WASP-Homology 2 domain (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. A common architecture, found in Spire, Cobl and VopL/VopF, consists of tandem W domains that bind three to four actin subunits to form a nucleus. Structural considerations suggest that NPFs-Arp2/3 complex can also be viewed as a specialized form of tandem W-based nucleator. The nucleation activities of these proteins vary significantly, and the most effective nucleators are not necessarily those with the largest number of W domains. We show that the inter-W linkers play a critical role in determining the nucleation activities of filament nucleators and the structures of the actin nuclei that they generate. Furthermore, we present evidence that a previously neglected factor, oligomerization, is a major determinant of filament nucleation activity and nuclei structure.

Differential Regulation of Actin Polymerization and Structure by Yeast Formin Isoforms

The budding yeast formins, Bnr1 and Bni1, behave very differently with respect to their interactions with muscle actin. However, the mechanisms underlying these differences are unclear, and these formins do not interact with muscle actin in vivo. We use yeast wild type and mutant actins to further assess these differences between Bnr1 and Bni1. Low ionic strength G-buffer does not promote actin polymerization. However, Bnr1, but not Bni1, causes the polymerization of pyrene-labeled Mg-G-actin in G-buffer into single filaments based on fluorometric and EM observations. Polymerization by Bnr1 does not occur with Ca-G-actin. By cosedimentation, maximum filament formation occurs at a Bnr1:actin ratio of 1:2. The interaction of Bnr1 with pyrene-labeled S265C Mg-actin yields a pyrene excimer peak, from the cross-strand interaction of pyrene probes, which only occurs in the context of F-actin. In F-buffer, Bnr1 promotes much faster yeast actin polymerization than Bni1. It also bundles the F-actin in contrast to the low ionic strength situation where only single filaments form. Thus, the differences previously observed with muscle actin are not actin isoform-specific. The binding of both formins to F-actin saturate at an equimolar ratio, but only about 30% of each formin cosediments with F-actin. Finally, addition of Bnr1 but not Bni1 to pyrene-labeled wild type and S265C Mg-F actins enhanced the pyrene- and pyrene-excimer fluorescence, respectively, suggesting Bnr1 also alters F-actin structure. These differences may facilitate the ability of Bnr1 to form the actin cables needed for polarized delivery of nutrients and organelles to the growing yeast bud.

Ultrastructural localization of actin and actin-binding proteins in the nucleus

Nuclear actin plays an important role in such processes as chromatin remodeling, transcriptional regulation, RNA processing, and nuclear export. Recent research has demonstrated that actin in the nucleus probably exists in dynamic equilibrium between monomeric and polymeric forms, and some of the actin-binding proteins, known to regulate actin dynamics in cytoplasm, have been also shown to be present in the nucleus. In this paper, we present ultrastructural data on distribution of actin and various actin-binding proteins (alpha-actinin, filamin, p190RhoGAP, paxillin, spectrin, and tropomyosin) in nuclei of HeLa cells and resting human lymphocytes. Probing extracts of HeLa cells for the presence of actin-binding proteins also confirmed their presence in nuclei. We report for the first time the presence of tropomyosin and p190RhoGAP in the cell nucleus, and the spatial colocalization of actin with spectrin, paxillin, and alpha-actinin in the nucleolus.

An integrative simulation model linking major biochemical reactions of actin-polymerization to structural properties of actin filaments

We report on an advanced universal Monte Carlo simulation model of actin polymerization processes offering a broad application panel. The model integrates major actin-related reactions, such as assembly of actin nuclei, association/dissociation of monomers to filament ends, ATP-hydrolysis via ADP-Pi formation and ADP-ATP exchange, filament branching, fragmentation and annealing or the effects of regulatory proteins. Importantly, these reactions are linked to information on the nucleotide state of actin subunits in filaments (ATP hydrolysis) and the distribution of actin filament lengths. The developed stochastic simulation modelling schemes were validated on: i) synthetic theoretical data generated by a deterministic model and ii) sets of our and published experimental data obtained from fluorescence pyrene-actin experiments. Build on an open-architecture principle, the designed model can be extended for predictive evaluation of the activities of other actin-interacting proteins and can be applied for the analysis of experimental pyrene actin-based or fluorescence microscopy data. We provide a user-friendly, free software package ActinSimChem that integrates the implemented simulation algorithms and that is made available to the scientific community for modelling in silico any specific actin-polymerization system.

Cytoskeleton, endoplasmic reticulum and nucleus alterations in CHO-K1 cell line after Crotalus durissus terrificus (South American rattlesnake) venom treatment

Snake venoms are toxic to a variety of cell types. However, the intracellular damages and the cell death fate induced by venom are unclear. In the present work, the action of the South American rattlesnake Crotalus durissus terrificus venom on CHO-K1 cell line was analyzed. The cells CHO-K1 were incubated with C. d. terrificus venom (10, 50 and 100g/ml) for 1 and 24 hours, and structural alterations of actin filaments, endoplasmic reticulum and nucleus were assessed using specific fluorescent probes and agarose gel electrophoresis for DNA fragmentation. Significant structural changes were observed in all analyzed structures. DNA fragmentation was detected suggesting that, at the concentrations used, the venom induced apoptosis.

Induced Microcracking Affects Osteoblast Attachment on Hydroxyapatite Scaffolds for Tissue Engineering

Bone microdamage caused by routine activity plays an important role in triggering targeted and nontargeted bone remodeling. Targeted remodeling occurs near localized areas of microdamage[1-4]. We hypothesize that bone remodeling may be directly and positively influenced by inducing microcracks in hydroxyapatite (HA) scaffolds for bone tissue engineering. A study by Case et al. showed microcracking in HA discs having >98% theoretical density[5]. These microcracks occurred without the application of external stress, likely as a result of thermal expansion anisotropy (TEA). TEA generates microcracks only when the specimen grain size exceeds a critical value (GCR). Due to conflicting data in the literature on the thermal expansion along the crystallographic axes of HA, it is difficult to estimate GCR precisely for HA, but GCR likely ranges from a few tenths of microns to several microns[6]. As the grain size of a polycrystalline specimen increases above the critical grain size, the number of microcracks also increases but the number density of microcracks is difficult to control. Therefore, in this study we used Vickers microindentation to induce controlled microcracks in >99% dense HA discs. The effect of microcracking on the osteoblast (OB) attachment was then quantified. Control HA discs and microcracked HA were seeded with MC3T3-E1 OBs and cultured for 4 hours. The OBs were then stained with Rhodamine-Phalloidin and Hoechst fluorescent dyes to identify the actin fibers and nuclei, respectively. OB attachment was quantified using fluorescent light microscopy. OB attachment at 4 hours was 29% ± 7.6% of the initial seeded OBs for the microcracked HA specimens and 18% ± 6.1% of initial seeded OBs for the non-microcracked control HA specimens. The difference in these OB attachment values was statistically significant as determined via the Student t-test (p = 0.004, with p < 0.05 taken to indicate significance).

Unpolymerized nuclear actin is involved in the activation of CSF-1 gene transcription.

Nuclear actin is a nuclear component in many kinds of eukaryotic cells but its function is still not clear. In this study, we overexpressed actin in nuclei and found that it promoted transcription of the CSF-1 gene, both exogenous and endogenous, approximately two-fold. Cytochalasin B did not affect this function of nuclear actin. Our results suggest that nuclear actin plays a role in regulating CSF-1 gene transcription, and this role does not depend on actin polymerization.

Fesselin, a synaptopodin-like protein, stimulates actin nucleation and polymerization.

Fesselin is a proline-rich actin binding protein that has recently been isolated from smooth muscle [Leinweber, B. D., Fredricksen, R. S., Hoffman, D. R., and Chalovich, J. M. (1999) J. Muscle Res. Cell Motil. 20, 539-545]. Fesselin is similar to synaptopodin [Mundel, P., Heid, H. W., Mundel, T. M., Krüger, M., Reiser, J., and Kriz, W. (1997) J. Cell Biol. 139, 193-204] in terms of its size, isoelectric point, and sequence although synaptopodin is not present in smooth muscle. The function of fesselin is unknown. Evidence is presented here that fesselin accelerates the polymerization of actin. Fesselin was effective on actin isolated from either smooth or skeletal muscle at low ionic strength and in the presence of 100 mM KCl. At low ionic strength, fesselin decreased the time for 50% polymerization to about 1% of that in the absence of fesselin. The lag phase characteristic of the slow nucleation process of polymerization was eliminated as the fesselin concentration was increased from very low levels. Fesselin did not alter the critical concentration for actin but did increase the rate of elongation by approximately 3-fold. The increase in elongation rate constant is insufficient to account for the total increase in polymerization rate. It is likely that fesselin stabilizes the formation of actin nuclei. Time courses of actin polymerization at varied fesselin concentrations and varied actin concentrations were simulated by increasing the rate of nucleation and both the forward and reverse rate constants for elongation.

Mechanisms of Cold-induced Platelet Actin Assembly

Various agonists but also chilling cause blood platelets to increase cytosolic calcium, polymerize actin, and change shape. We report that cold increases barbed end nucleation sites in octyl glucoside-permeabilized platelets by 3-fold, enabling analysis of the intermediates of this response. Although chilling does not change polyphosphoinositide (ppI) levels, a ppI-binding peptide completely inhibits cold-induced nucleation. The C terminus of N-WASp, which inhibits the Arp2/3 complex, blocks nucleation by 40%; GDPbetaS, N17Rac and N17Cdc42 have no effects. Some gelsolin translocates to the detergent-insoluble cytoskeleton after cooling. Chilled platelets from gelsolin-deficient mice have approximately 50% fewer new actin nuclei compared with platelets from wild-type mice. EGTA completely inhibits gelsolin translocation into the cytoskeleton, and the small amount of gelsolin initially there becomes soluble. Chilling releases adducin from the detergent-resistant cytoskeleton. We conclude that platelet actin filament assembly induced by cooling involves ppI-mediated actin filament barbed end uncapping and de novo nucleation independently of surface receptors or downstream signaling intermediates besides calcium. The actin-related changes occur in platelets at temperatures below 37 degrees C, suggesting that the platelet may be more activable at temperatures at the body surface than at core temperature, thereby favoring superficial hemostasis over internal thrombosis.