Recovery,visualization, and analysis of actin and tubulin polymer flow in live cells: a fluorescent speckle microscopy study

Fluorescent speckle microscopy (FSM) is becoming the technique of choice for analyzing in vivo the dynamics of polymer assemblies, such as the cytoskeleton. The massive amount of data produced by this method calls for computational approaches to recover the quantities of interest; namely, the polymerization and depolymerization activities and the motions undergone by the cytoskeleton over time. Attempts toward this goal have been hampered by the limited signal-to-noise ratio of typical FSM data, by the constant appearance and disappearance of speckles due to polymer turnover, and by the presence of flow singularities characteristic of many cytoskeletal polymer assemblies. To deal with these problems, we present a particle-based method for tracking fluorescent speckles in time-lapse FSM image series, based on ideas from operational research and graph theory. Our software delivers the displacements of thousands of speckles between consecutive frames, taking into account that speckles may appear and disappear. In this article we exploit this information to recover the speckle flow field. First, the software is tested on synthetic data to validate our methods. We then apply it to mapping filamentous actin retrograde flow at the front edge of migrating newt lung epithelial cells. Our results confirm findings from previously published kymograph analyses and manual tracking of such FSM data and illustrate the power of automated tracking for generating complete and quantitative flow measurements. Third, we analyze microtubule poleward flux in mitotic metaphase spindles assembled in Xenopus egg extracts, bringing new insight into the dynamics of microtubule assemblies in this system.     

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM). We describe the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes. Download video file.^{(229M, mp4)}     

The Schizosaccharomyces pombe cdc3+ gene encodes a profilin essential for cytokinesis

The fission yeast Schizosaccharomyces pombe divides by medial fission and, like many higher eukaryotic cells, requires the function of an F- actin contractile ring for cytokinesis. In S. pombe, a class of cdc- mutants defective for cytokinesis, but not for DNA replication, mitosis, or septum synthesis, have been identified. In this paper, we present the characterization of one of these mutants, cdc3-124. Temperature shift experiments reveal that mutants in cdc3 are incapable of forming an F-actin contractile ring. We have molecularly cloned cdc3 and used the cdc3+ genomic DNA to create a strain carrying a cdc3 null mutation by homologous recombination in vivo. Cells bearing a cdc3-null allele are inviable. They arrest the cell cycle at cytokinesis without forming a contractile ring. DNA sequence analysis of the cdc3+ gene reveals that it encodes profilin, an actin-monomer-binding protein. In light of recent studies with profilins, we propose that Cdc3-profilin plays an essential role in cytokinesis by catalyzing the formation of the F-actin contractile ring. Consistent with this proposal are our observations that Cdc3-profilin localizes to the medial region of the cell where the F-actin contractile ring forms, and that it is essential for F-actin ring formation. Cells overproducing Cdc3-profilin become elongated, dumbbell shaped, and arrest at cytokinesis without any detectable F-actin staining. This effect of Cdc3-profilin overproduction is relieved by introduction of a multicopy plasmid carrying the actin encoding gene, act1+. We attribute these effects to potential sequestration of actin monomers by profilin, when present in excess.     

How to Arm a Supervillin: Designing F-Actin Binding Activity into Supervillin Headpiece

Villin-type headpiece domains are compact motifs that have been used extensively as model systems for protein folding. Although the majority of headpiece domains bind actin, there are some that lack this activity. Here, we present the first NMR solution structure and ¹⁵N-relaxation analysis of a villin-type headpiece domain natively devoid of F-actin binding activity, that of supervillin headpiece (SVHP). The structure was found to be similar to that of other headpiece domains that bind F-actin. Our NMR analysis demonstrates that SVHP lacks a conformationally flexible region (V-loop) present in all other villin-type headpiece domains and which is essential to the phosphoryl regulation of dematin headpiece. In comparing the electrostatic surface potential map of SVHP to that of other villin-type headpiece domains with significant affinity for F-actin, we identified a positive surface potential conserved among headpiece domains that bind F-actin but absent from SVHP. A single point mutation (L38K) in SVHP, which creates a similar positive surface potential, endowed SVHP with specific affinity for F-actin that is within an order of magnitude of the tightest binding headpiece domains. We propose that this effect is likely conferred by a specific buried salt bridge between headpiece and actin. As no high-resolution structural information exists for the villin-type headpiece F-actin complex, our results demonstrate that through positive mutagenesis, it is possible to design binding activity into homologous proteins without structural information of the counterpart's binding surface.     

Real-time monitoring of actin polymerization in living cells using split luciferase

Real-time monitoring of actin polymerization in living cells is beneficial for characterizing cellular activities such as migration, proliferation, and death. We developed new bioluminescence-based probe proteins that enable the monitoring of actin polymerization in living cells. Unlike other ordinary split luciferase probes, our probes were incorporated in endogenous actin filament that enabled it to measure the actin polymerization quantitatively. The probe proteins exhibited a dose-responsive decrease in photon emission intensity in response to the filamentous (F)-actin-disrupting agent latrunculin A. This technique has a high sensitivity with a high signal-to-noise ratio and is nontoxic compared with other methods of monitoring actin polymerization in living cells. Using this technique, we succeeded in monitoring the F-actin level in living cells during apoptosis progression induced by UV irradiation continuously for 12 h. F-actin was transiently upregulated after UV irradiation. Since UV-induced cell death was enhanced by treatment with latrunculin A during the period which F-actin is increased, transient upregulation of F-actin after UV is likely a protective reaction against UV-induced cell death. Our novel technique is an effective tool for investigating actin polymerization in living cells.     

Mechanics of the F-actin cytoskeleton

Dynamic regulation of the filamentous actin (F-actin) cytoskeleton is critical to numerous physical cellular processes, including cell adhesion, migration and division. Each of these processes require precise regulation of cell shape and mechanical force generation which, to a large degree, is regulated by the dynamic mechanical behaviors of a diverse assortment of F-actin networks and bundles. In this review, we review the current understanding of the mechanics of F-actin networks and identify areas of further research needed to establish physical models. We first review our understanding of the mechanical behaviors of F-actin networks reconstituted in vitro, with a focus on the nonlinear mechanical response and behavior of “active” F-actin networks. We then explore the types of mechanical response measured of cytoskeletal F-actin networks and bundles formed in living cells and identify how these measurements correspond to those performed on reconstituted F-actin networks formed in vitro. Together, these approaches identify the challenges and opportunities in the study of living cytoskeletal matter.     

Actin filament reorganization in HL-60 leukemia cell line after treatment with G-CSF and GM-CSF

Currently, information regarding the influence of growth factors on the cytoskeleton, including G-CSF and GMCSF, remains limited. In the present study we show alterations in F-actin distribution and cell cycle progression in HL-60 promyelocytic leukemia cells, resulting from treatment with these cytokines in vitro. We found that both agents caused F-actin reorganization. Although multiple potential effects of various growth factors have been described previously, in our experimental conditions, we observed some rather subtle differences between the effects of G-CSF and GM-CSF on studied cells. The presence of these cytokines in the cell environment caused not only increased F-actin labeling in the cytoplasm, but also a weaker intensity of peripheral ring staining in comparison with control cells. In spite of the fact that HL60 cells exposed to G-CSF and GM-CSF contained different F-actin structures such as aggregates and F-actin network, the rate of actin polymerization was not significantly enhanced. Moreover, alterations were mainly related to considerable changes in the relative proportion of these different structures, what might be reflected by specific features of the differentiation process, with regard to the kind of stimulating factor used. Thus, reorganization of F-actin and other results obtained in our experimental conditions, might reflect unique characteristics of the differentiation process in HL-60 cells, involving low apoptosis frequency, the G1 to S phase transition in the cell cycle, as well as possible alternative ways of the cell death.     

Crystal structure of human coactosin-like protein at 1.9 A resolution

Human coactosin-like protein (CLP) shares high homology with coactosin, a filamentous (F)-actin binding protein, and interacts with 5LO and F-actin. As a tumor antigen, CLP is overexpressed in tumor tissue cells or cell lines, and the encoded epitopes can be recognized by cellular and humoral immune systems. To gain a better understanding of its various functions and interactions with related proteins, the crystal structure of CLP expressed in Escherichia coli has been determined to 1.9 A resolution. The structure features a central beta-sheet surrounded by helices, with two very tight hydrophobic cores on each side of the sheet. CLP belongs to the actin depolymerizing protein superfamily, and is similar to yeast cofilin and actophilin. Based on our structural analysis, we observed that CLP forms a polymer along the crystallographic b axis with the exact same repeat distance as F-actin. A model for the CLP polymer and F-actin binding has therefore been proposed.     

Cell Stress Promotes the Association of Phosphorylated HspB1 with F-Actin

Previous studies have suggested that the small heat shock protein, HspB1, has a direct influence on the dynamics of cytoskeletal elements, in particular, filamentous actin (F-actin) polymerization. In this study we have assessed the influence of HspB1 phosphorylation on its interaction(s) with F-actin. We first determined the distribution of endogenous non-phosphorylated HspB1, phosphorylated HspB1 and F-actin in neuroendocrine PC12 cells by immunocytochemistry and confocal microscopy. We then investigated a potential direct interaction between HspB1 with F-actin by precipitating F-actin directly with biotinylated phalloidin followed by Western analyses; the reverse immunoprecipitation of HspB1 was also carried out. The phosphorylation influence of HspB1 in this interaction was investigated by using pharmacologic inhibition of p38 MAPK. In control cells, HspB1 interacts with F-actin as a predominantly non-phosphorylated protein, but subsequent to stress there is a redistribution of HspB1 to the cytoskeletal fraction and a significantly increased association of pHspB1 with F-actin. Our data demonstrate HspB1 is found in a complex with F-actin both in phosphorylated and non-phosphorylated forms, with an increased association of pHspB1 with F-actin after heat stress. Overall, our study combines both cellular and biochemical approaches to show cellular localization and direct demonstration of an interaction between endogenous HspB1 and F-actin using methodolgy that specifically isolates F-actin.     

Rapid formation and high diffusibility of actin-cofilin cofilaments at low pH.

Cofilin is a small actin-binding protein that is known to bind both F-actin and G-actin, severing the former. The interaction of cofilin with actin is pH-sensitive, F-actin being preferentially bound at low pH and G-actin at higher pH, within the physiological range. Diffusion coefficients of F-actin with cofilin were measured by the fluorescence recovery after photobleaching (FRAP) technique. This has the potential for simultaneous and direct measurement of average polymer length via the average diffusion coefficient of the polymers (DLM) as well as the fraction of polymerized actin, fLM, present in solution. In the range of cofilin-actin ratios up to 1 : 1 and at both pH 6.5 and pH 8.0, the diffusion coefficients of the polymers increased with the amount of cofilin present in the complex, in a co-operative manner to a plateau. We interpret this as indicating co-operative binding/severing and that filaments less than a certain length cannot be severed further. Under the conditions used here, filaments were found to be more motile at pH 6.5 than at pH 8.0. At pH 8.0, some actin is expected to be sequestered as ADP-actin-cofilin complexes, with the remaining actin being present as long slowly diffusing filaments. At pH 6.5, however, cofilin binds to F-actin to form short rapidly diffusing cofilaments. These filaments form very rapidly from cofilin-actin monomeric complexes, possibly indicating that this complex is able to polymerize without dissociation. These findings may be relevant to the nuclear import of actin-cofilin complexes.     

Diffusing wave spectroscopy microrheology of actin filament networks.

Filamentous actin (F-actin), one of the constituents of the cytoskeleton, is believed to be the most important participant in the motion and mechanical integrity of eukaryotic cells. Traditionally, the viscoelastic moduli of F-actin networks have been measured by imposing a small mechanical strain and quantifying the resulting stress. The magnitude of the viscoelastic moduli, their concentration dependence and strain dependence, as well as the viscoelastic nature (solid-like or liquid-like) of networks of uncross-linked F-actin, have been the subjects of debate. Although this paper helps to resolve the debate and establishes the extent of the linear regime of F-actin networks' rheology, we report novel measurements of the high-frequency behavior of networks of F-actin, using a noninvasive light-scattering based technique, diffusing wave spectroscopy (DWS). Because no external strain is applied, our optical assay generates measurements of the mechanical properties of F-actin networks that avoid many ambiguities inherent in mechanical measurements. We observe that the elastic modulus has a small magnitude, no strain dependence, and a weak concentration dependence. Therefore, F-actin alone is not sufficient to generate the elastic modulus necessary to sustain the structural rigidity of most cells or support new cellular protrusions. Unlike previous studies, our measurements show that the mechanical properties of F-actin are highly dependent on the frequency content of the deformation. We show that the loss modulus unexpectedly dominates the elastic modulus at high frequencies, which are key for fast transitions. Finally, the measured mean square displacement of the optical probes, which is also generated by DWS measurements, offers new insight into the local bending fluctuations of the individual actin filaments and shows how they generate enhanced dissipation at short time scales.     

Disruption of blastomeric F-actin: a potential early biomarker of developmental toxicity in zebrafish

The expression of at least some biomarkers of toxicity is generally thought to precede the appearance of frank pathology. In the context of developmental toxicity, certain early indicators may be predictive of later drastic outcome. The search for predictive biomarkers of toxicity in the cells (blastomeres) of an early embryo can benefit from the fact that for normal development to proceed, the maintenance of blastomere cellular integrity during the process of transition from an embryo to a fully functional organism is paramount. Actin microfilaments are integral parts of blastomeres in the developing zebrafish embryo and contribute toward the proper progression of early development (cleavage and epiboly). In early embryos, the filamentous actin (F-actin) is present and helps to define the boundary of each blastomere as they remain adhered to each other. In our studies, we observed that when blastomeric F-actin is depolymerized by agents like gelsolin, the blastomeres lose cellular integrity, which results in abnormal larvae later in development. There are a variety of toxicants that depolymerize F-actin in early mammalian embryos, the later consequences of which are, at present, not known. We propose that very early zebrafish embryos (~5-h old) exposed to such toxicants will also respond in a like manner. In this review, we discuss the potential use of F-actin disruption as a predictive biomarker of developmental toxicity in zebrafish.     

The Formin Diaphanous Regulates Myoblast Fusion through Actin Polymerization and Arp2/3 Regulation

The formation of multinucleated muscle cells through cell-cell fusion is a conserved process from fruit flies to humans. Numerous studies have shown the importance of Arp2/3, its regulators, and branched actin for the formation of an actin structure, the F-actin focus, at the fusion site. This F-actin focus forms the core of an invasive podosome-like structure that is required for myoblast fusion. In this study, we find that the formin Diaphanous (Dia), which nucleates and facilitates the elongation of actin filaments, is essential for Drosophila myoblast fusion. Following cell recognition and adhesion, Dia is enriched at the myoblast fusion site, concomitant with, and having the same dynamics as, the F-actin focus. Through analysis of Dia loss-of-function conditions using mutant alleles but particularly a dominant negative Dia transgene, we demonstrate that reduction in Dia activity in myoblasts leads to a fusion block. Significantly, no actin focus is detected, and neither branched actin regulators, SCAR or WASp, accumulate at the fusion site when Dia levels are reduced. Expression of constitutively active Dia also causes a fusion block that is associated with an increase in highly dynamic filopodia, altered actin turnover rates and F-actin distribution, and mislocalization of SCAR and WASp at the fusion site. Together our data indicate that Dia plays two roles during invasive podosome formation at the fusion site: it dictates the level of linear F-actin polymerization, and it is required for appropriate branched actin polymerization via localization of SCAR and WASp. These studies provide new insight to the mechanisms of cell-cell fusion, the relationship between different regulators of actin polymerization, and invasive podosome formation that occurs in normal development and in disease.     

Non-muscle actin filament elongation from complexes of profilin with nucleotide-free actin and divalent cation-free ATP-actin

Using vertebrate cytoplasmic actin consisting of a mixture of beta and gamma isoforms, we previously characterized profilin and nucleotide binding to monomeric actin (Kinosian, H. J., et al. (2000) Biochemistry 39, 13176-13188) and F-actin barbed end elongation from profilin-actin (PA) (Kinosian, H. J., et al. (2002) Biochemistry 41, 6734-6743). Our initial calculations indicated that elongation of F-actin from PA was more energetically favorable than elongation of F-actin from monomeric actin; therefore, the overall actin elongation reaction scheme described by these two linked reactions appeared to be thermodynamically unbalanced. However, we hypothesized that the profilin-induced weakening of MgATP binding by actin reduces the negative free energy change for the formation of profilin-MgATP-actin from MgATP-actin. When this was taken into account, the overall reaction scheme was calculated to be thermodynamically balanced. In our present work, we test this hypothesis by measuring actin filament barbed end elongation of nucleotide-free actin (NF-A) and nucleotide-free profilin-actin (NF-PA). We find that the free energy change for elongation of F-actin by NF-PA is equal to that for elongation of F-actin from NF-A, indicating energetic balance of the linked reactions. In the absence of actin-bound divalent cation, profilin has very little effect on ATP binding to actin; analysis of elongation experiments with divalent cation-free ATP-actin and profilin yielded an approximately energetically balanced reaction scheme. Thus, the data in this present report support our earlier hypothesis.     

The lineaging of fluorescently-labeled Caenorhabditis elegans embryos with StarryNite and AceTree

Lineage analysis of Caenorhabditis elegans is a powerful tool for characterizing developmental phenotypes and embryonic gene-expression patterns. We present a detailed protocol for the lineaging of embryos by computational analysis of 4D images of embryos that ubiquitously express histone-GFP (green fluorescent protein) fusion proteins through the 350 cell stage followed by manual editing. We describe how to optimize imaging settings for this purpose, the use of the lineage-extraction software, StarryNite, and the lineage-editing software, AceTree. In addition, we describe a useful polymer bead mounting technique for C. elegans embryos that has several advantages compared with the standard agar pad mounting technique. The protocol requires about 1 h of user time spread over 2 days to generate the raw lineage, and an additional 2 or 4 h to edit the lineage to the 194- or 350-cell stage, respectively.     

Myosin-induced volume increase of the hyper-mobile water surrounding actin filaments

Microwave dielectric spectroscopy can measure the rotational mobility of water molecules that hydrate proteins and the hydration-shell volume. Using this technique, we have recently shown that apart from typical hydrating water molecules with lowered mobility there are other water molecules around the actin filaments (F-actin) which have a much higher mobility than that of bulk water [Biophys. J. 85 (2003) 3154]. We report here that the volume of this water component (hyper-mobile water) markedly increases without significant change of the volume of the ordinary hydration shell when the myosin motor-domain (S1, myosin subfragment-1) binds to F-actin. No hyper-mobile component was found in the hydration shell of S1 itself. The present results strongly suggest that the solvent space around S1 bound to F-actin is diffusionally asymmetric, which supports our model of force generation by actomyosin proposed previously [op. cit.].     

Myosin XI drives polarized growth by vesicle focusing and local enrichment of F-actin in Physcomitrium patens

In tip-growing plant cells, growth results from myosin XI and F-actin-mediated deposition of cell wall polysaccharides contained in secretory vesicles. Previous evidence showed that myosin XI anticipates F-actin accumulation at the cell’s tip, suggesting a mechanism where vesicle clustering via myosin XI increases F-actin polymerization. To evaluate this model, we used a conditional loss-of-function strategy by generating moss (Physcomitrium patens) plants harboring a myosin XI temperature-sensitive allele. We found that loss of myosin XI function alters tip cell morphology, vacuolar homeostasis, and cell viability but not following F-actin depolymerization. Importantly, our conditional loss-of-function analysis shows that myosin XI focuses and directs vesicles at the tip of the cell, which induces formin-dependent F-actin polymerization, increasing F-actin’s local concentration. Our findings support the role of myosin XI in vesicle focusing, possibly via clustering and F-actin organization, necessary for tip growth, and deepen our understanding of additional myosin XI functions.

Vesicle clustering by the molecular motor myosin XI enhances actin polymerization-dependent motility and polarized vesicle accumulation in tip-growing cells.     

Cell Elasticity Is Regulated by the Tropomyosin Isoform Composition of the Actin Cytoskeleton

The actin cytoskeleton is the primary polymer system within cells responsible for regulating cellular stiffness. While various actin binding proteins regulate the organization and dynamics of the actin cytoskeleton, the proteins responsible for regulating the mechanical properties of cells are still not fully understood. In the present study, we have addressed the significance of the actin associated protein, tropomyosin (Tpm), in influencing the mechanical properties of cells. Tpms belong to a multi-gene family that form a co-polymer with actin filaments and differentially regulate actin filament stability, function and organization. Tpm isoform expression is highly regulated and together with the ability to sort to specific intracellular sites, result in the generation of distinct Tpm isoform-containing actin filament populations. Nanomechanical measurements conducted with an Atomic Force Microscope using indentation in Peak Force Tapping in indentation/ramping mode, demonstrated that Tpm impacts on cell stiffness and the observed effect occurred in a Tpm isoform-specific manner. Quantitative analysis of the cellular filamentous actin (F-actin) pool conducted both biochemically and with the use of a linear detection algorithm to evaluate actin structures revealed that an altered F-actin pool does not absolutely predict changes in cell stiffness. Inhibition of non-muscle myosin II revealed that intracellular tension generated by myosin II is required for the observed increase in cell stiffness. Lastly, we show that the observed increase in cell stiffness is partially recapitulated in vivo as detected in epididymal fat pads isolated from a Tpm3.1 transgenic mouse line. Together these data are consistent with a role for Tpm in regulating cell stiffness via the generation of specific populations of Tpm isoform-containing actin filaments.     

Multifunctional roles of tropomodulin-3 in regulating actin dynamics

Tropomodulins (Tmods) are proteins that cap the slow-growing (pointed) ends of actin filaments (F-actin). The basis for our current understanding of Tmod function comes from studies in cells with relatively stable and highly organized F-actin networks, leading to the view that Tmod capping functions principally to preserve F-actin stability. However, not only is Tmod capping dynamic, but it also can play major roles in regulating diverse cellular processes involving F-actin remodeling. Here, we highlight the multifunctional roles of Tmod with a focus on Tmod3. Like other Tmods, Tmod3 binds tropomyosin (Tpm) and actin, capping pure F-actin at submicromolar and Tpm-coated F-actin at nanomolar concentrations. Unlike other Tmods, Tmod3 can also bind actin monomers and its ability to bind actin is inhibited by phosphorylation of Tmod3 by Akt2. Tmod3 is ubiquitously expressed and is present in a diverse array of cytoskeletal structures, including contractile structures such as sarcomere-like units of actomyosin stress fibers and in the F-actin network encompassing adherens junctions. Tmod3 participates in F-actin network remodeling in lamellipodia during cell migration and in the assembly of specialized F-actin networks during exocytosis. Furthermore, Tmod3 is required for development, regulating F-actin mesh formation during meiosis I of mouse oocytes, erythroblast enucleation in definitive erythropoiesis, and megakaryocyte morphogenesis in the mouse fetal liver. Thus, Tmod3 plays vital roles in dynamic and stable F-actin networks in cell physiology and development, with further research required to delineate the mechanistic details of Tmod3 regulation in the aforementioned processes, or in other yet to be discovered processes.