Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins

Courtemanche, Naomi; Pollard, Thomas D.; Qian, Chen

doi:10.1038/ncb3351

Cited by 127 publications

(204 citation statements)

References 42 publications

(62 reference statements)

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“…Our analysis was based on center-to-center measurements between filaments, which means that the sharp drop-off below 12 nm translates into a minimum distance of only 4.5 nm between two 7.5-nm-thick filaments. Note that these measurements are in large agreement with an estimate of ∼15 nm based on quantitative fluorescence microscopy (11).…”

supporting

confidence: 79%

“…On average, there were 34 (SD = 12) filaments per bundle cross-section, with individual numbers ranging between 14 and 60 filaments (Fig. 5), which was largely compatible with quantitative fluorescence microscopy that estimated there would be ∼50 filaments per ring cross-section (11). The majority of filaments traversed the entire thickness of the tomographic section, but many filament ends were visible as well (arrowheads in Fig.…”

mentioning

confidence: 51%

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Structure of the fission yeast actomyosin ring during constriction

Swulius

Nguyen

Ladinsky

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Cell division in many eukaryotes is driven by a ring containing actin and myosin. While much is known about the main proteins involved, the precise arrangement of actin filaments within the contractile machinery, and how force is transmitted to the membrane, remains unclear. Here we use cryosectioning and cryofocused ion beam milling to gain access to cryopreserved actomyosin rings in Schizosaccharomyces pombe for direct 3D imaging by electron cryotomography. Our results show that straight, overlapping actin filaments, running nearly parallel to each other and to the membrane, form a loose bundle of ∼150 nm in diameter that "saddles" the inward-bending membrane at the leading edge of the division septum. The filaments do not make direct contact with the membrane. Our analysis of the actin filaments reveals the variability in filament number, nearest-neighbor distances between filaments within the bundle, their distance from the membrane, and angular distribution with respect to the membrane.C ytokinesis, the final step of cell division in eukaryotic cells, is typically driven by a contractile actomyosin ring (AMR) primarily composed of actin (1) and myosin (2). Our understanding of the molecular mechanisms of cytokinesis is most detailed in the rodshaped unicellular eukaryote Schizosaccharomyces pombe (otherwise known as fission yeast), which shares a remarkably conserved set of cytokinesis genes with metazoans (3). In S. pombe, the AMR undergoes multiple phases known as assembly, maturation, constriction, and disassembly (4), with open questions in each of these four stages. Due to a lack of information about the precise arrangement of filamentous actin (F-actin) within the force-generating network of the AMR, we chose to focus on imaging the AMR during constriction.In S. pombe, glancing sections through plastic-embedded, dividing cells gave the first glimpse of actin filaments running parallel to the division plane at the front of the septum (5). Unfortunately, the study yielded limited examples and lacked 3D information for a full analysis. In an ambitious pioneering effort, Kamasaki et al. (6) produced 3D reconstructions of entire S. pombe AMRs by imaging serial sections through permeabilized cells decorated with myosin S1 fragments. The amount of F-actin and the size of the rings appeared significantly altered by the procedure used for preserving them (details in Discussion), but the continuous bundles that were reconstructed were composed of mixed polarity filaments running circumferentially around the cell.Here we sought to visualize the precise arrangement of F-actin within the AMR and its interface with the membrane by imaging intact cells in a cryopreserved state using electron cryotomography (ECT) (7). Because whole S. pombe cells are too thick for ECT, which is limited to specimens thinner than a few hundred nanometers, we overcame this obstacle by first rapid freezing dividing cells and then either cryosectioning them or using the recently developed method cryofocused ion beam (cryo-FIB) milling to ...

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supporting

confidence: 79%

mentioning

confidence: 51%

Structure of the fission yeast actomyosin ring during constriction

Swulius

Nguyen

Ladinsky

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…2B). We note that although a recent report suggests that LifeAct may affect actin dynamics (Courtemanche et al, 2016), we find that several LifeAct::TagRFP-expressing transgenes do not cause any EC phenotypes (Shaye and Greenwald, 2015), and LifeAct remains a reliable marker for measuring total Factin (Courtemanche et al, 2016; see also below). Venus::INFT-2, shown to function in the EC as described below, also colocalizes with F-actin, and colocalization was lost in cdc-42(0*) mutants (Fig.…”

Section: Introductionmentioning

confidence: 98%

“…4A,B; Materials and Methods). We note that although recent evidence suggests that LifeAct may affect actin polymerization kinetics, it remains a reliable marker for measuring the total number of polymerized actin molecules (Courtemanche et al, 2016).…”

Section: Cyk-1 Regulates Inft-2-dependent F-actin Levels and Inft-2 Amentioning

confidence: 99%

A network of conserved formins, regulated by the guanine exchange factor EXC-5 and the GTPase CDC-42, modulates tubulogenesis in vivo

Shaye

Greenwald

2016

Development

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The C. elegans excretory cell (EC) is a powerful model for tubulogenesis, a conserved process that requires precise cytoskeletal regulation. EXC-6, an ortholog of the diseaseassociated formin INF2, coordinates cell outgrowth and lumen formation during EC tubulogenesis by regulating F-actin at the tip of the growing canal and the dynamics of basolateral microtubules. EXC-6 functions in parallel with EXC-5/FGD, a predicted activator of the Rho GTPase Cdc42. Here, we identify the parallel pathway: EXC-5 functions through CDC-42 to regulate two other formins: INFT-2, another INF2 ortholog, and CYK-1, the sole ortholog of the mammalian diaphanous (mDia) family of formins. We show that INFT-2 promotes F-actin accumulation in the EC, and that CYK-1 inhibits INFT-2 to regulate F-actin levels and EXC-6-promoted outgrowth. As INF2 and mDia physically interact and cross-regulate in cultured cells, our work indicates that a conserved EXC-5−CDC-42 pathway modulates this regulatory interaction and that it is functionally important in vivo during tubulogenesis.

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“…It has been reported that, in contrast to expression in somatic cells or weak expression in the germline, a strong expression of LifeAct in the Drosophila germline results in severe actin remodeling and fertility defects (Spracklen et al, 2014). Moreover, it has been shown that LifeAct affects actin assembly during endocytosis and cytokinesis in fission yeast (Courtemanche et al, 2016). Although the underlying mechanisms are not well understood, both studies claim that the LifeAct-mediated effects on actin dynamics are concentration-dependent and thus suggest that the use of LifeAct requires a more elaborate optimization process.…”

Section: Live-cell Imaging Using Gfp-actin Derivativesmentioning

confidence: 99%

Actin visualization at a glance

Melak¹,

Plessner²,

Grosse³

2017

Development

127

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There was an error published in J. Cell Sci. 130,[525][526][527][528][529][530] Unfortunately, the diagram of the actin filament in the poster was accidentally reflected during the creation of the figure. As a result, the filament was illustrated as a left-handed rather than a right-handed F-actin helix in the original version of this article. The online version of the article has been corrected accordingly. This change has no impact on the concepts presented in this article.The authors apologise to the readers for any confusion that this error might have caused. 1688 Journal of Cell Science CELL SCIENCE AT A GLANCE Actin visualization at a glanceMichael Melak, Matthias Plessner and Robert Grosse* ABSTRACT Actin functions in a multitude of cellular processes owing to its ability to polymerize into filaments, which can be further organized into higher-order structures by an array of actin-binding and regulatory proteins. Therefore, research on actin and actin-related functions relies on the visualization of actin structures without interfering with the cycles of actin polymerization and depolymerization that underlie cellular actin dynamics. In this Cell Science at a Glance and the accompanying poster, we briefly evaluate the different techniques and approaches currently applied to analyze and visualize cellular actin structures, including in the nuclear compartment. Referring to the gold standard F-actin marker phalloidin to stain actin in fixed samples and tissues, we highlight methods for visualization of actin in living cells, which mostly apply the principle of genetically fusing fluorescent proteins to different actin-binding domains, such as LifeAct, utrophin and F-tractin, as well as antiactin-nanobody technology. In addition, the compound SiR-actin and the expression of GFP-actin are also applicable for various types of live-cell analyses. Overall, the visualization of actin within a physiological context requires a careful choice of method, as well as a tight control of the amount or the expression level of a given detection probe in order to minimize its influence on endogenous actin dynamics.

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Avoiding artefacts when counting polymerized actin in live cells with LifeAct fused to fluorescent proteins

Cited by 127 publications

References 42 publications

Structure of the fission yeast actomyosin ring during constriction

Structure of the fission yeast actomyosin ring during constriction

A network of conserved formins, regulated by the guanine exchange factor EXC-5 and the GTPase CDC-42, modulates tubulogenesis in vivo

Actin visualization at a glance

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