Focus on Actin detection and Actin binding proteins

Actin can exist in two forms: Globular subunit (G-actin) and Filamentous polymer (F-actin). Both forms of actin interact with a plethora of proteins in the cell. To date there are over 50 distinct classes of Actin-Binding Proteins (ABPs), and the inventory is still far from complete. Actin Binding Proteins allow the actin cytoskeleton to respond rapidly to cellular and extracellular signals and are integral to cytoskeletal involvement in many cellular processes. These include cell shape and motility, muscle contraction, intracellular trafficking, cell pathogenesis and signal transduction.

In the coming weeks I’d like to give you an overview of methods in actin research with validated R&D products and kits (actin polymerisation, and G-F actin ratio detection in cells); I also invite you to take a look at a post recently released about actin visualization: Focus on Actin staining and visualization.

In today’s post, let’s concentrate on a method which allows measuring actin binding capabilities of proteins of interest. But it’s not only about the simple fact that a given protein is binding to actin, with the method presented here, you’re also able to get an idea of the functionality of the protein – be it F-actin bundling activity, F-actin severing activity or G-actin binding activity. [Read more…]

Focus on Actin staining and visualization

Actin serves as one of the major cytoskeleton structures. It is a crucial component involved in a plethora of processes in cell biology:  stabilizing the cell shape, cell movements (e.g. cell migration)  and intracellular movements and transport mechanisms.

Actin is a 43 kDa protein that is very highly conserved between species. Actin has three main isotypes (α-actin, β-actin and γ-actin), which show >90% amino-acid (aa) homology between isotypes and >98% homology within members of a particular isotypic group.

A brief reminder: G-actin polymerizes to form F-actin

Untitled

Fig. 1: Double-helical structure of actin filaments (provided by Cytoskeleton Inc.)

Globular-actin (G-actin) readily polymerizes under physiological conditions to form Filamentous-actin (F-actin) with the concomitant hydrolysis of ATP. F-actin is a double-helical filament (Fig. 1).  Actin can polymerize from both ends in vitro. However, the rate of polymerization is not equal. This results in an intrinsic polarity in the actin filament. It has therefore become the convention to term the rapidly polymerizing end the plus-end or barbed-end (+) while the slow growing end is called the minus-end or pointed-end (-).

In the coming weeks I will give you an overview about methods in actin research with validated R&D products and kits (actin binding and actin binding proteins, actin polymerisation, and G-F actin ratio detection in cells).

 

Today I will focus on methods to visualize actin in fixed or living cells – which belong to basic experimental set-ups in Cell Biology.

Actin staining of fixed cells

Actistain photo

Fig. 2: Swiss 3T3 fibroblasts stained with ActiStain 488 (green), Dapi (blue) and Anti Vinuclin (orange). Provided by Cytoskeleton Inc.

Often fluorescent phalloidins are used to stain actin in fixed cells. Phalloidin belongs to the group of phallotoxins produced by the mushroom Amanita phalloides (death cap mushroom). The natural toxicity of Phalloidin is due to its stabilizing effect on F actin in cells. Based on its affinity for F-actin and coupled to a fluorescent dye, it can be used to visualize F-actin.

Cytoskeleton Inc. offers a set of phalloidin based stains (Acti-Stains) coupled to a number of different fluorophores compatible with popular filter sets such as FITC, TRITC and Cy5. The stains are exceptionally bright and stable and are indeed offered at very economical prices compared to other phalloidin based stains coupled to fluorophores of similar stability.

Results of staining of Swiss 3T3 cells with ActiStain 488 are shown in Fig. 2.

Live-cell imaging of Actin

Live-cell imaging of actin has been quite tricky as far as actin labelling is concerned – either cells had to be transfected with vectors carrying the genetic information for fluorescently tagged actin or actin binding proteins or, labelled actin had to be micro-injected to single cells.

Actin

Fig. 3: 3D-SIM microscopy image of labeled Actin stress fibers in human primary dermal fibroblasts. Provided by Spirochrome.

Together with Spirochrome, tebu-bio launched in Europe the first tool to directly label actin in living cells with no need to transfect or micro-inject anything.

SiR-Actin is a cell permeable compound which stains F-actin in living cells. The stain is composed of a photostable silicon rhodamine-like (SiR) dye which can be used with standard Cy5 settings and a component (Jasplakinolide) which specifically binds to F-actin (Fig. 3). SiR-actin is compatible with Super-Resolution Microscopy like Stimulated Emission Depletion [STED] and Structured Illumination Microscopy [SIM].

f you would like to get an overview about the results SiR-actin users got so far, please have a look at my recent blog: User experience of SiR-Actin and SiR-Tubulin Live Cell Imaging.

 

 

 

Interested in our Phalloidin and/or SiR-based stains?

Leave your comment or request in the form below.

 

Actin and Tubulin dynamics research studies in 2015

The world of cytoskeletal dynamics studies is in mutation. By example, the first synthetic chemical targeting actin and specifically triggering its growth has been recently released for research applications (BPA). A few weeks before, SiR stains were successfully launched for Actin and Tubulin Live Cell Imaging (SiR-actin Live cell Imaging was at the JBC frontcover in Feb. 2015).

Cellular and molecular biologists have now access to a unique range of discovery tools opening new perspectives for deciphering cytoskeletal events living cells.

Branched PolyAmines (BPA)

Until now, only molecules that stabilize or destroy the cytoskeleton of actin were available. Derived from supramolecular chemistry, Branched PolyAmines (BPA) rapidly enhances the growth of lamellar networks of actin filaments.

BPA-induced actin filaments

Actin filamentous network growth 10 minutes after addition of BPA to cell cultures.Source: Pr. Daniel Riveline, Laboratory of Cell Physics, ISIS/IGBMC, Strasbourg (France).

BPA is a specific modulator for in vitro and in vivo actin dynamics studies by regulating actin nucleation and turnover in cells. This compound complements currently available molecules to study actin dynamics such as Latrunculin A , Cytochalasin D, Jasplakinolide, WiskostatinActin Binding Proteins.

Transfection-free SiR Actin and Tubulin stains

SpiroChrome’s live cell SiR actin stains are non-toxic fluorescent stains for monitoring Actin and Tubulin changes in living cells. These cell permeable SiR-Actin and SiR-Tubulin compounds stain microtubules and F-actin respectively in living cells.

SiR Tubulin tebu-bio's fluorescent dye

3D-SIM microscopy image of labeled microtubules in primary rat cortex neuron body stained with SiR Tubulin.

Novel research tools aiming at better understanding cytoskeletal reorganization have radically changed discovery frontiers. Interestingly, and despite their degree of innovation, these new reagents remain very affordable and user friendly for research applications.

tebu-bio’s experts will follow on tracking emerging cystoskeleton-based technologies.

 

Staining Actin and Tubulin – from WB to Live Cell Imaging

Anti Anti Ab - IF photo

Fig. 1: Immunofluorescence images of mouse Swiss 3T3 cells stained with anti-actin antibody (027AAN01).

Actin and Tubulin, as the major cytoskeleton structures, are crucial components of a plethora of processes in cell biology. Both are very much involved in stabilizing the cell shape, and especially in cell movements (e.g. cell migration) and intracellular movements and transport mechanisms.

Thus visualizing Actin and Tubulin in fixed or living cells and detecting them in biological samples (e.g. in Western Blots) belongs to basic experimental set-ups in Cell Biology.

A broad range of tools is available for all kind of experimental levels, in this post we’ll take a look at some of these you can use from Western Blot to Live Cell Imaging. [Read more…]

User experience of SiR-Actin and SiR-Tubulin Live Cell Imaging

Very recently we launched new Live Cell Imaging tools: SiR-Actin and SiR-Tubulin, produced by Spirochrome.

These stains allow you to stain actin and tubulin in living cells without the need to transfect cells – as I described in my previous posts on these tools:

Today, I invite you to take a look at the brilliant results users of the stains have obtained. Some of them have already been published during the past months.

Most recent publications:

One of the most recent publications using SiR-Actin comes from the lab of the Nobel price winner Stefan W. Hell, who is one of the directors of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany:

STED Nanoscopy Reveals the Ubiquity of Subcortical Cytoskeleton Periodicity in Living Neurons, Elisa D’Este, Dirk Kamin, Fabian Göttfert, Ahmed El-Hady, and Stefan W. Hell, Cell Reports, 10, 8, 1246–1251 (2015)

By using STED (stimulated emission depletion) microscopy, Hell’s group could visualize the periodic subcortical actin structure in axons and dendrites in cultured hippocampal neurons. These results were obtained by staining Actin with SiR-Actin.

Another recent paper even brought an image of HEK293 cells co-expressing CaVβ and CaV1.2 L-type calcium channel and stained for actin filaments using SiR-Actin straight to the front cover of the respective JBC issue.

Direct Interaction of CaVβ with Actin Up-regulates L-type Calcium Currents in HL-1 Cardiomyocytes, Gabriel Stölting, Regina Campos de OliveiraRaul E. Guzman, Erick Miranda-Laferte, Rachel Conrad, Nadine Jordan, Silke Schmidt, Johnny Hendriks, Thomas Gensch,  and Patricia Hidalgo, Journal of Biological Chemistry, 290, 4561-4572 (2015)

The group around Patricia Hidalgo at the Institute of Complex Systems in Juelich, Germany, could show that the β-subunit (CaVβ) n of cardiac L-type calcium channels associates directly with actin filaments – again SiR-Actin was successfully used to get these results.

Videos showing the use of SiR-Tubulin

As SiR-stains are very photo stable and do not show toxic effects, even in long-term incubation, they are excellent tools for visualizing biological processes over time in videos.

We show here an example of dividing HeLa cell expressing mcherry-H2B (red) stained with SiR-Tubulin (green). Data have been collected by confocal imaging (Courtesy of Daniel Gerlich and Claudia Blaukopf, Institute of Molecular Biotechnology, Vienna, Austria).

Another video shows newborn mouse primary cardiac myocytes stained with SiR-actin. The authors used high speed (50fps) confocal imaging (Courtesy of Adam Kwiatkowski and Simon Watkins, Department of cell biology and center for biologic imaging, University of Pittsburgh, US).

Results provided by our customersHUVEC monolayer - Erik T. Valent and Geerten P. van Nieuw Amerongen - Amsterdam

HUVEC monolayer, stained with SiR-Actin. A ZEISS Axiovert 200 Marianas inverted microscope with custom ZEISS 40x air lens was used (Courtesy of Erik T. Valent and Geerten P. van Nieuw Amerongen, VU Medisch Centrum, Amsterdam , The Netherlands).

ACTIN MOD_J_Millan_2015_1Human endothelial cells B4G12 were grown on Ibidi® µ-Slide 4 Well dishes at confluence. Cells were labeled for 2h at 37°C/5% CO2 with 0.2 mM SiR-Actin and a confocal image was acquired exciting with a Laser Helio Neon of 637 nm with a Zeiss Confocal LSM510 META system (Courtesy of Cristina Ortega Muñoz and Jaime Millan, Centro de Biologica Molecular, Madrid, Spain).

 

SiR-Actin staining for transient labeling of breast cancer cells implanted into a xenogeneic zebrafish host. SiR-Actin stained cells (MDA-mb231B1 dsRED), stained overnight, followed for 6 days via confocal imaging (Leica TCS SPE) microscope (63x objective). Note the retention of SiR-Actin in vitro, and the absence of cellular artefacts with concentrations of SiR-Actin below 100 nM. Cytosolic CMV driven dsRED shown in red and Sir-Actin shown in cyan (Courtesy of Arwin Groenewoud and B. Ewa Snaar-Jagalska, Institute of Biology, Leiden, The Netherlands).

Sir-Actin (SC001)staining for transient labeling of breast cancer cells implanted into a xenogeneic zebrafish host - Arwin Groenewoud and B. Ewa Snaar-Jagalska, Leiden

STORM spines (1)Primary neurons derived from cortex + hippocampus of wild type mouse C57BL/6J were cultured for 19 days and subsequently stained with SiR-Actin and visualized with a Nikon NSTORM to shown spine formation (Courtesy of Oxana Klementieva, Gunnar Gouras (Lund University) and Catherine Kitts from Lund University Bioimaging Center [LBIC], Sweden).

We would like to thank all the users who provided the pictures and results!

And of course, we invite all researchers who would like to test the SiR stains in their laboratory to contact us through the form sheet below.

Lamellipodial BPA: A new actin dynamics investigation tool

Actin polymerisation and depolymerisation are crucial processes in cell biology. These assembly and disassembly dynamics generate protrusive forces involved in numerous cellular events (membrane scission, endocytosis or Golgi remodeling, lamellipodia and filopodia…). Other biological processes (adhesion, cytokinesis and cortex dynamics…) also involve such dynamics together with actin-myosin contractility in a higher level of complexity and interactions.

Cytoskeletal research and in particular the analysis of its dynamics is quite challenging. The need for deciphering more in details these physiological processes  and to evaluate the contribution of each of the “players” have generate huge expectations in terms of research tools.

In this post, I invite you to discover the Next Generation of research tools in Actin Dynamics studies with the new Lamellipodial Growth Promoter (BPA). [Read more…]

Live cell imaging tool SiR-actin on JBC frontcover

Not long ago,  in the summer of 2014, SiR-actin and SiR-tubulin to stain actin and tubulin in living cells were launched on the market by Spirochrome (represented across Europe by tebu-bio). Now, SiR-actin has already made its way to the front cover of the most recent issue of the Journal of Biological Chemistry.

By co-sedimentation assays and FRET experiments, Stölting et al. (1) could show that the ß-subunit of cardiac L-type calcium channels (CaVβ) directly interacts with actin filaments which are involved in intracellular trafficking. The front cover of the recent JBC issue shows spinning disk confocal images of HEK293 cells co-expressing CaVβ and CaV1.2 L-type calcium channel and stained for actin filaments using SiR-actin.

Are you also interested in directly staining actin (and/or tubulin) in living cells without any transfection step?

Take a look at our recent blogs on these reagents:

Any questions about how SiR-actin and SiR-tubulin (also available together in one Cytoskeleton Kit) could boost your research? Just leave your comments below!

Reference

(1) Stölting el al., The Journal of Biological Chemistry, 290: p. 4561-4572 (2015).

Capping, bundling, sequestering… the role of Actin Binding Proteins

Actin binding proteins (ABPs) have a wide variety of functions in regulating the cellular function of actin. They control G-actin polymerization but also drive actin filaments severing and cross-linking to form complex cytoskeleton networks. Here, I’d like to review the recent research reagents aimed at studying actin regulation.

[Read more…]

Verapamil can enhance live cell staining of Actin & Tubulin with SiR-dyes

SiR-actin and SiR-tubulin kits now contain Verapamil

Fig 3 c -

STED image (raw data) : axons of rat primary hippocampal neurons stained with SiR-actin at 16 days in vitro

Recently, we were pleased to launch highly innovative tools to stain actin and tubulin in living cells without the need to transfect cells with vectors coding for GFP- or RFP tagged proteins which bind to filamentous cytoskeletal structures. This makes the SiR stains produced by Spirochrome the only tools available on the market which allow direct live cell imaging of actin and tubulin. I introduced you to this technology, as well as the benefits of SiR-actin and SiR-tubulin, in a recent post 2 new Actin and Tubulin live-cell imaging stains – without transfection.

Quite a number of cell types have already been successfully stained with SiR dyes, e.g. HeLa cells, Vero cells, BHK cells and a lot more cell lines, as well as primary cells such as HUVECs cells, dermal fibroblasts, and hippocampal neurons.

However, it turned out that some cell types, especially cell lines, do not sufficiently take up the dye. In these cases, the addition of Verapamil usually increases the uptake efficiency significantly and results in satisfying staining. [Read more…]

Actin staining techniques in fixed and living cells

Actin can be stained in living and fixed cells to determine and follow the structure and function of the cytoskeleton. The actin cytoskeleton is a very dynamic and labile structure in the living cell, but it can be fixed by either cold methanol or paraformaldehyde prior to probing or staining for actin structures.

Actin staining in fixed cells

Phalloidin - Actin binding

Fluorescent phalloidin binding to F-Actin, Source: Cytoskeleton Inc.

In fixed cells, actin structures can be visualized by actin antibodies, fluorescent phalloidins, or even electron microscopy.
Antibodies recognize both monomer and polymer (filamentous or F-actin) actin and hence tend to have a high background compared to probes that bind only F-actin. Well designed fluorescent phalloidins only bind to the native quaternary structure of F-actin and therefore have a low background. To create the correct fixation conditions for phalloidin binding, paraformaldehyde must be used as the fixative because it retains the quanternary protein structure which is necessary for high affinity. Methanol destroys the native conformation and hence is not suitable for actin staining with phalloidin.  [Read more…]