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.

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!


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

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…]

Microtubule destabilization by suprafenacine: Template for novel anti-cancer drugs

Recently, B.-H. Choi et al. characterized a novel anti-mitotic molecule termed suprafenacine which destabilizes microtubules, resulting in cell cycle arrest in the G2/M phase and apoptotic cell death.  In silico screening identified several novel anti-cancer molecules based on their ability to inhibit in vitro cell proliferation and tubulin Tubulin cartoonpolymerization.  Structure Activity Relationship studies guided the synthesis of several analogues.  Of these analogues, suprafenacine was the most potent based on its in vitro ability to 1. specifically target cancer cells from multiple tumor types and 2. inhibit tubulin polymerization (IC50 = 0.38 mM).  [Read more…]

2 new Actin and Tubulin live-cell imaging stains – without transfection!

Cytoskeletal live-cell imaging is extremely powerful when investigating cellular processes such as cytokinesis, motility and organelle transport and organization. The current experimental procedures remain nevertheless cumbersome and long. This post demonstrates how cell permeable, transfection free, Tubulin and Actin red fluorescent dyes help Cell biologists in analysing cytoskeleton dynamics in living cells.

[Read more…]

Citrullination: Taking the Charge out of Arg

Protein citrullination (a.k.a. deimination) is a novel arginine-directed post-translational modification (PTM) that results in a permanent change in the targeted protein. PeptidylArginine Deiminases (PADs) mediate the calcium-dependent deimination of the guanidino group of Arginine side chains to form an ureido group and the non-standard amino acid citrulline.

Some biologically relevant proteins known to be citrullinated include Keratin, Filaggrin, Trichohyalin, Vimentin, Myelin Basic Protein (MBP), Histones, alpha-Enolase, Fibrinogen, Fibrins, Collagen type I and II, beta-Actin, and Tubulin 9-11… It is noteworthy that several of these proteins are part of the cytoskeleton and/or are structural in nature.


Citrullination of peptidyl-arginine by peptidylarginine deiminases (PADs).

In their October newsletter, Cytoskeleton Inc. presents an overview about the consequences of citrullination, especially referring to cytoskeleton proteins such as Vimentin.

Interested in this exciting new PTM mechanism?

Download your free copy of the review:
Citrullination: Taking the Charge out of Arg


5 Cytoskeletal Modeling Molecules

Cytoskeleton modeling molecules are relevant when trying to improve one’s understanding of cytoskeletal molecular modeling and associated mechanisms. Together with actin binding proteins, tubulin-based assays, small GTPase activation assays etc… these reagents are called small molecules, but they remain extremely potent in in vitro cell-based assays. Here, let’s take look at a selection of the most popular chemicals modifying actin or microtubule polymerization.

#1- Docetaxel

An antimitotic chemotherapeutic acting on the centrosome of the mitotic spindle via reversible high-affinity binding to microtubules. Docetaxel induces apoptosis in a variety of cancer cell lines.

#2- Epothilone B

A tubulin polymerization promoter inducing G2-M cell cycle arrest stabilizing microtubules and displaying potent cytotoxic activity in a variety of cell lines and mouse models.

#3- Latrunculins

Potent actin polymerization inhibitor disrupting microfilament organization.

#4- Nocodazole

A microtubule polymerization inhibitor used to induce mitotic arrest and cell synchronization. Nocodazol inhibits a number of cancer-related kinases including ABL, c-Kit, BRAF, MEK1, MEK2, and MET.

#5- Taxol

A chemotherapeutic agent for the treatment of breast, non-small cell lung and ovarian cancer. Taxol promotes tubulin polymerization, stabilizes microtubules in vitro and in vivo resulting in arrest of cells in the G2 and M phase of the cell cycle.

Many other well-qualified cytoskeleton modulators (Ansamitocin P-3, Cytochalasins, Colchicine, Vinblastine sulfate…) are available from various sources (my preference going to Focus Biomolecules for quality and price advantages!). Nevertheless, the 5 described here are among those most spontaneously cited by researchers.

What about you? Which ones would you recommend to study cytoskeleton dynamics?


How to probe motor-domains of kinesins?

In the past, molecular motor proteins like kinesins have been investigated with macromolecular approaches. Recent research on kinesins has been focused on resolving how kinesin is regulated by intramolecular dynamics. [Read more…]