SiR-actin and SiR-tubulin kits now contain Verapamil

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.

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

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. 

Today, I’d like to invite you to take a look at the 5 posts describing a pathway that saw the most visits on our blog in 2014. 

Just follow the links if you haven’t read them yet (or if you want to browse them again, feel free!).

Recently, R. Palanivel et al. investigated the role that RhoA-mediated re-organization of the actin cytoskeleton has in adiponectin-regulated glucose uptake in cardiomyocytes.  Adiponectin is a protein secreted by adipose tissue that modulates glucose and fatty acid metabolism.  In concert with APPL1, an adiponectin receptor binding partner, adiponectin carries out these functions which are important in obesity and type 2 diabetes, two diseases that reduce cardiac energy metabolism. The authors found that adiponectin (both full-length and globular) elevates RhoA activity which correlates with increased actin polymerization and glucose uptake.  Changes in the G-/F-actin ratio likely involve APPL1 as adiponectin increases colocalization of actin and APPL1.  Inhibition of actin polymerization or RhoA signaling significantly reduces the adiponectin-mediated increase in glucose uptake.  Thus, RhoA-mediated actin cytoskeleton remodeling is required for adiponectin-regulated glucose uptake in cardiomyocytes.  Increased glucose uptake is cardioprotective in diabetes. A number of products by Cytoskeleton Inc. (“The Protein Experts”) were essential in this study, providing accurate and sensitive assays for quantifying levels of activated RhoA and changes in G- and F-actin levels or binding partners in cardiomyocytes under conditions of RhoA inhibition.

• RhoA G-LISA activation assay 
• G-/F-actin In Vivo Assay Kit
• Anti-actin antibody
• Cell-permeable Rho inhibitor

These reagents are available in Europe through tebu-bio, who have also compiled useful selection tools:

• Actin Product Guide

• Small G Protein Product Guide

Reference: Palanivel et al. 2014. Adiponectin stimulates Rho-mediated actin cytoskeleton remodeling and glucose uptake via APPL1 in primary cardiomyocytes. Metabolism. 63, 1363-1373.