Versatile fluorescent probes for actin filaments based on the actin‐binding domain of utrophin

Abstract

Actin filaments (F-actin) are protein polymers that undergo rapid assembly and disassembly and control an enormous variety of cellular processes ranging from force production to regulation of signal transduction. Consequently, imaging of F-actin has become an increasingly important goal for biologists seeking to understand how cells and tissues function. However, most of the available means for imaging F-actin in living cells suffer from one or more biological or experimental shortcomings. Here we describe fluorescent F-actin probes based on the calponin homology domain of utrophin (Utr-CH), which binds F-actin without stabilizing it in vitro. We show that these probes faithfully report the distribution of F-actin in living and fixed cells, distinguish between stable and dynamic F-actin, and have no obvious effects on processes that depend critically on the balance of actin assembly and disassembly.

Actomyosin Contraction at the Cell Rear Drives Nuclear Translocation in Migrating Cortical Interneurons

“…The UTRCH-ABD probe has been shown to report the presence of F-actin without altering F-actin concentrations (Burkel et al, 2007). We obtained the same temporal and spatial fluorescence pattern with Utrophin-EGFP (see supplemental Fig.…”

Folate receptor 1 is necessary for neural plate cell apical constriction during Xenopus neural tube formation

“…Two-cell-stage embryos were bilaterally injected with 800 pg mCherrytagged utropin mRNA (mCherry-UtrCH, Addgene plasmid #26740, deposited by William Bement; Burkel et al, 2007) and unilaterally injected with 10 pmol Folr1-MO per cell along with Alexa 647-conjugated dextran. Medial neural plates of embryos at stage 14-14.5 were imaged for 15 min at 30-55 s intervals using 60× objective and z-stack confocal imaging (Sweptfield confocal, Nikon).…”

Osmotic surveillance mediates rapid wound closure through nucleotide release

“…1 b) integrate the functional contributions of all force-generative processes operant in an injured immersed in either normal, hypotonic bathing medium or bathing medium that had been adjusted to the ionic composition and/or tonicity of vertebrate interstitial fluid (with the addition of NaCl or sucrose). The actin cytoskeleton and plasma membranes were labeled using GFP-utrophin-calponin homology domain (GFPUtr-CH; Burkel et al, 2007), and AKT-pleckstrin homology domain (AKT-PH)-GFP (Kwon et al, 2007), respectively. Injection of mRNA into one-cell stage embryos led to ubiquitous labeling.…”

“…mrlc1-mKate2 was PCR amplified from pME and cloned into pCS2+ with XhoIXbaI. pCS2+GFP-Utrophin calponin homology domain (GFP-UtrCH) was created as described previously (26737; Addgene; Burkel et al, 2007). In brief, UtrCH (amino acids 1-261) was cloned into the BspE1-XhoI sites of the pCS2-EGFP plasmid (sp6 promoter).…”