Comparing lifeact and phalloidin for super-resolution imaging of actin in fixed cells

Abstract

Visualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a dSTORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments.

Modulated Start-Up Mode of Cancer Cell Migration Through Spinophilin-Tubular Networks

“…This process is called invadopodium (Revach et al, 2015;Eddy et al, 2017). Cells were stained with actin-rich invadopodia marker phalloidin (Wulf et al, 1979;Mazloom-Farsibaf et al, 2021) and the adhesion protein marker vinculin (Citi, 2019) in the presence of siSPL. The number of vinculin dots was enhanced (Figures 3A,B), with the intensity of phalloidin also enhanced in siSPL-treated A549 cells ( Figures 3A,C and low magnification images in Supplementary Figure 2).…”

Robust, fiducial-free drift correction for super-resolution imaging

“…Imaging was performed in TNG buffer mixed with Lifeact conjugated with dye. The blinking events for single molecule localization were due to the binding/unbinding of Lifeact to the actin filaments 39 , 40 . We used the same 25 mm coverslip chamber as above to mount the samples on the microscope.…”

Transient Fluorescence Labeling: Low Affinity—High Benefits

“…In addition, a common feature of cytoskeleton fibers is their composition of monomers which polymerize and depolymerize in response to cellular stimuli. Importantly, some high-affinity labels, such as phalloidin-based labels [82], stabilize the polymerized form, disrupting normal cellular functioning [83,84].…”

“…In addition, Lifeact exhibits a 10-fold higher affinity to G-actin, leading to background cytosolic actin labeling [14]. Nonetheless, the Lifeact-based imaging of actin is comparable or slightly better than dSTORM (direct STORM) with conventional phalloidin staining [82]. The list of actin-binding probes with low affinity includes Utr261 and F-tractin.…”