Live Fluorescence Imaging of F-Actin Organization in Chick Whole Embryo Cultures Using SiR-Actin.

Manuel Schmitz-Elbers,Gražvydas Lukinavičius,Theodoor H Smit

doi:10.3390/cells10071578

Manuel Schmitz-Elbers, Gražvydas Lukinavičius + Show 1 more

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https://doi.org/10.3390/cells10071578

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Abstract

Morphogenesis is a continuous process of pattern formation so complex that it requires in vivo monitoring for better understanding. Changes in tissue shape are initiated at the cellular level, where dynamic intracellular F-actin networks determine the shape and motility of cells, influence differentiation and cytokinesis and mediate mechanical signaling. Here, we stain F-actin with the fluorogenic probe SiR-actin for live fluorescence imaging of whole chick embryos. We found that 50 nM SiR-actin in the culture medium is a safe and effective concentration for this purpose, as it provides high labeling density without inducing morphological malformations.

Highlights

Embryogenesis is a complex process of tissue formation driven by the tightly coordinated behavior of numerous cells
We found that a SiR-actin concentration of 50 nM is both safe and effective for live fluorescent imaging of early chick embryos: it provides high labeling density without the occurrence of morphological abnormalities, such as head malformations or incomplete somite separations that may be caused by interference of the fluorescent probe with filamentous actin (F-actin) dynamics
While the initial live recordings of F-actin in the young chick embryo were successful, we found that culturing in the presence of 240 nM SiR-actin for more than 12 h induced morphological abnormalities

Summary

Introduction

Embryogenesis is a complex process of tissue formation driven by the tightly coordinated behavior of numerous cells. To proliferation and programmed cell death, this involves shape changes and relative cell movements [1]. Cell shape and motility as well as mechanotransduction, differentiation, and cytokinesis are mainly determined by the architecture and contractility of the intracellular filamentous actin (F-actin) network [2,3,4]. F-actin is often visualized by fluorescently labeled phalloidin, a filamentbinding toxin of low molecular weight assumed to “provide the most complete and accurate picture of the actin cytoskeleton” [5,6,7]. Phalloidin does not have the inherent disadvantages of antibody-based labeling techniques, such as non-specific binding, actin scavenging, high background, or epitope variations between actin species [8,9]. To understand the role of F-actin in morphogenesis, it is necessary to study it dynamically in vivo

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