Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

Summary

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Abstract

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM). We describe the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.

Protocol

Section 1: Obtaining your fluorescently labeled actin for FSM

Materials required: purified actin, fluorophore (Alexa, X-rhodamine recommended). G-buffer, ultracentrifuge

1. A key component to acquiring good FSM movies requires the proper labeling of actin (or other cytoskeletal proteins) with the fluorophore of your choice.
   We recommend using fluorophores of Alexa (488, 568 wavelengths) and X-rhodamine succinimidyl ester derivatives that target exposed lysine residues on the surface of actin.
2. When choosing your fluorophore and its appropriate wavelength, ensure that your microscope setup has the appropriate filters ....

Discussion

Several key factors are crucial for successfully obtaining speckle images, however good speckles begin and end with the quality of your fluorescently labeled actin. A high labeling ratio of dye to actin monomer, set around 0.4 to 0.7 will ensure that speckles will appear bright and discrete, while the labeled protein itself should be soluble and free of aggregates. Equally important is the microinjection procedure. To ensure normal cell homeostasis (and survival), cells need to be injected with a low flow pressure, int.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Injection buffer (IB) 50 mM potassium glutamate 0.5 M MgCl2 (APPENDIX 2A) Store up to 2 years at −20° C G-buffer 2 mM Tris⋅Cl, pH 8.0 (APPENDIX 2A) 0.2 mM CaCl2 (APPENDIX 2A) Add just before use: 0.2 mM ATP (see recipe for 100 mM) 0.5 mM 2-mercapt–thanol Store up to 1 day at 4° C