Involvement of Mechanical Strain in Actin Network Reorganization - Innovative Approaches to Cell Biomechanics - page 41

Other studies have also applied FMS and computer aided flow tracking to map
the dynamics of actin filament network during cell migration (Waterman-Storer
et al. 1998 ; Hu et al. 2007 ; Watanabe and Mitchison 2002 ; Ponti et al. 2004 ). For
instance, comprehensive mapping of F-actin velocity in epithelial cells revealed an
interesting correlation between meshwork contraction and depolymerization
(Vallotton et al. 2004 ; Okeyo et al. 2006 ). An elegant method for generating detailed
but scalar correlative maps of both actin and myosin II dynamics in migrating fish
keratocytes has also been described (Schaub et al. 2007 ).
For a comprehensive understanding of the actin network flow that accompany
cell migration in many cell types, it is important to evaluate actin network dynamics
both spatially and temporally and also to understand the nature of its various com-
ponents. We discuss how to do this in the following sections.

4.3 Labeling and Imaging Actin Network in Lamellipodia

Fish epidermal keratocytes isolated from the scale of a fish such as black tetra
(Gymnocorymbus ternetzi) can be cultured for days in typical culture media such
as DMEM (Dulbecco's modified eagle medium) supplemented with 10 % FBS
(fetal bovine serum) and 1 % antibiotics. Methods for isolating and culturing these
cells are well established in literature (Svitkina et al. 1997 ).
Since keratocytes do not divide, staining them by the transfection method is
challenging. For this reason, capillary microinjection and electroporation are more
convenient methods to deliver fluorescent dyes to live-stain the actin cytoskeleton in
these cells. In the case of microinjection, a fluorescently labelled phalloidin that
bind with high affinity to actin filaments is injected into cells in minute quantities.
Quantum dots are preferred labeling dyes because they give a high quantum yield
and they are more photostable compared with other commonly used dyes such as
rhodamine that are more susceptible to photobleaching. For instance, a minute
volume (<1 pl) of 8 ʼM Qdot-phalloidin in borate buffer can be injected to label
F-actin in fish keratocytes.
Phalloidin is a commonly used phallotoxins that preferentially binds to and
stabilizes F-actin (but not G-actin) and minute quantities of fluorescently-labelled
phallotoxins administered into keratocytes can enable the visualization of the actin
network in these cells (De La Cruz and Pollard 1994 ; Mahaffy and Pollard 2008 ).
Thus, the fluorescence of labeled phalloidin is much higher when bound to F-actin
than when free (Huang et al. 1992 ). Random attachment of Qdot-phalloidin yields
a random distribution of Qdots in the actin network and the speckle pattern observed
depends on the convolution of the distribution with the point-spread function of the
microscope (Danuser and Waterman-Storer 2006 ). Although widely used because
of its high specificity for actin filaments, phalloidin is known to induce stiffening of
F-actin, slightly altering the mechanical properties of bounded F-actin (Mahaffy
and Pollard 2008 ). For this reason, it is important to inject cells with only a very
small quantity (<1 pl) of a dilute solution of phalloidin to minimize the stiffening
effect and avoid any negative impact on cell migration.