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6. 0.5M EGTA
a. Add 190.175 g ethylene glycol bis(beta-aminoethyl ether) N,N,N
0
,N
0
-tetraa-
cetic acid (EGTA) to 1 L of dH
2
O. Stir vigorously on a magnetic stirrer. Adjust
the pH to 8.0 with NaOH. Dispense into aliquots and sterilize by either
autoclaving or filtering.
7. 1
PBS
a. 137 mM NaCl
b. 2.7 mM KCl
c. 10 mM Na
2
HPO
4
d. 2 mM KH
2
PO
4
Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na
2
HPO
4
, and 0.24 g KH
2
PO
4
in 800 mL
dH
2
O. Adjust the pH to 7.4 with HCl. Add dH
2
O to 1 L. Autoclave to sterilize.
(b) VAB-10ABD::GFP and other GFPs: Phalloidin staining is indispensible for
studying the F-actin cytoskeleton during morphogenesis. However, it is not
suitable for capturing rapid, dynamic movements involving the actin cyto-
skeleton. GFP-tagged versions of monomeric subunits of actin, such as act-2
(
Willis et al., 2006
) can be useful for some purposes, but the GFP likely
affects polymerization dynamics. An alternative approach for in vivo anal-
ysis in living embryo exploits F-actin-binding proteins. Two that have been
used during early development are moesin::GFP (
Velarde et al., 2007
) and
Lifeact::GFP (
Pohl and Bao, 2010
). For the study of morphogenesis, the
spectraplakin VAB-10, which contains both actin- and tubulin-binding
motifs, was exploited by the Labouesse laboratory to produce a construct
containing the actin-binding domain (ABD) of VAB-10 tagged with GFP or
mCherry (
Gally et al., 2009
). We have used this construct to image filopodial
dynamics during ventral enclosure (
Fig. 4A,B
), as well as circumferential
filaments network and junctional actin during elongation (
Fig. 4C, D
). Use of
the VAB-10 constructs requires care; we have found that excessive imaging
can lead to lethality in strains containing vab-10ABD transgenes. For short
periods of time, however, these constructs are invaluable for imaging rapid
actin dynamics. In addition, the original vab-10ABD strains are mosaically
expressed. For some applications, this is actually an advantage, but for others
it can be somewhat problematic.
ii Tubulin: Numerous antibodies can be used to visualize tubulin during
morphogenesis, using standard freeze-cracking (
Miller and Shakes,
1995
). Alternatively, there are several GFP constructs that have been
successfully used to visualize epidermal cells. A particularly useful set
of such constructs have recently been published that use the lbp-1 pro-
moter to drive expression of either tubulin subunits or plus-end tracking
proteins predominantly in epidermal cells. These include Plbp-1::gfp::
b
-
tubulin and Plbp-1::ebp-1/EB 1::gfp (
Fridolfsson and Starr, 2010
). These
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