Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

400 Török et al.
3 µM FL-calmodulin, 10 µM TA-calmodulin, 3 µM FL-calmodulin, and 10 µM
TA-calmodulin. For egg preparation see ref. 15.
2. The reagents are dissolved in an injection solution containing 0.5 M KCl, 20 mM
PIPES pH 7.2, and 100 µM EGTA.
3. Pulses of 0.1% of the cell volume are delivered using a high-pressure injector
system equipped with a Narishige manipulator.
4. Estimates of the volume of the injection pulse are measured through displacement
of cytoplasm and calculated by the relationship Vol = 4/3 × π × radius 3 . The
volume of the sea urchin egg is calculated to be roughly 500 pL, so the final
concentration injected can be estimated.
3.5. Confocal Microscopy and Imaging
Digital confocal imaging enables a quantitative analysis of areas of fluorescence
within cells. Here we use this technique to examine the mechanism of
localization of calmodulin during the first cell cycle of the sea urchin zygote.
However, because the small size of the microtubules prevents application of
this type of analysis to the association of calmodulin with microtubules, the
extent of calmodulin targeting to the nucleus and the mitotic apparatus is analyzed.
(See ref. 16 for a description on scanning confocal microscopy.)
Calmodulin images are analyzed off-line using Leica Lasertechnik software.
Analysis is performed after filtering the images independently with a low-pass
filter and then dividing the calmodulin-activation-sensitive channel pixel-by-pixel
by the insensitive channel. Resultant images are processed by individually
measuring pixel intensity values.
3.5.1. Imaging of Calmodulin Localization
1. FL-, Cy5-, and Texas Red-calmodulins report calmodulin localization in the cell.
We use the example of FL-calmodulin to illustrate calmodulin localization during
mitosis. FL-calmodulin is injected to a final concentration of 5 µM before the eggs
are fertilized. Prior to fertilization, the calmodulin is localized within the nucleus
whereas after fertilization the zygote nucleus centers and the astral microtubule
arrays form until nuclear envelope breakdown (NEB) and entry into mitosis. In this
period, calmodulin is localized in the nucleus and along the microtubule arrays.
After the breakdown of the nuclear envelope, calmodulin is localized mainly in the
spindle poles and can also be visualized in the location of the chromosomes. Finally,
calmodulin is present within the reformed nuclei of the daughter cells as the
embryo cleaves. This sequence of events can be seen in Fig. 13.
2. To test if the localization of FL-calmodulin was caused by specific targeting of
the protein, fluorescein conjugated to a 10,000 molecular weight Dextran is used
as a control. This is also injected before fertilization to a concentration of 5 µM.
FL-Dextran reports distributions in the cytoplasmic water space and perhaps
nonspecific binding. FL-dextran is mainly localized cytoplasmically, whereas
FL-calmodulin binds specifically to the astral tubule array. Also, the localization