Kristen M Johansen and J0rgen Johansen Introduction

One of the major foci in cell biology is to understand the process of nuclear division. In each cell cycle, the chromosomes must be faithfully replicated and the complex nuclear structure has to be duplicated and reorganized (1-4). Our understanding of the cell cycle and mitosis has increased dramatically in the last several years, as a result of cross-disciplinary approaches combining molecular, cell biological, and genetic techniques (reviewed in refs. 5-9). An organism offering a particularly advantageous model system for such studies of mitosis is the early embryo of Drosophila melanogaster. The cytoskeleton and mitotic spindle are large and easily visualized, thus facilitating structural analysis. The embryo undergoes 13 rapid and nearly synchronous nuclear divisions giving rise to about 5000 nuclei before cell boundaries form after 3 h of development (10-12). This syncytial organization of nuclei affords excellent accessibility for experimental perturbations (e.g., using antibodies [13-17] or pharmacological tools [15,18-20]).

In this chapter, we present approaches that have been optimized for a number of chromosomal and spindle matrix proteins that we have been studying in our laboratory (17,21-23). These immunostaining protocols are based on techniques developed by Zalokar and Erk (24) and Mitchison and Sedat (25). In addition, a number of related immunostaining protocols oriented toward analysis of cytoskeletal proteins (26), neural antigens (27), and embryonic proteins (28-30) have been published and may provide useful additional perspectives. However, it should be emphasized that for any new antigen of interest, it is necessary to optimize the chosen fixative and fixation conditions. Although it

From: Methods in Molecular Biology, vol. 247: Drosophila Cytogenetics Protocols Edited by: D. S. Henderson © Humana Press Inc., Totowa, NJ

is beyond the scope of this chapter to review the various principles and advantages of different fixatives, the reader is referred to one of several excellent histology texts for a detailed description of such considerations (e.g., refs. 31,32).

The strength of Drosophila as a model system lies in the wide range of molecular and genetic approaches that can be employed to study a cellular or developmental process. The presence, however, of maternal stores of mRNA and protein can complicate analysis of a protein's function during early development. Employing antibody perturbation approaches, especially in cases where it is not possible to recover germ-line clones, can allow one to block a protein's function during early development and assay the consequences of such perturbation. In these cases it is important to determine whether a particular antibody has function-blocking activity, and also whether the effects observed are indeed the consequence of loss of function or could result instead due to steric interference or indirect effect (note that this latter concern applies to analysis of genetic mutants as well!). A number of protocols have been adapted for injection of nuclei and pole cells (33), P-elements (34), and mRNA (35). In cases where dose-response information is required, injection approaches have been developed to facilitate measurement of the volume injected (36,37). The development of various Drosophila lines expressing GFP-tagged proteins that allow specific structures to be imaged in living embryos (e.g., microtubules, chromosomes, centrosomes) opens up the exciting prospect of analyzing the consequences of antibody perturbation in real time.

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