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Protoplasma

, Volume 249, Issue 2, pp 337–346 | Cite as

A pre-embedding immunogold approach reveals localization of myosin VI at the ultrastructural level in the actin cones that mediate Drosophila spermatid individualization

  • Marta LenartowskaEmail author
  • Mamiko Isaji
  • Kathryn G. Miller
Original Article

Abstract

Stable actin structures play important roles in the development and specialization of differentiated cells. How these structures form, are organized, and are used to mediate physiological processes is not well understood in most cases. In Drosophila testis, stable actin structures, called actin cones, mediate spermatid individualization, a large-scale cellular remodeling process. These actin cones are composed of two structural domains, a front meshwork and a rear region of parallel bundles. Myosin VI is an important player in proper actin cone organization and function. Myosin VI localizes to the cones' fronts and its specific localization is required for proper actin cone formation and function during individualization. To understand how these structures are organized and assembled, ultrastructural studies are important to reveal both organization of actin and the precise localization of actin regulators relative to regions with different filament organizations. In the present work, we have developed a novel pre-embedding immunogold-silver labeling method for high-resolution analysis of protein distribution in actin structures which allowed both satisfactory antibody labeling and good ultrastructural preservation. Electron microscopic studies revealed that myosin VI accumulated at the extreme leading edge of the actin cone and preferentially localized throughout the front meshwork of the cone where branched actin filaments were most concentrated. No myosin VI labeling was found adjacent to the membranes along the length of the cone or connecting neighboring cones. This method has potential to reveal important information about precise relationships between actin-binding proteins, membranes, and different types of actin structures.

Keywords

Actin cytoskeleton Drosophila melanogaster Myosin VI Pre-embedding immunogold technique Spermatid individualization 

Abbreviations

BSA

Bovine serum albumin

EM

Electron microscopy

JLA20

Anti-actin antibody

MAb

Monoclonal antibody

PB

Phosphate buffer

PBS

Phosphate-buffered saline

3C7

Anti-myosin VI antibody

Notes

Acknowledgments

We thank Michał Świdziński and Olga Narbutt for their technical assistance, and Deborah J Frank for the critical reading of this manuscript. This work was supported by a grant from Polish Ministry of Science and Higher Education, grant number no. N303 816240 and by the National Institutes of Health Grant R01GM-60494 (to K.G.M.).

Conflict of interest

None.

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Marta Lenartowska
    • 1
    Email author
  • Mamiko Isaji
    • 2
  • Kathryn G. Miller
    • 2
  1. 1.Laboratory of Developmental Biology, Institute of General and Molecular Biology, Faculty of Biology and Earth SciencesNicolaus Copernicus UniversityToruńPoland
  2. 2.Department of BiologyWashington University in St. LouisSt. LouisUSA

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