Immunolocalization of Proteins in Somatic Embryos

The Integrative Cell Biology Laboratory, Durham University, UK.
Methods in Molecular Biology (Impact Factor: 1.29). 02/2008; 427:157-71. DOI: 10.1007/978-1-59745-273-1_13
Source: PubMed


Plant embryogenesis requires a tight balance between cell proliferation and differentiation. In animals, embryogenesis is dependent on cell migrations, which is in contrast to plant embryogenesis where the rigid cell wall precludes migration. Therefore, plants have to position cells correctly by defining the direction of the division plane during proliferation and control cell shape by local cell expansion. Both these processes are reliant on the organization and dynamics of the cytoskeleton-actin filaments and microtubules. In previous work (7), we have shown that differentiation of the embryo suspensor is accompanied by reorientation of microtubules from random to transverse and reorganization of actin filaments from a fine filamentous network to bundled longitudinal cables. Here, we describe the technique for visualization of cytoskeletal components including actin filaments, microtubules and their associated proteins during the development of plant embryos in whole-mount specimens.

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    • "In addition, the slowness of chemical fixation, which is accentuated in higher plant cells by the presence of a rigid cellulosic cell wall, was suspected to give rise to artifactual cytoskeletal rearrangements [He and Wetzstein, 1995; Doris and Steer, 1996]. Therefore, continuous effort has been devoted to improve chemical fixation procedures and to develop alternative methods such as cryofixation [Vitha et al., 2000; Collings and Wasteneys, 2005; Wilsen et al., 2006; Smertenko and Hussey, 2008]. Despite the wide use of live cell imaging (see below), classical actin immunolocalization or labeling using appropriately fixed material yielded important results, especially in root and pollen tissues [Collings and Wasteneys, 2005; Wilsen et al., 2006]. "
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    ABSTRACT: Tight regulation of plant actin cytoskeleton organization and dynamics is crucial for numerous cellular processes including cell division, expansion and intracellular trafficking. Among the various actin regulatory proteins, actin-bundling proteins trigger the formation of bundles composed of several parallel actin filaments closely packed together. Actin bundles are present in virtually all plant cells, but their biological roles have rarely been addressed directly. However, decades of research in the plant cytoskeleton field yielded a bulk of data from which an overall picture of the functions supplied by actin bundles in plant cells emerges. Although plants lack several equivalents of animal actin-bundling proteins, they do possess major bundler classes including fimbrins, villins and formins. The existence of additional players is not excluded as exemplified by the recent characterization of plant LIM proteins, which trigger the formation of actin bundles both in vitro and in vivo. This apparent functional redundancy likely reflects the need for plant cells to engineer different types of bundles that act at different sub-cellular locations and exhibit specific function-related properties. By surveying information regarding the properties of plant actin bundles and their associated bundling proteins, the present review aims at clarifying why and how plants make actin bundles.
    Cell Motility and the Cytoskeleton 11/2009; 66(11):940-57. DOI:10.1002/cm.20389 · 4.19 Impact Factor