| Popis: |
The proper distribution of proteins is important for the development and function of both individual cells and whole organisms. Relatively little is known about the mechanisms of protein localization in plant cells. Protein body formation in maize endosperm provides a useful system in which to study these mechanisms. Maize endosperm is a specialized tissue that accumulates starch and storage proteins and ultimately provides nutrients for the germinating seedling. The storage proteins, called zeins, are cotranslationally inserted into the rough endoplasmic reticulum (RER) where they aggregate into spherical protein bodies. Previous studies have suggested that the cytoskeleton may play a role in protein body formation, since actin, the protein synthesis factor EF-1α (which associates with the cytoskeleton in other systems), and polysomes, including zein polysomes, were found associated with protein bodies following endosperm homogenization. To determine whether the cytoskeleton, EF-1α, and protein bodies are associated in situ, these components were visualized in intact endosperm cells. By using indirect immunofluorescence and confocal microscopy, changes were documented in the distributions of EF-1α, actin, and microtubules during development. The protein bodies are found enmeshed in EF-1α and actin and are juxtaposed with a multidirectional array of microtubules. In addition, actin and EF-1α appear to exist in a complex. Finally, actin bundling assays demonstrated that maize EF-1α is capable of bundling actin in vitro. Therefore, EF-1α may organize actin around protein bodies. One possible role of the cytoskeletal network around protein bodies is the transport and or anchoring of zein mRNAs to sites of protein body formation. To test this hypothesis, in situ hybridization experiments were conducted in the presence and absence of cytoskeleton disrupting agents. The results suggest that while the zein mRNAs may associate with the RER at random sites, a cytoskeleton-dependent transport mechanism may be utilized to traffick them to the RER surface. To more convincingly demonstrate a role of the cytoskeleton in zein mRNA localization, microinjection and visualization of zein mRNAs in the presence and absence of intact cytoskeletal elements is required. Methods for microinjecting maize endosperm cells with such mRNAs were devised and preliminary results are described. |
