Isolation of Actin and Actin-Binding Proteins.

Actin-binding proteins mediate and regulate the dynamics of actin and the organization of highly ordered structures of F-actin. Villin is generally expressed in plant cells and is associated with G-actin or F-actin dependent on Ca2+ concentrations. Using a DNase I affinity column chromatography approach, the villin and the G-actin can be isolated from plant material. An outline of this method including the preparation of crude protein extract from plant material, its application on the affinity column, and the successive elution of villin with a solution containing EGTA and then of G-actin with denatured reagents is presented.

The preprophase band is a localized center of clathrin-mediated endocytosis in late prophase cells of the onion cotyledon epidermis.

The preprophase band (PPB) marks the site on the plant cell cortex where the cell plate will fuse during the final stage of cytokinesis. Recent studies have shown that several cytoskeletal proteins are depleted at the PPB site, but the processes that bring about these changes are still unknown. We have investigated the membrane systems associated with the PPB regions of epidermal cells of onion cotyledons by means of serial thin sections and electron tomograms. In contrast with specimens preserved by chemical fixatives, our high-pressure frozen cells demonstrated the presence of large numbers of clathrin-coated pits and vesicles in the PPB regions. The vesicles were of two types: clathrin-coated and structurally related, non-coated vesicles. Quantitative analysis of the data revealed that the number of clathrin-coated pits and vesicles is higher in the PPB regions than outside of these regions. Immunofluorescent microscopy using anti-plant clathrin-antibody confirmed this result. In contrast, no differences in secretory activities were observed. We postulate that the removal of membrane proteins by endocytosis plays a role in the formation of PPB 'memory' structures.

21-kDa polypeptide, a low-molecular-weight cyclophilin, is released from pollen of higher plants into the extracellular medium in vitro.

Calcium ions play a key role in the elongation and orientation of pollen tubes. We found that significant amounts of 21-kDa polypeptide were specifically released into the extracellular medium when pollen grains of lily, Lilium longiflorum Thunb., were incubated in the presence of EGTA or at low concentrations of Ca2+. This phenomenon was also dependent on pH and on the concentrations of MgCl2 in the medium; the release of 21-kDa polypeptide from pollen was suppressed by increasing the MgCl2 concentration and by lowering pH. Germination of pollen grains was inhibited in the medium into which the 21-kDa polypeptide had been released. This inhibition was irreversible; germination did not occur on transfer of the pollen grains into basal culture medium. Immuno-electron microscopy using an antibody against 21-kDa polypeptide showed that this polypeptide was present in the cytoplasm, vegetative nucleus and generative cell. When the pollen was treated with a medium containing EGTA, the density of 21-kDa polypeptide in the cytoplasm significantly decreased, but its density in vegetative nuclei and the generative cell did not, suggesting that only cytoplasmic 21-kDa polypeptide was released into the extracellular medium. The 21-kDa polypeptide was also present in the pollen of other higher-plant species, such as Tradescantia virginiana L., Nicotiana tabacum L. (angiosperms), and Cryptomeria japonica D. Don. (gymnosperm), and was also released into the medium in the presence of EGTA. In the case of C. japonica, however, it was released from pollen at alkaline pH above 8.5. The expression of 21-kDa polypeptide was not pollen-specific, because 21-kDa components immunoreactive with the anti-21-kDa polypeptide serum also existed in vegetative organs and cells of lily or tobacco. However, the 21-kDa polypeptide was not released into the extracellular medium from cultured tobacco BY-2 cells, even in the presence of EGTA. Amino acid sequences of two peptide fragments derived from 21-kDa polypeptide matched well those of low-molecular-weight cyclophilin (CyP). The antiserum against 21-kDa polypeptide recognized the CyP A from calf thymus and that in A431 carcinoma cells. The 21-kDa polypeptide fraction purified from lily pollen possessed peptidyl-prolyl cis- trans isomerase activity, which was suppressed by cyclosporin A (CsA), an inhibitor of enzyme activities of CyPs. From these results, we concluded that the 21-kDa polypeptide is a low-molecular-weight CyP. The present study showed that CyP in the pollen of higher plants is released into the extracellular matrix under unfavorable conditions.