A comparative analysis of actin localization in Nicotiana pollen tubes has been made using a monoclonal actin antibody and rhodamine-phalloidin (Rh-Ph). The antibody, based on Western blotting of pollen tube extract, labels a polypeptide of 45 kD that comigrates with muscle actin. Immunofluorescence studies reveal a network of axially oriented strands of microfilaments (MFs) that distribute throughout the length of the tube except at the apex, where diffuse staining is usually observed. A similar pattern of MFs is evident after Rh-Ph staining. When pollen tubes are treated with cytochalasin B or D, cytoplasmic streaming is inhibited, as is tube elongation. Microscopic analysis reveals that the MF pattern is markedly altered; however, the antibody and Rh-Ph produce different staining patterns. The antibody reveals massive bundles of MFs that distribute throughout the tube length and extend into the very tip. In contrast, Rh-Ph shows mostly a diffuse staining pattern. Immunogold labeling of sections of pollen tubes prepared by rapid-freeze fixation and freeze substitution (RF-FS) also reveals many MF bundles after cytochalasin treatment. These results thus question the efficacy of Rh-Ph as a faithful F-actin indicator under all conditions. By using two monoclonal myosin antibodies, a myosin heavy chain polypeptide has been identified and localized in Nicotiana pollen tubes The epitopes of these antibodies were found to reside on myosin head and tail portion, and therefore were designated anti-S-1 and anti-LMM, respectively. On Western blots of pollen tube proteins, both antibodies label a polypeptide of approximately 175 kD. Immunofluorescence microscopy shows that both antibodies yield numerous fluorescent spots throughout the length of the tube, often with an enrichment in the apical region. These fluorescent spots are believed to represent the vesicles and organelles in the tubes. In addition to this common pattern, anti-S-1 stains both the generative cell and the vegetative nuclear envelope in a certain percentage of cells. The different staining patterns of the nucleus may be caused by some organization and/or anchorage state of the myosin molecules on the nuclear surface that differs from those on the vesicles and organelles.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-9055 |
| Date | 01 January 1995 |
| Creators | Tang, Xiaojing |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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