EM of the developing nematode sperm cytoskeleton. -- Nematode sperm activation involves the reorganization of specialized organelles and the rapid assembly of the MSP protein cytoskeleton in the amoeboid sperm. Because the Ascaris sperm cytoskeleton is visible in live cells with enhanced DIC, it is a valuable phenomenological model for amoeboid motility. Interest has developed in assembling a detailed 3D picture of the structure of this system in anticipation of the rapidly evolving biochemistry of MSP in Tom Robert's lab at Florida State University. The project requires the use of the Hitachi S-900 to demonstrate the association of cytoskeleton and plasma membrane, the 3D organization of the developing cytoskeleton (fiber complexes), and for the immunocytochernistry of assembling MSP and recently discovered CAP particles associated with the developing cytoskeleton. Dr. Sepsenwol has prepared a timed-activation series of anti-MSP and anti-CAP labeled """"""""wet-ripped"""""""" cells for this purpose. He has initiated cryo-SEM studies of fractured inactive and fully activated sperm with the high pressure firezer to demonstrate the presence of skeletal structures seen in fixed cells. Correlative 2-photon, IR laser scanning microscopy of activating and crawling nematode sperm -- LM of live Ascaris sperm has been limited to visible-light techniques (enhanced DIQ: the cell's exquisite sensitivity to short-wavelength illumination has precluded continuous fluorescence studies with dyes useful for studying the early activation events in living cells. This has now become possible with the prototype 2-photon IR laser scanning LM at the IMR. Dr. Sepsenwol has used the instrument to follow fluorescently-labeled five cells from the very first events in activation to their behavior during full motility for more than one hour (vs. seconds by epifluorescence or laser-confocal microscopy). In addition, Presumed cell-cell chernotaxis has been observed in packs of crawling sperm by taking advantage of 2-photon optical sectioning capability. Using the membrane probe FM4-64, the exact time of a critical and ephemeral event in pseudopod assembly -- membranous organelle [MO] fusion -- can be determined. MO fusion precedes the elaboration of new pseudopod membrane, and the polymerization of the first cytoskeleton. It has been postulated that the MO provides this new membrane. By timing fixation with these events, a series of whole-mount and plastic-embedded samples of early pseudopod formation have been generated for examination with the S-900 SEM. The first views of SEM whole mounts show giant pores forming in a vortex of (new) plasma membrane at the MO fusion sites. Later studies will look at whether the MO's provide the cytoskeleton polymerization sites known to exist in the mature sperm pseudopod membrane. Additional projects have been suggested by results obtained with other dyes that fluoresce wen with the IR 2-photon system: JC- 1, a potential-sensitive mitochondrial dye, shows a change in mitochondrial activity during activation, and Rhodamine 123 shows a dramatic compartmentalization between cell body and pseudopod, although there does not appear to be an anatomical barrier in EM studies.
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