Stage III RAFT
These video sequences were recorded (and displayed) in real time, using Nomarski (DIC) optics. The full frame width for each is 56 um. Stage III cells show dramtic changes in shape and adhesion to the substratum while gradually becoming more elongate and detaching. Their anterior flagellae (the shorter posterior one is only occasionally seen) become quite active. The ridge (a flattened "dorsal" protrusion" rich in actin microfilaments) is most obvious and active at this stage of the RAFT.
STAGE III (videosequences C-F): Two events appear to occur concurrently at the beginning of Stage III: 1. the nucleus becomes ovoid in shape and apparently fixed within what now appears as a tapered process from which the anterior flagellum protrudes, and 2. the cell body begins to elongate, defining an anterior-posterior axis. For kinetic studies, the ratio between cell length and width was defined as having a value between one and two to aid in distinguishing these cells from the similar, but more elongate, ones at stage IV. Stage III cells show a curvature of their long axis that frequently gives them a "comma shape." Sometime during this stage, the short, posteriorly-directed flagellum appears and, as will be presented below, a motile ridge also forms.
The transition from stage II to stage III also involves distinctive increases in motility. Relocation of the nucleus is often correlated with blebbing at the anterior surface; the blebs frequently propagate peripherally in a manner reminiscent of the "circus movements" seen on embryonic amphibian cells. Activity of the anterior flagellum commences as cells start to elongate. Stage III cells become increasingly motile as they gradually release themselves from the substratum. The anterior flagellum continues to elongate and also becomes more active. The increasingly tapered anterior process traces a circular path in the medium around the rest of the cell body, which is anchored to the substratum. Gradually, the entire cell rotates at rates that increase from less than 1 rpm to more than 10 rpm. Periods of rotational movement alternate with episodic thrashing of the anterior flagellum and shaking of the entire cell that may aid in detachment. Cells seem most adherent at a point near their posterior end and, just prior to release, some cells appear loosely tethered to the substratum by a short strand. [Although release usually occurs late in stage III, changes in adhesivity are somewhat variable; some stage II cells release as the peripheral movements of the forming flagellum begin and some stage IV cells remain adherent for many minutes after vigorous flagellar activity has begun.] After detachment, stage III cells swim in circles with little net displacement. Plasticity of morphology and movements is most apparent at stage III; cells that are in the early part of stage III appear frequently to "revert" to shape and behavior which is more characteristic of stage II cells, while the apparent length of a given stage III cell can vary in an oscillatory fashion. The number of stage III cells reaches a maximum about 10 minutes into the AFT II and declines gradually thereafter.
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Last modified:Friday, December 5, 2003
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