Mitosis in Barbulanympha. I. Spindle structure, formation, and kinetochore engagement

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Abstract
Successful culture of the obligatorily anaerobic symbionts residing in the hindgut of the wood-eating cockroach Cryptocercus punctulatus permits continuous observation of mitosis in individual Barbulanympha cells. Methods for culture of the protozoa, preparation of microscope slide cultures in which Barbulanympha survived and divided for up to 3 days, and an optical arrangement which permits observation and through-focus photographic recording of dividing cells sequentially, in differential interference contrast and rectified polarized light microscopy are presented. Prophase events and structures described are: development of the astral rays and large extranuclear central spindle from the tips of the elongate-centrioles; the fine structure of spindle fibers and astral rays, deduced in vivo from polarized light microscopy and seen as a particular array of microtubules in thin-section electron micrographs; formation of chromosomal spindle fibers by dynamic engagement of astral rays to the kinetochores embedded in the persistent nuclear envelope; and repetitive shortening of chromosomal spindle fibers which appear to hoist the nucleus to the spindle surface, cyclically jostle the kinetochores within the nuclear envelope, and churn the prophase chromosomes. These processes have implications for the evolution of mitosis and for general understanding of the mitotic process.