QUANTIFY MICROTUBULE NUMBERS ON SISTER KINETOCHORES OF CHROMOSOMES

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(Supported by NIH R01 GM 4019814 to C.L. Rieder) The mechanism by which bio-oriented chromosomes become positioned on the equator of the mitotic spindle during cell division is unknown. We do know that this "congression" process involves the production of poleward (P) forces that act on the sister kinetochores of each chromosome, and that these forces are roughly balanced so that no net force acts on the chromosome when it is positioned on the spindle equator. We have shown that the polar regions of forming vertebrate spindles exert an away-from-the-pole (AP) force on the chromosome arms, and that these "polar ejection" forces are involved in positioning chromosomes during vertebrate spindle formation. For example, the distance a mono-oriented chromosome resides relative to its pole is positively correlated with the numbers of Mts impacting the chromosome, but not the number of kinetochore-associated, and such chromosomes move closer to the pole when their arms are severed. When the tension-mediated directionally-unstable behavior of kinetochore motility is combined with the polar ejection forces, a mechanism of congression can be formulated that also explains the oscillatory behavior of chromosomes. In this model, kinetochores are "smart" in that they switch between persistent phases of P and AP motion in response to scalar and vectorial information within the spindle. A model of congression based on motile kinetochores and polar ejection forces predicts that chromosomes should congress closer to the "weaker" pole of a spindle, if a spindle is formed from two half spindles that differ significantly in Mt density. Such "asymmetrical" spindles do not occur in nature and cannot be created experimentally. However, a variant is often formed in newt lung cells when one or more chromosomes becomes bioriented between one of the spindle poles and a much weaker "ectopic" third pole (to form a "satellite spindle"). Live cell phase contrast and IFM observations on fixed cells (e.g., above) reveal that the third pole supports normal K fiber formation and anaphase chromosome motion, but that it is often severely depleted of astral and non K-fiber Mts. Thus, this system provides a unique opportunity to determine if the final congression position of chromosome achieves, relative to its two poles, is related to the density of Mts within the two half spindles and/or to the number of kinetochores Mts on opposing sister kinetochores. The latter information is needed because there is some data to suggest that the P force acting on the metaphase kinetochores depends on the number of KMts , although this data is also consistent with the notion that P force production is independent of KMt numbers but dependent on the number of P force produces in the kinetochores . Serial 1.0 (m sections are being cut from a newt lung cells followed in vivo that contain ectopic poles with congressed chromosomes. We are varying our fixation and staining conditions so that we can determine, using HVEM tomography of 1.0 (m thick sections, how many microtubules are associated with each sister kinetochore. Three single-tilt tomographic series have been recorded on the HVEM using the "low-dose" method, and two reconstructions were made. The results are very encouraging. We have found that by simply staining the sections in lead, and not uranyl, that we can directly count the Mts comprising each kinetochore fiber within these sections.