Meiosis I metaphase spindles were isolated from oocytes of the sea-star Pisaster ochraceus by a method that produced no detectable net loss in spindle birefringence. Some of the spindles were fixed immediately and embedded and sectioned for electron microscopy. Others were laminated between gelatine pellicles in a perfusion chamber, then fixed and sequentially and reversibly imbibed with a series of media of increasing refractive indices. Electron microscopy showed little else besides microtubules in the isolates, and no other component present could account for the observed form birefringence. An Ambronn plot of the birefringent retardation measured during imbibition was a good least squares fit to a computer generated theoretical curve based on the Bragg-Pippard rederivation of the Wiener curve for form birefringence. The data were best fit by the curve for rodlet index (n1) = 1.512, rodlet volume fraction (f) = 0.0206, and coefficient of intrinsic birefringence = 4.7 X 10(-5). The value obtained for n1 is unequivocal and is virtually as good as the refractometer determinations of imbibing medium index on which it is based. The optically interactive volume of the microtubule subunit, calculated from our electron microscope determination of spindle microtubule distribution (106/mum2), 13 protofilaments per microtubules, an 8 nm repeat distance and our best value for f, is compatible with known subunit dimensions as determined by other means. We also report curves fitted to the results of Ambronn imbibition of Bouin's-fixed Lytechinus spindles and to the Noll and Weber muscle imbibition data.

We have used micromanipulation to study the attachment of chromosomes to the spindle and the mechanical properties of the chromosomal spindle fibers . Individual chromosomes can be displaced about the periphery of the spindle, in the plane of the metaphase plate, without altering the structure ofthe spindle or the positions of the nonmanipulated chromosomes . From mid-prometaphase through the onset of anaphase, chromosomes resist displacement toward either spindle pole, or beyond the spindle periphery . In anaphase a chromosome can be displaced either toward its spindle pole or laterally, beyond the periphery of the spindle ; however, the chromosome resists displacement away from the spindle pole . When an anaphase half-bivalent is displaced toward its spindle pole, it stops migrating until the nonmanipulated half-bivalents reach a similar distance from the pole . The manipulated half-bivalent then resumes its poleward migration at the normal anaphase rate . No evidence was found for mechanical attachments between separating half-bivalents in anaphase . Our observations demonstrate that chromosomes are individually anchored to the spindle by fibers which connect the kinetochores of the chromosomes to the spindle poles. These fibers are flexible, much less extensible than the chromosomes, and are able to pivot about their attachment points. While the fibers are able to support a tensile force sufficient to stretch a chromosome, they buckle when subjected to a compressive force . Preliminary evidence suggests that the mechanical attachment fibers detected with micromanipulation correspond to the birefringent chromosomal spindle fibers observed with polarization microscopy . KEY WORDS birefringence -chromosome variety of cell types . However, the actual mechamovement micromanipulationnism of mitotic chromosome movement has not microtubules mitosis -spindle fibers been determined . Attempts to explain chromosome movement by such diverse mechanisms as The morphological events which comprise mitosis the depolymerization of microtubules (9, 20, 21), have been described in great detail for a wide the sliding of interdigitating microtubules (24,26, 528 J. CELL BIOLOGY

KEY WORDS birefringence -colcemid micromanipulation " microtubules " mitosis spindle fibers -vinblastine The results of the micromanipulation studies presented in the preceding paper (3) demonstrate that from mid-prometaphase through anaphase, flexible but relatively inextensible fibers attach the kinetochores of each bivalent to the spindle poles . These mechanically demonstrable attachment fibers are found to have the same spatial location as the optically detectable birefringent spindle fibers . Although these results suggest that the birefringent chromosomal fibers are the structures which attach the chromosomes to the spindle, they do not rule out the possible participation of a nonbirefringent component of the spindle fiber .Studies with various physical and chemical agents support the hypothesis that the birefringent chromosomal fibers anchor the chromosomes to J . CELL BIOLOGY

Equilibrium between mitotic microtubules and tubulin is analyzed, using birefringence of mitotic spindle to measure microtubule concentration in vivo. A newly designed temperature-controlled slide and miniature, thermostated hydrostatic pressure chamber permit rapid alteration of temperature and of pressure. Stress birefringence of the windows is minimized, and a system for rapid recording of compensation is incorporated, so that birefringence can be measured to 0.1 nm retardation every few seconds. Both temperature and pressure data yield thermodynamic values (delta H similar to 35 kcal/mol, delta S similar to 120 entropy units [eu], delta V similar to 400 ml/mol of subunit polymerized) consistent with the explanation that polymerization of tubulin is entropy driven and mediated by hydrophobic interactions. Kinetic data suggest pseudo-zero-order polymerization and depolymerization following rapid temperature shifts, and a pseudo-first-order depolymerization during anaphase at constant temperature. The equilibrium properties of the in vivo mitotic microtubules are compared with properties of isolated brain tubules.

This paper describes the development of a miniature, temperature-controlled, stainless steel pressure chamber which uses strain-free optical glass for windows. It is directly adaptable to standard phase-contrast and polarized-light microscopes and requires a minimum amount of equipment to generate and measure pressure. Birefringence retardations (BR) of 0.1 nm up to 3,000 psi, 0.4 nm up to 5,000 psi and 1.0 nm up to 10,000 psi can be detected over a 0.75-mm central field with two strain-free Leitz 20• UM objectives, one used as a condenser. In phase-contrast studies a Nikon DML 40• phase objective and Zeiss model IS long working-distance phase condenser were used, with little deterioration of image quality or contrast at pressures as high as 12,000 psi. The actual design process required a synthesis of various criteria which may be categorized under four main areas of consideration: (a) specimen physiology; (b) constraints imposed by available optical equipment and standard microscope systems; (c) mechanical strength and methods for generating pressure; and (d) optical requirements of the chamber windows. Procedures for using the chamber, as well as methods for shifting and controlling the temperature within the chamber, are included.Hydrostatic pressure is important as a physiological parameter in relation to life in the deep sea and as an experimental variable, especially in studies of the equilibria of labile cellular structures and the reaction rates of enzymatic processes. (For details, see reviews by Johnson et al.,