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Cell Division and the Mitotic Spindle'
The study of cell division spans the past full century. Lately,the field has blossomed, and exciting advances have beenmade, especially at the molecular and fine-structural levels .Yet as we commemorate the centennial of Flemming's discov-ery' of"indirect" cell division, or mitosis, many basic questionsstill remain unanswered or incompletely explained.The first half-century of study on cell division is synthesized
in Wilson's (2) classic treatise "The Cell in Development andHeredity ."' While laying a solid foundation for the cytology ofthe dividing cell and the genetic and developmental signifi-cance of mitosis and meiosis, Wilson (Chapter IX) also directsour attention to an important viewpoint regarding the struc-tural basis of cell function . Thus he quotes Brdcke :"We must therefore ascribe to living cells, beyond the mo-
lecular structure of the organic compounds that they contain,still another structure of different type of complication ; and itis this which we call by the name of organization ."It is this aspect of the dividing cell, its organization, especiallyin its dynamic attributes, that I shall stress in this briefhistoricalsketch . In particular, I shall focus on the organization of theephemeral mitotic spindle, which emerges cyclically at eachcell division . With it, the replicated, condensed chromosomesare separated and positioned forinclusion into the (two) daugh-ter cells.
The Mitotic Spindle
As we entered the early 1950s, evidence pointed to twomechanisms ofanaphase chromosome movement (summarized
' Dedicated to Professor KennethW. Cooper, University of California,Riverside, whose continued friendship and advice have added im-mensely to my work .z Translated and reproduced in 1965 . J. Cell Biol. 25(1 ; part 2) :1-69 .Flemming saw that the nucleus did not divide directly into two, butformed chromatin threads (hence mitosis) . The condensed chromatinthreads, or chromosomes, were moved apart and placed into two newcells by a transient, fibrillar achromatic apparatus, the "nuclear sp~-dle" (1) formed from the hyaline kinoplasm . r'Wilson's work is complemented in the botanical realm by Sharp (3) .Belai (4) provides a thorough, thought-provoking examination ofachromatic spindle components and varying patterns of mitosis inprotists . Morgan (5) illustrates and raises penetrating questions regard-ing the role of cell division in embryonic development and geneexpression .
Marine Biological Laboratory, Woods Hole, Massachusetts,and Department ofBiology, University of Pennsylvania, Philadelphia,Pennsylvania
THE JOURNAL OF CELL BIOLOGY " VOLUME 91 NO . 3 PT . 2 DECEMBER 1981 131s-147sC The Rockefeller University Press " 0021-9525/81/12/131s/17 $1 .00
by Schrader ) . Chromosomes were pulled toward the spindlepoles, via their kinetochores," by shortening of a traction fiberor the chromosomal spindle fiber. In addition, chromosomeswere "pushed" apart by the pole-to-pole lengthening of thecentral spindle to which the chromosomal fibers were an-chored . The notion of a musclelike contraction for polewardchromosome motion had been propounded by Flemming in1879 (1) and earlier workers, and questioned by Wilson (2) asnot being consistent with the "dynamic nature of the cytoplas-mic fibrillae" observed in living cells. As to the dual mecha-nism, Ris (8), in working with living grasshopper spermato-cytes, was able to inhibit the pole-to-pole elongation withoutaffecting chromosome-to-pole movement by exposing the cellsto a solution containing a few tenths of a percent chloralhydrate.Yet the nagging doubt, expressed by Wilson (e .g ., pages 178-
198 in reference 2) and others regarding the physical nature ofthe "achromatic" fibrous machinery of the mitotic spindle,which was believed to be responsible for chromosome move-ment, had not abated. Rather, the problems were compoundedby the late 1940s despite, and partly because of, the wealth ofstudies that had been made on carefully fixed and stained cellsand by the deductions drawn from observations of living cellbehavior (6) . In that atmosphere it was first necessary to learnwhether the mitotic figures seen in fixed cells in fact repre-sented, in living cells, a physically integral body capable ofmoving chromosomes or exerting force enough to deform cellshape.ISOLATION OF THE MITOTIC SPINDLE :
In1952,Ma-zia and Dan  succeeded in developing a method for the massisolation of "mitotic apparatuses," thereby identifying the mi-totic spindle, chromosomes, and asters as a coherent physicalbody separable from the rest of the cell (Fig . 1) . Although therewere earlier reports of expelling the spindle out of an intactcell (e .g ., Foot and Strobell  from earthworm eggs), thepioneering work of Mazia and Dan finally opened the way forthe mass isolation and characterization of the mitotic appara-tus. That same year, Carlson (11), in an extensive micromanip-ulation study on living grasshopper neuroblasts, demonstratedthe integrity and mechanical anisotropy of the metaphasespindle, as well as the "liquefaction" of the spindle mid-zoneobserved during anaphase .
"Chromosomes commonly possess a single spindle fiber attachmentpoint, or kinetochore. Some chromosomes have, or behave as thoughthey have, diffuse kinetochores along the length of their chromosomes(6, 7) . They are called holokinetic chromosomes .
on April 20, 2018jcb.rupress.org Downloaded from http://doi.org/Published Online: 22 February, 1981 | Supp Info:
FIGURE 1 Mitotic spindle in metaphase isolated from the egg of a sea urchin, Lytechinus variegatus. (Left) Observed with arectified polarizing microscope, spindle fibers and astral rays appear in light or dark contrast depending on their orientation . Theweak birefringence (measuring a few nanometers in retardation) of the fibers produces the sharp contrast observed . Microtubularbundles are responsible for the (positive form) birefringence of the fibers . Chromosomes display little birefringence and appear asgray bodies at the equator of the spindle. (Right) The same spindle in Nomarsky differential interference contrast . Chromosomesshow prominently. The microtubules in these clean spindles (isolated in a new medium devised by Salmon) depolymerize whenexposed to submicromoiarconcentrations of calcium ions once glycerol is removed from the isolation medium . In these isolates,which lack vesicular components, the chromosomal fibers shorten as they are depolymerized by micromolar concentrations ofcalcium ions. Unpublished figures, courtesy of Dr . E . D . Salmon, University of North Carolina . Bar, 10 Am .
Many improvements were made on the basic isolation tech-nique of Mazia and Dan. In particular, the work of Kane (12)that identified the pH and solute conditions (in effect, wateractivity) needed for mitotic apparatus isolation, helped shedlight on the basic physicochemical parameters that delineatedthe functioning cytoplasm. On the other hand, early attemptsat defining the chemical makeup of the mitotic spindle wereless successful . In retrospect, that is not so surprising becausethe fibers of the spindle and aster are immersed in (and spunout from) the hyaline cytoplasm that permeates the cell. Largecytoplasmic granules are excluded from the spindle, but ribo-somes and some membranes are not. Yolk and other granulesalso adhered to earlier isolates .SPINDLE FIBERS I N V I VO :
Whereas the isolated mitoticapparatus exhibited a physical coherence and clearly displayedspindle fibers, such fibers could have arisen by fixing oroverstabilizing the cell, as was suggested by many investigators(see 6) . Pollister (13), for example, argued that astral rays werenot fibers in the living cell but rather were channels offlow oforiented molecules belonging to the hyaloplasm. Our ownwork, which paralleled that of Mazia and Dan, focused on thedevelopment of sensitive polarized light microscopy, withwhich we hoped to study directly in living cells the nature ofthe anisotropically arrayed molecules that made up the spindlesand asters . From the 1930s to early 1950s, Schmidt (14), Hughesand Swann (15), Swann and Mitchison (16), Inoue and Dan(17), and Swann (18) had investigated how to optimize theperformance ofexisting polarized light microscopes. They alsoshowed that the mitotic spindle and astral rays in cleaving seaurchin eggs and cultured chick embryonic cells indeed dis-played a longitudinal positive birefringence consonant with thepresumed presence of molecules oriented parallel to the fiberaxes. Each of the workers also noted the striking emergence,rise, fall, and disappearance of spindle birefringence (retarda-
THE )OURNAL OF CELL BIOLOGY " VOLUME 91, 1981
tion) as a single cell progressed through prometaphase, meta-phase, anaphase, and telophase . Each interpreted the obser-vations in molecular terms, variously biased by the paradigmadopted .By 1953, I was able to demonstrate clearly with the polarizing
microscope (19) that "there is fibrous structure in living cellswhich in conformation is very close to what the cytologistshave long observed in well-fixed preparations . There are con-tinuous fibers, chromosomal fibers, and astral rays" (6). Cou-pled with Cleveland et al . (20) and Cooper's (21) earlierobservations of fibrous structures in the spindles of certainother living cells, the issue of the "reality" of spindle fibersseemed to be settled .Whereas these earlier studies vividly displayed some dy-
namic changes ofspindle birefringence in living cells, changeswhich should reflect events taking place at the molecular levelduring cell division, a clearer outlook depended on betteroptical resolution and broader experience gained through ob-servations and experimental manipulations of cells i