Quantitative Studies on the Polarization Optical Properties of Living Cells

Birefringence of the mitotic apparatus (MA) and its change during mitosis in sea urchin eggs were quantitatively determined using the birefringence detection apparatus reported in the preceding paper (Hiramoto et al ., 1981, J. Cell Biol. 89:115-120) . The birefringence and the form of the MA are represented by five parameters: peak retardation (SP), trough retardation (St), interpolar distance (D,), the distance (D2 ) between chromosome groups moving toward poles, and the distance (D 3) between two retardation peaks . Distributions of birefringence retardation and the coefficient of birefringence in the spindle were quantitatively determined in MAs isolated during metaphase and anaphase . The distribution of microtubules (MTs) contained in the spindle was determined in isolated MAs assuming that the observed birefringence of the spindle is attributable to the form birefringence caused by regularly arranged MTs. The distribution coincided fairly well with the distribution of MTs in isolated MAs determined by electron microscopy . Under the same assumption, the distribution of MTs in the spindle in living cells during mitosis was determined . The results show that the distribution of MTs and the total amount of polymerized tubulin (MTs) in the spindle change during mitosis, suggesting the assembly and disassembly of MTs as well as the dislocation of MTs during mitosis . Since the pioneering work of Runnstrom (26) and Schmidt (31, 32), the mitotic figures of many kinds of living cells have been known to be birefringent . Improvement of polarization microscope technique has made it possible to measure the variation of birefringence retardation within the mitotic apparatus (MA) and its change during mitosis (34, 35) . Inoue (13) found that the mitotic spindle consists of many birefringent fibers by polarization microscopy . Development of electron microscopy has revealed that a number of microtubules (MTs) are oriented in the pole-to-pole direction in the spindle and in radial direction in the aster, so that it has been generally assumed that the spindle fibers and astral rays observed by light microscopy consist of MTs . Sato et al . (30) demonstrated that the birefringence of the spindle isolated from the sea-star oocyte THE JOURNAL Of CELL BIOLOGY VOLUME 89 APRIL 1981 121-130 ©The Rockefeller University Press 0021-9525/81/04/0121/10 $1 .00 was mainly attributable to the MTs; they demonstrated the quantitative coincidence of observed and theoretical birefringence retardation of the spindle immersed in media of various refractive indices, based on the theories of Wiener (38) and Bragg and Pippard (1) on the birefringence of mixed bodies. However, it is undecided whether the spindle birefringence in living cells is solely attributable to MTs (e.g ., see references 4 and 25) . If the birefringence of the spindle is mainly attributable to MTs, it may be possible to follow the behavior of MTs in the spindle in living cells from birefringence measurements . MTs are considered to play important roles in mitotic events such as chromosome movement and spindle elongation, but they are invisible by ordinary light microscopy . We undertook the on A uust 8, 2017 jcb.rress.org D ow nladed fom present study to obtain quantitative data on the birefringence of mitotic apparatus and its change during mitosis in the sea urchin egg. We conclude that the birefringence of the isolated spindle results mainly from the form birefringence of aligned MTs . We have estimated changes in distribution ofthe number of MTs in the spindle during mitosis from our quantitative birefringence measurement of the spindle in living cells . MATERIALS AND METHODS