Fig. 13. 



meant to show the spindle fibres which in the 

 best preparations occupy, as you can see, a 

 rather small portion of the total area or volume 

 of the spindle. There are also chromosomes and 

 background or matrix material. This matrix is 

 very densely populated with vesicles, fragments 

 of membranes and a large number of particles. 

 The particles are of the same size and of the 

 same electron density as are the ribosomes 

 seen elsewhere in the cell, and there is no 

 reason to believe that they are not ribosomes. 

 If the characteristic spindle protein is what 

 makes these fibres, then one would conclude 

 from a picture like this that most of the protein 

 in the spindle is not the characteristic micro- 

 tubular protein. It is ribosomal and soluble 

 protein. 



A second consideration is relevant. If we 

 were to take a sample volume in an egg without 

 an organelle, like the mitotic spindle, we would 

 find that there were a certain number of yolk 

 particles in that volume. These yolk particles 

 are solid objects. They don't seem to have the 

 high degree of crystalline order in sea urchin 

 yolk that's seen in some other species, but the 



particles are nevertheless very dense and have 

 a high protein content. If the spindle or an 

 organelle like it is formed, the yolk particles 

 are extruded and indeed one can see large 

 particulates such as yolk and mitochondria 

 extruded from the forming spindle. Hence, the 

 mitotic apparatus has in it no particles of the 

 size of yolk and mitochondria. Soluble proteins, 

 on the other hand, are presumably not extruded 

 from the forming mitotic apparatus, because 

 ribosomes are not, and the soluble proteins are 

 smaller. Thus, if one were to measure the con- 

 centration of soluble proteins in the mitotic 

 apparatus, and in the region outside of it, one 

 would certainly find that the concentration of 

 soluble proteins is higher within the region 

 of the mitotic apparatus than it is in the peri- 

 phery, simply because the peripheral material 

 has in every volume element a large excluded 

 subelement occupied by the yolk. On this basis 

 alone, any report that something is localized 

 in the spindle should be viewed with caution. 

 For example, there are reports in the literature 

 on the cytochemical localization of enzymes 

 and certain thiol-rich proteins in the mitotic 

 apparatus, but I would venture to predict on the 

 basis of the argument just given that at least 

 some of the observed cytochemical localizations 

 are localizations by default and not the result 

 of active processes associated with the as- 

 sembly of the mitotic apparatus. I wanted to 

 make this argument clear because it suggests 

 that the radioactivity seen in the spindle may 

 have been included in that region in a passive 

 rather than an active fashion. One possibility 

 exists for testing this question further, and 

 that depends on the presence in the spindle 

 of fibres or microtubules that presumably 

 represent the definitive working part of the 

 organelle. 



Figure 14 is an optical autoradiogram of an 

 isolated spindle sectioned at one micron. This 

 spindle is a member of a population obtained 

 from eggs that have been pulsed with the amino 

 acid leucine and "chased" prior to the appear- 

 ance of the metaphase spindle. You might al- 

 ready see in the figure a suggestion that the 

 radioactivity, which is represented by the silver 

 grains, has a certain tendency to follow the 

 lines of the fibres. These fibres, which run in 

 tracts, are visible in sections of this thickness. 

 Figure 15 is an electron microscope auto- 

 radiogram made from the same material. At 

 low magnification, one sees tracts of fibres 

 running through the center of the spindle and 

 silver grains distributed over the whole area. 



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