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In the mitotic spindle, MT orientation remained a major question whose answer would help determine what role the microtubules played in lining up and separating chromosomes. Several in vitro studies revealed that MTs could be initiated from both kinetochores and centrosomes (Telzer et al., 1975; Gould and Borisy, 1977) and also that both kinetochore and centrosome MTs polymerized with their plus ends distal to the organizing center (Bergen et al., 1980).% K9 G s, W5 {) D) Q8 x& O
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Counterclockwise "hooks" of polymerized neurotubulin reveal that kinetochore microtubules have uniform polarity.5 ^9 W$ ]) j- \
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Trying to put all of this together into a model of mitosis, Richard McIntosh stuck to the law of parsimony. "If you could use simple ideas to explain complex phenomena, then the simplest idea would be right," he says. And the simplest explanation, given all of the above, was that the MTs in each half of the spindle were antiparallel. Furthermore, cross-bridges between opposing filaments would facilitate the sliding mechanism that could move kinetochore MTs (and their attached chromosomes) toward the spindle poles., a/ P' Z0 }3 o/ ^
. a5 B% I: b* K. H1 r0 G+ zA major prediction of the model was that in late anaphase, when chromatin moved to the poles, only minus ends of the centrosome MTs should be left at the midplate. In 1980, the McIntosh lab stumbled upon a technique to directly test MT polarity and thus the model. While testing a "very nonphysiological" cocktail of detergents and high molarity buffer to visualize how isolated mammalian spindles incorporated purified tubulin, the lab created "bushy-looking microtubules," McIntosh says. When he viewed these MTs in cross section, he saw that the bushy look was due to hooks of tubulin forming a pinwheel shape around each microtubule (Heidemann and McIntosh, 1980).* n; t4 U& G+ b3 P' V
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When his group tested the tubulin hooks on MTs of known polarity, they found that the direction of the curve of the hooks corresponded to MT polarity. With this serendipitous tool in hand, the group "went for the spindle midbody first to see if minus or plus ends were there." In the 1981 study, it turned out that in anaphase cells, 90–95% of the MTs in a half-spindle were oriented with their plus ends toward the middle (Euteneuer and McIntosh, 1981). Also, a look at just the kinetochore MTs confirmed that those MTs were also oriented with the plus ends distal to the spindle pole.
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! S- W- f/ |: c( k9 n/ AIn the same issue, Bruce Telzer and Leah Haimo published a study using dynein arms to form polarity-marking pinwheels on MTs in clam egg spindles (Telzer and Haimo, 1981). Their results also showed that the majority of MTs in a meiotic half-spindle were oriented with their plus ends distal to the poles. Together, the two studies sealed the idea that half-spindles contained parallel MTs.7 j& G- e, S7 @& A! R
5 f& C8 {; W/ aThat set others searching for the next most logical puzzle piece: did kinetochores "capture" centrosomal MTs or did they assemble MTs "upside-down" by adding subunits to the minus ends? Four years later, a group with a talent for in vitro MT manipulation found good evidence that kinetochores did indeed capture and stabilize the dynamically unstable MTs growing from the asters (Mitchison and Kirschner, 1985), a process that was later documented in vivo (Rieder and Alexander, 1990). KP# J, P- q( m. d1 J3 @- n4 P% K ^
" `# N2 L u1 X& Q2 JAllen, C., and G.G. Borisy. 1974. J. Mol. Biol. 90:381–402.) ?; q; `- `! B8 m
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Amos, L., and A. Klug. 1974. J. Cell Sci. 14:523–549.
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Bergen, L.G., et al. 1980. J. Cell Biol. 84:151–159.' p }( M8 x* u5 V( L
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Euteneuer, U., and J.R. McIntosh. 1981. J. Cell Biol. 89:338–345.! T! I @1 }. {! e
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Gibbons, I.R. 1966. J. Biol. Chem. 241:5590–5596.
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2 O% L0 Q$ m5 m1 w% H$ v7 w4 AGould, R.R., and G.G. Borisy. 1977. J. Cell Biol. 73:601–615.8 `4 D5 x; u' @/ ~! {6 d e2 J
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Heidemann, S.R., and J.R. McIntosh. 1980. Nature. 286:517–519." ]4 S# E6 E8 V8 W
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Mitchison, T.J., and M.W. Kirschner. 1985. J. Cell Biol. 101:766–777.1 E3 _. u1 T' i. u/ h8 f
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Rieder, C.L., and S.P. Alexander. 1990. J. Cell Biol. 110:81–95.8 }& p7 v; s+ V7 ?$ v1 n6 z
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Satir, P. 1968. J. Cell Biol. 39:77–94.1 X9 h2 f/ F( O% ?6 M, ]% U
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Telzer, B.R., and L.T. Haimo. 1981. J. Cell Biol. 89:373–378.(By the late 1970s, it was still unclear ) |
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发表于 2015-5-26 19:57
