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The studies were made possible by a new pharmacological tool. Cytochalasin B had been shown to inhibit motility (Carter, 1967), but without any real idea of mechanism. Schroeder (1969) then demonstrated that the drug also inhibited cytokinesis〞a process that, like the action of muscle, was thought to be contractile〞and he correlated this action with the disruption of the 50-? filament networks in cells.5 D! Q. e# ^' I% `* q3 I
3 Y j0 {# w' {: k |+ ? dWessells's group found a similar correlation between drug action and lack of filaments for cytochalasin B's inhibition of cell shape morphogenesis (Spooner and Wessells, 1970), and decided to see if the same held up when the drug was applied during neuron outgrowth and cell locomotion. "The lab was unusual in encouraging graduate students to wander off into unrelated projects," says Ken Yamada, then a student with Wessells and now at the NIH (Bethesda, MD). "So it was fascinating that everything fell together when we tested cytochalasin on the various systems."
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First the group added the drug to neurons in culture, which caused the axons' growing tips, or growth cones, to round up. The drug disrupted the filamentous networks in the growth cones and halted axon elongation (Yamada et al., 1970; Yamada et al., 1971). In a parallel study, the group described a network of microfilaments〞similar in organization to the one found in growth cones〞at the very leading edges of migratory glial cells. Cytochalasin B rapidly disrupted this network and halted cell migration (Spooner et al., 1971). When the drug was washed away in either system, the microfilament networks recovered and axon outgrowth or locomotion resumed.& y$ W5 V& ^3 _6 c C+ m0 U! i
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The very existence of the filament system was controversial. "It was quite peculiar to have such a meshwork associated with and forming the sole contents of protruding membranes," says Yamada. Coagulation after fixation was a concern, and one reviewer, recalls Yamada, "wondered if you would see the same if you fixed concentrated BSA." Still, the drug seemed to target the thin 50-? filaments specifically, as microtubules and intermediate filaments remained intact. But little was known about its mode of action. "We were using the agent without knowing what it was affecting, other than what we could see by electron microscopy," says Yamada.9 U" @+ B6 d) W# l- a
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A year later, experiments by Spudich and Lin (1972) showed that cytochalasin B specifically binds to purified muscle actin, supporting the conclusion that the microfilaments required for cell movement were indeed actin-like proteins. Their identity was, however, not confirmed until later studies demonstrated that the filaments could bind heavy meromyosin (Spooner et al., 1973) and again when actin antibodies became available (Spooner and Holladay, 1981). "In our original papers, we intentionally avoided using the word actin," says Yamada.2 U: j% X7 q1 D) ^* `+ U
/ Q0 g- P6 h8 W& t( d) a6 MHow actin might drive cell movement was even more obscure. Most of the speculation in the early papers was, by analogy with the known microfilament presence in muscle, centered on contractile possibilities. Contractile alignment of filaments might form microspikes, or contraction might pull the rearward cell contents forward, like an inchworm, to meet adhesions at the front. As yet there was no talk of pushing out the front of the cell with filament polymerization.' u! `( \6 e+ e
- A& Z( ~# y. iA filamentous network (FN; left), now known to be actin, disappears in the presence of cytochalasin B (right; R, ribosomes), as does motility.! K1 p! N1 v' E. l# `
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In their experiments, Spooner, Yamada, and Wessells also added the drug colchicine to cells to disassemble microtubules. This treatment did not affect growth cone elongation or glial cell migration. "Colchicine can collapse the axon, but the growth cone is still wriggling and trying to grow," explains Brian Spooner (Kansas State University), who was then a post-doc in Wessells's lab. "We now know that microtubules are involved in motility in all kinds of ways, but they are not required for cellular translocation. Our original conclusion has held up fairly well.". k% p% { A6 M3 ?2 O1 F2 u
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The thrill of finding part of the cell's motor was palpable. "It was a very exciting time," recalls Yamada. "Usually, 1 out of 10 experiments work; but at that time, 9 out of 10 would work."" [& j( B+ E0 R* i- U: `# \
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Carter, S.B. 1967. Nature. 213:261–264.' G3 _; L" t% u" H3 M* i4 J }
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Schroeder, T.E. 1969. Biol. Bull. 137:413.
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2 Y2 x; ]7 q) E, h, f& S; oSpooner, B.S., and N.K. Wessells. 1970. Proc. Natl. Acad. Sci. USA. 66:360–364.
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Spooner, B.S., et al. 1971. J. Cell Biol. 49:595–613.$ n8 J# U( ~/ @. y
0 ?/ ^/ G5 J8 F1 h% ?# }9 C" t6 pSpooner, B.S., et al. 1973. Tissue Cell. 5:37–46.
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* K1 D4 m' k0 b/ @) i+ R7 rSpudich, J.A., and S. Lin. 1972. Proc. Natl. Acad. Sci. USA. 69:442–446.9 y0 `; w ?) \/ O* {
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Yamada, K.M., et al. 1970. Proc. Natl. Acad. Sci. USA. 66:1206–1212.6 h [0 v& |* e1 O5 s$ U4 a( ?
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Yamada, K.M., et al. 1971. J. Cell Biol. 49:614–635.(By 1970, several ultrastructural studies) |
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发表于 2009-3-6 09:03
发表于 2015-5-25 13:18
