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Reorganization of microtubules and F-actin during development of branches from the growth cone and axon shaft. A, Lower-power time-lapse images of the same neuron at two time points showing a new axon, not present when the neuron was injected, forming from the distal tip of the large paused growth cone. The arrowhead indicates the growth cone at the tip of the developing axon (3:27hr). In A-D, F-actin is shown in red, microtubules in green, and their overlap in yellow. B, Series of higher-power time-lapse images of the developing branch (A, arrow at 3:27hr) on the growth cone shown in A. The branch begins as a filopodium containing few microtubules and F-actin (0:00hr). Later (1:13hr-3:27hr), microtubules and F-actin colocalize ( yellow) in the growth cone at the tip of the branch. C, Lower-power time-lapse images showing development of branches from the axon shaft. At each branch point, F-actin is concentrated in regions where microtubules splay apart. D, Series of higherpower images of the developing branch (arrow at 4:57hr) on the axon shown in C. Microtubules splay apart and invade the developing branch (4:57hr). At 21:13hr, a growth cone has formed on the tip of the branch. E, Electron micrograph from the boxed region of a growth cone drawn in the inset demonstrating that a microtubule (arrowheads) splays from the loop and is closely apposed to a bundle of actin filaments (arrows) in the lamellipodium. Scale bars: A, C, 10 m; B, D, 5 m; E, 0.5 m.

Reorganization of microtubules and F-actin during development of branches from the growth cone and axon shaft. A, Lower-power time-lapse images of the same neuron at two time points showing a new axon, not present when the neuron was injected, forming from the distal tip of the large paused growth cone. The arrowhead indicates the growth cone at the tip of the developing axon (3:27hr). In A-D, F-actin is shown in red, microtubules in green, and their overlap in yellow. B, Series of higher-power time-lapse images of the developing branch (A, arrow at 3:27hr) on the growth cone shown in A. The branch begins as a filopodium containing few microtubules and F-actin (0:00hr). Later (1:13hr-3:27hr), microtubules and F-actin colocalize ( yellow) in the growth cone at the tip of the branch. C, Lower-power time-lapse images showing development of branches from the axon shaft. At each branch point, F-actin is concentrated in regions where microtubules splay apart. D, Series of higherpower images of the developing branch (arrow at 4:57hr) on the axon shown in C. Microtubules splay apart and invade the developing branch (4:57hr). At 21:13hr, a growth cone has formed on the tip of the branch. E, Electron micrograph from the boxed region of a growth cone drawn in the inset demonstrating that a microtubule (arrowheads) splays from the loop and is closely apposed to a bundle of actin filaments (arrows) in the lamellipodium. Scale bars: A, C, 10 m; B, D, 5 m; E, 0.5 m.

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Cortical neurons innervate many of their targets by collateral axon branching, which requires local reorganization of the cytoskeleton. We coinjected cortical neurons with fluorescently labeled tubulin and phalloidin and used fluorescence time-lapse imaging to analyze interactions between microtubules and actin filaments (F-actin) in cortical growt...

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... reorganization of the F-actin and microtubule cytoskel- eton occurs during formation of branches from remnants of paused growth cones on the axon shaft. Lengthy imaging se- quences (n 26 sequences, 34 branches) show that branches often begin as a single filopodium containing F-actin and micro- tubules (Fig. 2 A,B). At branch points along the axon shaft (Fig. 2C,D), bundled microtubules splay apart, coincident with an ac- cumulation of F-actin, and microtubules invade the newly forming branch (Fig. 2 D). The growth cone at the tip of the branch has high levels of F-actin that overlap with microtubules, whereas F-actin has disappeared from ...
Context 2
... of the F-actin and microtubule cytoskel- eton occurs during formation of branches from remnants of paused growth cones on the axon shaft. Lengthy imaging se- quences (n 26 sequences, 34 branches) show that branches often begin as a single filopodium containing F-actin and micro- tubules (Fig. 2 A,B). At branch points along the axon shaft (Fig. 2C,D), bundled microtubules splay apart, coincident with an ac- cumulation of F-actin, and microtubules invade the newly forming branch (Fig. 2 D). The growth cone at the tip of the branch has high levels of F-actin that overlap with microtubules, whereas F-actin has disappeared from proximal regions of the branch. In living growth cones, ...
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... Lengthy imaging se- quences (n 26 sequences, 34 branches) show that branches often begin as a single filopodium containing F-actin and micro- tubules (Fig. 2 A,B). At branch points along the axon shaft (Fig. 2C,D), bundled microtubules splay apart, coincident with an ac- cumulation of F-actin, and microtubules invade the newly forming branch (Fig. 2 D). The growth cone at the tip of the branch has high levels of F-actin that overlap with microtubules, whereas F-actin has disappeared from proximal regions of the branch. In living growth cones, microtubules and F-actin appear to be closely apposed. We confirmed this with electron microscopy of fixed cortical growth cones (Fig. 2 E), ...
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... forming branch (Fig. 2 D). The growth cone at the tip of the branch has high levels of F-actin that overlap with microtubules, whereas F-actin has disappeared from proximal regions of the branch. In living growth cones, microtubules and F-actin appear to be closely apposed. We confirmed this with electron microscopy of fixed cortical growth cones (Fig. 2 E), which showed that micro- tubules splaying from the central loop ( Fig. 2 E, inset) extend into the lamellipodium, where they are closely apposed to bundles of actin filaments (Dailey and Bridgman, 1991;Rochlin et al., 1999). These results show that development of branches from a large paused growth cone or from its remnants along the ...
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... of F-actin that overlap with microtubules, whereas F-actin has disappeared from proximal regions of the branch. In living growth cones, microtubules and F-actin appear to be closely apposed. We confirmed this with electron microscopy of fixed cortical growth cones (Fig. 2 E), which showed that micro- tubules splaying from the central loop ( Fig. 2 E, inset) extend into the lamellipodium, where they are closely apposed to bundles of actin filaments (Dailey and Bridgman, 1991;Rochlin et al., 1999). These results show that development of branches from a large paused growth cone or from its remnants along the axon shaft is accompanied by local accumulation of F-actin, which coincides with ...
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... determine the nature of F-actin-microtubule interactions in living growth cones and at axon branch points, we used rapid acquisition (5-15 sec) of closely spaced (1-2 sec) sequential images of microtubules and actin filaments (n 36 sequences in six growth cones and five branch points) over periods of 10 -20 min. We chose large paused growth cones to visualize cytoskel- etal interactions associated with branching (Figs. 1, 2). Movies of sequential images of actin filaments and microtubules allowed us to analyze frame by frame the reorganization of each cytoskeletal 4 (Figure legend continued.) of F-actin ( 1) depolymerizes and moves rearward with the retrograde actin flow, whereas the other microtubule and associated bundle of F-actin ( 2) remains extended but turns perpendicular to the retrograde actin flow. ...

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