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First published online 30 July 2003
doi: 10.1242/jcs.00686

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Microtubule and Rac 1-dependent F-actin in growth cones

Department of Pharmacology and Center for Neurobiology and Behavior, Columbia University, New York, NY 10032, USA

View larger version (87K): [in a new window]   Fig. 1. Morphology and cytoskeleton of growth cones on different substrates. (A) A large growth cone on a polyamine substrate has a peripheral ribbed F-actin (green) network with microtubules (red) occupying the central region. (B) Growth cones on a laminin substrate are smaller and microtubules penetrate closer to the leading edge. Bars, 10 µm.

View larger version (67K): [in a new window]   Fig. 2. Induction of rapid growth by the acute addition of laminin. DIC recording of growth from a neuron plated on a fibronectin substrate that was then exposed to laminin. By 20 minutes after the addition of laminin, veils of membrane have appeared on the thickened shafts of filopodia (arrows). By 25 minutes, the large-spread growth cone has given rise to a thin process with a smaller growth cone. Bar, 10 µm.

View larger version (56K): [in a new window]   Fig. 3. Distribution of Rac 1. (A,B) Localization of Rac 1 in a growth cone cultured on a polyamine substrate. There is an extensive ribbed network of F-actin (A). Rac 1 in the same growth cone does not substantially localize to this peripheral network, but some colocalization is seen in the C region of the growth cone (B). Bar, 10 µm. (C,D) On a laminin substrate, Rac 1 is often colocalized with F-actin at the leading edge of the growth cone (arrows). Bar, 10 µm. (E) Detail of a part of a growth cone on a polyamine substrate that was acutely exposed to laminin. At the site of outgrowth of a streamlined growth cone, microtubules advance into a filopodium and colocalize with Rac 1 (arrow). A filopodium lacking microtubules does not contain Rac 1 (arrowhead). Bar, 2 µm.

View larger version (64K): [in a new window]   Fig. 4. Bundled dynamic microtubules associate with F-actin during outgrowth in the presence of CD. (A) After treatment with 125 nM CD for 20 minutes, a growth cone on a polyamine substrate shows an almost complete loss of F-actin (green), with microtubules (red) filling the growth cone. Bar, 10 µm. (B-D) 25 minutes after the addition of laminin to such a growth cone, microtubules have bundled at peripheral sites where protrusions form and then develop into neurites. (B) Merged image of tyrosinated tubulin (green) and total tubulin (red) in a protrusion shows an enrichment of tyrosinated tubulin. Bar, 2 µm. (C) An F-actin focus (green) is intimately associated with an accumulation of microtubules (red). Bar, 2 µm. (D) All outgrowths show F-actin foci associated with bundling microtubules. Bar, 10 µm.

View larger version (72K): [in a new window]   Fig. 5. Emergence of a process induced by the acute addition of laminin in the presence of CD is associated with actin. A growth cone labeled with fluorescent actin and growing on a polyamine substrate was treated with 125 nM CD for 15 minutes before exposure to laminin. Shown is a portion of the growth cone just before, and at indicated times after, the addition of laminin. Fluorescent actin (top row) and DIC images (bottom row) show the appearance of protrusions, which fuse and sprout a single filopodium. An intense focus of actin precedes a rapid elongation of the new process. Last panels show F-actin (phalloidin stain) and tubulin (immunocytochemical stain) in the same structure that was fixed at 27 minutes. Live actin staining is similar to F-actin staining. Tubulin is present and bundled in the shaft. Bar, 5 µm.

View larger version (15K): [in a new window]   Fig. 6. Outgrowth in the presence of CD and laminin is correlated with the presence of actin-based protrusions. The advance of a single process during a spurt of growth was recorded live by DIC video microscopy. The advance and appearance of actin-based structures were quantitated. A small area of lamellipodium appears immediately prior to advance and is present throughout advance. Single filopodia (three in all) extend during the growth spurt.

View larger version (13K): [in a new window]   Fig. 7. Microtubule dynamics are necessary for F-actin foci and rapid outgrowth on laminin. (A) Suppression of laminin-induced F-actin foci by nocodazole, taxol and Li+. Growth cones were induced to form F-actin foci by treatment with CD for 15 minutes followed by laminin for 30 minutes. Each drug was then added to cultures for 15 minutes before fixation and staining for F-actin. All three drugs significantly (P<0.00001) reduced the frequency of foci. Error bars for nocodazole and Li+ are too small to see. (B) Nocodazole inhibits the rapid growth of processes on laminin. Neurons were plated onto laminin or fibronectin and then exposed to 33 nM nocodazole. Recordings of the same growth cones over time were made to determine the increase in process length. Only the rapid outgrowth on laminin is substantially inhibited by this low concentration of nocodazole. (C) Dose-response curves for process outgrowth from neurons plated onto laminin or fibronectin and then exposed to CD. Cells were treated with CD for either 4 hours (fibronectin) or 2 hours (laminin) and the increase in process length measured. Error (s.e.m.) bars are too small to see. Results are representative of three separate experiments.

View larger version (44K): [in a new window]   Fig. 8. Association of Rac 1 with laminin-induced outgrowth in the presence of CD. (A) Localization of Rho family proteins in growth cones treated with CD for 15 minutes followed by laminin for 25 minutes. Cultures were fixed and double stained for each GTPase and actin with either phalloidin and antibody to Rac 1, or an antibody to actin together with antibodies to Rho and cdc42. Only Rac 1 co-concentrates with actin. Bar, 10 µm. (B) Rac 1 is intimately associated with bundled microtubules. Rac 1 (red) colocalizes with bundled microtubules (tubulin, green) at the leading edge of neurites induced by laminin. Bar, 10 µm.

View larger version (11K): [in a new window]   Fig. 9. Effects of laminin are dependent on Rac 1. Cells growing on a polyamine substrate were injected with either vehicle (veh.), wild-type Rac 1 (wt) or dominant-negative Rac 1 (N17). After determining which cells were alive and labeled, CD was added to the cultures for 15 minutes followed by treatment with laminin for 1 hour. CD was then washed out of the cultures and further observations made after 1 hour. (A) 25 minutes after the addition of laminin, F-actin foci have formed in cells injected with wt Rac 1 but typically not in cells injected with N17 Rac 1. (B) 1 hour after the addition of laminin, N17 Rac 1 has also inhibited neurite outgrowth. (C) After washout of CD, N17 Rac 1 continues to inhibit outgrowth compared to the wt control.

View larger version (27K): [in a new window]   Fig. 10. How microtubule and Rac 1-dependent actin might contribute to rapid growth on laminin. (A) On a polyamine or fibronectin substrate, there is an incremental advance of the leading edge of the growth cone by polymerization onto the existing actin network, well ahead of the microtubules. (B) An additional mechanism that could contribute to normal growth on laminin. Microtubules penetrate filopodia (1). Multiple microtubules penetrate filopodia and peripheral regions (2). Rac 1-dependent F-actin colocalized with bundled microtubules produces `splashes' of veil at the distal edge (3) and facilitates rapid advancement.

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