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First published online 27 February 2007
doi: 10.1242/jcs.03384

Journal of Cell Science 120, 1113-1125 (2007)
Published by The Company of Biologists 2007
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Kinetic-structural analysis of neuronal growth cone veil motility

Anne K. Mongiu*, Elizabeth L. Weitzke, Oleg Y. Chaga and Gary G. Borisy{ddagger}

Department of Cell and Molecular Biology, Northwestern University Feinberg School of Medicine, and Marine Biological Laboratory, 303 E. Chicago Avenue, Chicago, IL 60611, USA


Figure 1
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  		Fig. 1. Growth cone advance and veil dynamics. (A) Growth cone advance. Blue line traces path traveled by growth cone during a 3-hour time-lapse observation. * indicates start point. (B) Veil dynamics. Kymographs (right) of veils in growth cone front (top, left) and rear (bottom, left) show alternation of protrusion and retraction. Black line denotes path along which kymograph was obtained. (C) Orientation of protrusion and retraction phases. `Other' denotes veil dynamics – collapse, ruffling or stalling – not readily categorized as protrusion or retraction. n=151 veils; 20 individual growth cones; 352 minutes observation; 329 transitions recorded.

 

Figure 2
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  		Fig. 2. Veil protrusion and retraction are primarily filopodia associated. (A) Time-lapse merges illustrate veil categories. In this and all succeeding figures, protruding and retracting regions are indicated by red and cyan, respectively, by merging the later time point (red channel) with the earlier time point (blue and green channels). (a,b) Filopodia-associated protrusion – protrusion between two established filopodia. Veil edges can be convex (a) or concave (b). (c) Filopodia-independent protrusion – veil protrusion not connected to established filopodia. (d) Filopodia-associated retraction – retraction between two filopodia, retaining direct connection throughout retraction. (e, arrowhead) Filopodia-independent retraction is seen less than 3% of the time. (e, asterisk) Single filopodium-associated protrusion – protrusion along a single filopodium. (f, asterisk) Veil protrusion arising on a filopodium shaft. (B) Tabulation of frequency and width of veils in each category.

 

Figure 3
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  		Fig. 3. Correlative light and electron microscopy of filopodia-associated veil protrusion and retraction. (A) Phase-contrast panels show a low magnification image of the growth cone with a white box denoting the region of interest, followed by four consecutive images from the last 9 seconds of the time-lapse sequence before the cell was extracted, a live-live phase-contrast merge of the first and last images, the same region after extraction `lysed', and a phase-contrast merge of the lysed and the last live image. In all merges, red regions indicate protrusion, cyan regions indicate retraction. The veil in the left panel was protruding at 4.2 µm/minute, the veil in the right panel was retracting at 1.7 µm/minute. (B) Electron micrographs of region of interest from the time lapses in A. (C) Higher magnification of the boxed regions in B.

 

Figure 4
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  		Fig. 4. Actin distribution in protruding and retracting veils. (A) Veil dynamics merge (left) and filamentous actin distribution (right) in a growth cone. The merge combines the last live phase-contrast image before extraction and the one obtained 6 seconds earlier. Enlarged insets (p, r) show regions of veil protrusion (red) and retraction (cyan). (B) Line scans of Texas Red phalloidin fluorescence for protrusion and retraction derived from the regions of the growth cone shown in A (dotted lines). Fluorescence was normalized to 100 at peak of protrusion distribution. Protruding veils have more and deeper filamentous actin than retracting veils. n=4 growth cones; 9 retracting veils; 22 protruding veils.

 

Figure 5
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  		Fig. 5. Phase transitions and local autonomy of veils. Panel construction as in Fig. 4. (A) Left: nascent protrusion (3.6 µm/minute) characterized by dense branched network is demarcated from surrounding sparse network. Right: protruding (7.6 µm/minute) and retracting veil (2.8 µm/minute) adjacent to same filopodium show distinct filament organization: dense branched vs sparse, respectively. (B) Electron micrographs of the regions of interest from the time lapses in A. (C) Higher magnification of the boxed region in B. The new protrusion (left) is sharply demarcated from the local network (dashed line). Adjacent filopodia associated veils (right) have independent behavior.

 

Figure 6
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  		Fig. 6. Alternate forms of veil protrusion. The upper panel of each subsection contains a low magnification image of the growth cone, with the region of interest boxed, a single phase-contrast overlay, and a low magnification EM of the region of interest. (A) Nascent veil protrusion (6.8 µm/minute) along a single filopodium. Veil contains a dense network of filaments connected to filopodial shaft. (B) Veil formation (8.3 µm/minuts) isolated from lamellipodium. Dense network interconnects two filopodia shafts. (C) Established Filopodia `independent' protrusion (8.1 µm/minute). Veil organization is a mixture of dense network and loosely bundled long filaments. Boxed regions (a-c) show branched filaments in the network, pseudo-colored cyan for visualization. (D) Nascent filopodia-`independent' protrusion (1.1 µm/minute). Veil displays multiple finger-like protrusions containing dense network and some long filaments.

 

Figure 7
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  		Fig. 7. Distribution of Arp2/3 in veils. (A) Growth cone immunostained against Arp3 subunit and phalloidin. Inset shows an enrichment of Arp3 (green) at the leading edge that co-localizes with phalloidin stained actin (red). (B) Low magnification growth cone (veil protrusion at 5.3 µm/minute) followed by time lapse and merges of boxed region. (C) Left. EM of region in lysed-live in B immunostained against Arp3 subunit. Right. Same EM overlaid with a 0.5 µm grid colored to reflect the number of gold particles in each box; heat map indicates number of gold particles per 0.25 µm2 box. Similar to A, Arp3 staining co-localizes with actin and is highest at the leading edge of protruding veils. (D) High magnification of the boxed region from C. Gold particles have been pseudo-colored yellow.

 

Figure 8
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  		Fig. 8. Correlation of Arp3 location with veil dynamics. The upper panel of each subsection contains a low magnification image of the growth cone, with the region of interest boxed, a single phase-contrast overlay, and a low magnification EM immunostained against Arp3 in the region of interest. (A) Protruding filopodia (5.2 µm/minute)-associated veil with a concave leading edge. (B) Retracting filopodia (1.7 µm/minute)-associated veil with sparse actin network. (C) De novo veil initiation from filopodial shaft (*), which travels (11.3 µm/minute) toward the growth cone. (D) Representative example of a veil arising de novo from a phase-dense spot along the shaft of a filopodium.

 

Figure 9
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  		Fig. 9. Modes of veil dynamics in the neuronal growth cone. (1) Veil protrusion associated with filopodia at the front and rear of the growth cone. Branched filaments can arise from the veil network and merge into the filopodia or can occur off existing filaments in the filopodia. (2) Veil protrusion along a single filopodium, either crawling along the side of the filopodium (2a) or arising de novo off the shaft of the filopodium (2b). (3) Veil protrusion independent of filopodia. The veil network appears to be similar to lamellipodial network seen in nonneuronal cells. (4) Retraction at the front and rear of the growth cone. The veil network becomes very sparse and some filaments are seen aligned and bundled, parallel to the membrane.

 




© The Company of Biologists Ltd 2007