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First published online 29 January 2003
doi: 10.1242/jcs.00335

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Retrograde flow rate is increased in growth cones from myosin IIB knockout mice

Washington University School of Medicine, Department of Anatomy and Neurobiology, Box 8108, 660 S. Euclid Avenue, St Louis, MO, USA

View larger version (32K): [in a new window]   Fig. 1. Quantitative analysis of retrograde flow. (A) A sequence (13 second intervals) showing the movement of a peripherally located bead (top bead) towards the wt growth cone central zone. A second bead (bottom bead) in the central zone remains stationary. The line through the image is to aid detection of displacement. Beads are 1.5 µm in diameter. (B) A similar sequence (but at 6.5 second intervals) as in A, from a KO growth cone. Three beads are undergoing rearward movement (towards the bottom of the image) independently of a single growth cone. (C) Regression analysis of the steepest portion of a bead displacement versus time curve from a wt cone. The slope of the regression line was used as the maximum rate of bead displacement. (D) The same analysis as in C, but from a KO cone. Note that in general KO cones showed slightly more deviation from linearity (lower r2 values) than wt. (E) Comparison of wt and KO maximum rates of bead displacement (n=10 for each). The difference was significant (t-test, P<0.01).

View larger version (58K): [in a new window]   Fig. 2. Tracking retrograde flow using GFP–myosin-IIA fluorescent spots. (A) A KO growth cone from a SCG neuron expressing GFP–myosin-IIA (the barely visible neurite is on the left side of the field). The spots of fluorescence sometimes align presumably along actin bundles. (B) A high magnification sequence from the KO cone showing the displacement of spots (the pair indicated by the arrowhead) over time. The lines through the images are for position reference and allow one to see the retrograde displacement (from top to bottom of image). Because many of the spots changed intensity, appeared and disappeared at different times, only a subset of spots could be identified with certainty in multiple frames. The elapsed time between images is 13 seconds. (C) Comparison of the average rate (±s.e.m.) of GFP–myosin-IIA spot displacement from wt and KO growth cones. The spots in the KO growth cones (n=11) move more than twice the speed of those in the wt (n=15). The individual rates were calculated from the total distance that spots moved over a minimum of three frames divided by the elapsed time (13 second intervals). A, bar, 12 µm; b, bar, 2 µm.

View larger version (68K): [in a new window]   Fig. 3. Comparison of filopodia protrusion from wt (A) and KO (B) growth cones. (A) A sequence (10 second intervals) showing the extension of a filopodia near the leading edge of a wt growth cone. The filopodia grows in length, but some retraction of the lamellipodia at the base of the filopodia adds to the appearance of length increase. (B) A sequence as in A, but from a KO growth cone. The filopodium extends at a similar rate to the wt filopodium in A. A common feature of growing KO filopodia are the small bumps or protrusions that form. They sometimes lead to branching and appear to undergo retrograde movements. Bar, 3 µm.

View larger version (85K): [in a new window]   Fig. 4. Comparison of microtubule distribution in wt (A) and KO (B) growth cones. (A) A wt growth cone triple stained for actin (red), myosin IIA (green) and microtubules (blue). The insert shows only the microtubule staining. Microtubules penetrate the base of F-actin-rich structures. (B) A KO growth cone stained as in A. Microtubules penetrate into peripheral structures, but these often appear to have reduced staining for F-actin compared with wt. (C) Higher magnification example of multiple (1-4) microtubule ends interacting with actin containing protrusive structures. The average distance between microtubule ends and the tips of protrusive structures is greater for the KO examples. The overlap of color makes identification of microtubules difficult so the microtubule staining is also shown by itself (black and white image; contrast enhancement has produced a thickening of microtubules). B, Bar, 4.3 µm; C, the vertical Bar in No. 4=1.8 µm.

View larger version (36K): [in a new window]   Fig. 5. Comparison of myosin 1C (green) and myosin IIA (red) staining in wt and KO growth cones (cold methanol fixed). (A) A KO growth cone from a SCG neuron. The myosin IC staining is mostly confined to the neurite, whereas the myosin IIA staining is distributed throughout the growth cone. Wt SCG growth cones showed a similar pattern, although the growth cones were usually larger. (B) A wt DRG growth cone shows a similar pattern of staining compared to that seen in SCG neurons (a relatively small cone was selected for comparison purposes). The intensity of the myosin IC staining appears to be elevated in the neurite compared with that observed in SCG neurons. This was consistently observed, but it is unclear if this results from thickness differences in the two types of neurites. (C) A KO DRG growth cone also shows a similar pattern and intensity of staining to the wt DRG growth cone. C, bar, 6.8 µm.

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