Myosin IIB is required for growth cone motility - PubMed (original) (raw)
Myosin IIB is required for growth cone motility
P C Bridgman et al. J Neurosci. 2001.
Abstract
Growth cones are required for the forward advancement and navigation of growing axons. Modulation of growth cone shape and reorientation of the neurite are responsible for the change of outgrowth direction that underlies navigation. Change of shape involves the reordering of the cytoskeleton. Reorientation of the neurite requires the generation of tension, which is supplied by the ability of the growth cone to crawl on a substrate. The specific molecular mechanisms responsible for these activities are unknown but are thought to involve actomyosin-generated force combined with linkage to the cell surface receptors that are responsible for adhesion (Heidemann and Buxbaum, 1998). To test whether myosin IIB is responsible for the force generation, we quantified shape dynamics and filopodial-mediated traction force in growth cones from myosin IIB knock-out (KO) mice and compared them with neurons from normal littermates. Growth cones from the KO mice spread less, showed alterations in shape dynamics and actin organization, and had reduced filopodial-mediated traction force. Although peak traction forces produced by filopodia of KO cones were decreased significantly, KO filopodia occasionally developed forces equivalent to those in the wild type. This indicates that other myosins participate in filopodial-dependent traction force. Therefore, myosin IIB is necessary for normal growth cone spreading and the modulation of shape and traction force but acts in combination with other myosins for some or all of these activities. These activities are essential for growth cone forward advancement, which is necessary for outgrowth. Thus outgrowth is slowed, but not eliminated, in neurons from the myosin IIB KO mice.
Figures
Fig. 1.
Examples of video-enhanced DIC time-lapse sequences taken from WT (Control;A–F) and knock-out SCG growth cones (KO; G–L). Protrusions (arrowheads) in the WT occur mainly at the leading edge. In contrast, protrusions (arrowheads) in the KO occur both at the leading edge and at other areas around the perimeter of the cone. Frames are at 15 sec intervals. A, B, Inset, Immunoblot for myosin IIB in brains from the mice that were used for these cultures. A 200 kDa band specific for myosin IIB is seen only in the control. Scale bar, 6 μm.
Fig. 2.
An example of the analysis of protrusion and retraction in growth cones from WT (A, C, E) and myosin IIB KO (B, D, F) animals. A, B, The perimeter of the growth cone traced from the previous time point is indicated by the red outline. Areas of protrusions are indicated in green overlays, whereas areas of retractions are indicated in blue. The sequential_numbering_ around the perimeter of the cones indicates each area of either protrusion or retraction that had been identified in a 1 min time interval and then used as a single time point for plots of protrusion/retraction number versus the area shown in_C_ and D and for the multiple time points shown in E and F. _C_represents the WT cone shown in A; _D_represents the KO cone shown in B. In C_and D the positive values indicate protrusion areas, and the negative values indicate retraction areas. E, F, Examples of the time series analysis of growth cone protrusion and retraction taken from time-lapse records of WT (as in A) and myosin IIB KO (as in B) cones of approximately equal area. Note that, as in C and D, protrusion areas are indicated as positive values (on the_z_-axis), whereas retraction areas are indicated as negative values. The sequential numbering around the cone perimeter for each time point is indicated on the_y_-axis. A single 1 min time point (identified with different colors; x_-axis) in E or_F represents an individual set of area measurements as shown in C or D. The sizes of protrusions and retractions tend to be larger in the WT cone but occur less frequently over the 10 min period. Thus the differences in area and frequency of protrusion and retraction that are observed in_C and D persist over time.
Fig. 3.
F-actin organization is abnormal in growth cones from myosin IIB KO animals. A, The distribution of F-actin that is stained with rhodamine phalloidin in a control (WT) growth cone. Note that the palm of the cone contains numerous actin bundles (arrowheads). B–D, The distribution of F-actin in three KO cones is shown. Although actin bundles are still present in filopodia (arrows), they are absent or decreased in the narrowed palm of the cone. Scale bar, 4 μm.
Fig. 4.
Interaction of growth cone filopodia and lamellipodia with the 3.75% acrylamide gel surface causes displacement of fluorescent beads (white dots) embedded in the gel.A–H, Filopodia in a control (WT) growth cone cause displacement of three (1–3) adjacent beads at different time intervals. The beads move (arrowheads) toward the growth cone palm. Bead 1 appears to move initially (B–D) without filopodia shortening. Later, the filopodia appear to move laterally (E) and then shorten (note increased phase density in F–H). Both beads 2 and 3 appear to move as the filopodia shorten. Time between frames is 20 sec. I–L, Bead movement in response to filopodial shortening in a KO growth cone. A single bead (arrowhead) moves toward the growth cone palm as the filopodia appear to shorten. Time between frames was 20 sec, except between I and J when the time was 40 sec. M, Comparison of bead movement induced by WT filopodial or lamellipodial interaction. Both examples start as 0,0 coordinates. Points indicate the bead centroid. A straight line along the _x_-axis would be parallel with the neurite. Positive values along the _x_-axis indicate movement toward the growth cone palm. In response to the lamellipodium a bead first moves to the side and then is pushed out (negative value along _x_-axis). Then the bead is pulled inward toward the growth cone palm but also moves laterally. In response to a filopodium a bead moves inward toward the growth cone palm with less lateral movement. Scale bar (in H): 9.5 μm.
Fig. 5.
Examples of the change in force over time because of individual filopodial interactions with the gel substrate.Inset, The macroscopic elastic property of a 3.75% acrylamide gel is shown. The two traces indicate the force-versus-length relationship during gel stretching and relaxation. The curves are essentially the same. The least-squares best fit line through the data is indicated. A, The force exerted by filopodia from WT growth cones is depicted. The force tends to increase quickly (approximately half-maximal by 50 sec) and then stabilizes or decreases. B, The force exerted by filopodia from KO growth cones is shown. The force tends to develop slower and usually reaches a lower maximum value before decreasing. However, in filopodia number 5 the force-versus-time relationship is very similar to that seen in WT cones.
Fig. 6.
WT (A) and myosin IIB KO (B) radial outgrowth from explants on a relatively adhesive substrate (laminin plus 0.1 mg/ml polyornithine). The KO outgrowth is slow compared with WT. Insets, Comparison of growth cone morphology. The cone from the WT animals has a spread morphology. Retraction fibers can be seen at the rear of the cone and along the neurite. Most retraction fibers lie approximately parallel to the neurite axis. In contrast, the cone from the myosin IIB KO animal is narrow. It has a much smaller area in contact with the adhesive substrate. Retraction fibers are seen approximately perpendicular to the neurite axis. C, D, Localization and expression levels of myosin IIA (green) appear relatively normal in growth cones from myosin IIB KO animals.C, Immunofluorescent localization of myosin IIA (green) and F-actin (red) in a WT cone. D, Localization of myosin IIA (green) and actin (red) in a myosin IIB KO cone. In both cones myosin IIA staining is brightest in the central region of the cone. Punctate staining also can be seen more peripherally along actin bundles, which include bundles in filopodia. Quantitative analysis of myosin IIA staining brightness did not reveal any significant differences between WT and KO cones. Scale bars:B, 114 μm; D, 4 μm.
References
- Bentley D, Toroian-Raymond A. A disoriented pathfinding by pioneer neuron growth cones deprived of filopodia by cytochalasin treatment. Nature. 1986;323:712–715. - PubMed
- Chien CB, Rosenthal DE, Harris WA, Holt CE. Navigational errors made by growth cones without filopodia in the embryonic Xenopus brain. Neuron. 1993;11:237–251. - PubMed
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