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BACKGROUND: Interactions between microtubules and actin filaments (F-actin) are critical for cellular motility processes ranging from directed cell locomotion to cytokinesis. However, the cellular bases of these interactions remain poorly understood. We have analyzed the role of microtubules in generation of a contractile array comprised of F-actin and myosin-2 that forms around wounds made in Xenopus oocytes.
RESULTS: After wounding, microtubules are transported to the wound edge in association with F-actin that is itself recruited to wound borders via actomyosin-powered cortical flow. This transport generates sufficient force to buckle and break microtubules at the wound edge. Transport is complemented by local microtubule assembly around wound borders. The region of microtubule breakage and assembly coincides with a zone of actin assembly, and perturbation of the microtubule cytoskeleton disrupts this zone as well as local recruitment of the Arp2/3 complex and myosin-2.
CONCLUSIONS: The results reveal transport of microtubules in association with F-actin that is pulled to wound borders via actomyosin-based contraction. Microtubules, in turn, focus zones of actin assembly and myosin-2 recruitment at the wound border. Thus, wounding triggers the formation of a spatially coordinated feedback loop in which transport and assembly of microtubules maintains actin and myosin-2 in close proximity to the closing contractile array. These results are surprisingly reminiscent of recent findings in locomoting cells, suggesting that similar feedback interactions may be generally employed in a variety of fundamental cell motility processes.
Figure 1. Microtubule Organization around Wound Borders
All times are in min:sec.
(A) Confocal image of a laser-wounded oocyte. Microtubules (red), F-actin (green), and myosin-2 (blue) show complementary distribution
around the wound.
(B) Individual channels (MT, microtubule; FA, F-actin; and M2, myosin-2). F-actin and myosin-2 concentrate inside the area of highest
microtubule density.
(C) Image from a movie of an OG-Tax-injected oocyte. Microtubules are organized into a radial array around the wound (W). See Movie 1
(Movies are available in the Supplemental Data available with this article on line).
(D) Image from a movie of a wound in an OG-Tax-injected oocyte. Microtubules are perpendicular to the wound border and translocate to
the wound. See Movie 2.
(D) Time course of microtubule movement toward the wound (indicated by an arrow). Asterisks mark the leading ends of individual microtubules,
and arrowheads mark the trailing ends.
(E) Kymograph of OG-Tax-labeled microtubule motility toward the wound edge. Horizontal arrow is 45 s; vertical arrow is 5 m. Microtubules
accelerate as they approach the wound border (w), then slow at its immediate edge.
(F) Images from a movie of an OG-Tax-injected oocyte. An arrowhead shows a sharply bent microtubule moving toward the wound.
(G) Images from a movie of an OG-Tax-injected oocyte. Microtubules buckle (arrowhead) at the wound border. The arrow indicates direction
of flow.
Figure 2. Microtubule Transport is F-Actin Dependent
(A) Plot of instantaneous rates of microtubules before and after treatment with latrunculin B (20 M). Microtubule translocation to the wound
border was arrested after latrunculin treatment. For each point, the rates of movement of a minimum of 10 microtubules were quantified; bars
indicate standard error of the mean (SEM).
(B) Images from a movie of an OG-Tax-injected oocyte. Arrowheads show a microtubule translocating to the wound (W), until latrunclin
application (asterisk). See Movie 3.
(C) Double-labeled image from a movie of an oocyte injected with OG-Tax (green) and Alexa 568-phalloidin (red). See Movie 5.
(C) Microtubules move to the wound (arrow) in association with F-actin and buckle upon reaching the wound edge (arrowheads). See Movie 6.
(D) Dual-label images showing end-end association between F-actin (FA, red) and microtubules (MT, green); the overlap of the two is indicated
in two frames by arrowheads. F-actin is localized to the tip of microtubules moving to the wound. The horizontal arrow is 30 s; the vertical
arrow is 15 m. See Movie 7.
(E) Dual-label image from a movie showing microtubules (green) aligned with F-actin (red; alignment indicated by arrowheads).
Figure 3. Cotransport of F-Actin and Microtubules (A) Dual-label images showing examples of bent microtubules.
Arrows
indicate direction of movement. Microtubules (green) bend
away from the direction ofmovement where they are not associated
with F-actin (red). -
(B) Images from a movie showing F-actin (red) and microtubules
(green). F-actin appears to link the ends of several microtubules
and drag them through the cortex. See Movie 8.
(C) Dual-label and split-channel images showing buckling and
breakage of a microtubule that is associated with F-actin at both
leading and trailing edges. F-actin at the trailing edge accelerates
between the 00:45 and 01:00 time points, whereas the F-actin at
the leading edge does not (white lines). Microtubule buckling is
observed in precisely this time window. Later (at 01:30), the microtu-
bule breaks (arrow). See Movie 9.
(D) Quantification of specific microtubule-F-actin overlap (see Supplemental Data for details). The ratio of yellow pixels in unrotated:
rotated images is significantly higher in the region where microtu-
bule-F-actin cotransport occurs (10–30 m) than at the wound edge
(0–10 m) or in areas more than 30 m away from wound. Results
are mean SEM from four independent experiments; an asterisk
indicates 0.01.
(E) Double-labeled kymograph. F-actin (red) and microtubules
(green) show same patterns of acceleration and deceleration. W
indicates the wound; the horizontal arrow is 90 s; the vertical arrow
is 10 m.
Figure 5. Microtubule Perturbation Defocuses
the Actin Assembly Zone.
(A) Images from movies of oocytes injected
with OG-actin and wounded after no treatment
(control), nocodazole treatment (Nocodazole;
5 m; 1 hr), or taxol treatment (Taxol;
20 m; 1 hr). In controls, the zone of F-actin
polymerization remains tightly focused around
wounds; in nocodazole-treated oocytes it becomes
uneven and broadens; in taxol-treated
samples it spreads and then splits into two
rings (arrows). See Movies 13, 14, and 15.
(B) Confocal fluorescence images showing
distribution of F-actin (red) and microtubules
(green) in a taxol-treated oocyte subjected to
wounding. The microtubule and actin arrays
have split into concentric rings.
Figure 7. Schematic Diagram of Microtubule-Actomyosin Interac-tions during Oocyte Wound Healing
