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Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling.

Waterman-Storer CM, Salmon ED - J. Cell Biol. (1997)

Bottom Line: Occasionally "pioneering" MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow.Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that approximately 80% of the MTs in the lamella are not centrosome bound.We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, 607 Fordham Hall, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA. waterman@email.unc.edu

ABSTRACT
We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally "pioneering" MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated fluorescent marks on perpendicular MTs move rearward at the 0.4 mircon/min rate of retrograde flow in the lamella. MT rearward transport persists when MT dynamic instability is inhibited by 100-nM nocodazole but is blocked by inhibition of actomyosin by cytochalasin D or 2,3-butanedione-2-monoxime. Rearward flow appears to cause MT buckling and breaking in the lamella. 80% of free minus ends produced by breakage are stable; the others shorten and pause, leading to MT treadmilling. Free minus ends of unknown origin also depolymerize into the field of view at the lamella. Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that approximately 80% of the MTs in the lamella are not centrosome bound. We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.

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Rearward flow of assembly-inhibited MT plus ends in  the lamella. (A) Selected fluorescence micrographs from a series  of a cell that was injected with X-rhodamine–labeled tubulin and  then mounted in media containing 100 nM nocodazole to inhibit  MT plus end assembly dynamics. Elapsed time after addition of  nocodazole shown in the upper right of each panel. The positions  of three MT plus ends are highlighted. (B) Dynamic life history  plots of the distance of the plus ends of the three MTs marked in  A from the leading edge of the cell versus time (images acquired  at 7-s intervals). The y axis is inverted for clarity. The MT plus  ends do not exhibit typical plus end dynamic instability but instead move slowly away from the cell edge at ∼0.4 μm/min (B).  While moving rearward, the MTs maintain characteristic bending  patterns (A), indicating that the movement of the plus end is not  due to depolymerization. Bar, 10 μm.
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Figure 6: Rearward flow of assembly-inhibited MT plus ends in the lamella. (A) Selected fluorescence micrographs from a series of a cell that was injected with X-rhodamine–labeled tubulin and then mounted in media containing 100 nM nocodazole to inhibit MT plus end assembly dynamics. Elapsed time after addition of nocodazole shown in the upper right of each panel. The positions of three MT plus ends are highlighted. (B) Dynamic life history plots of the distance of the plus ends of the three MTs marked in A from the leading edge of the cell versus time (images acquired at 7-s intervals). The y axis is inverted for clarity. The MT plus ends do not exhibit typical plus end dynamic instability but instead move slowly away from the cell edge at ∼0.4 μm/min (B). While moving rearward, the MTs maintain characteristic bending patterns (A), indicating that the movement of the plus end is not due to depolymerization. Bar, 10 μm.

Mentions: Although the lattice of perpendicular MTs and parallel MTs move rearward in the lamella at similar rates, their movement could be driven by different mechanisms. One possibility is that perpendicular MTs are pushed rearward in the lamella by net plus end growth that exerts a pushing force against the leading edge of the cell or a barrier at the base of the lamellipodia. To test this hypothesis, we treated cells previously injected with X-rhodamine tubulin with 100 nM nocodazole to block MT plus end assembly/ disassembly dynamics (Vasquez et al., 1997). The positions and assembly dynamics of MT plus ends in the lamella were then monitored by acquiring images at 7-s intervals for 30 to 40 min (Fig. 6). Distance versus time plots of the movement of MT ends showed that the MTs did not undergo the growth and shortening characteristic of dynamic instability, but were in a continuous state of pause, as expected from Vasquez et al. (1997; Fig. 6 B). The assembly/ disassembly-inhibited plus ends moved slowly away from the leading edge at 0.39 ± 0.12 μm/min, similar to the rate of MT retrograde transport in untreated cells (Figs. 3 C and 4; Table II). It is possible that 100 nM nocodazole caused the MT plus ends to steadily depolymerize at this rate, giving the appearance of rearward flow. We rule this out because bends and curves in nocodazole-inhibited MTs moved rearward at the same rates as the plus ends (Fig. 6 A). After 40 min in 100-nM nocodazole, retrograde movement had cleared the 15–30-μm-wide lamella of all MTs, while regions near the nucleus still contained many sinuous MTs (not shown).


Actomyosin-based retrograde flow of microtubules in the lamella of migrating epithelial cells influences microtubule dynamic instability and turnover and is associated with microtubule breakage and treadmilling.

Waterman-Storer CM, Salmon ED - J. Cell Biol. (1997)

Rearward flow of assembly-inhibited MT plus ends in  the lamella. (A) Selected fluorescence micrographs from a series  of a cell that was injected with X-rhodamine–labeled tubulin and  then mounted in media containing 100 nM nocodazole to inhibit  MT plus end assembly dynamics. Elapsed time after addition of  nocodazole shown in the upper right of each panel. The positions  of three MT plus ends are highlighted. (B) Dynamic life history  plots of the distance of the plus ends of the three MTs marked in  A from the leading edge of the cell versus time (images acquired  at 7-s intervals). The y axis is inverted for clarity. The MT plus  ends do not exhibit typical plus end dynamic instability but instead move slowly away from the cell edge at ∼0.4 μm/min (B).  While moving rearward, the MTs maintain characteristic bending  patterns (A), indicating that the movement of the plus end is not  due to depolymerization. Bar, 10 μm.
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Figure 6: Rearward flow of assembly-inhibited MT plus ends in the lamella. (A) Selected fluorescence micrographs from a series of a cell that was injected with X-rhodamine–labeled tubulin and then mounted in media containing 100 nM nocodazole to inhibit MT plus end assembly dynamics. Elapsed time after addition of nocodazole shown in the upper right of each panel. The positions of three MT plus ends are highlighted. (B) Dynamic life history plots of the distance of the plus ends of the three MTs marked in A from the leading edge of the cell versus time (images acquired at 7-s intervals). The y axis is inverted for clarity. The MT plus ends do not exhibit typical plus end dynamic instability but instead move slowly away from the cell edge at ∼0.4 μm/min (B). While moving rearward, the MTs maintain characteristic bending patterns (A), indicating that the movement of the plus end is not due to depolymerization. Bar, 10 μm.
Mentions: Although the lattice of perpendicular MTs and parallel MTs move rearward in the lamella at similar rates, their movement could be driven by different mechanisms. One possibility is that perpendicular MTs are pushed rearward in the lamella by net plus end growth that exerts a pushing force against the leading edge of the cell or a barrier at the base of the lamellipodia. To test this hypothesis, we treated cells previously injected with X-rhodamine tubulin with 100 nM nocodazole to block MT plus end assembly/ disassembly dynamics (Vasquez et al., 1997). The positions and assembly dynamics of MT plus ends in the lamella were then monitored by acquiring images at 7-s intervals for 30 to 40 min (Fig. 6). Distance versus time plots of the movement of MT ends showed that the MTs did not undergo the growth and shortening characteristic of dynamic instability, but were in a continuous state of pause, as expected from Vasquez et al. (1997; Fig. 6 B). The assembly/ disassembly-inhibited plus ends moved slowly away from the leading edge at 0.39 ± 0.12 μm/min, similar to the rate of MT retrograde transport in untreated cells (Figs. 3 C and 4; Table II). It is possible that 100 nM nocodazole caused the MT plus ends to steadily depolymerize at this rate, giving the appearance of rearward flow. We rule this out because bends and curves in nocodazole-inhibited MTs moved rearward at the same rates as the plus ends (Fig. 6 A). After 40 min in 100-nM nocodazole, retrograde movement had cleared the 15–30-μm-wide lamella of all MTs, while regions near the nucleus still contained many sinuous MTs (not shown).

Bottom Line: Occasionally "pioneering" MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow.Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that approximately 80% of the MTs in the lamella are not centrosome bound.We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, 607 Fordham Hall, University of North Carolina, Chapel Hill, North Carolina 27599-3280, USA. waterman@email.unc.edu

ABSTRACT
We have discovered several novel features exhibited by microtubules (MTs) in migrating newt lung epithelial cells by time-lapse imaging of fluorescently labeled, microinjected tubulin. These cells exhibit leading edge ruffling and retrograde flow in the lamella and lamellipodia. The plus ends of lamella MTs persist in growth perpendicular to the leading edge until they reach the base of the lamellipodium, where they oscillate between short phases of growth and shortening. Occasionally "pioneering" MTs grow into the lamellipodium, where microtubule bending and reorientation parallel to the leading edge is associated with retrograde flow. MTs parallel to the leading edge exhibit significantly different dynamics from MTs perpendicular to the cell edge. Both parallel MTs and photoactivated fluorescent marks on perpendicular MTs move rearward at the 0.4 mircon/min rate of retrograde flow in the lamella. MT rearward transport persists when MT dynamic instability is inhibited by 100-nM nocodazole but is blocked by inhibition of actomyosin by cytochalasin D or 2,3-butanedione-2-monoxime. Rearward flow appears to cause MT buckling and breaking in the lamella. 80% of free minus ends produced by breakage are stable; the others shorten and pause, leading to MT treadmilling. Free minus ends of unknown origin also depolymerize into the field of view at the lamella. Analysis of MT dynamics at the centrosome shows that these minus ends do not arise by centrosomal ejection and that approximately 80% of the MTs in the lamella are not centrosome bound. We propose that actomyosin-based retrograde flow of MTs causes MT breakage, forming quasi-stable noncentrosomal MTs whose turnover is regulated primarily at their minus ends.

Show MeSH
Related in: MedlinePlus