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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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MT breakage induced by local MT  buckling in the lamella. Selected images from series of fluorescence micrographs of cells injected  with X-rhodamine–labeled tubulin. Time in min/ sec is shown in the upper left of each panel. (A)  A MT with local buckling broke at 11 s. The  newly formed minus end (large arrowhead)  shortened immediately after breakage, while the  new plus end (small arrowhead) formed by the  break shortened slightly before beginning to undergo dynamic instability (times 00:19–1:57). (B)  A MT with local buckles broke (time = 00:10),  forming a new minus end (large arrowhead) that  remained stable and a new plus end that shortened before undergoing dynamic instability  (times 00:20–01:59). Bar, 10 μm.
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Figure 8: MT breakage induced by local MT buckling in the lamella. Selected images from series of fluorescence micrographs of cells injected with X-rhodamine–labeled tubulin. Time in min/ sec is shown in the upper left of each panel. (A) A MT with local buckling broke at 11 s. The newly formed minus end (large arrowhead) shortened immediately after breakage, while the new plus end (small arrowhead) formed by the break shortened slightly before beginning to undergo dynamic instability (times 00:19–1:57). (B) A MT with local buckles broke (time = 00:10), forming a new minus end (large arrowhead) that remained stable and a new plus end that shortened before undergoing dynamic instability (times 00:20–01:59). Bar, 10 μm.

Mentions: Local bending and buckling of otherwise straight or slightly curved MTs often was observed in the lamella as a result of continuous rearward transport of perpendicular MTs in the lamella. During retrograde flow of MTs, local buckling led to very tight bends and curves in the MT, which often broke (Fig. 8) at an average radius of curvature of 0.6 ± 0.15 μm (n = 7). We have never witnessed straight MTs breaking, indicating that breakage is only associated with local buckling, and not directly caused by photodamage (Vigers et al., 1988).


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)

MT breakage induced by local MT  buckling in the lamella. Selected images from series of fluorescence micrographs of cells injected  with X-rhodamine–labeled tubulin. Time in min/ sec is shown in the upper left of each panel. (A)  A MT with local buckling broke at 11 s. The  newly formed minus end (large arrowhead)  shortened immediately after breakage, while the  new plus end (small arrowhead) formed by the  break shortened slightly before beginning to undergo dynamic instability (times 00:19–1:57). (B)  A MT with local buckles broke (time = 00:10),  forming a new minus end (large arrowhead) that  remained stable and a new plus end that shortened before undergoing dynamic instability  (times 00:20–01:59). Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139796&req=5

Figure 8: MT breakage induced by local MT buckling in the lamella. Selected images from series of fluorescence micrographs of cells injected with X-rhodamine–labeled tubulin. Time in min/ sec is shown in the upper left of each panel. (A) A MT with local buckling broke at 11 s. The newly formed minus end (large arrowhead) shortened immediately after breakage, while the new plus end (small arrowhead) formed by the break shortened slightly before beginning to undergo dynamic instability (times 00:19–1:57). (B) A MT with local buckles broke (time = 00:10), forming a new minus end (large arrowhead) that remained stable and a new plus end that shortened before undergoing dynamic instability (times 00:20–01:59). Bar, 10 μm.
Mentions: Local bending and buckling of otherwise straight or slightly curved MTs often was observed in the lamella as a result of continuous rearward transport of perpendicular MTs in the lamella. During retrograde flow of MTs, local buckling led to very tight bends and curves in the MT, which often broke (Fig. 8) at an average radius of curvature of 0.6 ± 0.15 μm (n = 7). We have never witnessed straight MTs breaking, indicating that breakage is only associated with local buckling, and not directly caused by photodamage (Vigers et al., 1988).

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