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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 minus ends depolymerize into the field of view in  the lamella. Fluorescence images from a series acquired at 7-s intervals of a cell injected with X-rhodamine–labeled tubulin.  Elapsed time in min/sec is in the upper left of each panel. The  leading edge of the cell is visible in negative image near the top of  each panel. The positions of two different MT minus ends are  highlighted with white and black arrowheads, respectively, in  each panel. The minus end at the white arrow was present at the  start of the sequence. The MT rapidly depolymerizes from the  minus end (times 00:00–00:55) which then stabilizes and remains  so (times 00:55–02:32) until the MT is consumed by plus end depolymerization (time 03:28). The minus end at the white arrow  enters the field of view by rapid depolymerization (times 00:14– 00:55), which it continues until the MT is nearly consumed (times  00:55–03:28). Note that the plus end of this MT grows (times  02:12–03:28); thus a MT piece appears to move in the lamella as  the minus end continues to depolymerize during the same time  period. Bar, 10 μm.
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Figure 9: MT minus ends depolymerize into the field of view in the lamella. Fluorescence images from a series acquired at 7-s intervals of a cell injected with X-rhodamine–labeled tubulin. Elapsed time in min/sec is in the upper left of each panel. The leading edge of the cell is visible in negative image near the top of each panel. The positions of two different MT minus ends are highlighted with white and black arrowheads, respectively, in each panel. The minus end at the white arrow was present at the start of the sequence. The MT rapidly depolymerizes from the minus end (times 00:00–00:55) which then stabilizes and remains so (times 00:55–02:32) until the MT is consumed by plus end depolymerization (time 03:28). The minus end at the white arrow enters the field of view by rapid depolymerization (times 00:14– 00:55), which it continues until the MT is nearly consumed (times 00:55–03:28). Note that the plus end of this MT grows (times 02:12–03:28); thus a MT piece appears to move in the lamella as the minus end continues to depolymerize during the same time period. Bar, 10 μm.

Mentions: MT breakage created a new free plus end on the MT that broke and a portion of MT with a new free minus end and an old plus end. Just after breakage, 100% of the newly formed plus ends analyzed shortened at least slightly before beginning to exhibit growth and shortening behavior typical of the bulk population of plus ends (Fig. 8 and Table III). In contrast, 82% of the free minus ends formed by breakage neither grew nor shortened, but remained stable (Fig. 8 B). The other 18% of newly formed minus ends shortened immediately after breaking at an average rate of 6.43 ± 5.85 μm/min (Fig. 8 A, and Tables I and III) similar to the shortening rate of plus ends. Free minus ends never grew. For the most part, stabilized minus ends formed by breaking remained stable until the end of the recorded sequence of images or until rearward flow swept them into regions crowded with MTs and they were no longer visible. In a few cases minus ends that were stable after breakage began to rapidly shorten a few minutes later. If a free MT minus end produced by breakage began to shorten, it never restabilized but continued to shorten with intermittent pauses. Minus end shortening continued until the MT was fully consumed by minus end shortening catching up with the dynamic plus end (Fig. 9). Thus, minus ends produced by MT breaking were either stabilized or dynamic, with their dynamics consisting of only two states: shortening, which occupied 77.8% of their time, and pause, which occupied 22.2% (Table I, total analysis time = 167.3 min on 32 MTs in 15 cells).


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 minus ends depolymerize into the field of view in  the lamella. Fluorescence images from a series acquired at 7-s intervals of a cell injected with X-rhodamine–labeled tubulin.  Elapsed time in min/sec is in the upper left of each panel. The  leading edge of the cell is visible in negative image near the top of  each panel. The positions of two different MT minus ends are  highlighted with white and black arrowheads, respectively, in  each panel. The minus end at the white arrow was present at the  start of the sequence. The MT rapidly depolymerizes from the  minus end (times 00:00–00:55) which then stabilizes and remains  so (times 00:55–02:32) until the MT is consumed by plus end depolymerization (time 03:28). The minus end at the white arrow  enters the field of view by rapid depolymerization (times 00:14– 00:55), which it continues until the MT is nearly consumed (times  00:55–03:28). Note that the plus end of this MT grows (times  02:12–03:28); thus a MT piece appears to move in the lamella as  the minus end continues to depolymerize during the same time  period. Bar, 10 μm.
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Figure 9: MT minus ends depolymerize into the field of view in the lamella. Fluorescence images from a series acquired at 7-s intervals of a cell injected with X-rhodamine–labeled tubulin. Elapsed time in min/sec is in the upper left of each panel. The leading edge of the cell is visible in negative image near the top of each panel. The positions of two different MT minus ends are highlighted with white and black arrowheads, respectively, in each panel. The minus end at the white arrow was present at the start of the sequence. The MT rapidly depolymerizes from the minus end (times 00:00–00:55) which then stabilizes and remains so (times 00:55–02:32) until the MT is consumed by plus end depolymerization (time 03:28). The minus end at the white arrow enters the field of view by rapid depolymerization (times 00:14– 00:55), which it continues until the MT is nearly consumed (times 00:55–03:28). Note that the plus end of this MT grows (times 02:12–03:28); thus a MT piece appears to move in the lamella as the minus end continues to depolymerize during the same time period. Bar, 10 μm.
Mentions: MT breakage created a new free plus end on the MT that broke and a portion of MT with a new free minus end and an old plus end. Just after breakage, 100% of the newly formed plus ends analyzed shortened at least slightly before beginning to exhibit growth and shortening behavior typical of the bulk population of plus ends (Fig. 8 and Table III). In contrast, 82% of the free minus ends formed by breakage neither grew nor shortened, but remained stable (Fig. 8 B). The other 18% of newly formed minus ends shortened immediately after breaking at an average rate of 6.43 ± 5.85 μm/min (Fig. 8 A, and Tables I and III) similar to the shortening rate of plus ends. Free minus ends never grew. For the most part, stabilized minus ends formed by breaking remained stable until the end of the recorded sequence of images or until rearward flow swept them into regions crowded with MTs and they were no longer visible. In a few cases minus ends that were stable after breakage began to rapidly shorten a few minutes later. If a free MT minus end produced by breakage began to shorten, it never restabilized but continued to shorten with intermittent pauses. Minus end shortening continued until the MT was fully consumed by minus end shortening catching up with the dynamic plus end (Fig. 9). Thus, minus ends produced by MT breaking were either stabilized or dynamic, with their dynamics consisting of only two states: shortening, which occupied 77.8% of their time, and pause, which occupied 22.2% (Table I, total analysis time = 167.3 min on 32 MTs in 15 cells).

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