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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 movement of the lattice of perpendicular MTs in the lamella.  (A) Digitally overlaid and  pseudocolored micrographs  of a cell that was injected  with a mixture of X-rhodamine (red)- and caged fluorescein (yellow–green)-labeled  tubulins (captured at 3-min  intervals within 1.5 s of each  other). The cell was exposed  to a 2.5-μm-wide bar of UV  light to activate the fluorescein label just before the first  image. Elapsed time (in min/ sec) is in the upper left of  each panel. (B) Plots of relative fluorescence intensity  (after background subtraction) versus position along the white line in A. Fluorescence loss due to  photobleaching of C2CF during the total exposure time is <5% under similar conditions (not shown). The green lines represent intensity of uncaged  fluorescein, and the red lines represent intensity of X-rhodamine. Time at  which the scan was taken is denoted by the thickness of the plotted line.  The position of the fluorescein-labeled subunits in the primarily perpendicular MTs moves rearward through the lamella over time (A, white arrowheads in B over the green scanlines mark the peak fluorescein intensity),  while the level of the X-rhodamine–labeled MT polymer remains relatively  constant across the lamella over time (red scan lines in B). Loss in intensity  of the fluorescein signal over time is due to depolymerization of MTs  through the marked region and photobleaching. Note that the fluorescein-labeled subunits move rearward as a relatively coherent bar (A), and the  width of the bar increases very little over time (B). Bar, 10 μm.
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Figure 5: Rearward movement of the lattice of perpendicular MTs in the lamella. (A) Digitally overlaid and pseudocolored micrographs of a cell that was injected with a mixture of X-rhodamine (red)- and caged fluorescein (yellow–green)-labeled tubulins (captured at 3-min intervals within 1.5 s of each other). The cell was exposed to a 2.5-μm-wide bar of UV light to activate the fluorescein label just before the first image. Elapsed time (in min/ sec) is in the upper left of each panel. (B) Plots of relative fluorescence intensity (after background subtraction) versus position along the white line in A. Fluorescence loss due to photobleaching of C2CF during the total exposure time is <5% under similar conditions (not shown). The green lines represent intensity of uncaged fluorescein, and the red lines represent intensity of X-rhodamine. Time at which the scan was taken is denoted by the thickness of the plotted line. The position of the fluorescein-labeled subunits in the primarily perpendicular MTs moves rearward through the lamella over time (A, white arrowheads in B over the green scanlines mark the peak fluorescein intensity), while the level of the X-rhodamine–labeled MT polymer remains relatively constant across the lamella over time (red scan lines in B). Loss in intensity of the fluorescein signal over time is due to depolymerization of MTs through the marked region and photobleaching. Note that the fluorescein-labeled subunits move rearward as a relatively coherent bar (A), and the width of the bar increases very little over time (B). Bar, 10 μm.

Mentions: As a MT with a bend moved rearward in the lamella or lamellipodia, the vertex of the bend moved rearward at the same rate (Fig. 3, times 6:17–7:28), suggesting that the portion of the MT that was perpendicular to the cell edge and proximal to the bend was also moving rearward. This observation suggested that all MTs in the lamella, both parallel and perpendicular in orientation, are continuously transported rearward, in spite of the appearance of a relatively constant distance between the bulk of the plus ends and the leading edge of the cell. To test this hypothesis directly, we used photoactivation methods (Mitchison, 1989) to mark subunits in the lattice of perpendicularly oriented MTs in the lamella and monitor the position of the marked region relative to the cell edge. Cells were microinjected with a mixture of X-rhodamine tubulin and caged-fluorescein tubulin (1:10 X-rhodamine tubulin:C2CF tubulin, 5 mg/ml total), and a narrow (∼2.5 μm wide) bar of fluorescence was photoactivated by exposure to 3 s of unattenuated UV (360 nm) light, parallel to and ∼8–12 μm from the leading edge of the cell. This exposure to UV had no apparent effect on the ruffling activity or advancement of the cell edge. By taking pairs of rhodamine and fluorescein images at 3-min intervals after photoactivation, we found that fluorescein marks on MTs moved rearward from the leading edge at 0.30 ± 0.11 μm/min (n = 8; Fig. 5, A and B), somewhat slower on average but not significantly different (P > 0.5) than the rates of rearward movement of either parallel MTs, DIC refractile ridges, or surface-coupled beads (Table II). Although the photoactivated fluorescein marks in the lamella moved rearward, the positions of the MT plus ends remained nearly constant relative to the cell's edge, and the length of the of the X-rhodamine–labeled portion of the MTs between the mark and the cell edge increased (Fig. 5, A and B), indicating that the bulk of plus ends maintain a net growth rate of 0.3 μm/min.


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 movement of the lattice of perpendicular MTs in the lamella.  (A) Digitally overlaid and  pseudocolored micrographs  of a cell that was injected  with a mixture of X-rhodamine (red)- and caged fluorescein (yellow–green)-labeled  tubulins (captured at 3-min  intervals within 1.5 s of each  other). The cell was exposed  to a 2.5-μm-wide bar of UV  light to activate the fluorescein label just before the first  image. Elapsed time (in min/ sec) is in the upper left of  each panel. (B) Plots of relative fluorescence intensity  (after background subtraction) versus position along the white line in A. Fluorescence loss due to  photobleaching of C2CF during the total exposure time is <5% under similar conditions (not shown). The green lines represent intensity of uncaged  fluorescein, and the red lines represent intensity of X-rhodamine. Time at  which the scan was taken is denoted by the thickness of the plotted line.  The position of the fluorescein-labeled subunits in the primarily perpendicular MTs moves rearward through the lamella over time (A, white arrowheads in B over the green scanlines mark the peak fluorescein intensity),  while the level of the X-rhodamine–labeled MT polymer remains relatively  constant across the lamella over time (red scan lines in B). Loss in intensity  of the fluorescein signal over time is due to depolymerization of MTs  through the marked region and photobleaching. Note that the fluorescein-labeled subunits move rearward as a relatively coherent bar (A), and the  width of the bar increases very little over time (B). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 5: Rearward movement of the lattice of perpendicular MTs in the lamella. (A) Digitally overlaid and pseudocolored micrographs of a cell that was injected with a mixture of X-rhodamine (red)- and caged fluorescein (yellow–green)-labeled tubulins (captured at 3-min intervals within 1.5 s of each other). The cell was exposed to a 2.5-μm-wide bar of UV light to activate the fluorescein label just before the first image. Elapsed time (in min/ sec) is in the upper left of each panel. (B) Plots of relative fluorescence intensity (after background subtraction) versus position along the white line in A. Fluorescence loss due to photobleaching of C2CF during the total exposure time is <5% under similar conditions (not shown). The green lines represent intensity of uncaged fluorescein, and the red lines represent intensity of X-rhodamine. Time at which the scan was taken is denoted by the thickness of the plotted line. The position of the fluorescein-labeled subunits in the primarily perpendicular MTs moves rearward through the lamella over time (A, white arrowheads in B over the green scanlines mark the peak fluorescein intensity), while the level of the X-rhodamine–labeled MT polymer remains relatively constant across the lamella over time (red scan lines in B). Loss in intensity of the fluorescein signal over time is due to depolymerization of MTs through the marked region and photobleaching. Note that the fluorescein-labeled subunits move rearward as a relatively coherent bar (A), and the width of the bar increases very little over time (B). Bar, 10 μm.
Mentions: As a MT with a bend moved rearward in the lamella or lamellipodia, the vertex of the bend moved rearward at the same rate (Fig. 3, times 6:17–7:28), suggesting that the portion of the MT that was perpendicular to the cell edge and proximal to the bend was also moving rearward. This observation suggested that all MTs in the lamella, both parallel and perpendicular in orientation, are continuously transported rearward, in spite of the appearance of a relatively constant distance between the bulk of the plus ends and the leading edge of the cell. To test this hypothesis directly, we used photoactivation methods (Mitchison, 1989) to mark subunits in the lattice of perpendicularly oriented MTs in the lamella and monitor the position of the marked region relative to the cell edge. Cells were microinjected with a mixture of X-rhodamine tubulin and caged-fluorescein tubulin (1:10 X-rhodamine tubulin:C2CF tubulin, 5 mg/ml total), and a narrow (∼2.5 μm wide) bar of fluorescence was photoactivated by exposure to 3 s of unattenuated UV (360 nm) light, parallel to and ∼8–12 μm from the leading edge of the cell. This exposure to UV had no apparent effect on the ruffling activity or advancement of the cell edge. By taking pairs of rhodamine and fluorescein images at 3-min intervals after photoactivation, we found that fluorescein marks on MTs moved rearward from the leading edge at 0.30 ± 0.11 μm/min (n = 8; Fig. 5, A and B), somewhat slower on average but not significantly different (P > 0.5) than the rates of rearward movement of either parallel MTs, DIC refractile ridges, or surface-coupled beads (Table II). Although the photoactivated fluorescein marks in the lamella moved rearward, the positions of the MT plus ends remained nearly constant relative to the cell's edge, and the length of the of the X-rhodamine–labeled portion of the MTs between the mark and the cell edge increased (Fig. 5, A and B), indicating that the bulk of plus ends maintain a net growth rate of 0.3 μm/min.

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