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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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Bending, reorientation, and retrograde flow of a MT in  the lamellipodia. (A) A series of micrographs in which the fluorescence image of X-rhodamine–labeled MTs (pseudocolored  red) has been digitally superimposed onto the DIC image (in  grayscale) of the lamella. Pairs of fluorescence and DIC images  were captured within 1.5 s of each other at 9-s intervals; elapsed  time in min/sec is in the lower right of each panel. The base of the  lamellipodia can be seen as a slightly diffuse staining of X-rhodamine–labeled subunits ∼5 μm from the leading edge. Because  the cell is slightly thicker in this region (not shown), the increased  volume produces a higher amount of fluorescent label. (B) Dynamic life history plot of the distance of the MT end at the arrowhead in A from the origin (the position of the plus end at time 00:00)  versus time. (C) Plot of the distance of a point on the MT (square  in A) from the leading edge (directly in front of the point) versus  time. The y axis is inverted for clarity. Initially, the MT plus end  (arrowhead) perpendicular to the leading edge exhibits little net  growth (section 1 of graph B). The MT then grows from within  the lamella into the lamellipodia and touches the plasma membrane (time 00:00–01:56 in A; section 2 of graph B) and then undergoes dynamic instability as it “probes” the leading edge (times  01:56–03:52 in A; section 3 of graph B) and then bends within the  lamellipodia (times 03:52–05:32 in A; section 4 of graph B), reestablishing its axis of growth parallel to the leading edge. The plus  end then undergoes rapid net growth (times 05:32–07:28 in A; section 5 of graph B). The parallel portion of the MT (black square)  then moves rearward away from the leading edge (times 6:17–8:58  in A; graph C). Bar, 10 μm.
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Figure 3: Bending, reorientation, and retrograde flow of a MT in the lamellipodia. (A) A series of micrographs in which the fluorescence image of X-rhodamine–labeled MTs (pseudocolored red) has been digitally superimposed onto the DIC image (in grayscale) of the lamella. Pairs of fluorescence and DIC images were captured within 1.5 s of each other at 9-s intervals; elapsed time in min/sec is in the lower right of each panel. The base of the lamellipodia can be seen as a slightly diffuse staining of X-rhodamine–labeled subunits ∼5 μm from the leading edge. Because the cell is slightly thicker in this region (not shown), the increased volume produces a higher amount of fluorescent label. (B) Dynamic life history plot of the distance of the MT end at the arrowhead in A from the origin (the position of the plus end at time 00:00) versus time. (C) Plot of the distance of a point on the MT (square in A) from the leading edge (directly in front of the point) versus time. The y axis is inverted for clarity. Initially, the MT plus end (arrowhead) perpendicular to the leading edge exhibits little net growth (section 1 of graph B). The MT then grows from within the lamella into the lamellipodia and touches the plasma membrane (time 00:00–01:56 in A; section 2 of graph B) and then undergoes dynamic instability as it “probes” the leading edge (times 01:56–03:52 in A; section 3 of graph B) and then bends within the lamellipodia (times 03:52–05:32 in A; section 4 of graph B), reestablishing its axis of growth parallel to the leading edge. The plus end then undergoes rapid net growth (times 05:32–07:28 in A; section 5 of graph B). The parallel portion of the MT (black square) then moves rearward away from the leading edge (times 6:17–8:58 in A; graph C). Bar, 10 μm.

Mentions: When pairs of fluorescence images of MTs and high resolution DIC images of the lamella and lamellipodia were acquired at 9-s intervals, we were able to clearly see the relation between MT growth patterns and the retrograde flow near the cell edge (Fig. 3 A). Often as a pioneer MT grew into the lamellipodia, it appeared to “probe” the plasma membrane as its plus end grew and shortened (Fig. 3 A, time 0:00–1:56, and b, section 3 of graph). If the leading edge did not protrude, the tip of pioneer MTs became bent near the leading edge in the rapid rearward flow in the lamellipodia (Fig. 3 A, times 1:56–4:56, and B, section 4 of graph). This established the axis of growth fully parallel to the cell's edge (Fig. 3 A, times 5:32–7:28, and B, section 5 of graph), similar to observations on the formation of “parallel” MTs in migrating NRK cells (Mikhailov and Gundersen, 1995).


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)

Bending, reorientation, and retrograde flow of a MT in  the lamellipodia. (A) A series of micrographs in which the fluorescence image of X-rhodamine–labeled MTs (pseudocolored  red) has been digitally superimposed onto the DIC image (in  grayscale) of the lamella. Pairs of fluorescence and DIC images  were captured within 1.5 s of each other at 9-s intervals; elapsed  time in min/sec is in the lower right of each panel. The base of the  lamellipodia can be seen as a slightly diffuse staining of X-rhodamine–labeled subunits ∼5 μm from the leading edge. Because  the cell is slightly thicker in this region (not shown), the increased  volume produces a higher amount of fluorescent label. (B) Dynamic life history plot of the distance of the MT end at the arrowhead in A from the origin (the position of the plus end at time 00:00)  versus time. (C) Plot of the distance of a point on the MT (square  in A) from the leading edge (directly in front of the point) versus  time. The y axis is inverted for clarity. Initially, the MT plus end  (arrowhead) perpendicular to the leading edge exhibits little net  growth (section 1 of graph B). The MT then grows from within  the lamella into the lamellipodia and touches the plasma membrane (time 00:00–01:56 in A; section 2 of graph B) and then undergoes dynamic instability as it “probes” the leading edge (times  01:56–03:52 in A; section 3 of graph B) and then bends within the  lamellipodia (times 03:52–05:32 in A; section 4 of graph B), reestablishing its axis of growth parallel to the leading edge. The plus  end then undergoes rapid net growth (times 05:32–07:28 in A; section 5 of graph B). The parallel portion of the MT (black square)  then moves rearward away from the leading edge (times 6:17–8:58  in A; graph C). Bar, 10 μm.
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Related In: Results  -  Collection

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Figure 3: Bending, reorientation, and retrograde flow of a MT in the lamellipodia. (A) A series of micrographs in which the fluorescence image of X-rhodamine–labeled MTs (pseudocolored red) has been digitally superimposed onto the DIC image (in grayscale) of the lamella. Pairs of fluorescence and DIC images were captured within 1.5 s of each other at 9-s intervals; elapsed time in min/sec is in the lower right of each panel. The base of the lamellipodia can be seen as a slightly diffuse staining of X-rhodamine–labeled subunits ∼5 μm from the leading edge. Because the cell is slightly thicker in this region (not shown), the increased volume produces a higher amount of fluorescent label. (B) Dynamic life history plot of the distance of the MT end at the arrowhead in A from the origin (the position of the plus end at time 00:00) versus time. (C) Plot of the distance of a point on the MT (square in A) from the leading edge (directly in front of the point) versus time. The y axis is inverted for clarity. Initially, the MT plus end (arrowhead) perpendicular to the leading edge exhibits little net growth (section 1 of graph B). The MT then grows from within the lamella into the lamellipodia and touches the plasma membrane (time 00:00–01:56 in A; section 2 of graph B) and then undergoes dynamic instability as it “probes” the leading edge (times 01:56–03:52 in A; section 3 of graph B) and then bends within the lamellipodia (times 03:52–05:32 in A; section 4 of graph B), reestablishing its axis of growth parallel to the leading edge. The plus end then undergoes rapid net growth (times 05:32–07:28 in A; section 5 of graph B). The parallel portion of the MT (black square) then moves rearward away from the leading edge (times 6:17–8:58 in A; graph C). Bar, 10 μm.
Mentions: When pairs of fluorescence images of MTs and high resolution DIC images of the lamella and lamellipodia were acquired at 9-s intervals, we were able to clearly see the relation between MT growth patterns and the retrograde flow near the cell edge (Fig. 3 A). Often as a pioneer MT grew into the lamellipodia, it appeared to “probe” the plasma membrane as its plus end grew and shortened (Fig. 3 A, time 0:00–1:56, and b, section 3 of graph). If the leading edge did not protrude, the tip of pioneer MTs became bent near the leading edge in the rapid rearward flow in the lamellipodia (Fig. 3 A, times 1:56–4:56, and B, section 4 of graph). This established the axis of growth fully parallel to the cell's edge (Fig. 3 A, times 5:32–7:28, and B, section 5 of graph), similar to observations on the formation of “parallel” MTs in migrating NRK cells (Mikhailov and Gundersen, 1995).

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