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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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Dynamics of MTs in the lamella and lamellipodia during protrusion of the leading edge. (A) A series of fluorescence  micrographs of the leading edge of a cell that had been injected  with X-rhodamine–labeled tubulin, elapsed time in min/sec in the  upper right of each panel. The boundary of the cell can be seen in  negative image because labeled tubulin subunits are diffusely fluorescent within the cell but not outside of the cell. Before protrusion of the cell edge (times 00:00–00:39) most MTs extend only as  far as the one marked with an arrowhead, however some MTs,  such as the one marked P, extend closer to the leading edge. As  the leading edge advances (times 00:39–04:49), the plus end of the  P MT approximately maintains its distance from the leading  edge, while the MT at the arrowhead does not extend into the  new protrusion until advancement of the leading edge has ceased  (times 4:49–5:29). (B) Dynamic life history plots of the plus ends  of the MTs marked in A in relation to the position of the cell  edge. Distance from the origin (a point at the bottom edge of the  micrograph at time 00:00) was plotted against time for images  captured at 7-s intervals. The position of the edge was determined from fluorescence intensity linescans perpendicular to and  across the leading edge. The “pioneer” MT steadily grows at  about 2 μm/min during advancement of the leading edge (∼0–5  min). The proximal perpendicular MT undergoes very little net  growth until advancement of the leading edge ceases (∼5 min),  whereupon the MT rapidly grows at ∼8 μm/min. Bar, 10 μm.
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Figure 2: Dynamics of MTs in the lamella and lamellipodia during protrusion of the leading edge. (A) A series of fluorescence micrographs of the leading edge of a cell that had been injected with X-rhodamine–labeled tubulin, elapsed time in min/sec in the upper right of each panel. The boundary of the cell can be seen in negative image because labeled tubulin subunits are diffusely fluorescent within the cell but not outside of the cell. Before protrusion of the cell edge (times 00:00–00:39) most MTs extend only as far as the one marked with an arrowhead, however some MTs, such as the one marked P, extend closer to the leading edge. As the leading edge advances (times 00:39–04:49), the plus end of the P MT approximately maintains its distance from the leading edge, while the MT at the arrowhead does not extend into the new protrusion until advancement of the leading edge has ceased (times 4:49–5:29). (B) Dynamic life history plots of the plus ends of the MTs marked in A in relation to the position of the cell edge. Distance from the origin (a point at the bottom edge of the micrograph at time 00:00) was plotted against time for images captured at 7-s intervals. The position of the edge was determined from fluorescence intensity linescans perpendicular to and across the leading edge. The “pioneer” MT steadily grows at about 2 μm/min during advancement of the leading edge (∼0–5 min). The proximal perpendicular MT undergoes very little net growth until advancement of the leading edge ceases (∼5 min), whereupon the MT rapidly grows at ∼8 μm/min. Bar, 10 μm.

Mentions: To examine MT dynamics in the lamella and lamellipodia, we microinjected peripheral cells of the epithelial sheet with X-rhodamine–conjugated tubulin and allowed incorporation of fluorescent subunits into MTs for 1 to 2 h. At the end of this time, the MT cytoskeleton was fully labeled with fluorescent subunits, as determined by fixation of injected cells and processing for immunolocalization of tubulin (not shown). We then obtained a time-lapse series of fluorescence images of the lamella at 7-s intervals. The plus ends of most perpendicular MTs in the lamella extended to the base of the lamellipodia, ∼5–10 μm from the leading edge. There they exhibited dynamic instability, asynchronously and frequently alternating between very short phases of growth and shortening (∼1–3 μm) and spending ∼40% of their time in “pause,” neither growing nor shortening (Table I). Thus, the bulk of the perpendicular MTs exhibited little net change in length over time, and their ends maintained a relatively constant position at the base of the lamellipodia (Fig. 2, MT at arrowhead, time 00:00–4:49).


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)

Dynamics of MTs in the lamella and lamellipodia during protrusion of the leading edge. (A) A series of fluorescence  micrographs of the leading edge of a cell that had been injected  with X-rhodamine–labeled tubulin, elapsed time in min/sec in the  upper right of each panel. The boundary of the cell can be seen in  negative image because labeled tubulin subunits are diffusely fluorescent within the cell but not outside of the cell. Before protrusion of the cell edge (times 00:00–00:39) most MTs extend only as  far as the one marked with an arrowhead, however some MTs,  such as the one marked P, extend closer to the leading edge. As  the leading edge advances (times 00:39–04:49), the plus end of the  P MT approximately maintains its distance from the leading  edge, while the MT at the arrowhead does not extend into the  new protrusion until advancement of the leading edge has ceased  (times 4:49–5:29). (B) Dynamic life history plots of the plus ends  of the MTs marked in A in relation to the position of the cell  edge. Distance from the origin (a point at the bottom edge of the  micrograph at time 00:00) was plotted against time for images  captured at 7-s intervals. The position of the edge was determined from fluorescence intensity linescans perpendicular to and  across the leading edge. The “pioneer” MT steadily grows at  about 2 μm/min during advancement of the leading edge (∼0–5  min). The proximal perpendicular MT undergoes very little net  growth until advancement of the leading edge ceases (∼5 min),  whereupon the MT rapidly grows at ∼8 μm/min. Bar, 10 μm.
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Figure 2: Dynamics of MTs in the lamella and lamellipodia during protrusion of the leading edge. (A) A series of fluorescence micrographs of the leading edge of a cell that had been injected with X-rhodamine–labeled tubulin, elapsed time in min/sec in the upper right of each panel. The boundary of the cell can be seen in negative image because labeled tubulin subunits are diffusely fluorescent within the cell but not outside of the cell. Before protrusion of the cell edge (times 00:00–00:39) most MTs extend only as far as the one marked with an arrowhead, however some MTs, such as the one marked P, extend closer to the leading edge. As the leading edge advances (times 00:39–04:49), the plus end of the P MT approximately maintains its distance from the leading edge, while the MT at the arrowhead does not extend into the new protrusion until advancement of the leading edge has ceased (times 4:49–5:29). (B) Dynamic life history plots of the plus ends of the MTs marked in A in relation to the position of the cell edge. Distance from the origin (a point at the bottom edge of the micrograph at time 00:00) was plotted against time for images captured at 7-s intervals. The position of the edge was determined from fluorescence intensity linescans perpendicular to and across the leading edge. The “pioneer” MT steadily grows at about 2 μm/min during advancement of the leading edge (∼0–5 min). The proximal perpendicular MT undergoes very little net growth until advancement of the leading edge ceases (∼5 min), whereupon the MT rapidly grows at ∼8 μm/min. Bar, 10 μm.
Mentions: To examine MT dynamics in the lamella and lamellipodia, we microinjected peripheral cells of the epithelial sheet with X-rhodamine–conjugated tubulin and allowed incorporation of fluorescent subunits into MTs for 1 to 2 h. At the end of this time, the MT cytoskeleton was fully labeled with fluorescent subunits, as determined by fixation of injected cells and processing for immunolocalization of tubulin (not shown). We then obtained a time-lapse series of fluorescence images of the lamella at 7-s intervals. The plus ends of most perpendicular MTs in the lamella extended to the base of the lamellipodia, ∼5–10 μm from the leading edge. There they exhibited dynamic instability, asynchronously and frequently alternating between very short phases of growth and shortening (∼1–3 μm) and spending ∼40% of their time in “pause,” neither growing nor shortening (Table I). Thus, the bulk of the perpendicular MTs exhibited little net change in length over time, and their ends maintained a relatively constant position at the base of the lamellipodia (Fig. 2, MT at arrowhead, time 00:00–4:49).

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