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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 at the centrosome. Fluorescence images from a series (taken at 7-s intervals) of a cell injected with X-rhodamine tubulin. Elapsed time in min/sec is in the upper right of each panel. The centrosome in this cell was positioned at the edge of the  nucleus, so that half of the MTs emanating from the centrosome were visible beneath the nucleus. A MT was nucleated from the centrosome (small arrowhead) and grew radially out of the field of view (times 00:29–00:59). Another MT depolymerized from outside the  field of view (large arrowhead) and was consumed by complete depolymerization all the way back to the centrosome (times 00:59–01: 59). Bar, 10 μm.
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Figure 11: Dynamics of MTs at the centrosome. Fluorescence images from a series (taken at 7-s intervals) of a cell injected with X-rhodamine tubulin. Elapsed time in min/sec is in the upper right of each panel. The centrosome in this cell was positioned at the edge of the nucleus, so that half of the MTs emanating from the centrosome were visible beneath the nucleus. A MT was nucleated from the centrosome (small arrowhead) and grew radially out of the field of view (times 00:29–00:59). Another MT depolymerized from outside the field of view (large arrowhead) and was consumed by complete depolymerization all the way back to the centrosome (times 00:59–01: 59). Bar, 10 μm.

Mentions: To image MT dynamics at the centrosome, peripheral cells in which the centrosome was positioned beneath the nucleus were chosen because the overlying nucleus provided both a clear image as well as a flattened array of MTs (Fig. 11). Images were acquired at 7-s intervals and time-lapse series examined for evidence of MT ejection from the centrosome. In 121.4 min of analysis time in six cells, only three MTs were ejected from the centrosome, a frequency of 0.02 ± 0.04/cell/min (Table IV). In spite of this low ejection frequency, free minus ends depolymerized into the ∼30 × 30 μm field of view at the lamella from the direction of the cell body at a frequency of 0.3 ± 0.2/min (Table IV). Thus, for a typical cell with ∼90 × 30 μm lamella, about one minus end depolymerizes into the lamella per minute. Since only 0.02 minus end is released from the centrosome per minute, the centrosome is not responsible for generating most of the free minus ends seen in the lamella.


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 at the centrosome. Fluorescence images from a series (taken at 7-s intervals) of a cell injected with X-rhodamine tubulin. Elapsed time in min/sec is in the upper right of each panel. The centrosome in this cell was positioned at the edge of the  nucleus, so that half of the MTs emanating from the centrosome were visible beneath the nucleus. A MT was nucleated from the centrosome (small arrowhead) and grew radially out of the field of view (times 00:29–00:59). Another MT depolymerized from outside the  field of view (large arrowhead) and was consumed by complete depolymerization all the way back to the centrosome (times 00:59–01: 59). Bar, 10 μm.
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Related In: Results  -  Collection

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

Figure 11: Dynamics of MTs at the centrosome. Fluorescence images from a series (taken at 7-s intervals) of a cell injected with X-rhodamine tubulin. Elapsed time in min/sec is in the upper right of each panel. The centrosome in this cell was positioned at the edge of the nucleus, so that half of the MTs emanating from the centrosome were visible beneath the nucleus. A MT was nucleated from the centrosome (small arrowhead) and grew radially out of the field of view (times 00:29–00:59). Another MT depolymerized from outside the field of view (large arrowhead) and was consumed by complete depolymerization all the way back to the centrosome (times 00:59–01: 59). Bar, 10 μm.
Mentions: To image MT dynamics at the centrosome, peripheral cells in which the centrosome was positioned beneath the nucleus were chosen because the overlying nucleus provided both a clear image as well as a flattened array of MTs (Fig. 11). Images were acquired at 7-s intervals and time-lapse series examined for evidence of MT ejection from the centrosome. In 121.4 min of analysis time in six cells, only three MTs were ejected from the centrosome, a frequency of 0.02 ± 0.04/cell/min (Table IV). In spite of this low ejection frequency, free minus ends depolymerized into the ∼30 × 30 μm field of view at the lamella from the direction of the cell body at a frequency of 0.3 ± 0.2/min (Table IV). Thus, for a typical cell with ∼90 × 30 μm lamella, about one minus end depolymerizes into the lamella per minute. Since only 0.02 minus end is released from the centrosome per minute, the centrosome is not responsible for generating most of the free minus ends seen in the lamella.

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