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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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Model of MT arrangement and turnover in the lamella  and lamellipodia of migrating newt lung epithelial cells. Thick  lines represent MTs; dotted lines, the border between the lamella  and lamellipodia; thin arrows, MT growth or shortening; arrowheads, sites of MT breakage; thick arrows, the direction of retrograde flow; dotted area, the putative zone of MT breakage at the  base of the lamella; and circles, the centrosome. The numbers in  the diagram refer to findings and hypotheses from this study. The  cell is migrating to the right. 1, MTs in the lamella oriented perpendicular to the leading edge extend to the base of the lamellipodia, exhibit frequent and short dynamic instability, and show  little net change in length. 2, Parallel MTs within the lamellipodia  undergo catastrophe less often and exhibit net growth. 3, Parallel  MTs and photoactivated marks on perpendicular MTs in the  lamella (stars) move continuously towards the cell center at ∼0.4  μm/min. 4, F-actin (beaded lines) crosslinked to MTs is postulated  to be moved rearward by myosin, which is bound to an unknown  stationary structure (question mark) in the lamella. 5, MT breakage occurring at sites of local MT buckling. 6, Free minus ends  formed by breakage are specifically capped (asterisks). 7, Treadmilling of MTs by net plus end growth and net minus end shortening. 8, <25% of all MTs in the cell are bound at their minus  ends to the centrosome. 9, Cytoplasmic dynein bound to a membranous organelle (question mark) or other MT crosslinking  proteins, are proposed to organize noncentrosomal MTs in the  lamella.
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Figure 12: Model of MT arrangement and turnover in the lamella and lamellipodia of migrating newt lung epithelial cells. Thick lines represent MTs; dotted lines, the border between the lamella and lamellipodia; thin arrows, MT growth or shortening; arrowheads, sites of MT breakage; thick arrows, the direction of retrograde flow; dotted area, the putative zone of MT breakage at the base of the lamella; and circles, the centrosome. The numbers in the diagram refer to findings and hypotheses from this study. The cell is migrating to the right. 1, MTs in the lamella oriented perpendicular to the leading edge extend to the base of the lamellipodia, exhibit frequent and short dynamic instability, and show little net change in length. 2, Parallel MTs within the lamellipodia undergo catastrophe less often and exhibit net growth. 3, Parallel MTs and photoactivated marks on perpendicular MTs in the lamella (stars) move continuously towards the cell center at ∼0.4 μm/min. 4, F-actin (beaded lines) crosslinked to MTs is postulated to be moved rearward by myosin, which is bound to an unknown stationary structure (question mark) in the lamella. 5, MT breakage occurring at sites of local MT buckling. 6, Free minus ends formed by breakage are specifically capped (asterisks). 7, Treadmilling of MTs by net plus end growth and net minus end shortening. 8, <25% of all MTs in the cell are bound at their minus ends to the centrosome. 9, Cytoplasmic dynein bound to a membranous organelle (question mark) or other MT crosslinking proteins, are proposed to organize noncentrosomal MTs in the lamella.

Mentions: In this study, we have discovered several novel features of MT dynamics in living migrating cells that have implications for the arrangement of MTs and the mechanism of polymer turnover in these cells that differ considerably from the prevailing view in which MTs are bound to centrosomes at their minus ends and turn over by dynamic instability at their free plus ends. These findings are outlined in Fig. 12, and below we discuss them in sequence, considering possible underlying mechanisms, related phenomena, and the implications for MT turnover in migrating 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)

Model of MT arrangement and turnover in the lamella  and lamellipodia of migrating newt lung epithelial cells. Thick  lines represent MTs; dotted lines, the border between the lamella  and lamellipodia; thin arrows, MT growth or shortening; arrowheads, sites of MT breakage; thick arrows, the direction of retrograde flow; dotted area, the putative zone of MT breakage at the  base of the lamella; and circles, the centrosome. The numbers in  the diagram refer to findings and hypotheses from this study. The  cell is migrating to the right. 1, MTs in the lamella oriented perpendicular to the leading edge extend to the base of the lamellipodia, exhibit frequent and short dynamic instability, and show  little net change in length. 2, Parallel MTs within the lamellipodia  undergo catastrophe less often and exhibit net growth. 3, Parallel  MTs and photoactivated marks on perpendicular MTs in the  lamella (stars) move continuously towards the cell center at ∼0.4  μm/min. 4, F-actin (beaded lines) crosslinked to MTs is postulated  to be moved rearward by myosin, which is bound to an unknown  stationary structure (question mark) in the lamella. 5, MT breakage occurring at sites of local MT buckling. 6, Free minus ends  formed by breakage are specifically capped (asterisks). 7, Treadmilling of MTs by net plus end growth and net minus end shortening. 8, <25% of all MTs in the cell are bound at their minus  ends to the centrosome. 9, Cytoplasmic dynein bound to a membranous organelle (question mark) or other MT crosslinking  proteins, are proposed to organize noncentrosomal MTs in the  lamella.
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Figure 12: Model of MT arrangement and turnover in the lamella and lamellipodia of migrating newt lung epithelial cells. Thick lines represent MTs; dotted lines, the border between the lamella and lamellipodia; thin arrows, MT growth or shortening; arrowheads, sites of MT breakage; thick arrows, the direction of retrograde flow; dotted area, the putative zone of MT breakage at the base of the lamella; and circles, the centrosome. The numbers in the diagram refer to findings and hypotheses from this study. The cell is migrating to the right. 1, MTs in the lamella oriented perpendicular to the leading edge extend to the base of the lamellipodia, exhibit frequent and short dynamic instability, and show little net change in length. 2, Parallel MTs within the lamellipodia undergo catastrophe less often and exhibit net growth. 3, Parallel MTs and photoactivated marks on perpendicular MTs in the lamella (stars) move continuously towards the cell center at ∼0.4 μm/min. 4, F-actin (beaded lines) crosslinked to MTs is postulated to be moved rearward by myosin, which is bound to an unknown stationary structure (question mark) in the lamella. 5, MT breakage occurring at sites of local MT buckling. 6, Free minus ends formed by breakage are specifically capped (asterisks). 7, Treadmilling of MTs by net plus end growth and net minus end shortening. 8, <25% of all MTs in the cell are bound at their minus ends to the centrosome. 9, Cytoplasmic dynein bound to a membranous organelle (question mark) or other MT crosslinking proteins, are proposed to organize noncentrosomal MTs in the lamella.
Mentions: In this study, we have discovered several novel features of MT dynamics in living migrating cells that have implications for the arrangement of MTs and the mechanism of polymer turnover in these cells that differ considerably from the prevailing view in which MTs are bound to centrosomes at their minus ends and turn over by dynamic instability at their free plus ends. These findings are outlined in Fig. 12, and below we discuss them in sequence, considering possible underlying mechanisms, related phenomena, and the implications for MT turnover in migrating 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