Tensile stress stimulates microtubule outgrowth in living cells
Irina Kaverina1,
Olga Krylyshkina1,
Karen Beningo2,
Kurt Anderson3,
Yu-Li Wang2 and
J. Victor Small1,*
1 Institute of Molecular Biology of the Austrian Academy of Sciences, A-5020,
Austria
2 Department of Physiology, University of Massachusetts Medical School,
Worcester, Massachusetts 01605, USA
3 Max Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstr.
108, Dresden, D-01307, Germany
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Fig. 1. Cell body displacement stimulates growth of peripheral adhesions in B16
melanoma cells and the polymerisation of microtubules towards the cell edge.
(A) Video frames show a motile cell, expressing GFP-VASP, whose cell body was
displaced by a microneedle in the direction indicated in the phase contrast
image. Panel 0'00'' in this and subsequent figures corresponds to
the video frame before tension application. Boxed insets in the fluorescence
images show enlargement of peripheral adhesion sites in the region
diametrically opposite the cell body. The continued protrusion of the cell
edge is indicated by the persistence of the line of GFP-VASP at the tip of the
lamellipodium. An example of one from five cells is shown. Times are in
minutes and seconds. Bar, 10 µm. (B) The conditions used were the same as
in A for a B16 melanoma cell expressing GFP-tubulin. Arrows in the phase
contrast and fluorescent images of the video sequence indicate the direction
of stress application. Insets show invasion of microtubules into lamella
region in the line of applied stress. An example of one from seven cells is
shown. (C) The conditions used were the same as in A for a B16 melanoma cell
expressing GFP-CLIP-170. Arrows indicate the direction of stress application.
Note the increase in number of polymerising microtubules in peripheral lamella
(ellipse), which are marked by GFP-CLIP-170 at their tips. An example from one
from 15 cells is shown.
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Fig. 2. (A,B) Quantification of the increase in the number of microtubules
extending into anterior lamella regions of B16 melanoma cells in response to
increased stress imposed by cell body manipulation (A, 22 cells) and
stretching of a flexible growth substrate (B, 19 cells). (C) Quantification of
the increase of CLIP-170-associated polymerising microtubule tips in lamella
regions of B16 melanoma cells in response to increased stress imposed by cell
body manipulation (blue, 15 cells) and stretching of a flexible growth
substrate (magenta, four cells).
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Fig. 3. Microtubules induced to polymerise by increased stress target the adhesion
sites that simultaneously enlarge at the cell periphery. The figure shows a
B16 melanoma cell that was transfected with GFP-zyxin and GFP-tubulin and
subjected to cell body displacement in the direction indicated by the arrows.
Upper phase images show the cell just before (left) and 6 minutes 15 seconds
(6'15'') after tension application (right) with the microneedle.
The area boxed in the left phase image corresponds to the region shown in
fluorescence in the lower video frames. An example of one of 15 cells is
shown.
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Fig. 4. Cell body displacement stimulates formation of radial bundles of actin
filaments that terminate at the cell periphery. Video sequences show a B16
melanoma cell expressing either GFP-actin (A, example from nine cells) or
GFP-calponin h1 (B, example from five cells.) that was subjected to cell body
manipulation at time 0. The chevrons in (A) indicate regions of bundle
formation. Boxed regions in (B) are shown at higher magnification in the right
hand panels.
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Fig. 5. Tension applied via stretching of the growth substrate beyond the cell
front induces microtubule growth at the cell periphery. The figure shows a B
16 melanoma cell that was transfected with GFP-tubulin and plated onto a
flexible, polyacrylamide substrate impregnated with rhodamine-tagged
fluorescent beads. The upper left and right panels (boxed insets enlarged)
show video frames of the cell before tension application. The lower frames
show the corresponding regions after tension application [at 3 minutes 30
second (3'40'')] by a needle applied to the substrate around 20
µm beyond the cell edge. The direction and magnitude of tension is
indicated by the shift of fluorescent beads (middle panel, left), which
corresponds to the smaller boxes regions in the lower, left panels. One
example from 19 cells is shown.
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Fig. 6. Recovery from brief, local inhibition of contractility by ML-7 is
associated with the repolymerisation of microtubules to peripheral adhesions
and enhanced actin bundle formation. (A) A fish fibroblast expressing
GFP-tubulin that was injected with TAMRA vinculin. The phase contrast image
(left) indicates the region of application of ML-7 (ellipse) via a
micropipette (chevron). The period of application was 3 minutes. Fluorescent
images show video frames of the region of application at the time points
indicated. One example from seven cells is shown. (B) A fish fibroblast was
treated as in A but was transfected with GFP-calponin to highlight actin
bundles. Arrowheads show peripheral bundles disassembling during application.
Arrows indicate stress fibres enhancing during recovery. One example from
eight cells is shown. (C) The retraction of microtubules from the cell edge on
ML-7 treatment is caused by depolymerisation and not by bulk withdrawal of
microtubules. A fish fibroblast expressing GFP-tubulin was photobleached in a
narrow region across the base of lamella (arrow) and then exposed to local
ML-7 application. Arrowheads indicate depolymerising microtubules in
subsequent frames. One example from five cells is shown.
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Fig. 7. Mechanical restraint of the keratocyte cell body stimulates growth of
adhesion sites and penetration of microtubules into the lamellipodium. A trout
keratocyte cell body, injected with rhodamine vinculin, was arrested at time 0
by a micropipette (phase image). The fluorescence images and insets (of boxed
regions) are shown at the initiation of cell body arrest (0'0'')
and 1 minute later. Note the incorporation of vinculin into multiple, new
focal complexes in response to stress (at 1'00''). One example of
five cells is shown. (B) The same protocol was used as in A for a black molly
keratocyte injected with Cy-3 tubulin. The cell body was released 70 seconds
before the last frame (4'50''). The right panels show, in the
fluorescence channel, the regions boxed in the phase contrast images (left).
An example from 15 cells is shown.
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© The Company of Biologists Ltd 2002
