force was developed by the lamellipodium and the lamellipodium was compliant
(the elastic constant of lamellipodia of fibroblast has been measured to be ~0.5 kPa;
[165]). A previous study using a glass microneedle found the forces on the similar
magnitude required to stall the locomotion of keratocyte (% protrusive force; [166]).
What was remarkable in the AFM study was that the measured force-velocity curve
was similar to what has been predicted from the elastic ratchet mechanism. Thus, the
velocity slowly decreased in the small force regime, but it decreased sharply when
the force reached ~1 nanonewton; the relation was qualitatively different from the
prediction of the thermal ratchet mechanism, which showed a concave dependence
of the velocity on the force. The magnitude of the force on the order of nanonewton
is indeed expected from the thermodynamically determined force developed by
polymerizing actin filament (~a few piconewton) and the number density of the
filaments estimated for the edge of fibroblast (roughly 150/μm cell edge; [127, 167]
and the width of ~5 microns for the region of the lamellipodium of keratocyte, which
pushes the AFM cantilever). Thus, the elastic ratchet model can explain both
qualitatively and quantitatively the protrusion of keratocyte lamellipodia.
7.18.2 Fibroblasts: Measurement with Optical Trapping
Technique
However, in some cases, much lower forces have been shown to affect the
lamellipodial protrusion. For exmaple, lamellipodium extending from a fibroblast
cell has been shown to be halted by the water flow from a glass capillary with a
micron-sized opening [168]. Hydrodynamic calculation indicated that the force
exerted on the lamellipodium is a few piconewtons/μm of the front edge. The
experiment has shown that the hydrodynamic force immediately halted the protrusion. The interference contrast microscopy has suggested that this was due to the
dissociation of the cell edge, which would have resulted in the incomplete adhesion
of the extended lamellipodium, not the counterforce against the polymerization
force. This result demonstrates that the lamellipodial protrusion is a complex process
in which not only the magnitude of the force, but also the conversion of the
protrusive motion to the advancement of the cell edge through the attachment is
important.
In another case, the optical trapping technique was used to exert piconewton
forces on extending lamellipodium (Fig. 7.25; [169]). In this experiment, a micronsized polystyrene bead (probe-bead) was held by the optical trap that was created by
focusing an infra-red laser beam of up to a few mW. As described in Sect. 3.7.2, the
trap is characterized by a spring constant trap stiffness, k. With this technique,
lamellipodial activity of fibroblast cells was measured. The cell was observed by
phase-contrast video microscopy and a bead held in the trap (the k value ranged from
0.01 pN/nm to 0.1 pN/nm) was contacted the edge of a lamellipodium to probe the
movement of the lamellipodium.
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7 Moving Life
(the elastic constant of lamellipodia of fibroblast has been measured to be ~0.5 kPa;
[165]). A previous study using a glass microneedle found the forces on the similar
magnitude required to stall the locomotion of keratocyte (% protrusive force; [166]).
What was remarkable in the AFM study was that the measured force-velocity curve
was similar to what has been predicted from the elastic ratchet mechanism. Thus, the
velocity slowly decreased in the small force regime, but it decreased sharply when
the force reached ~1 nanonewton; the relation was qualitatively different from the
prediction of the thermal ratchet mechanism, which showed a concave dependence
of the velocity on the force. The magnitude of the force on the order of nanonewton
is indeed expected from the thermodynamically determined force developed by
polymerizing actin filament (~a few piconewton) and the number density of the
filaments estimated for the edge of fibroblast (roughly 150/μm cell edge; [127, 167]
and the width of ~5 microns for the region of the lamellipodium of keratocyte, which
pushes the AFM cantilever). Thus, the elastic ratchet model can explain both
qualitatively and quantitatively the protrusion of keratocyte lamellipodia.
7.18.2 Fibroblasts: Measurement with Optical Trapping
Technique
However, in some cases, much lower forces have been shown to affect the
lamellipodial protrusion. For exmaple, lamellipodium extending from a fibroblast
cell has been shown to be halted by the water flow from a glass capillary with a
micron-sized opening [168]. Hydrodynamic calculation indicated that the force
exerted on the lamellipodium is a few piconewtons/μm of the front edge. The
experiment has shown that the hydrodynamic force immediately halted the protrusion. The interference contrast microscopy has suggested that this was due to the
dissociation of the cell edge, which would have resulted in the incomplete adhesion
of the extended lamellipodium, not the counterforce against the polymerization
force. This result demonstrates that the lamellipodial protrusion is a complex process
in which not only the magnitude of the force, but also the conversion of the
protrusive motion to the advancement of the cell edge through the attachment is
important.
In another case, the optical trapping technique was used to exert piconewton
forces on extending lamellipodium (Fig. 7.25; [169]). In this experiment, a micronsized polystyrene bead (probe-bead) was held by the optical trap that was created by
focusing an infra-red laser beam of up to a few mW. As described in Sect. 3.7.2, the
trap is characterized by a spring constant trap stiffness, k. With this technique,
lamellipodial activity of fibroblast cells was measured. The cell was observed by
phase-contrast video microscopy and a bead held in the trap (the k value ranged from
0.01 pN/nm to 0.1 pN/nm) was contacted the edge of a lamellipodium to probe the
movement of the lamellipodium.
136
7 Moving Life