Reader Comments

Post a new comment on this article

Referee Comments: Referee 2

Posted by PLOS_ONE_Group on 30 Oct 2007 at 23:07 GMT

Reviewer 2's Review

-----

In this study, the authors utilized laser optical tweezers to measure the tiny mechanical forces generated by elongating DRG growth cones and their filopodia and lamellipodia. Briefly, a microbead was first trapped by a beam of 1064-nm laser and then moved close to a growth cone. The subsequent contact between the bead and the growth cone often displaced the bead from its trap, in which growth cone must have exerted a force to counterbalance a laser force applied to the bead to pull it back to the trap. The force exerted by the growth cone can be calculated by the formula F=kD, where k is a force constant (designated trap stiffness in the manuscript) and D is the distance of the displacement. The authors found that DRG growth cones could produce a force up to 20 pN in magnitude. With the same experimental strategy, the forces exerted by filopodia and lamellipodia were also measured respectively. Finally, the dependence of force generation on cytoskeleton, specifically actin and microtubule polymerization, was examined by using pharmacologically specific drugs.

In general, this is an interesting study that provides some insights into the mechanic properties of filopodia and lamellipodia, two most prominent motile structures of the growth cone. The manuscript can be much strengthened if the following comments were addressed.

General comment:

Growth cones, as well as their filopodia and lamellipodia, are highly dynamic in both morphology and motility, and their "wandering" movements are not as predictable as those of the lamellipodia of migrating keratocytes (Prass et al., 2006), which makes it more difficult in force characterization and accurate measurement. Moreover, the patterns of contacts between growth cones and trapped microbeads in this study varied so that the measured forces were affected by many factors, such as the size of contact area, the contact positions on both growth cones and microbeads, and the initial movement velocities and directions of growth cones and their filopodia and lamellipodia. It is reasonable to believe that the measured forces may not fully represent the ability that each growth cone or filopodium/lamellipodium has, because at least in some cases only a fraction of the forces exerted by growth cones and their components was picked up by the beads. In this regard, it would be necessary to perform another experiment in which microbeads are first stably attached to growth cone/filopodium/lamellipodium and trapped, and then their displacements are measured. In this way, the movements of growth cone/filopodium/lamellipodium would be monitored reliably and hence the force measurements. Finally, in this study there is no measurement on the forces specifically during filopodia retraction, which is one of the significant features of filopodia.

With respect to the physiological significance of the findings, it would be important for the authors to examine the growth cones subject to different adhesion substrates or extracellular factors that can either accelerate or inhibit growth cone motility. Furthermore, it would be extremely interesting if the beads are coated with certain guidance molecules and examine the mechanic responses.


-----

N.B. These are the general comments made by the reviewer when reviewing this paper in light of which the manuscript was revised. Specific points addressed during revision of the paper are not shown.