1. Substrate Deformation Predicts Neuronal Growth Cone Advance
Ahmad I.M. Athamneh, Alexander X. Cartagena-Rivera, Arvind Raman, Daniel M. Suter 
Biophysical Journal 
Volume 109, Issue 7, Pages (October 2015)
DOI: /j.bpj
Copyright © 2015 Biophysical Society Terms and Conditions

2. Figure 1
Schematic of AFM-based force measurement approach. Illustration depicts the side view of an AFM cantilever modified with apCAM-coated bead interacting with the P domain of a neuronal growth cone (A) before and (B) after active coupling between the bead and the growth cone cytoskeleton. Before coupling, the net external forces, F0, acting on the cantilever via the bead are the normal and adhesion forces. After coupling, the net external force F acting on the cantilever via the bead changes because of the introduction of the retrograde traction force f.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2015 Biophysical Society Terms and Conditions

3. Figure 2
Measuring the temporal traction force profile in Aplysia growth cones using AFM. (A) Phase contrast images showing AFM cantilever modified with apCAM-coated 5 μm bead (white dashed circle) interacting with a growth cone at 0, 12, and 19 min following engagement of the cantilever onto the growth cone. C domain boundary (black dashed line) started to reorient toward the bead after ∼12 min, and reached the bead after 19 min. Leading edge is marked by white dashed line. (B) Traction force felt by the apCAM cantilever over time was calculated using Eq. 14. This plot (solid line) shows the temporal profile of traction force development in the P domain of the growth cone shown in the sequence of panel (A). Also shown are the force profiles for an incomplete growth cone response to an apCAM-coated cantilever (dotted line) and a BSA-coated cantilever (gray line). (C) Comparison of traction force measured at the transition between latency and traction phase for complete interactions and maximum traction force recorded after a corresponding amount of time during incomplete interactions. Box and whiskers plot shows the median, 25th, and 75th percentiles and minimum and maximum values. Asterisk indicates significant difference.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2015 Biophysical Society Terms and Conditions

4. Figure 3
Growth cone traction force measured by apCAM microneedles. (A) DIC images showing an apCAM-coated microneedle with N/m stiffness interacting with a growth cone. The three images show the microneedle when first touching the growth cone (0 min), the C domain beginning reorientation toward the microneedle contact point (6 min; end of latency period), and C domain reaching the microneedle tip (15 min; end of traction period). (B) Kymograph taken along the white line in (A) and showing the position of the microneedle throughout the time course of the growth cone response. (C) Retrograde force exerted by the growth cone on the microneedle over time (solid circles) and F-actin retrograde flow rate during latency (open circles). (D) Comparison of F-actin retrograde flow rates for complete and incomplete growth cone responses early in latency and traction periods, respectively. Middle graph shows flow rates measured between the leading edge and the needle tip, whereas left and right graphs show flow rates measured between the needle tip and the C domain. Mean values ± SE; paired t-test.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2015 Biophysical Society Terms and Conditions

5. Figure 4
Measured force is proportional to microneedle stiffness. Relationship between traction force measured immediately before the C domain advanced toward the microneedle and the stiffness of the microneedle used to perform the measurement. Separate correlations are shown for apCAM- and Con A-coated microneedles.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2015 Biophysical Society Terms and Conditions

6. Figure 5
Microneedle deflection is a better predictor of growth cone advance than absolute traction force values. (A) Comparison of force measured for complete and incomplete growth cone responses for apCAM and Con A microneedles. For incomplete growth cone response, the value reported is the maximum force value within 10 min from the time the microneedle was lowered onto the growth cone, which corresponds to the mean latency time of apCAM-induced responses. (B) Comparison of microneedle deflection observed for complete and incomplete growth cone responses for apCAM and Con A microneedles, respectively. (C) Comparison of traction force measured for complete and incomplete growth cone responses using microneedles with a very narrow range of stiffness (0.018 ± N/m, n = 24). Box and whiskers plots show the median, 25th and 75th percentiles, and minimum and maximum values. Asterisk indicates significant difference.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2015 Biophysical Society Terms and Conditions

7. Figure 6
The same growth cone produces different amounts of traction force during adhesion-mediated advance. (A) DIC images of a growth cone interacting with a N/m Con A-coated microneedle at the beginning of the experiment, at the end of latency and traction phases. (B) The same growth cone shown in (A) interacting with N/m Con A-coated microneedle. Needle stiffness (k), needle deflection, and traction force are indicated. (C) Kymographs showing the position of the microneedle throughout the time course of the experiments shown in (A) and (B). Arrows indicate the end of latency phase.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2015 Biophysical Society Terms and Conditions

8. Figure 7
Hypothesis: development of traction force and the origin of the observed micrometer-scale substrate (microneedle) deformation. Upon contact with the microneedle tip, the nascent apCAM-mediated actin-based elastic adhesions establish linkage between the flowing actin network and microneedle tip. Individually, elastic adhesions are too weak to hold the connection, resulting in a very low traction force. Over time, dynamic and transient adhesions become more abundant and capable of bearing increasing tension, resulting in higher traction force and reduced retrograde flow. The magnitude of microneedle tip movement is related to the micrometer scale length to which elastic adhesions can be stretched before breaking (34). To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2015 Biophysical Society Terms and Conditions