This research was supported in part by AMED under grant number 21gm0810011h0005 (N.I. and Y.S.), JSPS KAKENHI (JP19H03223, N.I.) and JSPS Grants-in-Aid for Early-Career Scientists (JP19K16258, T.M.), the Osaka Medical Research Foundation for Incurable Diseases (T.M.), and NAIST Next Generation Interdisciplinary Research Project (Y.S.).

The authors have nothing to disclose.

The protocols described in this study use commercially available materials and microscopy equipment routinely found in all the laboratories, institutes, and universities. Therefore, investigators can easily adopt the present single speckle imaging and traction force microscopy in their studies.
The speckle imaging can analyze actin polymerization and clutch coupling. In addition, speckle imaging can monitor the retrograde flow of clutch molecules such as shootin1 and cortactin, which interact with F-actin retrograde flow. By using a TIRF microscope, the retrograde flow of the cell adhesion molecule L1-CAM can also be monitored23,41; L1-CAM undergoes grip and slip behaviors that reflect the clutch coupling efficiency23,41. Although the present study employs the TMR-HaloTag system for speckle imaging, other fluorescent proteins, such as EGFP and monomeric red fluorescent protein, are also available in the analysis16,18,20,23,24,27,39. The essentials for visualizing actin speckles are a low expression level of fluorescent actin and the illumination of a minimum area (Figure 2). In this protocol, Lifeact and HaloTag-actin signals are sequentially acquired. Because actin retrograde flow is relatively slow (4.5 &#177; 0.1 &#181;m/min)24, analysis of F-actin retrograde flow and actin polymerization are unaffected by sequential image acquisition of different fluorescent channels (~1 s interval). Lifeact is a widely-used F-actin marker, but can compete with actin binding proteins47. Of further importance, Lifeact can alter actin dynamics, thereby affecting F-actin structures and the cell morphology47,48,49.
Traction force microscopy can detect forces to drive growth cone advance. By mounting the neurons within an extracellular matrix, investigators can also analyze forces generated in a semi-3D environment11. High-magnification imaging is important for accurate quantification of traction force because growth cones generate weak traction forces7. Although other methods with nanopillars or stress-sensitive biosensors are also used to measure traction force50,51, PAA gel-based method is highly adaptable and allows for the adjustment of substrate rigidity by varying the concentrations of acrylamide and bis-acrylamide41,44,52. In this protocol, the PAA gel is prepared at a final concentration of 3.75% acrylamide and 0.03% bis-acrylamide; Young&#39;s modulus is ~270 Pa22 and this stiffness is within the range of brain tissue (100-10,000 Pa)53,54,55. Due to the thickness of the PAA gel (~100 &#181;m), this method limits the use of high-magnification lenses during microscopy. To obtain high-magnification images, investigators should use the zoom function in a laser scanning confocal microscope.
In conclusion, the present speckle imaging and traction force microscopy enable quantitative analyses of the key events in force generations. This information will be invaluable for improving understanding of the mechanisms that underlie growth cone advancement and navigation.

In the developing vertebrate brain, neurons undergo elaborately organized migrations and project axons toward appropriate synaptic partners to establish functional neuronal networks1,2,3. Growth cones, which are sensory and motile structures located at the tip of neurites, determine the speed and direction of neuronal migration and axon outgrowth3,4,5. Since neurons are surrounded by tightly packed environments, growth cones must exert forces against their environment to move forward6,7. To understand the mechanisms underlying neuronal migration and axonal guidance, analyses of the molecular mechanics for growth cone advance are essential.
Decades of analysis have revealed that traction force to drive growth cone advance is generated by the &#39;clutch&#39; mechanism; this mechanism is thought to function not only in the axonal growth cone but also in the leading process growth cone of migrating neurons8,9,10,11,12. Namely, actin filaments (F-actins) in growth cones polymerize at the leading edge and depolymerize proximally, pushing out the leading-edge membrane13,14,15. The resultant force, in conjunction with actomyosin contraction, induces rearward movement of F-actins called retrograde flow7,11,16,17,18,19,20,21. Clutch- and cell adhesion molecules mediate mechanical coupling between F-actin retrograde flow and the adhesive substrate and transmit the force of F-actin flow onto the substrate, thereby generating traction force for growth cone advance7,8,9,11,12,22. Concurrently, the actin-substrate coupling reduces the F-actin flow velocity and converts actin polymerization into the force to protrude the leading-edge membrane9,10.
Axonal growth cones sense local chemical cues and transduce them into a directional driving force for growth cone navigation3,23,24,25. For example, an axon guidance molecule netrin-1 stimulates its receptor deleted in colorectal cancer (DCC), and activates the Rho guanosine triphosphate (GTP)-binding proteins cell division control protein 42 (Cdc42) and Ras-related C3 botulinum toxin substrate 1 (Rac1), and their downstream kinase p21-activated kinase 1 (Pak1)26. Cdc42 and Rac1 promote 1) actin polymerization, and Pak1 phosphorylates a clutch molecule shootin122,26. Shootin1 interacts with F-actin retrograde flow via an actin-binding protein cortactin27. Shootin1 also interacts with L1 cell adhesion molecule (L1-CAM)20,24. Shootin1 phosphorylation increases the binding affinities for cortactin and L1-CAM, and enhances shootin1-mediated 2) clutch coupling24,27. Within the growth cone, asymmetrical activations of actin polymerization and clutch coupling increase 3) traction force on the side of the netrin-1 source, thereby generating directional driving force for growth cone turning (Figure 1)24. Intensive research over the last few decades with respect to neuronal migration and axon guidance has enhanced the understanding of guidance molecules, their receptors, and associated downstream signaling cascades2,10,28,29,30. However, the molecular machineries to generate forces for growth cone advance are just beginning to be elucidated; this may be attributed to the limited usage of the protocols for mechanobiological analyses.
The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging16,18. Monitoring of F-actin retrograde flow has been extensively performed using super-resolution microscopy, spinning-disk confocal microscopy and total interference reflection fluorescence (TIRF) microscopy25,31,32,33,34,35,36,37,38. The protocol in the present study, however, uses a standard epifluorescence microscope and is thus readily adoptable11,16,18,20,22,23,24,27,39,40,41,42. When combined with F-actin labelling by Lifeact43,&#160;single speckle imaging allows for quantifications of the actin polymerization rate and the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate39,42. The present study further describes a detailed protocol of traction force microscopy using a fluorescent bead-embedded polyacrylamide (PAA) gel11,22,23,24,27,39,41,42,44. This method detects and quantifies traction force under the growth cone by monitoring force-induced bead movements44,45. An open-source traction force analysis code is provided, and the method for quantifying traction force during growth cone migration is explained in detail. With the aid of single speckle imaging and traction force microscopy, understanding the molecular mechanics underlying growth cone migration and navigation will be facilitated. These techniques are also applicable for analyzing the molecular mechanics underlying dendritic spine enlargement, which is known to be important in learning and memory42.

<table><tbody><tr><td>0.5% trypan blue stain solution</td><td>Nacalai</td><td>29853-34</td><td></td></tr><tr><td>3-aminopropyltrimethyoxysilane</td><td>Sigma</td><td>281778-100ML</td><td></td></tr><tr><td>Acrylamide monomer</td><td>Nacalai</td><td>00809-85</td><td></td></tr><tr><td>Ammonium persulphate</td><td>Cytiva</td><td>17-1311-1</td><td></td></tr><tr><td>Axio Observer Z1</td><td>Zeiss</td><td>431007-9902-000</td><td>Epi-fluorescence microscope (single speckle imaging)</td></tr><tr><td>B-27 supplement (50x)</td><td>Thermo Fisher Scientific</td><td>17504-044</td><td></td></tr><tr><td>Bovine serum albmine</td><td>Sigma</td><td>A7906-10G</td><td></td></tr><tr><td>C-Apochromat 63x/1.2 W Corr</td><td>Zeiss</td><td>421787-9970-799</td><td>Objective lens (traction force microscopy)</td></tr><tr><td>Coverslip (diameter 18 mm)</td><td>Matsunami</td><td>C018001</td><td></td></tr><tr><td>D-glucose</td><td>Nacalai</td><td>16806-25</td><td></td></tr><tr><td>DNaseI</td><td>Sigma</td><td>DN25-100MG</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Thermo Fisher Scientific</td><td>10270-106</td><td></td></tr><tr><td>Fiji</td><td>Open source software package</td><td>https://imagej.net/software/fiji/</td><td></td></tr><tr><td>FluoSpheres carboxylate-modified 0.2 mm, red (580/605), 2% solid</td><td>Thermo Fisher Scientific</td><td>F8810</td><td>carboxylate-modified microspheres</td></tr><tr><td>Glass bottom dish (14 mm diameter)</td><td>Matsunami</td><td>D1130H</td><td></td></tr><tr><td>Glass bottom dish (27 mm diameter)</td><td>Matsunami</td><td>D1140H</td><td></td></tr><tr><td>Glutaraldehyde solution</td><td>Sigma</td><td>G5882-10X10ML</td><td></td></tr><tr><td>HaloTag TMR ligand</td><td>Promega</td><td>G8251</td><td></td></tr><tr><td>HBO103 W/2</td><td>Osram</td><td>4050300382128</td><td>Mercury lamp (single speckle imaging)</td></tr><tr><td>Image Processing Toolbox</td><td>MathWork</td><td>https://www.mathworks.com/products/image.html</td><td></td></tr><tr><td>Laminin solution from mouse EHS tumor</td><td>Wako</td><td>120-05751</td><td></td></tr><tr><td>Leibovitz&amp;rsquo;s L-15 medium</td><td>Thermo Fisher Scientific</td><td>11415064</td><td></td></tr><tr><td>L-glutamine</td><td>Nacalai</td><td>16919-42</td><td></td></tr><tr><td>LSM710</td><td>Zeiss</td><td>N/A</td><td>Conforcal laser microscope (traction force microscopy)</td></tr><tr><td>MATLAB2018a</td><td>MathWork</td><td>https://www.mathworks.com/products/new_products/release2018a.html</td><td></td></tr><tr><td>Mouse C57BL/6</td><td>Japan SLC</td><td>N/A</td><td></td></tr><tr><td>Mouse neuron nucleofector kit</td><td>Lonza</td><td>VPG-1001</td><td></td></tr><tr><td>N,N,N&amp;rsquo;,N&amp;rsquo;-tetramethylethylenediamine (TEMED)</td><td>Nacalai</td><td>33401-72</td><td></td></tr><tr><td>N,N&amp;rsquo;-methylenebisacrylamide</td><td>Nacalai</td><td>22402-02</td><td></td></tr><tr><td>Neurobasal medium</td><td>Thermo Fisher Scientific</td><td>21103-049</td><td></td></tr><tr><td>Nucleofector I</td><td>Amaxa</td><td>AAD-1001</td><td>Electroporation apparatus</td></tr><tr><td>ORCA Flash 4.0 V2</td><td>Hamamatsu</td><td>C11440-22CU</td><td>CMOS camera (single speckle imaging)</td></tr><tr><td>Papain</td><td>Nacalai</td><td>26036-34</td><td></td></tr><tr><td>Parallel Computing Toolbox</td><td>MathWork</td><td>https://www.mathworks.com/products/parallel-computing.html</td><td></td></tr><tr><td>pEGFP-C1</td><td>Clontech</td><td>1528177</td><td></td></tr><tr><td>Penicillin-streptomycin (100x)</td><td>Nacalai Tesque</td><td>26253-84</td><td></td></tr><tr><td>pFN21A-HaloTag-actin</td><td>(Minegishi et al., 2018)</td><td>N/A</td><td></td></tr><tr><td>Phosphate buffered saline (PBS) pH 7.4 (10x)</td><td>Thermo Fisher Scientific</td><td>70011-044</td><td></td></tr><tr><td>Plan-Apochromat 100x/1.4 Oil</td><td>Zeiss</td><td>420790-9901-000</td><td>Objective lens (single speckle imaging)</td></tr><tr><td>pmNeonGreen-N1-Lifeact</td><td>(Kastian et al., 2021)</td><td>N/A</td><td></td></tr><tr><td>Poly-D-lysine hydrobromide</td><td>Sigma</td><td>P6407-5MG</td><td></td></tr><tr><td>Slulfo-SAMPHA</td><td>Thermo Fisher Scientific</td><td>22589</td><td></td></tr><tr><td>Sodium dodecyl sulfate</td><td>Nacalai</td><td>08933-05</td><td></td></tr><tr><td>Sodium hydrate (NaOH)</td><td>Nacalai</td><td>31511-05</td><td></td></tr><tr><td>Steel ball</td><td>Sako tekkou</td><td>N/A</td><td>Microshpere to determin PAA gel rigidity. 0.6 mm diameter, 7.87 g/cm&lt;sup&gt;3&lt;/sup&gt;.</td></tr><tr><td>ZEN2009</td><td>Zeiss</td><td>https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html#introduction</td><td>Image acquisition software (traction force microscopy)</td></tr><tr><td>ZEN2012</td><td>Zeiss</td><td>https://www.zeiss.com/microscopy/us/products/microscope-software/zen.html#introduction</td><td>Image acquisition software (single speckle imaging)</td></tr></tbody></table>

analyses of actin dynamics, clutch coupling and traction force for growth cone advance

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes depend on the advances of growth cones that located at the tips of neurites. Axonal growth cones generate driving forces by sensing their local microenvironment and modulating cytoskeletal dynamics and actin-adhesion coupling (clutch coupling). Decades of research have led to the identification of guidance molecules, their receptors, and downstream signaling cascades for regulating neuronal migration and axonal guidance; however, the molecular machineries required for generating forces to drive growth cone advance and navigation are just beginning to be elucidated. At the leading edge of neuronal growth cones, actin filaments undergo retrograde flow, which is powered by actin polymerization and actomyosin contraction. A clutch coupling between F-actin retrograde flow and adhesive substrate generates traction forces for growth cone advance. The present study describes a detailed protocol for monitoring F-actin retrograde flow by single speckle imaging. Importantly, when combined with an F-actin marker Lifeact, this technique can quantify 1) the F-actin polymerization rate and 2) the clutch coupling efficiency between F-actin retrograde flow and the adhesive substrate. Both are critical variables for generating forces for growth cone advance and navigation. In addition, the present study describes a detailed protocol of traction force microscopy, which can quantify 3) traction force generated by growth cones. Thus, by coupling the analyses of&#160;single speckle imaging and traction force microscopy, investigators can monitor the molecular mechanics underlying growth cone advance and navigation.

Single speckle imaging to quantify actin polymerization rate and clutch coupling efficiency 
A high Lifeact expression permits F-actin visualization within the growth cone; a low HaloTag-actin expression allows the monitoring of F-actin retrograde flow (Figure 3, Supplemental File 2). Tracing of the actin speckles allows measurement of F-actin flow velocity (Figure 3C,D). Since the mechanical coupling of F-actin retrograde flow and the adhesive substrate reduces the F-actin flow velocity, the clutch coupling efficiency can be estimated from the velocity. Furthermore, F-actin labeling with Lifeact aids visualization of F-actin extension and is useful for quantifying the actin polymerization rate (Figure 3E,F).
Quantitative analysis of traction force 
Strict adherence to the methodology presented here will reveal the movements of fluorescent beads under the growth cone (Figure 6C, Supplemental File 4). F-actin retrograde flow onto the substrate generates traction force causing the fluorescent beads to move rearward under the growth cone. The traction force analysis code estimates traction force from fluorescent bead displacement, and expresses the calculated traction force as a force vector. The direction and magnitude of the traction force are determined from the x- and y-components of the force vector (Figure 8C,D). The red box in Figure 8D represents the x- and y-components of a force vector; Figure 8C depicts the corresponding growth cone. In terms of x-y coordinates, the vector points to -93.8&#176; against the x-axis; this orientation is directed toward the rear of the growth cone (Figure 8C). The magnitude of the traction force F was calculated as follows:
<img alt="Equation 2" src="/files/ftp_upload/63227/63227eq02.jpg" />
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63227/63227fig01.jpg" /> 
Figure 1: A growth cone machinery for force generation and growth cone navigation. A netrin-1 chemoattractant gradient induces asymmetrical stimulation of its receptor DCC on an axonal growth cone. This activates Rac1 and Cdc42, and their downstream kinase Pak1. Rac1 and Cdc42 promote (1) actin polymerization, whereas Pak1 phosphorylates shootin1, enhancing shootin1-mediated (2) clutch coupling. The asymmetric activation of actin dynamics and the clutch coupling within the growth cone increases (3) traction force on the side of the netrin-1 source, thereby generating a directional driving force for growth cone attraction. The protocols presented here allow for the quantification of the key variables (1)-(3) for growth cone navigation. <a href="https://www.jove.com/files/ftp_upload/63227/63227fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63227/63227fig02.jpg" /> 
Figure 2: Fluorescence images of a neuronal growth cone under a fully opened and narrowed diaphragm. The growth cone expresses Lifeact and HaloTag-actin. (A) A high Lifeact expression permits visualization of growth cone morphology. On the other hand, HaloTag-actin expression levels are very low, with dim signals when the diaphragm is fully opened. (B) When the diaphragm is appropriately narrowed, background signals diminish, and single actin speckles appear in the growth cone. Scale bars: 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63227/63227fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63227/63227fig3v2.jpg" /> 
Figure 3: Steps for quantifying F-actin flow velocity and actin polymerization rate using an image processing and analysis software Fiji. (A) Adjust the angle of the image for analysis. (1) Find and select an actin speckle (arrowhead) that flows in an F-actin bundle. (2) Select Image &#62; Transform &#62; Rotate. (3) Set the angle so that the F-actin bundle is directed upward. (4) The angle of the image will be changed. (B) Demarcate the region, including the actin speckle and the F-actin bundle. (1) Click on Rectangle on the toolbar. (2) Delineate a region on the image. The brightness and contrast of the image are increased to allow clear visualization of the actin speckle and the tip of the F-actin bundle. (3) Select Image &#62; Duplicate. (4) Input the five time-frames that show actin speckle flow in the F-actin bundle. (5) The selected stack image will appear on the screen. (C) Overlay circles on the actin speckle. (1) Click on Oval on the toolbar. (2) Draw a circle on an actin speckle. (3) Select Image &#62; Overlay &#62; Add Selection. (4) The circle is overlaid. 5) Repeat overlaying the circles on the remaining actin speckles. (D) Measure the translocation distance of actin speckles during the five time-frames. (1) Click on Straight on the toolbar. (2) Draw a line which links the centers of the circles. (3) Select Analyze &#62; Measure. The result, indicated by the parameter Height (red box), relaying the actin speckle translocation distance. (E) Measure the change in the length of F-actin protrusion during the five time-frames. (1) Draw a line which links the tips of the F-actin protrusion. (2) Select Analyze &#62; Measure. The result (red box), Height, indicates the extension length of the F-actin bundle. (F) The actin polymerization rate is calculated from the sum of the F-actin flow velocity and the extension rate.&#160;See also Supplemental File 2. Supplemental File 3 assists investigators to practice the methodology described above. <a href="https://www.jove.com/files/ftp_upload/63227/63227fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63227/63227fig04.jpg" /> 
Figure 4: Steps for the PAA gel preparation. Please see step 7.1 for a detailed description. <a href="https://www.jove.com/files/ftp_upload/63227/63227fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63227/63227fig05.jpg" /> 
Figure 5: Determination of PAA gel rigidity. (A) A microsphere indentation method. When a microsphere is placed on a fluorescent bead-embedded PAA gel, the weight of the microsphere causes an indentation in the gel. The indentation depth is calculated by subtracting the z-positions of the PAA gel surface from the bottom of the microsphere (B,C) Fluorescence images of a PAA gel indented by a microsphere and containing fluorescent beads. A laser scanning confocal microscope was used to capture images of the gel surface (B) and the bottom of the microsphere (C). Signals from the fluorescent&#160;beads are not visible at the gel surface in the indented region (B, circle). They can, however, be observed at the bottom of the microsphere. Scale bars: 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/63227/63227fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63227/63227fig06.jpg" /> 
Figure 6: Force mapping of a neuronal growth cone. (A-C) Fluorescence images of beads embedded in a PAA gel and a neural growth cone visualized with EGFP. Following the acquisition of time-lapse images (A), the neuron was released from the gel substrate by applying SDS solution, and an image of the beads in the unstrained substrate was captured (B). (C) The image of the beads in the unstrained substrate shows the beads in their original (green) and displaced (red) positions. The EGFP signal of the growth cone is shown in blue. Kymographs (right) show the movements of beads at 3 s intervals for a duration of 147 s, indicated by the arrows in boxed areas 1 and 2. The bead in area 2 is a reference bead. Scale bars: 2 &#181;m for (A), (B) and (C, left), and 1 &#181;m for (C, middle). See also Supplemental File 4. <a href="https://www.jove.com/files/ftp_upload/63227/63227fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/63227/63227fig07.jpg" /> 
Figure 7: Steps for the correcting x-y position of the bead image in unstrained substrate using Fiji. (A) Concatenate the bead image in unstrained substrate with the time-lapse stack image of fluorescent beads. (1) Select Image &#62; Stacks &#62; Tools &#62; Concatenate. (2) Select the beads image in unstrained substrate and the time-lapse stack image of fluorescent beads in Image1 and Image2, respectively. Click on&#160;OK. (3) The bead image in the unstrained substrate is added to the time-lapse stack image. (B) Correct the x-y position of the fluorescent bead image. (1) Use the scroll bar (red frame) to select the second frame (red arrow) in the image stack. (2) Select Plugins &#62; StackReg. (3) Select Rigid Body from the drop-down list (red frame) and click on OK. The correction of the x-y position will begin. (C) Save the x-y position-corrected bead image. After selecting the first frame of the x-y position-corrected stack image, (1) select Image &#62; Duplicate. (2) input 1 into the Range and deselect Duplicate stack. Then click on OK. (3) the x-y position-corrected bead image in the unstrained substrate will appear on the screen. Save this image as a tiff file. Supplemental File 6 assists investigators to practice the methodology described above. <a href="https://www.jove.com/files/ftp_upload/63227/63227fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/63227/63227fig08.jpg" /> 
Figure 8: Analysis of traction force under a neuronal growth cone using an open-source traction force analysis code. (A) GUI for analyzing traction force. Time-lapse images selected on the GUI can be confirmed from the drop-down list and with the slider indicated (red box). (B) Select a region that includes the growth cone. (1) Click on ROI on the GUI. With the mouse cursor, specify two points (arrowheads) on the cell image. (2) A red box will appear on the cell image. Two clicks determine the locations of two corners. (C) Select the detected beads (white dots) under the growth cone. (1) On the GUI, click on Select beads, and demarcate a polygonal region that includes the growth cone by clicking. Press the Enter key. (2) The white dots within the polygonal region will change into a red color. (D) Calculated results of the direction and magnitude of the traction force. The red box in the spreadsheet represents the x- and y-components of the force vector, estimated by traction force analysis. On the x-y coordinate in the right panel, the force vector generated by the growth cone points to -93.8&#176; against the x-axis; this orientation is directed toward the rear of the growth cone in (C). Supplemental File 6 assists investigators to practice the methodology described above. <a href="https://www.jove.com/files/ftp_upload/63227/63227fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1: Recipes of solutions and media used in this study. See text for detailed usage. <a href="https://www.jove.com/files/ftp_upload/63227/JoVE Review - Supplemental File 1-63227R1.xlsx" target="_blank">Please click here to download this File.</a>
Supplemental File 2: Fluorescence imaging of Lifeact and fluorescent speckle imaging of HaloTag-actin in a nerve growth cone. Lifeact (green) and HaloTag-actin (magenta). Images were acquired every 3 s for a total duration of 147 s. Scale bar: 2 &#181;m. See also Figure 3. <a href="https://www.jove.com/files/ftp_upload/63227/JoVE Review - Supplemental File 2.mp4" target="_blank">Please click here to download this File.</a>
Supplemental File 3: Practice data for quantifying F-actin flow velocity and the actin polymerization rate. A multichannel time-lapse stack image of Lifeact (green) and HaloTag-actin (magenta). See also Figure 3. <a href="https://www.jove.com/files/ftp_upload/63227/Supplemental file 3.zip" target="_blank">Please click here to download this File.</a>
Supplemental File 4: A force-mapping video detecting traction force at a neuronal growth cone. The original (green) and displaced (red) positions of the beads. The EGFP signal in the growth cone is shown in blue. Images were acquired every 3 s for 147 s. Scale bar: 2 &#181;m. See also Figure 6. <a href="https://www.jove.com/files/ftp_upload/63227/JoVE Review - Supplemental File 4.mp4" target="_blank">Please click here to download this File.</a>
Supplemental File 5: Traction force analysis code. Please see Step 8.12 for detailed usage. <a href="https://www.jove.com/files/ftp_upload/63227/JoVE Review - Supplemental file 5.zip" target="_blank">Please click here to download this File.</a>
Supplemental File 6: Practice data for quantifying traction force. A single-channel RGB image of the beads in unstrained substrate and single-channel time-lapse stack RGB images of fluorescent beads under a growth cone, EGFP, and bright-field. See also Figures 7 and 8. <a href="https://www.jove.com/files/ftp_upload/63227/JoVE Review - Supplemental file 6.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance at JoVE.com

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance.

To establish functional networks, neurons must migrate to their appropriate destinations and then extend axons toward their target cells. These processes ...

analyses-actin-dynamics-clutch-coupling-traction-force-for-growth

Research

JoVE Journal

Neuroscience

3.3K Views.  Nara Institute of Science and Technology. To advance, growth cones must exert traction forces against the external environment. The generation of traction forces is dependent on actin dynamics and clutch coupling. The present study describes methods for analyzing actin dynamics, clutch coupling and traction forces for growth cone advance. 

Video: Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Bioengineering

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally expressed molecular guidance cues that bind growth cone receptors and regulate the dynamics and organization of the growth cone cytoskeleton3-6. The main target of these navigational signals is the actin filament meshwork that fills the growth cone periphery and that drives growth cone motility through continual actin polymerization and dynamic remodeling7. Positive or attractive guidance cues induce growth cone turning by stimulating actin filament (F-actin) polymerization in the region of the growth cone periphery that is nearer the source of the attractant cue. This actin polymerization drives local growth cone protrusion, adhesion of the leading margin and axonal elongation toward the attractant.
Actin filament polymerization depends on the availability of sufficient actin monomer and on polymerization nuclei or actin filament barbed ends for the addition of monomer. Actin monomer is abundantly available in chick retinal and dorsal root ganglion (DRG) growth cones. Consequently, polymerization increases rapidly when free F-actin barbed ends become available for monomer addition. This occurs in chick DRG and retinal growth cones via the local activation of the F-actin severing protein actin depolymerizing factor (ADF/cofilin) in the growth cone region closer to an attractant8-10. This heightened ADF/cofilin activity severs actin filaments to create new F-actin barbed ends for polymerization. The following method demonstrates this mechanism. Total content of F-actin is visualized by staining with fluorescent phalloidin. F-actin barbed ends are visualized by the incorporation of rhodamine-actin within growth cones that are permeabilized with the procedure described in the following, which is adapted from previous studies of other motile cells11, 12. When rhodamine-actin is added at a concentration above the critical concentration for actin monomer addition to barbed ends, rhodamine-actin assembles onto free barbed ends. If the attractive cue is presented in a gradient, such as being released from a micropipette positioned to one side of a growth cone, the incorporation of rhodamine-actin onto F-actin barbed ends will be greater in the growth cone side toward the micropipette10.
Growth cones are small and delicate cell structures. The procedures of permeabilization, rhodamine-actin incorporation, fixation and fluorescence visualization are all carefully done and can be conducted on the stage of an inverted microscope. These methods can be applied to studying local actin polymerization in migrating neurons, other primary tissue cells or cell lines.

A method to visualize and quantify F-actin barbed ends in neuronal growth cones is described. After culturing neurons on glass coverslips, cells are permeabilized with a saponin-containing solution. Then, a short incubation with the saponin buffer containing rhodamine-actin incorporates fluorescent actin onto free actin barbed ends.

The motile tips of growing axons are called growth cones. Growth cones lead navigating axons through developing tissues by interacting with locally ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Developmental Biology

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Biology

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is facilitated by actin-rich protrusions called invadopodia that proteolytically degrade the extracellular matrix (ECM). Invadopodia activity has been shown to be dependent on ECM rigidity and cancer cell contractile forces suggesting that rigidity signals can regulate these subcellular structures through actomyosin contractility. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. Invasive and contractile properties of cancer cells can be correlated in vitro using invadopodia and traction force assays based on polyacrylamide gels (PAAs) of different rigidities. While some variations between the two assays exist, the protocol presented here provides a method for creating PAAs that can be used in both assays and are easily adaptable to the user&#8217;s specific biological and technical needs.

Mechanical rigidity in the tumor microenvironment plays a crucial role in driving malignant behavior by increasing invadopodia activity and actomyosin contractility. Using polyacrylamide gels (PAAs), invadopodia and traction force assays can be utilized to study the invasive and contractile properties of cancer cells in response to matrix rigidity.

Rigid tumor tissues have been strongly implicated in regulating cancer cell migration and invasion. Invasive migration through cross-linked tissues is ...

Medicine

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical gradient, known as chemotaxis, plays an important role in development, the immune response, wound healing, and cancer metastasis. While chemotaxis modulates the migration of single cells as well as collections of cells in vivo, in vitro research focuses on single-cell chemotaxis, partly due to the lack of the proper experimental tools. To fill that gap, described here is a unique experimental system that combines microfluidics and micropatterning to demonstrate the effects of chemical gradients on collective cell migration. Furthermore, traction microscopy and monolayer stress microscopy are incorporated into the system to characterize changes in cellular force on the substrate as well as between neighboring cells. As proof-of-concept, the migration of micropatterned circular islands of Madin-Darby canine kidney (MDCK) cells is tested under a gradient of hepatocyte growth factor (HGF), a known scattering factor. It is found that cells located near the higher concentration of HGF migrate faster than those on the opposite side within a cell island. Within the same island, cellular traction is similar on both sides, but intercellular stress is much lower on the side of higher HGF concentration. This novel experimental system can provide new opportunities to studying the mechanics of chemotactic migration by cellular collectives.

Collective cell migration in development, wound healing, and cancer metastasis is often guided by the gradients of growth factors or signaling molecules. Described here is an experimental system combining traction microscopy with a microfluidic system and a demonstration of how to quantify the mechanics of collective migration under biochemical gradient.

Cells change migration patterns in response to chemical stimuli, including the gradients of the stimuli. Cellular migration in the direction of a chemical ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out shape. Following the cell-substrate attachment, the cell forms a thin sheet of lamellipodia emanating from the cell body. In the lamellipodia, globular actin (G-actin) monomers polymerize into a dense filamentous actin (F-actin) meshwork that pushes against the plasma membrane, thereby providing the mechanical forces required for the cell to spread. Notably, the molecular players that control the actin polymerization in lamellipodia are essential for many other cellular processes, such as cell migration and endocytosis. 
Since spreading cells form continuous lamellipodia that span the entire cell periphery and persistently expand outward, cell spreading assays have become an efficient tool to assess the kinetics of lamellipodial protrusions. Although several technical implementations of the cell spreading assay have been developed, a detailed description of the workflow, which would include both a step-by-step protocol and computational tools for data analysis, is currently lacking. Here, we describe the experimental procedures of the cell spreading assay and present an open-source tool for quantitative and unbiased analysis of cell edge dynamics during spreading. When combined with pharmacological manipulations and/or gene-silencing techniques, this protocol is amenable to a large-scale screen of molecular players regulating lamellipodial protrusions.

In this protocol, we present the experimental procedures of a cell spreading assay that is based on live-cell microscopy. We provide an open-source computational tool for the unbiased segmentation of fluorescently labeled cells and quantitative analysis of lamellipodia dynamics during cell spreading.

Cell spreading is a dynamic process in which a cell suspended in media attaches to a substrate and flattens itself from a rounded to a thin and spread-out ...

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.

This protocol aims to measure the dynamic parameters (protrusions, retractions, ruffles) of protrusions at the edge of spreading cells.

The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the ...

Analyses of Actin Dynamics, Clutch Coupling and Traction Force for Growth Cone Advance

Summary

Explore More Videos

Preparation of Complaint Matrices for Quantifying Cellular Contraction

Labeling F-actin Barbed Ends with Rhodamine-actin in Permeabilized Neuronal Growth Cones

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Polyacrylamide Gels for Invadopodia and Traction Force Assays on Cancer Cells

Traction Microscopy Integrated with Microfluidics for Chemotactic Collective Migration

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Quantitative Analysis of Cell Edge Dynamics during Cell Spreading

Measuring Cell-Edge Protrusion Dynamics during Spreading using Live-Cell Microscopy