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.
Cell migration is a critical process that controls the development and function of all living organisms. Cell migration occurs in both physiological conditions, such as embryogenesis, wound healing, and immune response, and in pathological conditions, such as cancer metastasis and autoimmune disease. Despite differences in cell types that take part in different migratory events, all cell motility events share similar molecular mechanisms, which have been conserved in evolution from protozoa to mammals, and involve common cytoskeletal control mechanisms that can sense the environment, respond to signals, and modulate cell behavior in response1.
An initial stage in cell migration can be the formation of highly dynamic protrusions at the leading edge of the cell. Behind the lamellipodium one can find the lamella, which couples actin to myosin II-mediated contractility and mediates adhesion to the underlying substrate. Lamellipodia are induced by extracellular stimuli such as growth factors, cytokines, and cell adhesion receptors and are driven by actin polymerization, which provides the physical force that pushes the plasma membrane forward2,3. Many signaling and structural proteins have been implicated in this; among them are Rho GTPases, which act coordinately with other signals to activate actin-regulating proteins such as the Arp 2/3 complex, WASP family proteins, and members of the Formin and Spire families in lamellipodia2,4,5.
In addition to actin polymerization, myosin II activity is required for generating contractile forces at the lamellipodium and the anterior lamella. These contractions, also defined as cell-edge retractions, can also result from depolymerization of dendritic actin at the cell periphery and are critical for developing the lamellipodial leading edge and allowing the protrusion to sense the flexibility of the extracellular matrix and other cells and determine the direction of migration6,7,8. Cell edge protrusions that cannot attach to the substrate will form peripheral membrane ruffles, sheet-like structures that appear on the ventral surface of lamellipodia and lamella and move backward relative to the direction of migration. As the lamellipodium fails to attach to the substrate, a posterior lamellipodium forms underneath it and mechanically pushes the first lamellipodium toward the upper ventral surface. The actin filaments in the ruffle that were formerly parallel to the substrate now become perpendicular to it, and the ruffle is now positioned above the advancing lamellipodium. The ruffle that moves backward falls back into the cytosol and represents a cellular mechanism for recycling lamellipodial actin9,10.
Here, we describe an assay for the measurement of cell-edge protrusion dynamics. The protrusion assay uses time-lapse video microscopy to measure single cell-edge protrusion dynamics for 10 min during the spreading phase of the cell. Protrusion dynamics are analyzed by generating kymographs from these movies. In principle, a kymograph imparts detailed quantitative data of moving particles in a spatiotemporal plot to yield a qualitative understanding of cell edge dynamics. The intensity of the moving particle is plotted for all image stacks in a time versus space plot, where the X-axis and the Y-axis represent time and distance, respectively11. This method uses a manual kymograph analysis with ImageJ to get detailed quantitative data, enabling retrieving information from movies and images in case of low signal-to-noise ratio and/or high feature density, and the analysis of images acquired in phase-contrast light microscopy or poor image quality.
The cell-edge protrusion dynamics assay described herein is a fast, simple, and cost-effective method. As such, and because it has been shown to directly correlate with cell migration11,12, it can be used as a preliminary method for testing cytoskeletal dynamics involved in cell motility before deciding to perform more resource-demanding methods. Moreover, it also enables quantitative measurement of how genetic manipulations (knockout, knockdown, or rescue constructs) of cytoskeletal proteins impact cytoskeletal dynamics using a straightforward platform. The assay is an instructive model for exploring cytoskeletal dynamics in the context of cell migration and could be used for elucidation of the mechanisms and molecules underlying cell motility.
All methods described in this protocol have been approved by the institutional Animal Care and Use Committee (IACUC) of Bar-Ilan University.
NOTE: A step-by-step graphical depiction of the procedure described in this section appears in Figure 1.
1. Cell culture
NOTE: The cells used in the protocol are mouse embryonic fibroblasts (MEFs) that were generated from E11.5-13.5 embryos of wild-type C57BL/6 mice. Primary MEFs were generated according to the Jacks laboratory protocol13. Cells from five different embryos were pooled together and immortalized by infection with a retroviral vector expressing SV40 large T antigen followed by selection with 4 mM Histidinol for 3 weeks.
- Culture cells in tissue culture plates containing Dulbecco's Modified Eagle Medium (DMEM) containing 1 g/L glucose, 1% glutamine, 1% penicillin-streptomycin, and 10% fetal bovine serum (FBS) (see Table of Materials), in a humidified incubator with 5% CO2 at 37 °C.
- Culture the cells until 90%-95% confluency and split at a ratio of 1:5 every 2-3 days.
2. Glass-bottom dish coating
NOTE: Glass-bottom dish coating should be performed in the tissue culture hood in sterile conditions.
- Add 2 mL of 1N HCl solution at the center of a glass-bottom dish (Table of Materials) and incubate for 20 min at room temperature (RT).
NOTE: This stage is meant to remove residue from the glass that may interrupt imaging later.
- Wash the glass-bottom dish three times with 2 mL of 1x PBS (Table of Materials) each.
- Dilute fibronectin (see Table of Materials) to 10 µg/mL in 1x PBS. Add 200 µL of the diluted fibronectin solution to the glass center of the dish. Incubate at 37 °C (tissue culture incubator) for 1 h.
NOTE: Alternatively, the glass-bottom dish can be incubated overnight at 4 °C on a flat surface.
- During fibronectin coating incubation time, prepare 1% BSA (Table of Materials) solution in 1x PBS, filter through 0.2 µm, and denature by incubating at 70 °C for 30 min in a pre-warmed water-bath.
- Wash the coated glass-bottom dish three times with 2 mL of 1x PBS each.
- Add 2 mL of denatured BSA solution to the glass center and incubate for 1 h at 37 °C (tissue culture incubator).
NOTE: Alternatively, the glass-bottom dish can be incubated overnight at 4 °C on a flat surface.
- Wash the glass-bottom dish 3 times with 2 mL of 1x PBS each.
NOTE: Incubation of glass-bottom dishes with fibronectin should be performed for at least 1 h at 37 °C to coat the surface properly. Shorter incubation time may not produce proper coating, and as a result, cell phenotype may not be related to integrin activation. The BSA layer is inert and will not affect protrusion dynamics and is meant to block free potential sites for integrin-independent cell adhesion. The BSA must be denatured before coating to prevent it from inducing cell apoptosis, which can influence cell-edge dynamics.
3. Preparation of cells for imaging
- Sixteen to eighteen hours before the experiment, plate the cells to reach 70%-80% confluence on the following day. For MEFs, plate 0.7 x 106 cells per 10 cm diameter tissue culture plate a day before performing the experiment.
NOTE: For a successful protrusion experiment, it is important that the cells will be used during their logarithmic growth phase. To achieve this, cells should reach 70%-80% confluence on the day of the experiment. A higher density of cells will result in a longer spreading time and/or impediment in attachment during the experiment.
- On the experiment day, add 2 mL of trypsin solution (Table of Materials) per 10 cm diameter tissue culture plate and incubate for 2-3 min in a tissue culture incubator until cells detach. Inactivate trypsin by adding 5 mL of the complete medium.
- Count the cells using a hemocytometer and plate 20,000 cells in 2 mL of the complete medium on a glass-bottom dish coated as above (section 2).
NOTE: The number of cells for plating depends on the size and type of cells. For larger or more spread cells, plate only 10,000 cells to have enough cells for imaging. It is important to pick single cells that do not touch as cell-to-cell contact can alter cell-intrinsic spreading behaviors.
- Incubate the glass-bottom dishes with plated cells in the tissue culture incubator for 15 min.
NOTE: During this stage, cells adhere to the fibronectin substrate and spread over it. When measuring protrusions during cell spreading, cells should be allowed to spread for 15 min after plating and before imaging. Imaging can be performed within a time window between 15 min to ~1 h following plating, and in any case, before the cells start migrating.
4. Microscope setup and imaging
NOTE: Various live-cell microscopy systems are available. The system used here is a Leica AF6000 inverted microscope equipped with CO2 and heating units and is attached to an ORCA-Flash 4.0 V2 digital CMOS camera.
- Turn on the heating unit at least 1 h before imaging and set it at 37 °C.
- Turn on the CO2 unit at least 10 min before imaging and set it at 5% CO2.
- Switch on the microscope, camera, and computer.
- Open the microscope acquisition software (see Table of Materials). Choose the folder to save captured images and type a file name. Save every movie as a new file.
- Set the magnification on a 40x dry lens, phase-contrast. Set the time interval on 5 s, total movie duration 10 min.
- After 15 min incubation of the plated cells, place the glass-bottom dish with adhered cells into the adapter and fix it. Insert the adapter with the dish into its slot in the microscope stage.
- Take off the dish cover and place the CO2 lid instead. Open the CO2 valve.
NOTE: Make sure the CO2 cover is clean before placing it on top of the glass-bottom dish. A dirty cover will reduce the quality of movies. Wipe the underside of the CO2 lid with a lint-free wipe soaked with 70% ethanol to remove dust and dirt. Wipe a second time with a dry lint-free wipe.
- View the cells and find an appropriate cell for imaging. Make sure the cell is in focus and start movie acquisition.
5. Image analysis
NOTE: Image analysis is performed using ImageJ (Table of Materials) as following:
- Open the acquired movie in ImageJ.
- Using the Straight tool in the main toolbar, make eight lines of 20 arbitrary units each perpendicular to the protrusions, including the lamella and cell edge, in a radial arrangement every 45°, as shown in Figure 2A.
- In the main toolbar, go to Image > Stacks > Reslice. This will yield a kymograph picture, which describes the movement of single points within the cell membrane (Figure 2B). This action should be performed for every line out of the eight lines separately.
- Extract and manually count from the respective kymograph images the number of protrusions, retractions, and ruffles in each of the eight regions in the cell, marked by the grid lines. These numbers represent the frequency of protrusions, retractions, and ruffles per 10 min (Figure 2C, D).
NOTE: Ruffles can be distinguished from other structures based on their dark appearance in phase-contrast microscopy and their centripetal movement, which starts at the cell edge and ends at the border of the cell body, which can be observed in the acquired movies. Of note, when quantifying protrusion, retraction, and ruffle frequency, movies should be observed as a control for quantification and especially for defining ruffles.
- Determine the protrusion persistence, distance, and velocity by kymography analysis. For each of the generated kymographs, the X-axis represents distance, and the Y-axis represents time.
- To measure protrusion distance, follow steps 5.6.1-5.6.2.
- Draw a perpendicular line from the base of the protrusion to the highest peak of the protrusion. Press M in ImageJ to measure the length of the line in pixels.
- To convert the length from pixels to μm, ensure that the pixel to μm ratio is known.
NOTE: The μm to pixel ratio is the physical length of a pixel on the CCD camera / total magnification. The pixel size is characteristic of each type of camera. For example, for the camera used in this study, the pixel size is 6.5 μm x 6.5 μm, the physical length is 6.5 μm and the magnification we used is 40x. Therefore, the μm to pixel ratio of our camera is 0.1625 μm/pixel. In the analysis, for a line in the length of 30 pixels, the protrusion distance would be 30 pixels x 0.1625 μm/pixel = 4.875 μm (Figure 3).
- To measure protrusion time (persistence; the amount of time a protrusion spends protruding before retracting), follow steps 5.7.1-5.7.2.
- Draw a horizontal line from the beginning of the protrusion (left to right) to the region of the highest peak. Press M in ImageJ to measure the length of the line in pixels.
- To convert the length from pixels to minutes, calculate the pixel to min ratio. This value depends on the interval between images.
NOTE: In this example, the interval between images is 5 s, the min/pixel ratio is 0.0833, and the horizontal line length is 8 pixels. Therefore, the protrusion time is 8 pixels x 0.0833 min/pixel = 0.6664 min.
- Measure and calculate the retraction time similarly to protrusion time for a line drawn horizontally at the base of the protrusion from where the peak of the protrusion is to its base on the right (line X2 in Figure 3).
- Calculate the protrusion velocity by dividing protrusion distance by protrusion time. Calculate the retraction velocity by dividing protrusion distance by retraction time. In this example, protrusion velocity is calculated as 4.875 μm/0.6664 min = 7.315 μm/min., and the retraction velocity is identical because the line representing time is at the same length.
NOTE: In comparing different cell types, i.e., cells expressing wild type and mutant constructs of the same protein, it is imperative to perform a blinded analysis, so no bias is introduced.
In the experiment described in Figure 2, immortalized MEFs were plated on glass-bottom dishes pre-coated with fibronectin to activate integrin-mediated signaling, blocked by denatured BSA, to block free potential sites for cell adhesion which is not dependent on integrin activation. To reach the logarithmic growth phase at 70%-80% confluence of cells on the day of the experiment, 0.7 x 106 MEFs were plated in a 10 cm diameter tissue culture plate 16 h before the experiment. On the experiment day, cells were trypsinized and counted, and 20,000 cells were plated on a fibronectin/BSA-coated glass-bottom dish. The dish was incubated for 15 min at 37 °C to allow attachment and spreading of the cells before imaging. Following incubation, the plate was placed in a microscope incubator chamber (37 °C, 5% CO2), and single cells were imaged using a 40x dry lens in phase light. Imaging was performed between 15 min to 1 h following plating before the cells started migrating. Images were acquired every 5 s for 10 min, which yielded 121 images per cell (Supplementary Movie 1).
Image analysis was performed using ImageJ. Using the Straight tool in the main toolbar, 20 arbitrary unit-long straight lines were made on a radial grid in the same places every 45 degrees in all cells (Figure 2A). To generate a kymograph, we used the Image > Stack > Reslice commands, which yielded a kymograph picture describing the movement of single points within the cell membrane (Figure 2B-D). The number of protrusions, retractions, and ruffles formed during 10 min of the movie in each of the eight regions in the cells, which are marked by the grid lines, was extracted, manually counted from the respective kymograph images, and plotted in a graph as protrusions/retractions/ruffles frequencies per 10 min. The average frequencies obtained were 5.1/10 min for protrusions and retractions and 2.1/10 min for ruffles (Figure 2E).
The protrusion distance, protrusion time, retraction time, protrusion and retraction velocities were calculated from the generated kymographs. In the representative kymograph in Figure 3, protrusion distance was 30 pixels x 0.1625 μm/pixel = 4.875 μm, protrusion time was 8 pixels x 0.0833 min/pixel = 0.6664 min, retraction time was 8 pixels x 0.0833 min/pixel = 0.6664 min. Protrusion and retraction velocities were calculated as 4.875 μm/0.6664 min = 7.315 μm/min.
When measuring cell-edge protrusion, it is important to choose cells that are in their spreading phase. An example of a proper cell for analysis appears in Figure 2A and Supplementary Movie 1. Following kymography analysis, the protrusions, retractions, and ruffles can be easily distinguished in this experiment. An example of a wild-type fibroblast that is not appropriate for analysis is shown in Figure 4. Following kymography analysis, in lines (slices) 1, 3, 5, 7, for instance, clear protrusions cannot be distinguished. In this case, the cell finished spreading but did not start moving yet, and therefore not many membrane movements can be observed.
Figure 1: Experimental stages of the protrusion assay. (A) A 1N HCl solution is added to the glass-bottom dish for 20 min. (B) Following washes in PBS, 10 µg/mL fibronectin solution is added to the glass part of the dish and incubated for 1 h at 37 °C. (C) The dish glass bottom is blocked by incubation in 1% denatured BSA for 1 h at 37 °C. (D) A tissue culture plate of 70%-80% confluent fibroblasts is trypsinized and counted (E) 20,000 cells are plated in a glass-bottom dish and (F) incubated for 15 min at 37 °C to allow cells to spread. (G) The plate is placed in a microscope humid chamber with 37 °C in 5% CO2, and live imaging is performed by phase-contrast light microscopy. (H) Cell movies and images are subjected to kymography analysis by Image J. Please click here to view a larger version of this figure.
Figure 2: Protrusion assay image analysis. (A) Representative image of MEF in ImageJ with indicated eight membrane cross-sections for quantification during 10 min. Scale bar, 20 µm. (B) Generation of a kymograph in Image J. (C) Representative kymograph from cross-section on which protrusions, retractions, and ruffles can be distinguished. (D) Resulting kymographs after image analysis during 10 min in Image J using the Reslice command. (E) Quantification of protrusions, retractions, and ruffles frequencies per 10 min from the analyzed movie and kymograph. Average protrusion frequency per 10 min = 5.1, average retraction frequency per 10 min = 5.125, average ruffles frequency per 10 min = 2.1. Eight kymographs were analyzed from one movie. Please click here to view a larger version of this figure.
Figure 3: Analysis and quantification of protrusion persistence, distance, and velocity. In the representative kymograph, the X-axis represents time in min (left to right), and the Y-axis shows the distance in μm. X1 represents protrusion time (persistence), X2 represents retraction time, and Y represents the protrusion distance. Protrusion velocity is calculated by dividing protrusion distance (Y) by protrusion time (X1). Retraction velocity is calculated by dividing protrusion distance (Y) by retraction time (X2). In this example, protrusion distance was 30 pixels x 0.1625 μm/pixel = 4.875 μm, protrusion time was 8 pixels x 0.0833 min/pixel = 0.6664 min., retraction time was 8 pixels x 0.0833 min/pixel = 0.6664 min. Protrusion/retraction velocity was calculated as 4.875 μm/0.6664 min. = 7.315 μm/min. Please click here to view a larger version of this figure.
Figure 4: Example of a cell that should be excluded from the analysis. (A) An example of a cell that is not appropriate for analysis. Scale bar, 20 µm. (B) The kymography analysis of this cell shows, especially in re-slice 1,3,5,7, no clear protrusions. In this case, the cell finished spreading but did not start moving yet, and therefore not many membrane protrusions can be observed. Please click here to view a larger version of this figure.
Supplementary Movie 1. MEF was plated on a fibronectin-coated glass-bottom dish and imaged using time-lapse phase-contrast video microscopy for 10 min using 40x/1.4 NA dry objective. Time is indicated in seconds. Scale bar, 10 μm. Please click here to download this Movie.
Cell-edge protrusion dynamics, comprised of protrusions, retractions, and ruffles, is both a prerequisite and a potential rate-limiting event in cell motility. Here we describe a fast and simple method for measuring the dynamics of cell-edge protrusions during spreading. This method enables short-time imaging, generates a significant amount of data, does not require fluorescent labeling of cells or expensive fluorescent microscopy equipment, and could be used as a preliminary method for testing cytoskeletal dynamics involved in cell motility before deciding to perform more resource-demanding methods. Moreover, one can use knockout or knockdown cells and/or protein mutants in this assay as a fast and simple tool to identify critical proteins and potential signaling mechanisms involved in cytoskeletal dynamics.
Of note, it is important to choose the correct cells for protrusion analysis. Cells are incubated for 15 min before movies are acquired to allow spreading. If one decides to measure protrusion during spreading (as opposed to measuring protrusions during migration), then only cells that are in their spreading phase during movie acquisition should be imaged. Cells that did not start spreading not be appropriate for kymography analysis. Cells that completed their spreading phase but did not start moving (Figure 4) will not be appropriate for analysis as well. The movement of their nucleus can distinguish these cells: during spreading, the nucleus is stationary, while during cell migration, the nucleus is dynamic and localizes at the rear side of the cell to construct a leading edge-centrosome-nucleus axis towards the direction of migration. Another common issue in the later stages of imaging is a situation in which cells touch each other. Such movies should be excluded from analysis, as interactions and signals from neighboring cells can interfere with cell-edge protrusion dynamics.
In this manuscript, we describe the analysis of cell-edge protrusion dynamics using phase-contrast light microscopy. This method can be expanded to measure the dynamics of intracellular components with fluorescence microscopy as well. Such common usage of fluorescent kymography is often described for measuring the dynamics of cytoskeletal structures within cells. For example, Dogget and Breslin have used kymography of GFP-actin transfected HUVEC cells to analyze actin stress fiber dynamics and turnover14.
This protocol and several other previous papers used fibroblasts plated on fibronectin for the cell-edge protrusion assay as well as for two-dimensional cell motility assays. Fibroblasts are commonly used for motility assays and other related assays such as the cell-edge protrusion dynamics assay because they are mesenchymal and motile and have clear cytoskeletal structures such as lamellipodia, filopodia, and focal adhesions. Although we do not describe the assay for other cell types and substrates, this method could easily be modified. For example, in the first documentation of the lamella dynamics assay, which we and others modified to become the cell-edge protrusion dynamics assay11, the authors used keratinocytes stimulated to migrate by EGF in a scratch assay, demonstrating that other cell types and other stimulations could be applied to this assay. Moreover, although we describe the measurement of cell-edge protrusion dynamics during cell spreading, the same method could be used by measuring the dynamics of protrusions during migration of cells, as demonstrated, for example, in Bear et al.12 and Hinz et al.11.
Indeed, several labs have used this assay on MEFs to elucidate cytoskeletal dynamics and signaling mechanisms in the past. For example, Miller et al. had previously used the protrusion assay to demonstrate that Abl2/Arg mediates the contact between actin and microtubules at the cell-edge15. Bryce et al. demonstrated using the assay that cortactin knockdown cells have impaired cell motility which co-insides with impairment in the persistence of lamellipodial protrusions. This defect results from impairment in the assembly of new adhesions in protrusions16. Lapetina et al. used the cell-edge protrusion assay in Abl2/Arg knockout and cortactin knockdown cells rescued with mutants of the two proteins to elucidate an Abl2/Arg-mediated regulation mechanism cell-edge protrusions17. Using the same assay, Miller et al. have also demonstrated that Arg regulates N-WASP-mediated actin polymerization and consequent cell-edge protrusion dynamics18. We have recently used the cell edge protrusion assay to demonstrate that the non-receptor tyrosine kinase Pyk2 regulates the dynamics of protrusions and subsequent cell migration via direct and indirect interactions with the adaptor protein CrkII. In this paper, we have used Pyk2-WT and Pyk2-/- and Crk-WT and knockdown MEF, rescue mutants, and epistasis experiments to elucidate the molecular interactions and signaling hierarchy between the two proteins during cell-edge protrusion. This novel complex regulation mechanism enables fine-tuning of cell-edge protrusion dynamics and consequent cell migration on the one hand together with tight regulation on cell motility on the other hand19. Using the cell-edge protrusion assay, the above papers and others that followed have significantly increased our knowledge of the regulation mechanisms of cell-edge protrusions in particular and cell migration in general.
The authors have no conflict of interests to disclose.
This work was supported by grants NIH MH115939, NS112121, NS105640, and R56MH122449-01A1 (to Anthony J. Koleske) and from the Israel Science Foundation (grants number 1462/17 and 2142/21) (to Hava Gil-Henn).
|10 cm cell culture plates||Greiner||P7612-360EA|
|Bovine serum albumin (BSA)||Sigma-Aldrich||A7906|
|Dulbecco’s modified Eagle medium (DMEM)||Biological Industries, Israel||01-055-1A||Medium contains high glucose (4.5 g/L D-glucose)|
|Dulbecco’s phosphate buffered saline (1xDPBS)||Biological Industries, Israel||02-023-1A|
|Fetal bovine serum (FBS)||Biological Industries, Israel||04-001-1A|
|Fibronectin from human plasma, liquid, 0.1%, suitable for cell culture||Sigma-Aldrich||F0895|
|Glass bottom dishes||Cellvis||D35-20-1.5-N||35mm glass bottom dish, dish size 35 mm, well size 20mm, #1.5 cover glass (0.16-0.19 mm).|
|ImageJ software||NIH||Feely available at: https://imagej.nih.gov/ij/download.html|
|LAS-AF Leica Application Suite 3.2||Microscope acquisition software equipped with an ORCA-Flash 4.0 V2 digital CMOS|
|Leica AF6000||Leica||Inverted bright field microscope (40x, NA 1.3 ) equipped with phase-contrast optics, an incubator, and CO2 unit with LAS AF acquisition software equipped with an ORCA-Flash 4.0 V2 digital CMOS camera .|
|L-glutamine solution||Biological Industries, Israel||03-020-1B|
|ORCA-Flash 4.0 V2 digital CMOS camera||Hamamatsu Photonics|
|Penicillin-streptomycin solution||Biological Industries, Israel||03-031-1B|
|Trypsin-EDTA solution B (0.25%), EDTA (0.05%)||Biological Industries, Israel||03-052-1A|
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