1. Coverslip Washing and Sterilization
2. Treatment of Cells, Transfection, and Seeding onto Coverslips
3. Assembly of Microscopy Imaging Chamber
4. Microinjection Procedure
5. FRAP Procedure
6. Photoactivation Procedure
NOTE: Software, microscope setup, and settings, except for the laser power, are similar to those for FRAP. In photoactivation, an important difference as compared to FRAP, is that a 405nm-laser power significantly lower than that employed for photobleaching must be used, to activate PA-GFP without simultaneously photobleaching it.
7. Data Analysis and Presentation of FRAP Results
NOTE: The method presented is used for investigating the turnover of a protein accumulating at sites of dynamic actin assembly, in this case VASP, which associates with adhesion sites and the tips of protruding lamellipodia. We are analyzing its turnover at the lamellipodium tip, but the same principles of analysis can be applied for investigating the turnover of VASP or any other protein and other subcellular compartments.
8. Determining the Lamellipodial Actin Polymerization Rate by FRAP
9. Analysis of Protein Diffusion and Mobility Upon Photoactivation
NOTE: The method presented here describes the analysis of actin monomer mobility by employing photoactivation of actin fused to PA-GFP, as illustrated by visualization and quantification of protein diffusion through the cytosol.
Figure 1g, h show phase contrast images of an NIH3T3 fibroblast cell prior and 10 min post-microinjection of Rac1, which is a small Rho-family GTPase capable of inducing lamellipodia formation through its interaction with the WAVE complex. The cell is first visualized before the microinjection (Figure 1g), to confirm its viability and morphology, e.g., lack of lamellipodia. At 10 min post-microinjection, the cell has clearly changed its morphology, which is expected from this treatment, and indicates a successful injection (Figure 1h).
For simplicity and clarity, we next provide exemplary results for FRAP and photoactivation analysis in cells, which have not been additionally microinjected.
Analysis of the turnover of EGFP-tagged VASP at the lamellipodium tip is shown in Figure 2a-f. Note that VASP in addition targets to nascent and focal adhesions, small and elongated dots in the cell interior18,19. The fluorescence intensity of a lamellipodial region with a clear VASP accumulation at the tip was bleached and measured for each time frame, by following the contour of the ROI before, during, and after bleaching as the lamellipodium protrudes forwards. As bleached EGFP-VASP proteins are being recycled by non-bleached molecules at these sites, gradual recovery of fluorescence is observed (Figure 2b). The FRAP recovery curve obtained in this fashion and normalized to the pre-bleach intensity (expressed as 1) can be seen in Figure 2c. Photobleaching efficiency can vary and was approximately 20% of the value before bleaching in this example, as determined from the value at t0 (the first frame after photobleaching). The increase of fluorescence reaches a plateau in the example shown at roughly 80% of the fluorescence before bleaching. In a static structure during the time course of the experiment, such as a focal adhesion, the difference between the pre-bleach intensity and the plateau fluorescence reached after recovery is defined as the immobile fraction (IF, red arrow in Figure 2c, e), whereas the amount of fluorescence recovered between the time of bleaching and full recovery is defined as the mobile fraction (green double-headed arrow in Figure 2c, e). Note that in a dynamically changing structure such as the lamellipodium tip analyzed here, the extent of the IF might not only represent immobile molecules, but also derive from a reduction of protrusion speed, as EGFP-VASP intensity is known to depend on this parameter18. To calculate the half-time of recovery, a fit curve was created on Sigma plot (Figure 2d). In this case, the value of the "b" parameter extracted from solving Equation 2 is equal to 0.0754, which when applied to the logarithmic function (Equation 4) results in an estimated half-time of recovery of 9.19 s (Figure 2d, far right panel), which is relatively fast in this particular cell as compared to the average published previously5. It must be noted that recovery half-times may sometimes vary significantly from cell to cell within the same population. Therefore, for obtaining representative results, we recommend determining this parameter as an average from at least 15-20 cells. To illustrate the degree of variance, arithmetic means of EGFP-VASP recovery averaged from 15 cells for each time-point were generated (Figure 2e), and average curve fits created and displayed in an analogous fashion (Figure 2f).
The polymerization rate of the lamellipodial actin network comprises the sum of forward network protrusion and retrograde flow. FRAP can be applied for measuring the actin polymerization rate by transfecting cells (in this case B16-F1) with EGFP-tagged β-actin and photobleaching a protruding lamellipodial region (Figure 2g). For analysis of lamellipodial actin network polymerization, the fluorescence recovery upon bleaching of EGFP-tagged β-actin is assessed over time. As the polymerization of actin monomers progresses at the barbed ends of lamellipodial actin filaments (which all point towards the front20), the network is constantly translocated rearwards and progressing forwards, the rates of which can be easily obtained through polarized recovery of fluorescence upon photobleaching. Fluorescence recovery of the lamellipodium is complete as soon as the bleached zone has reached the transition zone between the rear part of the lamellipodium and the lamella, which is characterized by a lower density of more horizontally-arranged filament bundles turning over much more slowly than what is observed in the lamellipodium. As illustrated in Figure 2g, fluorescence recovery can be visualized as a line horizontal to the edge and flowing backwards towards the lamella, which allows measuring the distances of protrusion and retrograde flow (individually represented in the far right panel of Figure 2g as orange and red double-headed arrows, respectively).
We have also applied photoactivation in B16-F1 cells transfected with PA-GFP-actin to track the mobility of actin monomers within the cytosol and the rate of their incorporation within protruding lamellipodia. As illustrated in Figure 3a, b, a cytosolic region was photoactivated by exposure to a 405 nm laser, while images were acquired on the GFP channel every 1.5 s for visualizing the distribution of GFP-tagged, photoactivated actin. Photoactivated GFP-actin can be seen diffusing out of the cytosolic region in Figure 3b. The rate of fluorescence intensity decrease in the photoactivated cytosolic region is represented as the percentage of the initial intensity at t0 (first frame after photoactivation; Figure 3c). Photoactivated actin also integrates at the tips of lamellipodia, where new actin monomers are added to the growing barbed ends of elongating actin filaments during protrusion. To estimate the rate of lamellipodial incorporation, we measured the fluorescence intensity over time of a two-dimensional contour/region of approximately 5 µm in width and 1 µm in height; the region was constantly re-positioned at the tip of the lamellipodium as it protruded. Actin incorporation was represented as the percentage of fluorescence intensity of the photoactivated cytosolic region at t0 (Figure 3d). As elongation of actin filaments progressed, new actin monomers were incorporated at the lamellipodial front. A fraction of these actin monomers was stochastically derived from the cytosolic pool where monomers were photoactivated. This results in the rapid increase of fluorescence in lamellipodia in the first 20 s after photoactivation. As new monomers are being added to the lamellipodial front, previously incorporated actin monomers flow with filaments towards the lamella by retrograde flow. Over time, the ROI is completely filled with fluorescent monomers and a plateau in fluorescence is reached (Figure 3d). A gradual drop in fluorescence is then observed when, following diffusion of photoactivated monomers throughout the cell, non-photoactivated actin monomers are increasingly being re-added to the lamellipodial front. This decrease in fluorescence will find a new plateau, which will be reached as soon as a balance in the entire cell between photoactivated and non-photoactivated monomers is reached (data not shown).
The mobility of actin monomers throughout the cytosol was derived by measuring fluorescence intensities in regions of equal size positioned distally from the photoactivated region (exemplified on Figure 3a by color coded regions labeled R1-R5). As illustrated in Figure 3e, fluorescence intensity in each of these regions is gradually decreasing away from the cytosolically photoactivated region, as the fraction of photoactivated actin monomers becomes increasingly diluted with non-activated (i.e., non-fluorescent) monomers. Furthermore, the peak of fluorescence is reached later: the more distant the measured region is located from the photoactivated region, the longer the time that is required for actin monomers to diffuse into these regions. A representative value for the degree of actin monomer infiltration into each region can be derived by quantifying the half time of reaching the fluorescence plateau. The more distant the region, the longer it takes for the photoactivated actin to diffuse into it, and thus more time is required for the fluorescent plateau to be reached, ultimately leading to a higher t1/2 value (Figure 3e).
Figure 1: Imaging chamber assembly and microinjection procedure. (a) Imaging chamber components. (b) Silicone grease is carefully smeared around the opening of a plastic sealer. (c) The coverslip is positioned with the cell-side facing up into the center of the imaging chamber opening. (d) A secure seal is established by positioning the plastic sealer on top of the coverslip and by tightening the side clamps. (e) Microscopy medium is pipetted into the chamber slot. (f) The imaging chamber is positioned on the microscope stage, heat detector and electrodes are linked to a heating unit pre-set to 37 °C, and cells are allowed to adapt for at least 30 min before microscopy is initiated. In this example, the microscope stage is also equipped with a micromanipulator for performing microinjections, and the microinjection needle is dipped into the medium covering the cell layer in the imaging chamber. (g) An NIH3T3 fibroblast cell is visualized before microinjection by phase-contrast microscopy. The red cross in the perinuclear compartment indicates the location of the future microinjection, which corresponds to a high cytoplasmic region due to the close proximity to the bulky nucleus. (h) 10 min following microinjection with Rac1, the cell reacts by prominent formation of lamellipodia around the entire cell periphery (indicated by green arrows). Please click here to view a larger version of this figure.
Figure 2: FRAP allows determining rates of protein turnover or lamellipodial actin polymerization. (a) Representative example of B16-F1 cell expressing EGFP-VASP before photobleaching of a lamellipodial region as indicated. Differently colored contours/shapes are labeled to indicate which regions were considered for fluorescence intensity measurements over time. Note the red contour marked with an exclamation mark, which labels a cytosolic region positioned in an area containing multiple vesicles and cell surface ruffles. Dynamic areas like this should be avoided for selecting regions of fluorescence reference, as they are characterized by strong short-term fluctuations of fluorescence, potentially causing inaccurate results. (b) Lamellipodial region of the EGFP-VASP expressing cell before and after photobleaching. The recovery of fluorescent signal after photobleaching within the region marked in purple is visualized over time. Arrow indicates the tip of a microspike, enriched for VASP likely due to the high density of actin filaments polymerizing there19. (c) An example of a FRAP recovery curve as derived from quantifying the fluorescent intensity of the photobleached lamellipodium (purple contour) in b. Red and green lines on the right indicate, respectively, immobile and mobile fractions. (d) A fit of the FRAP recovery curve in c (left panel) and an example of the calculation method used to derive the recovery half time (right panel). (e) An example of a FRAP recovery curve derived from averaging the fluorescence recovery curves of 15 cells, with SEM bars indicating the degree of variability within the sample population. (f) A curve fit derived from averaging the FRAP recovery curve fits of 15 cells (left panel) and an example of the calculation method used to derive the recovery half time (right panel). (g) Time-lapse panels of protruding lamellipodium of a B16-F1 cell expressing EGFP-tagged β-actin before and after bleaching of a lamellipodial region as indicated, followed by fluorescence recovery in the lamellipodium over time. On the far right panel, values measured for protrusion and retrograde distances are provided (in orange and red, respectively). Calculations under the image panels reveal how the sum of protrusion and retrograde distances are used to derive polymerization rate of the lamellipodial actin network. Please click here to view a larger version of this figure.
Figure 3: Photoactivation of PA-GFP-actin for monomer tracking throughout the cell. (a) A representative example of a B16-F1 cell expressing PA-GFP-actin before triggering photoactivation in a cytosolic region as indicated by the red circle (PA). Differently colored contours are labeled to indicate which regions were considered for fluorescence intensity measurements over time. (b) An illustration of the temporal distribution of PA-GFP-actin following photoactivation. Note the gradual reduction of fluorescence in the photoactivated, cytosolic region (red circle), as the photoactivated actin diffuses away from it. Due to their diffusion to the front and assembly into the network, photoactivated actin monomers are gradually enhanced in lamellipodia (cyan region) and throughout the cytosol (different color-coded regions) in a distance- and time-dependent fashion. (c) Representative, temporal decline of fluorescence within the photoactivated cytosolic region (red contour in b). (d) Temporal changes in fluorescence intensity in the lamellipodial region (cyan contour in b). (e) Curves representative of the temporal changes in fluorescence intensity of cytosolic regions (color-coded in b) due to positioning in variable distances from the area of photoactivation. Note how half-times of reaching the fluorescence plateau (indicated in legend on the right) increase with the distance of given region to the area of photoactivation, likely correlating with the increased times needed for diffusion of actin monomers into the respective region. Please click here to view a larger version of this figure.
B16-F1 mouse skin melanoma cells | American Type Culture Collection, Manassas, VA | CRL-6323 | |
NIH-3T3 cells | American Type Culture Collection, Manassas, VA | CRL-1658 | |
DMEM 4.5g/L glucose | Life Technologies, Thermno Fisher Scientific, Germany | 41965-039 | |
Ham’s F-12 medium | Sigma-Aldrich | N8641 | |
Fetal calf serum (FCS) | PAA Laboratories, Linz, Austria | A15-102 | |
Fetal bovine serum (FBS) | Sigma-Aldrich, Germany | F7524 | Lot054M3396 |
MEM Non essential amino acids | Gibco, ThermoFisher Scientific, Germany | 11140035 | |
L-Glumatine 200mM (100x) | Life Technolgies | 25030-024 | |
Pen-Strep 5000 U/mL | Life technologies | 15070063 | |
Sodium Pyruvate (100 mM) | Gibco, ThermoFisher Scientific, Germany | 11360-039 | |
Laminin | Sigma-Aldrich | L-2020 | |
Laminin coating buffer | Self-made: 50mM Tris ph7.4, 150mM NaCl | ||
Fibronectin from human plasma | Roche Diagnostics, Mannheim, Germany | 11 051 407 001 | |
Jetpei | Polyplus Transfection, Illkirch, France | 101-10N | |
JetPei buffer | Polyplus Transfection, Illkirch, France | 702-50 | 150mM NaCl |
PA-GFP-actin plasmid DNA | described in Koestler et al.2008 | ||
pEGFP-actin plasmid DNA | Clontech, Mountain View, CA, USA | ||
Rac1 protein for microinjection | Purified as GST-tagged version, and cleaved from GST prior to injection | ||
Microinjection buffer | Self-made: 100mM NaCl, 50mM Tris-HCl ph7.5, 5mM MgCl2, 1mM DTT | ||
Dextran, Texas Red, 70,000 MW, Lysine Fixable | Molecular Probes, Thermno Fisher Scientific, Germany | D1864 | |
Microscope circular cover glasses 15mm, No.1 | Karl Hecht, Aisstent, Sondheim, Germany | 1001/15 | |
Eppendorf Femtotips Microloader Tips | Eppendorf, Hamburg, Germany | 5242 956 003 | |
Eppendorf Femtotip Microinjection Capillary Tips | Eppendorf, Hamburg, Germany | 930000035 | |
Silicone Grease | ACC Silicones, Bridgewater, England | SGM494 | |
Aluminium Open Diamond Bath Imaging Chamber | Warner instruments | RC-26 | |
Automatic temperature controller | Warner Instruments | TC-324B | |
Microscope: Axio Observer | Carl Zeiss, Jena, Germany | ||
CoolSnap-HQ2 camera | Photometrics, Tucson, AZ | ||
Lambda DG4 light source | Sutter Instrucment, Novato, CA | ||
Laser source | Visitron Systems | ||
Eppendorf FemtoJet microinjector | Eppendorf, Hamburg, Germany | With built-in compressor for pressure supply | |
Nikon Narishige Micromanipulator system | Nikon Instruments, Japan | ||
Visiview software v2.1.4 | Visitron Systems, Puchheim, Germany | ||
Metamorph software v7.8.10 | Molecular Devices, Sunnyvale, CA | ||
Sigma Plot v.12 | Systat Software Inc. |
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 β-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 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.
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 β-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 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.
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 β-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 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.