For rhodamine-actin labeling, neurons are cultured on glass coverslips placed in the bottom of 35 mm plastic dishes, or on coverslips glued into “video” dishes.
1. Preparation of Coverslips or “Video” Dishes
2. Preparation of Neuronal Cultures
3. Preparation of Rhodamine-actin Permeabilization Buffer
4. Permeabilization of Neuronal Cultures and Incorporation of Rhodamine-actin onto F-actin Barbed Ends
VARIATIONS OF THIS PROCEDURE
5. Variation One: Collect Live Cell Images Prior to Rhodamine-actin Labeling
6. Variation Two: Immunochemistry to Co-label Additional Proteins
7. Variation Three: Assessment of the Effects of Guidance Cues on F-actin Free Barbed Ends
8. Variation Four: F-actin Free Barbed End Labeling on Transfected Neurons
9. Representative Results
Figure 1. Global addition of NGF increases total F-actin and F-actin barbed ends at the growth cone leading margin. When a DRG growth cone is stimulated with nerve growth factor (NGF) for 5 minutes, actin polymerization is stimulated at the leading margin, and a bright band of rhodamine-actin labeling is seen around the growth cone periphery. Figure 1 compares rhodamine-actin incorporation into an unstimulated DRG growth cone and an NGF-stimulated DRG growth cone. The green fluorescence in the merged images is phalloidin labeling for F-actin in the growth cone and the red is rhodamine-actin labeling. A good control for the barbed end labeling is to add 10-6 M or higher cytochalasin B or D in the permeabilization buffer in steps 4.2 and 4.3. Cytochalasins cap F-actin barbed ends and inhibit rhodamine-actin binding to barbed ends. This should severely reduce or eliminate incorporation of rhodamine-actin into the cell. Scale bars, 10 μm.
Figure 2. An NGF gradient locally increases F-actin barbed ends. A micropipette that releases NGF is brought to one side of a DRG growth cone for 2 minutes, followed by labeling of F-actin free barbed ends. The images below show that exposure to a gradient of NGF locally stimulates an increase in F-actin free barbed ends in the growth cone region closer to the pipette. The merged image shows phalloidin in green and rhodamine-actin in red. Scale bar, 10 μm.
Figure 3. Activated ERM proteins accumulate at the growth cone leading margin. Immunocytochemical labeling of phospho-Ezrin/Radixin/Moesin (pERM) with F-actin free barbed ends. Gluteraldehyde (0.05%) and paraformaldehyde (0.05%) were added to the initial permeabilization buffer for one min to preserve ERM localization. Rhodamine-actin, red; pERM, green; phalloidin, blue. Scale bar, 10 μm.
Figure 4. Stimulation of actin polymerization requires active ERM proteins. Dissociated DRGs were co-transfected with GFP-actin and a GFP control plasmid or a dominant-negative Ezrin/Radixin/Moesin (DN ERM) construct. The use of GFP-actin allows identification of transfected cells after permeabilization. Here, NGF was added 5 min prior to labeling of F-actin barbed ends. Note the growth cone transfected with DN ERM has reduced barbed end levels at the growth cone leading margin (lower middle panel), which contrasts with a nearby untransfected growth cone (lower panels), or with the GFP control (upper middle panel). Rhodamine-actin, red; GFP-actin, green. Scale bars, 10 μm.
Material Name | Tipo | Company | Catalogue Number | Comment |
---|---|---|---|---|
18×18 mm coverslip | Gold Seal | 3305 | ||
35mm Petri dishes | Falcon | 351008 | ||
Aquarium cement | DAP 100% silicone aquarium sealant | Any hardware store | ||
Poly-D-lysine (mw > 300,000) | Sigma | P1024 | ||
Natural mouse laminin | Invitrogen | 23017-015 | ||
L1 CAM | R & D systems | 777-NC | ||
Alexa-fluor 350 phalloidin | Invitrogen (Molecular Probes) | A22281 | ||
Rhodamine non-muscle actin | Cytoskeleton, Inc. | APHR-A | ||
F12 Culture medium | Invitrogen (Gibco) | 21700-075 | ||
B27 | Invitrogen (Gibco) | 17504-044 | ||
Slowfade | Invitrogen | 536937 |
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.
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.
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.