The overall goal of the following experiment is to visualize the location and numbers of free f actin, barbed ends that are available for actin polymerization in neuronal growth cones that are exposed to axonal guidance cues. This is achieved by first bathing neurons cultured on cover slips in a saponin containing buffer to make the growth cones permeable. The cells are next exposed to a rumine actin containing buffer for four minutes to allow rumine actin to enter the growth cones and polymerize onto free f actin barbed ends.
Finally, the cells are chemically fixed and rinsed to preserve the distribution of the F actin. Barbed ends that have incorporated rumine actin. Based on fluorescence microscopy and quantitative image analysis results are obtained that show that attractive guidance cues increase the number of free f effectin.
Barbed ends at the leading edge of migrating growth cones. Hi, I am Paul Erno from the Department of Neuroscience at the University of Minnesota Medical School. Today we're gonna demonstrate preparing cultures of chick neuronal growth cones.
We'll demonstrate a method to visualize in growth cones, the barbed ins of Acton filaments, which are the dynamic cytoskeletal components that drive axons forward as they navigate through embryos to their synaptic targets. Though we use this method to investigate actin dynamics and nerve growth cones, it can be applied to other motile neural cells such as glia, neural crest cells, or migrating neurons. Demonstrating the procedures.
Today will be laboratory members, Bonnie Marek, a postdoctoral fellow and Jose San Miguel Ruiz, an MD PhD student To prepare video dishes used for labeling neuronal actin with rumine rinse 18 millimeters square or 24 millimeter circular glass cover slips with distilled water and bake them in dry heat for three months or longer. Other methods of cleaning cover slips can be substituted for the oven baking drill circular holes of the appropriate diameter into the bottoms of 35 or 50 millimeter plastic Petri or tissue culture dishes using a cork, boar or other instrument. Then scrape the edges of the drilled holes with scissors to create a smooth surface on both sides.
Next, a cleaned cover slip is glued over the hole in each dish with silicon aquarium cement and the dishes are rinse several times with sterile distilled water. The dishes are now ready for substrate coating. Apply 100 micrograms per milliliter solution of PDL and PBS to cover slips or video dishes and incubate for two to four hours in a humidified 38 degrees Celsius incubator.
Aspirate PDL solution and rinse cover slips three times in sterile distilled water air dry. Next, make a 1%nitrocellulose solution by dissolving five milligrams of nitrocellulose in five milliliters of amyl acetate. Apply 10 microliters of this solution to the top of a cover slip and tilt to spread it over the surface air dry approximately 10 minutes.
Finally, apply 250 microliters of 25 micrograms per milliliter laminin, or four micrograms per milliliter L one cam and PBS and spread it over the nitrocellulose surface. Incubate six to eight hours in a humidified 38 degrees Celsius incubator. Then in a tissue culture hood, aspirate the substrate and immediately add culture medium, ensuring that the substrate is not allowed to dry.Explan.
Cultures of dorsal root ganglia or DRGs or neuronal retinal tissues are prepared by dissecting embryonic. Day seven chick embryos. DRG implants are half a whole ganglion and retinal explants are one millimeter or less in diameter.
Under a dissecting microscope, use forceps to place the explan onto the cover slip. Then press it gently into the center of the cover slip to prevent movement while transporting it to the incubator. The dishes with cover slips are carefully moved and placed in a warm humidified incubator for overnight or for 18 hours.
During this period, axons of several hundred microns are extended from the explan. Prepare the rumine actin permeation buffer for use by adding the following components, saponin A TP and LOR three 50 fein. The composition of this buffer is described in the written protocol.
Prepare a separate solution using the same components plus 0.45 micromolar ermine non-muscle actin T rate and vortex to mix to perme and label the neuronal cells. Carefully remove from the incubator a culture dish with DRG or retinal implants and perform the following steps at room temperature. Using a pipette carefully remove the culture medium and add enough permeation buffer to cover the cells incubate for one minute.
Gently remove the permeation buffer and replace it with the buffer containing 0.45 micromolar rumine non-muscle actin incubate for four minutes. Finally, remove the rumine actin containing buffer and fix the neurons with 4%paraform aldehyde and 0.05%glutaraldehyde for five minutes. Gently rinse with PBS and mount with slow fade on three inch glass slides.
Use an epi fluorescence microscope equipped with a 60 x oil immersion objective lens to visualize the incorporation of R domine actin into f actin, barbed ends, and fluorescent fain labeling of f actin in growth cones for best quantification of R domine actin incorporation. All images should be collected in a single session using the same settings for the digital camera sensitivity gain and exposure for each image. Analyze the labeling from equivalent regions using metamorph image J or a similar image analysis software.
Several variations to this procedure can be performed as well. One variation involves immunochemistry to localize other proteins in addition to F fact and barbed ends. After fixing the incorporated rumine actin and rinsing with PBS antibodies against another growth cone component can be added, followed by rinsing blocking an incubation with a fluorescent secondary antibody appropriate for triple labeling with r domine actin and fluorescent fallin.
A third variation assesses the effects of guidance cues on F FACT and free barbed ends at a growth factor or guidance cue to culture dishes in the incubator for five to 15 minutes before removing the dish from the incubator and beginning the permeation procedure. This allows assessment of the short-term effects of growth factors or guidance skews on fac and free barbed ends. Alternatively, place a video dish with neuronal cultures on a warmed microscope stage and use a micropipet to release growth factors or guidance skews in in gradient neuro growth cone before beginning the permeation and rumine actin incorporation.
The growth factors can be introduced for as little as one to two minutes before beginning the permeation procedure. The permeation and fixation can be done while leaving the video dish on the microscope stage. This allows assessment of the local effects of a guidance cue gradient on the formation of F ACT and free barbed ends in growth cones.
This video shows the rapid expansion of the modal growth cone margin in response to the global addition of NGF. When A DRG growth cone is globally treated with nerve growth factor for five minutes, actin polymerization is stimulated at the leading margin and a bright band of rumine actin incorporation is seen around the growth cone periphery. This video shows a growth cone turning toward a micro pipette that releases NGF and creates an NGF gradient shown here.
When one side of A DRG growth cone is exposed to an NGF gradient for two minutes, it locally stimulates an increase in R domine actin incorporation into F act and free barbed ends. In this example, the distribution of phospho RIN ridin moin is revealed by immunochemistry. In addition to labeling F ACT and free barbed ends by R Domine actin incorporation when NGF is added globally here, DRG neurons were cot transfected with GFP actin and a dominant negative ezrin ridin moisan construct NGF was added five minutes prior to labeling of f actin barbed ends.
Note the growth cone transfected with the dominant negative ERM construct has few f actin barbed ends the growth cone leading margin, which contrasts with a nearby unresected growth cone. By combining this procedure with immunochemistry, the localization of f and bardens can be related to the distribution of other proteins that regulate actin dynamics and organization. While conducting this procedure, it's important to be careful not to disrupt the delicate structure of the motel growth cones.
