In this protocol, we describe the selection and preparation of appropriate cells for micromanipulation and the use of a piezoelectric micromanipulator to reposition chromosomes within those cells.
This method can help answer key questions in cell biology, such as, how important is tension in satisfying the spindle checkpoint, and what is the role on spindle attachment in chromosome behavior in miosis? The main advantage of this technique is that it is a nonlethal technique that allows the experimenter to apply tension to, or reposition chromosomes in a living cell. To begin, prepare a 75 by 25 millimeter slide with a 20 millimeter diameter circular hole cut out of the center.
Then, run a 25 by 25 millimeter number 1.5 cover slit through a bunsen burner flame for two seconds. Apply vacuum grease around the edge of the hole in the glass slide. Next, place the cover slip over the hole and press it to form a tight seal.
Flip the slide over, and fill the newly created dissection well with halocarbon oil. After obtaining some adult male crickets, use dissecting scissors to cut through the dorsal surface parallel to the long axis of the abdomen, directly behind the wing buds. Then, gently squeeze the abdomen to push the testes through the cut in the exoskeleton.
Using forceps, place the isolated testes into the dissection well. Under the dissecting microscope, use fine pointing forceps to divide the testes into smaller pieces, and remove any fat. Then, spread the contents of the testes under the oil on the surface of the cover slit.
Continue to spread the contents of the testes until the spread portion is barely visible to the naked eye. Additional oil loaded dissection wells may be used if necessary. First, place the end of a glass tube in the flame of bunsen burner.
Hold the end of the tubing to create a 150 degree angle and an extended area of narrow glass tubing. Break the tubing in the thin region so that the thin region extending from the previously created angle is approximately 10 millimeters long. Using the microforge, melt the tip of the glass needle, forming a 45 degree angle between the needle and the platinum wire of the microforge.
Then, pull the glass away from the wire while simultaneously turning off the heat. Place the previously prepared slide onto the stage of an inverted phase contrast microscope. Then, find the dividing cells and center them in the field of view using the lowest magnification possible.
Place the microneedle into the needle holder of the micromanipulator. Then, manually position the microneedle in the light path of the microscope. Focus the microscope several focal planes above the plane containing the cells.
Next, use the joystick controller to reposition the microneedle several times to find the shadow of the needle in the x and y axes. Readjust the needle position until the position of the tip is visible, then, adjust the position of the needle along the z axis until the tip is in focus. Next, refocus on the cells, and focus the microscope just above the cell plane.
Then, readjust the position of the needle so that the tip is in focus in this focal plane. Repeat this process using the higher magnification objective and a higher sensitivity setting. Refocus on the cells, and adjust their position, so they remain in the center of the field of view.
Use the joystick to control the microneedle as it pushes chromosomes around inside the cell. Ensure the needle tip remains in the plane above the cells. To move the chromosome, focus on a chromosome near the top of the cell.
Then, adjust the z axis on the joystick to bring the needle tip into focus, and move the needle with the joystick in x and y. Place the tip of the needle directly on the chromosome of interest, and push it in the desired direction. Apply enough tension to separate the chromosome from the spindle.
Finally, place the chromosome anywhere within the cell. Using this protocol, it is possible to reposition, apply tension to, and completely detach a chromosome from a spindle using micromanipulation. In this example, two manipulated chromosomes were kept from reattaching to the spindle by being continually nudged with a micromanipulation needle.
Once mastered, samples can be prepared for manipulation, and the micromanipulator can be positioned in 15 to 20 minutes. Micromanipulation experiments can take anywhere from a few seconds to several hours. While attempting this procedure, it's important to remember to manipulate chromosomes on the side of the cell farthest from the cover slip.
After its development, this technique paved the way for researchers in the field of cell biology to explore the spindle checkpoint in grasshoppers and praying mantis.
