Micromanipulation of Chromosomes in Insect Spermatocytes

Summary

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.

Abstract

The micromanipulation of chromosomes has been an essential method for illuminating the mechanism for chromosome congression, the spindle checkpoint, and anaphase chromosome movements, and has been key to understanding what controls chromosome movements during a cell division. A skilled biologist can use a micromanipulator to detach chromosomes from the spindle, to reposition chromosomes within the cell, and to apply forces to chromosomes using a small glass needle with a very fine tip. While perturbations can be made to chromosomes using other methods such as optical trapping and other uses of a laser, to date, no other method allows the repositioning of cellular components on the scale of tens to hundreds of microns with little to no damage to the cell.

The selection and preparation of appropriate cells for the micromanipulation of chromosomes, specifically describing the preparation of grasshopper and cricket spermatocyte primary cultures for the use in live-cell imaging and micromanipulation, are described here. In addition, we show the construction of a needle to be used for moving chromosomes within the cell, and the use of a joystick-controlled piezoelectric micromanipulator with a glass needle attached to it to reposition chromosomes within dividing cells. A sample result shows the use of a micromanipulator to detach a chromosome from a spindle in a primary spermatocyte and to reposition that chromosome within the cell.

Introduction

Micromanipulation has revealed parts of the mechanism for a chromosome congression, the spindle checkpoint, and anaphase chromosome movements. The earliest publication describing the results of micromanipulation experiments was by Robert Chambers¹. Chambers used a mechanical micromanipulator with an attached glass needle to probe the cytoplasm of a number of different cell types. Unfortunately, contrast methods that allowed the visualization of chromosomes and many other cellular components in living cells were not available at the time, so Chambers' experiments could not show the effects of repositioning such cellular components. Early micromanipulations that altered the chromosome position used the Chambers apparatus to sweep the spindle midzone in anaphase cells, showing that such manipulations could alter the position of chromosome arms in anaphase grasshopper neuroblasts². Nicklas and his collaborators were the first to perform fine micromanipulations of chromosomes, stretching the chromosomes³, detaching them from the spindle and inducing a reorientation^3,4, stabilizing a malorientation by applying tension to the chromosomes^5,6,7, and measuring the forces produced by spindles in anaphase^8,9. Other work by the Nicklas lab showed that cytoplasmic granules could also be manipulated¹⁰ and that centrosomes could be repositioned by micromanipulation¹¹. Micromanipulation is not just useful for moving chromosomes and other cellular components. A micromanipulation needle can cleanly cut through a mitotic spindle in demembranated cells¹² or can be used to dissolve the nuclear envelope¹³. In addition, adjacent cells can be fused by micromanipulation^14,15.

With such a wide variety of interesting experiments that can be done using micromanipulation, it is at first glance surprising that micromanipulation experiments have been done by very few chromosome biologists. One reason for this deficiency is that the mitotically-dividing cultured cells that are derived from vertebrate tissues and are commonly used for studying chromosome movements are extremely difficult to micromanipulate. These tissue culture cells generally have a cortical cytoskeleton that "gets in the way" of the micromanipulation needle, and chromosomes either cannot be reached by the needle or the needle grinds through the cell, leading to a cell rupture and death. We, and other experimenters who use micromanipulation, have found arthropod cells to be amenable to micromanipulation. Arthropod spermatocytes are easily spread under a layer of halocarbon oil and appear to have a much less robust cortical cytoskeleton underlying the cell membrane during a cell division. Thus, arthropod testes provide a good source of meiotically-dividing cells (spermatocytes) and mitotically-dividing cells (spermatogonia) with easily accessible chromosomes for micromanipulation. A serial sectioning of a grasshopper spermatocyte fixed during a manipulation revealed that the needle never penetrates the cell membrane; the cell membrane deforms around the needle (Nicklas R.B., personal communication). Spermatocytes from a number of insect and spider taxa have been micromanipulated successfully, including grasshoppers, praying mantids, fruit flies, crane flies, crickets, spittlebugs, moths, black widow spiders, cellar spiders, and orb-weaving spiders^{3,7,17,18,19,20,21,22}. Cultured, mitotically-dividing cells from insects can be micromanipulated. For example, the chromosomes in grasshopper neuroblasts in a primary culture have chromosomes that can be readily micromanipulated^2,23. We suspect that the available cultured lines derived from Drosophila and other insects will also be micromanipulatable, though we have not tested the technique with these cells. We will show how dividing cells from grasshoppers and crickets can be prepared for a micromanipulation. Crickets are easy to obtain from most pet stores at any time of the year. Grasshoppers are only easily obtainable in the summer unless the researcher has access to a laboratory colony, but the species used (Melanoplus sanguinipes) has easily flattened cells, and long, easy-to-manipulate chromosomes.

Another reason why micromanipulation experiments have been done by a small handful of biologists is that micromanipulators that move chromosomes well are rarely available in the marketplace. We have found that a joystick-controlled piezoelectric micromanipulator controls the needle movement with no vibration, drift, or lag between the joystick movement and the needle movement, but other types of manipulators can also successfully push chromosomes around in the cell. The micromanipulators designed by Ellis and Begg^25,26 are ideal for the micromanipulation of chromosomes, though they use older technology. Piezoelectric micromanipulators are currently available and commonly used in electrophysiology; however, these micromanipulators are not typically joystick-controlled. Joystick control is key to the smooth movements required for a successful micromanipulation, and so a custom joystick should be constructed to make the currently-available piezoelectric micromanipulators work for a chromosome micromanipulation. The joystick-controlled piezoelectric micromanipulators that work best have direct position control, in which the movement of the joystick translates directly to a needle movement.

A newly-designed piezoelectric micromanipulator can be constructed from commercially-available parts that can be easily replaced and from some small 3-D printed components, and it works well for chromosome micromanipulation²⁴. The micromanipulator has adjustable sensitivity, manual coarse positioning, and no vibration, drift, or lag in the needle movement, and direct position control of the needle. Scientists can construct the micromanipulator using instructions available online²⁴. Below are the methods for preparing a primary spermatocyte cell culture and for micromanipulating the chromosomes within the cells in that culture.

Protocol

1. Preparation of Primary Insect Spermatocyte Cell Culture for Micromanipulation

1. Slide preparation
   1. Obtain a 75 mm x 25 mm glass slide with a 20 mm diameter circular hole cut out in the center of the slide.
      NOTE: These were cut from a single sheet of window glass to be the size of a glass slide with a hole in the center.
   2. Run a 25 mm x 25 mm #1.5 coverslip through a Bunsen-burner flame for 2 s.
   3. Apply vacuum grease around the edge of the hole in the glass slide
   4. Place the coverslip on the hole as in Figure 1. Press it and make sure to form a tight seal.
   5. Flip the glass slide and fill the dissection well with halocarbon oil.
2. Spermatocyte culture preparation for viewing on microscope
   NOTE: Here, we describe the culture preparation method using grasshopper or cricket cells. Testes contents of other arthropods can be cultured using a similar method.
   1. Obtain subadult male crickets (or grasshoppers). Place a living insect in a refrigerator for approximately 2 min to cold-anesthetize it. Using dissecting scissors, quickly cut through the dorsal surface parallel to the long axis of the abdomen directly behind the wing buds. Manually squeeze the abdomen gently to push the testes through the cut in the exoskeleton (Figure 2).
   2. Using forceps, place the isolated testes in a dissection well containing halocarbon oil.
   3. If needed, view the testes under a dissecting microscope. Divide them into smaller pieces using fine-pointed forceps, removing any fat covering the testes, and use fine-pointed forceps to break up the testes (Figure 3A and 3B). Spread the testes contents under the oil, on the surface of the coverslip (Figure 3C).
   4. Spread the testes contents until the spread portion of the testis is barely noticeable to the eyes (Figure 3C). Use additional oil-loaded dissociation wells if needed.

2. Micromanipulation

1. Production of the microneedle
   1. Place one end of a (0.85 mm in outer diameter, 0.65 mm in inner diameter) glass tube in the flame of a Bunsen burner. Pull the end of the glass tubing in such a way that a 150° angle is formed, and an extended area of narrow glass tubing is created. Break the tubing in the thin region so that the thin region extending from the angle is approximately 10 mm long (Figure 4A).
   2. Using a microforge (either custom-made according to Powell²⁷ or commercially available-see Table of Materials), touch the tip of the glass needle to the hot platinum wire to melt the glass at the tip. Form an approximately 45° angle between the needle and the platinum wire of the microforge (Figure 4B). Pull the glass away from the hot wire, while shutting off the heat to the wire, forming a thin tip of ≤1.5 mm at the end of the glass needle.
      NOTE: The tip diameter will be difficult to measure, but a tip of this length is likely to produce a firm but flexible needle that will be appropriate for micromanipulation. Alternatively, a micropipette puller could be used to create glass tubing with an appropriate tip diameter (the experimenter will have to test different pulling recipes to produce an appropriately firm but flexible microneedle for the job). The needle could be placed in a holder that is shaped similarly to the manipulation needle created according to the method described above.
2. Positioning the micromanipulator
   1. Place the prepared slide on the stage of an inverted, phase-contrast microscope. Find the dividing cells and center them in the field of view. Focus on the cells using the lowest magnification possible.
      NOTE: We used a 16X phase-contrast objective.
   2. Place the microneedle in the needle holder on the micromanipulator.
   3. Manually position the microneedle in the light path of the microscope so that the tip of the needle is illuminated by the light (Figure 5B).
   4. Focus the microscope several focal planes above the plane in which the cells lie.
   5. Reposition the microneedle using the joystick controller at a low sensitivity several times to find the shadow of the needle in the X- and Y-axes. Continue to readjust the position until the position of the tip is visible. Adjust the position of the needle along the Z-axis until the needle tip is in focus.
   6. Refocus on the cells, then focus the microscope above the cell plane so that cells are just out of focus and invisible. Readjust the position of the needle so that its tip is in focus in this focal plane.
   7. Adjust the magnification of the microscope as needed.
      NOTE: We used a 100X 1.4 numerical aperture (NA) phase-contrast objective for the micromanipulation.
   8. After focusing on the cells using the higher-magnification objective, again focus the microscope above the cell plane so that the cells are just out of focus and invisible. Using a higher sensitivity setting, readjust the position of the needle so that its tip is in focus and centered in the focal plane above the plane in which the cells lie.
   9. Refocus on the cells, adjusting their position so they remain in the center of the field of view. The needle is now ready for the micromanipulation.
3. Micromanipulation of chromosomes
   1. Using the same sensitivity setting as for the fine positioning at a high magnification, use the joystick to control the microneedle and push the chromosomes around inside the cell. Keep the needle tip above the plane of the cells.
   2. To pull on or move a chromosome, focus on a chromosome near the top of the cell.
      NOTE: These chromosomes are easier to manipulate-manipulating chromosomes close to the coverslip makes it likely that the needle will grind into the coverslip, bursting the cell and ending the experiment.
   3. Adjust the Z-axis on the joystick to bring the needle tip into focus, and then move the needle tip with the joystick in X and Y. Place the tip of the needle directly on the chromosome to be manipulated and push it in the desired direction to allow the manipulator to reposition the chromosome.
   4. Depending on how far the chromosome is pushed, either apply tension to the chromosome or apply sufficient tension to detach a chromosome from the spindle. Once a chromosome is removed from a spindle, place it anywhere within the cell.

Results

Figure 6 shows a sample micromanipulation of 2 adjacent grasshopper primary spermatocytes in several examples of the possible uses of micromanipulation. This experiment was done using an inverted, phase-contrast microscope. The 0:00 (times shown are in min:s) image shows both cells prior to the manipulation. One chromosome in the bottom cell is shown under tension applied by the micromanipulation needle (0:05; black arrow) and then completely detached from th...

Discussion

With practice, moving chromosomes around in the cell can become second nature. Needles that are both sufficiently stiff and sufficiently thin-tipped are difficult to "get the knack of" fabricating, but this ability also comes with practice. Needles that are so fine that they deform when moved in the halocarbon oil will not be useful for pushing chromosomes in the cell. Needles that are so blunt that their tips are visible and as large as 1/3 of the width of a chromosome (or larger) are very likely to kill the cel...

Materials

Name	Company	Catalog Number	Comments
VWR micro cover glass	VWR	48366 249	25 mm x 25 mm, no 1.5
Dow Corning High Vacuum Grease	VWR	AA44224-KT
KEL-F Oil #10	Ohio Valley Specialty Chemical	10189
Microdissecting Scissors, Stainless Steel	Sigma-Aldrich	S3271-1EA
Dumont #5 fine foreceps	Fine Science Tools	11254-20
0.85 mm outer diameter, 0.65 mm inner diameter Pyrex glass tube	Drummond Scientific	Custom order--call to request
Inverted, Phase contrast microscope with 10X or 16X low magnification objective and 60X or 100X high magnification objective	Any brand
microforge	either custom built or Narashige	MF-900
micromanipulator	either custom built or Burleigh PCS-6000 with custom piezo-controlling joystick	PCS-6300