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 mic....

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....

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

List of materials used in this article

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