Assembly and Micromanipulation of Xenopus In Vitro–Assembled Mitotic Chromosomes

Sébastien Almagro1, Stefan Dimitrov1

1 Institut Albert Bonniot, Grenoble, null
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.9
DOI:  10.1002/0471143030.cb2209s26
Online Posting Date:  April, 2005
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Abstract

Mitotic chromosomes have fascinated scientists for several decades. Despite the numerous microscopy studies, chromosome structure is, however, still poorly understood. This is due to both the high complexity of the mitotic chromosomes and the lack of other appropriate techniques suitable for studying their organization. This unit describes a novel physical approach based on measurements of mitotic chromosome elasticity. The elasticity properties are determined by the underlying structure, and knowledge of them has allowed a description of the organization of the mitotic chromosomes and critical analysis of the available models. In this unit, a detailed protocol for the measurements of the elastic response of in vitro assembled mitotic chromosomes in Xenopus egg extract is presented.

Keywords: Chromosome; Xenopus; Micromanipulation; Elasticity

     
 
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Table of Contents

  • Basic Protocol 1: Stretching a Mitotic Xenopus Chromosome
  • Support Protocol 1: Preparing Xenopus Sperm Nuclei
  • Support Protocol 2: Assembling Xenopus Chromosomes
  • Support Protocol 3: Building the Experimental Setup for Measurement of Chromosome Elasticity
  • Support Protocol 4: Designing the Micropipets
  • Support Protocol 5: Calibrating Pixel Size
  • Support Protocol 6: Measuring the Spring Constant of the Micropipet
  • Support Protocol 7: Preparation of the Force Calibration Reference
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Stretching a Mitotic Xenopus Chromosome

  Materials
  • Xenopus chromosome suspension (see protocol 3)
  • Stain buffer: buffer to be used in experiment (usually EB buffer; see recipe), containing 1 × 10−5 Hoechst 33258
  • EB buffer (see recipe)
  • Experimental equipment setup, isolated from vibration (also see protocol 4, Fig. , and Fig. ) consisting of:
    • 3 to 4 reservoirs (see protocol 4)
    • Inverted microscope equipped for phase‐contrast and fluorescence microscopy using Hoechst 33258 excitation and emission filters (see protocol 4 for greater detail)
    • Objectives: Olympus LCPlanFl 20× (NA = 0.40; air; phase 1) and Olympus long‐working‐distance LCplanFl 40× (NA = 0.60; air; phase 2)
    • Micromanipulators (see protocol 4 for greater detail)
    • Motor‐driven injector, syringe pump, nitrogen source, connectors, and other necessary hardware (see protocol 4 for greater detail)
    • CCD camera (see protocol 4 for greater detail)
    • S‐VHS videocasette recorder (AG‐TL‐700, Panasonic) with S‐VHS tape and TV monitor
    • Computer (see protocol 4 for greater detail) with PCI bus frame grabber (DT‐3155, Datatranslation, Inc.)
    • UTHSCSA ImageTool image analysis software with plug‐ins installed (see )
  • 20 to 30 rigid micropipets and the same number of flexible micropipets (see protocol 5)
  • Micropipet filling needles (MicroFil MF34G, World Precision Instruments)
  • 10‐ml syringes
  • 24‐mm × 24‐mm × 170‐µm glass coverslips
  • Additional reagents and equipment for pixel calibration (see protocol 6) and measuring the spring constant of a micropipet (see protocol 7)

Support Protocol 1: Preparing Xenopus Sperm Nuclei

  Materials
  • Two or three male Xenopus frogs
  • Buffer T (see recipe), 4°C
  • Buffer S (see recipe), 4°C
  • Fix/stain solution (see recipe)
  • Buffer R (see recipe), 4°C
  • Instruments necessary for frog dissection: forceps, scalpel, scissors
  • Petri dishes
  • 20‐ml glass beaker
  • 15‐ml conical centrifuge tubes (e.g., Falcon)
  • Tabletop centrifuge (e.g., IEC Clinical)
  • Fluorescence microscope with excitation and emission filters for Hoechst 33258 fluorescent dye and oil‐immersion objective (NA ∼1.3) with 60×/100× magnification
  • Additional reagents and equipment for counting cells using a hemacytometer (unit 1.1)

Support Protocol 2: Assembling Xenopus Chromosomes

  Materials
  • Mitotic egg extract (de la Barre et al., ; unit 11.10)
  • EB buffer (see recipe)
  • 10× ATP regenerating system (see recipe)
  • Coverslips
  • Fluorescence microscope with excitation and emission filters for Hoechst 33259 fluorescent dye

Support Protocol 3: Building the Experimental Setup for Measurement of Chromosome Elasticity

  Materials
  • 50‐mm × 26‐mm × 170‐µm glass coverslips
  • O‐rings (rubber; internal diameter = 13 mm, external diameter = 19 mm)
  • Metal cylinders (height = 10 cm, diameter = 19 mm; weight = ∼250 g; custom‐prepared in machine shop)
  • Dow Corning 732 adhesive/sealant
  • 24 × 24‐mm × 170‐µm glass coverslips
  • Table with pneumatic vibration isolators (TLC, Integrated Dynamics Engineering)
  • Inverted microscope (IX70, Olympus) with left‐side connector tube for camera and 1.25× lens adapted to the connector tube, motorized stage (Newport, cat. no. 860–C2) and two filters for the Hoechst dye (excitation, 365 nm; emission, 465 nm)
  • Two micromanipulators: one (MP‐285, Sutter Instrument Company) with a displacement precision of about 40 nm and a second (DC3 XYZ, World Precision Instrument) with a lower precision, and corresponding controllers
  • Motor–driven injector (IM–300, Narishige) connected to a N 2 gas bottle.
  • Syringe pump (World Precision Instruments, model no. SP100i), equipped with 50‐ml syringe
  • Taps: two 2‐way valves and one 3‐way valve (available in aquarium supply stores)
  • Several meters (length depending on distance between components of setup) of plastic tubing (internal diameter, ∼1 mm)
  • CCD camera (Micam VHR–2000 from Digital Vision Technologies, 512 × 512 pixels, 8 bits)
  • S–VHS VCR (AG–TL700, Panasonic)
  • Personal computer (PC), with Microsoft Windows 2000 or XP installed, sufficient RAM and hard drive space to work with stacks of pictures, and a free PCI bus
  • PCI bus frame grabber (DT3155 from Data Translation Inc.)
  • Spirit level
  • Strong bungee cords

Support Protocol 4: Designing the Micropipets

  Materials
  • Hellmanex II detergent (Helma; store in dust‐free location)
  • Ultrapure H 2O (conductivity ∼12 MΩ), filtered with a 0.22‐µm filter immediately before use
  • EB buffer (see recipe)
  • Capillaries (GC100–T10; Harvard Apparatus Ltd; internal diameter= 0.78 mm; external diameter = 1 mm)
  • 60°C water bath
  • Micropipet filling needles (MicroFil MF34G, World Precision Instruments)
  • 10‐ml syringes
  • Micropipet puller (P–97) with through filament (FT320B), both available from Sutter Instrument Company
  • Experimental setup for measuring chromosome elasticity (see protocol 4; also see protocol 1)
  • UTHSCSA ImageTool image analysis software with plug‐ins installed (see )
  • Additional reagents and equipment for calibrating pixel size (see protocol 6) and measuring the spring constant of micropipets (see protocol 7)

Support Protocol 5: Calibrating Pixel Size

  • Stage micrometer (graduated slide) with 100 divisions, 10 µm per division (Graticules Ltd., model PS 8)

Support Protocol 6: Measuring the Spring Constant of the Micropipet

  • Experimental buffer (usually EB, see recipe) containing 50 to 100 mM NaCl
  • Calibrated needle (see protocol 8)

Support Protocol 7: Preparation of the Force Calibration Reference

  Materials
  • Thin copper electric wire, 50 cm
  • Balance capable of precision within 1 mg
  • Mirror (at least 10 cm wide)
  • Transparent plastic ruler (graduated in millimeters)
  • Needle (MicroFil MF34G, World Precision Instruments)
  • Binocular microscope, preferably with zoom from 5× to 20×
  • Tweezers
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Figures

  •   FigureFigure 22.9.1 Schematic presentation of the pressurization/depressurization system. The pressurization system comprises an N2 gas bottle linked to a motor‐driven microinjector. A tap is present to isolate the pressurization system from the rest of the system. The depressurization system comprises a syringe mounted on a syringe pump along with another isolation tap. The two systems are linked together with a “Y” connector, and feed into a third tap (a three‐way valve) that gives the user the ability either to isolate the global system from the atmosphere (under‐pressure position, i.e., system closed, with the micropipet linked to the microinjector/syringe pump) or to vent it to the atmosphere (no pressure position, i.e., the system is opened to the outside and is not linked to the microinjector/syringe pump) This latter tap is the so‐called pipet tap.
  •   FigureFigure 22.9.2 General scheme of the experimental set‐up used for chromosome elasticity measurements. A reservoir containing the chromosome solution is placed on the motorized stage of an inverted microscope. The two micromanipulators holding the micropipets are located on each side of the microscope. The experiment is filmed with the help of a CCD camera and the video sequence is stored using an S‐VHS VCR. A computer is linked to the VCR in order to analyze the results.
  •   FigureFigure 22.9.3 Positions of the two micropipets and the chromosome during a stretching experiment. The two micropipets are positioned upon on the y‐axis, and each one has caught one end of the chromosome.
  •   FigureFigure 22.9.4 Graphic representation of the results of a chromosome‐stretching experiment. The force applied on the chromosome during the stretching experiment is plotted as a function of its relative extension. Data points are represented as circles. The line represents the linear fit of the experimental data and its slope is the stretch modulus.
  •   FigureFigure 22.9.5 Diagram of the reservoir used for chromosome stretching experiments. An O‐ring is attached to a coverslip with adhesive and the chromosome suspension is poured into the cavity formed by the O‐ring.
  •   FigureFigure 22.9.6 Correct geometry of the filament of the micropipet puller. The filament must have a trapezoidal shape with the length of the smallest base being 2/3 of the length of the largest base.
  •   FigureFigure 22.9.7 Size calibration of the pixel. The graduations of the stage micrometer are 10 µm apart. In order to calibrate the size of the pixel on the image it is necessary to measure the number of pixels between graduations.
  •   FigureFigure 22.9.8 Schematic of the needle calibration apparatus. Three stands are aligned and a mirror, a needle, and a transparent plastic ruler are aligned on top of them. A binocular microscope is placed vertically above the mirror and adjusted to allow observation of both the needle and the plastic ruler.
  •   FigureFigure 22.9.9 Graphic depiction of the spring constant of the needle. The force applied to the needle is plotted as a function of its deflection. The data points (circles) are fitted (gray line) to a line through zero. The slope of the gray line is the spring constant of the needle.

Literature Cited

   Almagro, S., Riveline, D., Hirano, T., Houchmandzadeh, B., and Dimitrov, S. 2003a. The mitotic chromosome: An assembly of rigid elastic axes, organized by SMC proteins and surrounded by a soft chromatin envelope. J. Biol. Chem. 279:5118‐5126.
   Almagro, S., Dimitrov, S., Hirano, T., Vallade, M., and Riveline, D. 2003b. Individual chromosomes as viscoelastic copolymers. Europhys. Lett. 63:908‐914.
   de la Barre, A.E., Robert‐Nicoud, M., and Dimitrov, S. 1999. Assembly of mitotic chromosomes in Xenopus egg extract. Methods Mol. Biol. 119:219‐229
   DuPraw, E.J. 1966. Evidence for a “folded‐fibre” organization in human chromosomes. Nature 209:577‐581
   Earnshaw, W.C., Halligan, B., Cooke, C.A., Heck, M.M., and Liu, L.F. 1985. Topoisomerase II is a structural component of mitotic chromosome scaffolds. J. Cell. Biol. 100:1706‐1715
   Houchmandzadeh, B. and Dimitrov, S. 1999. Elasticity measurements show the existence of thin rigid cores inside mitotic chromosomes. J. Cell. Biol. 145:215‐223.
   Lohka. M.J. and Masui, Y. 1983. Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220:719‐721.
   Manuelidis, L. 1990. A view of interphase chromosomes. Science 250:1533‐1540.
   Paulson, J.R. and Laemmli, U.K. 1977. The structure of histone‐depleted metaphase chromosomes. Cell 12:817‐828.
   Poirier, M., Eroglu, S., Chatenay, D., and Marko, J.F. 2000. Reversible and irreversible unfolding of mitotic newt chromosomes by applied force. Mol. Biol. Cell. 11:269‐276.
   Smythe, C. and Newport, J.W. 1991. Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts. Methods Cell. Biol. 35:449‐468
Internet Resources
   http://curry.edschool.virginia.edu/go/frog/
  Detailed video, audio and pictures explaining dissection of Xenopus.
   http://ddsdx.uthscsa.edu/dig/itdesc.html
  Web site where ImageTool version 3 and its plug‐ins can be downloaded.
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