3.3　 Observation of mitosis in a protonemal cell

Yuji Hiwatashi

Introduction

This protocol gives you the opportunity to observe cell divisions of protonemal apical cells.　

Procedure

(1) Pre-culture

1. Sub-culture protonemata on cellophane-overlaid BCDATG* plates under continuous white light (30-40 µmol m-2 sec-1) at 25˚C every 5~7 days.

2. Inoculate ~7-day-old protonema tissue on BCDATG plates (3 cm-diameter dish) and cover them with a sterile cover slip (18 x18 mm). Place protonema tissue in a line (figure).

3. Incubate the plates under unilateral red light (15-20 µmol m-2 sec-1) at 25˚C for 7 days.　 Cover a fluorescent tube light with a red plastic sheet, and use as a red light (red plastic sheet: shikolite 102; Mitsubishi Rayon, Japan, http://www.mrc.co.jp/shinkolite/index.html). New protonema grow to the direction of the red light.

(2) Time-lapse observation

1. Keep room temperature at 25C.

2. Remove the cover slip from the plate and cut an agar block containing protonemata with a scalpel.

3. Place the agar block up-side down in a 35 mm glass-bottom dish (IWAKI 3910-039: a 27 mm diameter opening in the center of a dish, http://www.atgc.co.jp/div/rika/hbine/index_e.html), and protonemata attach to the bottom of the dish.　 Seal the dish with parafilm.

4. Place the dish on a stage of an inverted microscope.

5. Seek a protonemal apical cell just before cell division and focus its nucleus. The apical cell before cell division is much longer than its neighboring subapical cell and its cytoplasm is localized to more apical end. One of the signs of mitosis is a transition of a nuclear shape. Just before entering prophase, a nucleus becomes spherical rather than oval.

6. Carry out the time-lapse observation. Mitosis ends within ~30 min in this condition, and take an image at every 60 sec. Emission light should be as weak as possible especially when GFP signals are observed. Strong light inhibits cell division and fades GFP fluorescence.

Solution required

・ BCDATG

1 stock solutions

solution A (x 100 )

Ca(NO3)2·4H2O	118 g
FeSO4·7H2O	1.25 g
H2O	fill up to 1000 ml

SolutionB (x 100 )

MgSO4·7H2O	25 g
H2O	fill up to 1000 ml

Autoclaved

SolutionC (x 100 )

KH2PO4	25 g
	adjust pH to 6.5 with 4 M KOH
H2O	fill up to 1000 ml

Autoclaved

Solution D (x 100)

KNO3	101 g
FeSO4·7H2O	1.25 g
H2O	fill up to 1000 ml

Alternative TES (x 1000)

CuSO4·5H2O	55 mg
H3BO3	614 mg
CoCl2·6H2O	55 mg
Na2MoO4·2H2O	25 mg
ZnSO4·7H2O	55 mg
MnCl2·4H2O	389 mg
KI	28 mg
H2O	fill up to 1000 ml

Autoclaved

500mM Ammonium Tartrate (x 100 )

Ammonium Tartrate	92.05 g
H2O	fill up to 1000 ml

Autoclaved

50mM CaCl2 (x 50 )

CaCl2·2H2O	7.35 g
H2O	fill up to 1000 ml

Autoclaved

2.BCDATG

H2O	900 ml
Stock B	10 ml
Stock C	10 ml
Stock D	10 ml
Alternative TES	1 ml
500mM Ammonium tartrate	10ml (= 5 mM)
50mM CaCl2 2H2O (powder)	20 ml (= 1 mM) 　(0.15 g)
Glucose	5 g
Agar (Sigma, A6924)	8 g (= 0.8%)
	Fill up to 1000 mL with H2O

Autoclaved