Calidad

Exponentially growing culture of S.pombe were harvested

by centrifugation in a Beckman GS-6R at 3 000rpm for 3min at

room temperature. All subsequent steps were carried out at 4°C. Pellets were washed twice in a protein extraction buffer (15mM EGTA, 15mM MgClg.6H2O, 25mM Trizma base, ImM DTT; pH 7.5) in the presence of protease inhibitors (40/xgml'

ruptured to 70% breakage by vortexing the sample with an equal volume of ice-cold acid-washed glass beads (425- 600/xm) in approximately ten 60s bursts with 60s cooling on

ice between each burst. The resultant lysate was separated

from the glass beads by centrifugation and beads were

washed in buffer. The combined supernatants were

recentrifuged to remove any cell debris and protein concentration determined using the Bio-Rad Protein Assay.

Samples were stored at -2 0°C.

b.Sample Preparation for Electrophoresis.

For Sodium-Dodecyl-Sulphate-Polyacrylamide Gel

Electrophoresis (SDS-PAGE; Laemmli, 1970), samples were solubilized by boiling for 3min in an equal volume of

sample buffer (125mM Tris.HCl, 2% SDS, 10% glycerol, 5% (3-

mercaptoethanol, 0.001% bromophenol blue, pH6 .8 ).

c.(SDS-PAGE).

One dimensional protein separation was performed

according to Laemmli (1970) using SDS-PAGE. Resolving gels were routinely used at 8.25%, 12.5%, or 15% [eg. 12.5%

acrylamide/bis-acrylamide, 0.37M Tris.HCl (pH8.9), 3.75%

glycerol, 0.2% SDS] . Stacking gels were 3% or 5% in 0. 5M

Tris.HCl/ 0.2% SDS (pH6 .8 ). Gel polymerization was

accelerated using ammonium persulphate and TEMED solution. Molecular weights were calibrated using Sigma MW-200 high

molecular weight markers which included: myosin (2 05kD) , (3-

galactosidase (116kD), phosphorylase B (97.4kD), bovine

serum albumin (6 6 kD), egg albumin (45kD) and carbonic

anhydrase (29kD). Minigels were run at 50mA (constant

current) in a running buffer of 25mM Tris, 192mM glycine,

0.1% SDS, pH8 .3, for approximately Ih. Large gels were run

similarly but took approximately 4h at 70mA.

Protein bands were visualised by one of two methods: Coomassie blue staining for 3h (40% methanol, 5% acetic acid, 0.05% Coomassie brilliant blue) followed by rinsing in distilled H^O (dHgO) and destaining with destain (40%

methanol, 5% acetic acid); or alternatively, silver

staining (Protostain, National Diagnostics). For silver

staining, gels were fixed by immersion for lOmin in 50% ethanol, 5% acetic acid followed by soaking twice (lOmin) in 5% ethanol, 5% acetic acid and washing 4 times (Imin)

with dHgO. The gel was then sensitised by soaking for Smin

in Protostain Sensitiser, washed 4 times (Smin) in dHgO, and exposed to fluorescent light while immersed in Silver Stain

for 2 0min. After further rinsing in dHgO, the gel staining

was then developed by 3 immersions in Protostain developer. Development was stopped by the removal of developer and

replacement with 2 % acetic acid.

d.Western Blotting.

Protein was transferred to 0.45/xm nitrocellulose

filters (Schleicher and Schnell) using a cooled tank

(Transphor Electrophoresis Unit, Hoefer Scientific

Instruments). Gels were washed for 3 0min in transfer buffer

(see below) then assembled into the electrophoresis unit and run in transfer buffer for 3h at lOOV or overnight at

4°C at 40V (constant voltage). If the protein of interest

was <150kD transfer buffer A (2 0mM Tris, pH8 .0, 0.01% SDS, ISOmM glycine, 20% methanol) was used. Larger proteins were transferred using transfer buffer B (12.4mM Tris pH8.4,

0.01% SDS, 96mM glycine, 0.07% /3-mercaptoethanol) . After

transfer, the nitrocellulose blot was soaked (protein-side down) in 5% milk powder ("Marvel", Premier Brands, UK) in PBS-Tween (0.15M NaCl, O.OIM NaPi pH7.2, 0.05% Tween) for

Ih at room temperature to block reactive sites. The

nitrocellulose was then washed twice (30min) in PBS-Tween

and incubated overnight at room temperature, with

agitation, in the primary antibody diluted in PBS-Tween (as

specified) . After removal of the primary antibody, the blot

was then washed twice (5min) in PBS-Tween and incubated for

3h at room temperature in a phosphatase conjugated

secondary antibody, diluted 1:250 in PBS-Tween. Upon

further 5 times (5min) in PBS-Tween followed by two washes

(Smin) in 0.15M veronal acetate pH9.6 . Bands were

visualised by incubation for approximately 3min in 0.15M

veronal acetate pH9.6, 10 % nitroblue tétrazolium, 1 % bromo-

chloro-indoylphosphate, 4mM MgClz. Development of bands was

terminated by washing the blot with PBS-Tween (Blake et al.,1984) .

Materials and methods pertaining to specific chapters are detailed in those sections.

c h a p t e r 3.

3.1: INTRODUCTION. a .AIM.

The aim of this chapter was to purify actin from

S.pombe in a functionally CLctcVe form and to identify

different actin pools within the cell. Two routes of investigation were taken to this effect:

a) Purification of actin in an active form from

S.pombe by conventional biochemical techniques.

Activity was monitored using a DNase-I inhibition assay.

b) Pharmacological manipulation of the actin cytoskeleton using cytochalasins.

b.DNase-I and the G-actin Assay.

G-actin is a natural and highly specific inhibitor of

DNase-I (Lazarides and Lindberg, 1974), binding by

hydrophobic interaction to DNase-I with a stochiometry of

1:1. Due to the tendency of G-actin to polymerize,

crystallization (a pre-requisite for X-ray crystallography) of G-actin alone was unsuccessful. Researchers who had previously worked out the 3D structure of DNase-I exploited

the tenacious binding of G-actin to DNase-I and

crystallized the complex. As the structure of DNase-I was known, they were able to solve the crystal structure of actin (both G-actin-ADP and G-actin-ATP) to a resolution of

2.8A (Kabsch et al., 1990) . Whether the association of the

two proteins has any physiological relevance or not remains undetermined. Suggestions have been made that:

a) The primary function of DNase-I may not be to degrade DNA but to sequester actin.

b) actin may control the enzymatic activity of DNase-I during the cell cycle.

c) the complex may contribute some other function in controlling DNA metabolism (Kabsch et al., 1990).

The presence of G-actin in cell extracts can be

detected using a DNase-I activity inhibition assay

conditions. The principle of the assay exploits the association of G-actin to DNase-I preventing the ability of DNase-I to break down an introduced DNA substrate. It operates by steric hinderance at the DNA binding site (Kabsch et al., 1990). When no G-actin is present, DNase-I breaks down the DNA into its constituent nucleotides and, correspondingly there is an increase in absorbance at ODzg^nm as the bases are released. Therefore, a decreased rate of absorbance is indicative of the presence of G-actin.

3.2: MATERIALS AND METHODS.