Normas de Seguridad para el uso de Balancines.

In an attempt to further characterise the apparent association between membranes and the PI-TP(3 isoform, fractions prepared from HL60 cells disrupted using a ball-bearing cell-cracker and resolved into distinct membrane fractions were examined by SDS-PAGE and Western blotting. This however failed to reveal the presence o f PI-TP in any fraction other than the cytosol, with no immunoreactivity being detected in any o f the membrane fractions investigated. The material for screening in Western blots was provided by Dr. J. Whatmore from this lab. The results o f this investigation are illustrated in Fig. 7.9. The cytosolic portion o f the gradient is represented by fractions 15-17.

It is suggested that the interaction o f this protein with the membranes under investigation is such that it is disrupted by preparing membranes in this fashion and the protein is lost from the membrane to the cytosol. Certainly there is reason to believe that the observed association o f PI-TPP with the membrane is not an artefact, as comparable effects have been observed in studies o f the intracellular distribution o f the tw o PI-TP isoforms by immunofluorescence (167), i.e. that the p isoform o f PI-TP is observed to be retained in the peimeabilised cell to a significantly greater degree than the alternative isoform. That this observation is not purely an artefact o f the low levels o f P I-T P a expressed in HL60 cells resulting in poor detection with the antibody is supported by the observation that PI-TPP is quite clearly detectable in the membranes o f P C I2 and RBL 3H3 cells, while P I-T P a which is expressed at comparable levels is not found in washed membranes. The preparation o f membrane fraction using the cell cracker and sucrose density gradient centrifugation, while providing an efficient means o f resolving many o f the intracellular membranes into distinct fractions represents a long and protracted protocol involving a rigorous physical disruption o f the cell coupled to a 16 hour centrifugation step. Since PI-TP is not an intrinsic membrane protein and by virtue o f the hypothesised mechanisms by which it operates, it is suggested that any association with membranes is fairly weak and transient. This protracted handling o f the material prior to examination

A Fraction no. 1 B. r— P P I - T P 10 15 17 4-ve

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# * 6 6 kDa 45 kDa 29 kDa

Fig 7.9 W este rn blotting o f su cro se density g ra d ie n t fra c tio n a te d H L 60 cells - p ro b in g fo r P I-T P p in d istin ct m e m b ra n e

co m p artm en ts.

3.5x10^ cells were cultured in RPM l as per the normal protocol. The cells were thendisrupted using a ball bearing cell cracker. The disrupted cells were exam ined with a microscope, and w ere found to be disrupted in excess o f 90%. The material was then resolved on a sucrose density gradient from 10.4% to 50% at 20x1 O^g for 16 hours at 4°C . This fractionation was prepared by Dr. J.W hatm ore in this lab.

6 6pl samples o f fractions from the gradient were treated w ith 3x sample buffer, and resolved by SDS PAGE. The gel was then W estern blotted, and probed with monoclonal antibodies specific for PI-TPp. As can be seen, a strong signal for PI-TP was observed corresponding to that portion o f the gradient expected to contain cytosolic com ponents but at no other points in the gradient (Fig. 7.9A.). This probably reflects the relatively weak, transient nature w ith which it is hypothesised that PI-TP interacts with m em branes. Fig. 7.9 B shows a Coomassie stained gel o f the material.

gives ample opportunity for the dissociation o f the protein and its loss to the cytosolic phase. Another consideration in this analysis is that in the course o f fractionating the various cellular membranes, the degree o f dilution which the sample undergoes is considerably greater than in the studies utilising crude preparations o f membranes.

7.4 T ran slocatio n of PI-TP(3 to m em branes and leakage experim ents:

Having established the presence o f PI-TPp within membrane fractions o f the cell it was decided to investigate whether stimulation o f the cell using a variety o f agents affected the distribution o f the protein between membrane and cytosolic components.

7.4.1 Stim ulation of H L60 cells with PMA

W ork conducted in the laboratory of K.Wirtz had already demonstrated the fact that stimulation o f the Swiss 3T3 cells with PMA led to elevated levels o f phosphorylation o f PI-TP and was associated with a redistribution o f the protein into the Golgi complex. On the basis o f these data it was decided to examine the influence o f PMA on the kinetics with which PI-TPP exited from SLO peimeabilised cells.

Earlier attempts to examine any effects on the behaviour o f PI-TP using the polyclonal antibody failed to reveal differences in the kinetics o f the leakage process. Given the later demonstration o f the fact that this reagent very much favours the recognition o f the

a

isoform o f PI-TP (Demonstrated by Dr. N. Goldring in this lab - data not shown ) it was considered possible that any retained PI-TPp within the cell may have been below the threshold of detection. Previous work in this lab had demonstrated that by the end o f the first five minutes o f permeabilisation, all o f the immunoreactive material detectable using the polyclonal antibodies had exited from the cell.

For the purposes o f these experiments, 50x10^ HL60 cell were prepared as per the methods described in Chapter 2. The cells were resuspended in the usual permeabilisation buffer, to the appropriate volume, divided into two 2.5 ml aliquots

and allowed to stand for 15 minutes at 37°C to recover. At the end o f this period, one portion o f the cells was stimulated by pre-treatm ent with lOnM PM A for five minutes prior to permeabilisation. At the end o f this period, 180pl cells w ere dispensed into individual E p pen do rf tubes maintained on ice. 20pl o f permeabilisation cocktail w as then added and the assay transferred to a 37°C w ater bath. The samples w ere rem oved from the incubation at time points o f t=0, 30s, 60s, 120s, 180s, 300s, 600s and 900s and the solid cellular material removed from the permeabilisation supernatant by a 5s pulse spin in a microfuge. The samples o f supernatant and the cell pellet w ere then maintained on ice until the end o f the assay, when lOOpl o f 3x concentrated SDS PA G E sample buffer was added to the supernatant, while the cell pellet w as treated w ith 100|il o f RIPA buffer for 15 minutes on ice with occasional mixing using a vortexer. At the end o f the RIPA extraction, the insoluble material w as pelleted by centrifugation at lOxlO^g for 15 mins at 4“C, the solubilised material retained and treated w ith 50|il o f concentrated sample buffer. The material prepared in this way w as then resolved by SDS PA G E and blotted onto PVD F before being probed w ith PI- T P p specific antibodies.

The results o f these experiments are shown in Fig. 7.10. As can be seen, the accum ulation o f PI-TPp in the supernatant is accompanied by a concom itant lose o f P I-T P from the permeabilised cell. Results from stimulated cell pellets are shown adjacent to unstimulated cells and the supernatants from stimulated cells are show n in juxtaposition with those from the unstimulated sample. As can be seen, the accum ulation o f PI-TP in the supernatant in both sets o f samples is similar and the loss o f P I-T P from the permeabilised cell appears to follow identical tim e courses. D ensitom etric analysis o f the blot using TIN A softw are showed that in both cases approxim ately 20% o f PI-TPP was retained in the cells pellet.

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