88 MONODISPERSOS Y PARA MEZCLAS BINARIAS entre diferentes especies está dado como

In view of the results obtained using fixed cells, the redistribution of NAII-GFP in live, fully intact cells was investigated during stimulated secretion. Data suggesting NAII- GFP becomes tightly associated with the plasma membrane during stimulated secretion using chemical fixation and confocal microscopy agreed with published results (Chasserot Golaz et al., 1996) and indicated that a portion o f cytoplasmic NAII-GFP becomes tightly associated with the subplasmalemma on stimulation. Furthermore NAII-GFP was seen to be associated with vesicular structures adjacent to the plasma membrane in stimulated cells. Evanescent Wave Microscopy (EWM - see Figure 2.2) was used to investigate NAII-GFP plasma membrane association, and the vesicular structures, in live cells. EWM allows measurement of fluorescence at the base of a cell in a thin, peri-plasmalemmal layer illuminated by the evanescent wave. One would predict that translocation of NAII-GFP to the plasma membrane would lead to an increase in the concentration of GFP molecules in the evanescent wave and a corresponding increase in fluorescence.

RBL cells transiently expressing either GFP or NAII-GFP and sensitised with anti- DNP IgE were mounted for EWM at 37®C and stimulated in situ with 100 ng/ml DNP- albumin. Images were acquired at 1 frame every 6 s and analysis of the resulting image

sequences revealed numerous dark vesicles backlit against NAII-GFP in the cytoplasm (data not shown). NAII-GFP was not concentrated around these vesicles, and so the identity o f the peri-plasma membrane vesicles seen in fixed IgE/antigen stimulated cells remains unknown. The image sequences generated were subsequently analysed to quantify changes both in area and fluorescence (Figure 3.13, overleaf).

(a) (i) G F P i <u y 6 0 6 0 F lu o r e s c e n c e Area 5 0 5 0 4 0 4 0 3 0 3 0 2 0 2 0 1 0 0 1 0 2 . 5 0 . 0 2 . 5 5 . 0 7 . 5 1 0 . 0 1 2 . 5 1 5 . 0 1 7 . 5 2 0 . 0

E lapsed tim e (mins)

(b ) (1) oJ

I

I

I

/

/

E lapsed tim e (mins)

F ig u r e 3 . 1 3 ; S tim u la tio n o f G F P ( + ) o r N A I I - G F P ( + ) R B L c e l l s w ith I g E /D N P -a lb u m in le a d s to c e ll s p r ea d in g an d an in c r e a s e in average f l u o r e s c e n c e .

R B L c e lls e x p r e s s in g e ith e r G F P o r N A I I -G F P w ere s e n s itis e d w ith Ig E , im a g e d at 3 7 ° C u s in g E W M a n d stim u la te d in situ w ith 1 0 0 n g /m l D N P - a lb u m in . ( a ) ( i) P lo t o f th e a v e r a g e f lu o r e s c e n c e o f an e x a m p le c e ll e x p r e s s in g G F P illu stra te s that b o th area a n d a v e r a g e f lu o r e s c e n c e in c r e a se o n stim u la tio n . ( b ) ( i) S im ila r in c r e a se s in area a n d a v e r a g e f lu o r e s c e n c e are se e n in c e lls e x p r e s s in g a n n e x in II-G F P . In b o th (a ) a n d (b ) e x a m p le f r a m e s p rior to s tim u la tio n ( ii) a n d 1 m in u te a fter stim u la tio n ( iii) illu stra te th e m o r p h o lo g ic a l c h a n g e s t r ig g e r e d b y a d d itio n o f D N P - a lb u m in at T = 0 . ( S c a le bar = 2 0 p m )

These data (Figure 3.13) yielded two pieces o f information. Firstly, cells expressing GFP or NAII-GFP increased the size of their ‘footprint’ when stimulated with IgB/DNP-albumin, as reported previously for untransfected RBL cells (Pfeiffer et al., 1985; Spudich, 1994). Expression of GFP or NAII-GFP did not appear to have any gross effect on the morphological changes elicited by stimulation.

Secondly, cells expressing NAII-GFP or GFP both showed an increase in fluorescence when stimulated (Figure 3.14, overleaf). Unlike NAII-GFP, GFP alone was never seen to associate with membranes under any conditions in either live or fixed cells, hence the observed increase in fluorescence was not due to membrane translocation of either GFP or NAII-GFP.

Initially there appeared to be two explanations for the measured increase in cellular fluorescence. Firstly, and most simply, an increase in fluorescence could be explained through closer adhesion of stimulated cells to the coverslip since this would effectively introduce more fluorophore into the brightest part o f the evanescent wave, immediately adjacent to the glass. A second, perhaps more intriguing explanation, is that the cells may undergo a volume change on stimulation. Using the soluble cytoplasmic fluorophore calcein, it has been possible to accurately measure volume changes in single N lB l-1 1 5 neuroblastoma cells (Crowe et al., 1995) and show that a decrease in cell volume is elicited by both hyperosmotic shock or high intracellular free Ca^^.

The fluorescence changes seen on stimulation of G FP and NAII-GFP expressing cells would thus be consistent with a decrease in cell volume. Such volume changes have previously been suggested to occur, since stimulation o f RBL cells with IgB/DNP leads to a decrease in plasma membrane tension consistent with a decrease in cell volume (Dai et al., 1997). Furthermore recent data demonstrated that an increase in membrane tension in chromaffin cells causes a dramatic decrease in exocytosis (70 fold) without irreversibly impairing secretory capability (Solsona et al., 1998). Together these results suggest that membrane tension might play key role in modulating exocytosis, which could be influenced by membrane addition (exocytosis), membrane retrieval (endocytosis) and volume changes.

To test the hypothesis that the observed increases in fluorescence could be explained by cell shrinkage, RBL cells expressing NAII-GFP were imaged using BWM during challenge with HBS supplemented with 150 mM sucrose (Figure 3.14, overleaf).

As expected, challenge with mildly hyperosmotic HBS induced a similar (though more rapid) increase in fluorescence as seen in stimulated cells, without inducing any morphological changes (data not shown). Although these preliminary experiments proved inconclusive in themselves, the investigation o f hyperosmolarity induced volume changes led directly to the discovery of a second, unprecedented phenomenon induced by

hyperosmotic challenge. This was the appearance of a novel class of motile vesicle, as discussed in Chapter 4, 1 0 0 (a) « 1000 & 9 0 0 8 0 0 7 0 0 2 0 2 4 6 8 (b) N A II-G F P T im e (m in s)

Figure 3.14: The flu orescence increase seen in N A II-G F P (+ ) RBL c e ll s when stimulated to secrete can be mimiced hy hyperosmotic shock.

T o test w h e th e r th e in c r e a se in flu o r e s c e n c e se e n in stim u la te d c e lls m ig h t b e c a u s e d b y a d e c r e a s e in c e ll v o lu m e , R B L c e lls e x p r e s s in g N A II-G F P , w er e stim u la te d w ith H B S + 1 5 0 m M s u c r o s e at 37"C to in d u c e c e ll sh r in k a g e . C e lls w e r e im a g e d u s in g E W M (1 fr a m e e v e r y 6 s ) an d c h a lle n g e d w ith h y p e r o s m o la r H B S after 2 m in . (a) M e a su r e m e n t o f th e a v e r a g e f lu o r e s c e n c e o f an e x a m p le c e ll d e m o n str a te s that a rapid in c r e a se in f lu o r e s c e n c e , c o n s is te n t w ith c e ll sh r in k a g e , is se e n on c h a lle n g e w ith h y p e r o s m o la r H B S . (b ) E x a m p le fra m es fro m the im a g e s e q u e n c e im m e d ia te ly b e fo r e c h a lle n g e (i) and 1 m in p o st c h a lle n g e ( ii) illu stra te that n o rad ica l c h a n g e in m o r p h o lo g y o c c u r s im m e d ia te ly a fter c h a lle n g e w ith h y p e r o s m o la r H B S .

S c a le bar: 2 0 p m .