Chapter discussion

The aim o f this study was to use biochemical approaches to identify potential

regulators o f exocytic fusion in the eosinophil and to investigate the RBL-2H3 cell

line as a model system for studying degranulation in haematopoietic cells. Relatively

pure preparations o f eosinophil granules were obtained by subcellular firactionation o f

purified eosinophils, but no components o f the synaptic vesicle fusion complex, or

homologues o f these components were identified by W estern blotting in these

fractions. Direct sequencing o f granule protein bands also proved to be problematic.

membrane proteins, but coomassie staining o f this extract after gel electrophoresis

highlighted how little protein is present in this extract relative to the soluble fraction.

At the time o f this study protein sequencing techniques were not sensitive enough to

identify proteins directly fi’om this sample. Accumulating enough material to attempt

further sequencing was not an approach judged likely to be successful in the lifespan

o f this project, given the progress o f the emergent sequencing technologies. However,

the approach outlined here has been applied to the identification o f granule membrane

proteins from the granulocyte cell line AML-DIO. Again using this approach no

components o f the synaptic vesicle fusion complex were identified. However, novel

proteins, localising to the endosomal-lysosomal pathway were identified (C. Teahan,

personal communication). However, a larger proportion o f the proteins identified in

this study proved to be mitochondial, highlighting the discrepancy between

estimations o f purity from both enzyme marker assays and electron microscopy

compared to the abundance o f mitochondrial proteins present in the AML-DIO

granule fraction. Clearly, the goal o f characterisation o f organelles, by the direct

sequencing o f purified fractions, will prove challenging. To date few examples o f

characterisation o f the protein composition o f organelles have been published,

although interestingly, given the abundance o f mitochondrial proteins in the AML-

DIO granule preparations, one o f the few examples is the identification and targeting

analysis o f chloroplast lumenal and peripheral thylakoid proteins (Peltier et a l, 2000). A growing area for the use o f the types o f techniques employed here is that o f

identification o f protein complexes (Pfannschmidt et a l, 2000) and key components o f the synaptic fusion machinery were identified using comparable approaches. The

major problem to be overcome in the search for components o f the granulocyte

granule exocytotic machinery is that o f abundance o f source material. Brain tissue is a

rich source o f synaptic vesicle components, but other cell types undergoing regulated

secretion do not contain comparable numbers o f vesicles or granules undergoing

regulated secretion.

Western blotting approaches presented here did not demonstrate the presence

o f heterotrimeric Ga subunits on the granule membrane, but did confirm the presence

o f Gja3, Gas possibly Gao ill the eosinophil. The presence o f small GTP-binding

Although detection o f small GTP-binding proteins in specific subcellular fractions by

this method could not unequivocally identify a particular G-protein, it could be used

to narrow down possible candidates. The presence o f GTP-binding proteins with low

pi in the granule fractions for example was strongly suggestive o f Rab3 isoforms,

already implicated in granulocyte regulated secretion. Unfortunately, efforts in this

laboratory could not identify the specific isoform involved. This finding did highlight

however the need for a model cell line to test the function o f any candidate gene

involved in granulocyte regulated secretion.

The data presented here demonstrates the suitability o f the RBL-2H3 cell line

as a model cell line for these studies. It contains lysosomal granules that can be

stimulated to release their granule contents upon application o f the appropriate trigger

for example activation o f the IgE receptor. Evidence presented here also suggests that

overexpression o f wild-type Rab3a inhibits the regulated release o f granule contents

from RBL-2H3 cells. More recently Roa et al., (1997), have observed an inhibitory effect on regulated secretion in RBL cells by overexpression o f wild-type and N135I

Rab3d, but no effect was observed with Rab3a. It is unclear why these discrepancies

were observed, although it is possible that expression levels o f the Rab isoforms are

critical to the observed phenotype. Recent evidence has suggested that the Rab3d

isoform is implicated in exocytosis through the regulation o f actin polymerization

around secretory granules (Valentijn et al., 2000). In contrast Rab3a, in neurons has been demonstrated to play a role in later fusion events possibly by a mechanism

limiting vesicle release to a single quantum, by regulation o f the docking and/or

fusion complex (Geppert et al., 1997). If Rab3a performs this function in the RBL, changing expression levels even o f wild-type protein may effect granule release.

One explanation for the inhibitory effect o f wild-type Rab3a overexpression is

the formation o f a complex o f Rab3a with the effector Rabphillin3a. It has been

suggested that overexpression o f wild-type Rab3a might be expected to increase both

GTP- and GDP-bound Rab3a in the cell. This increase in GTP-Rab3a could sequester

Rabphillin3a and block exocytosis by inhibiting the dissociation o f the Rab3a-

Rabphillin3a complex thought to be a prerequisite for exocytosis. However, recent

evidence argues against this model. Rabphillin3a knock-out mice do not display the

mutants, unable to bind Rabphillin are still capable o f inhibiting exocytosis (Chung et a l, 1999). Other Rab3 effectors, which may play a role in granulocyte exocytosis include the zinc-finger protein Noc2 which binds Rab3a in a nucleotide-dependant

manner. Overexpression o f Noc2 has been demonstrated to inhibit exocytosis in

permeabilised P C I2 cells suggestive o f a role as a negative effector for Rab3a

(Haynes et al., 2000). Demonstration that expression o f a GTPase deficient Rab3a, modified to display reduced binding to calmodulin, retained binding capacity to other

Rab3a effectors such as RIM and Rabphillin, but failed to inhibit Ca^^-triggered

secretion also implicates calmodulin as a possible Rab3a effector (Coppola et al., 1999). Further work is necessary to dissect the relevance o f the interactions o f Rab3a

with its various binding partners and their roles in regulated secretion.

Evidence presented here highlights the problems associated with searching for

known mediators o f synaptic vesicle release or their homologues in other regulated

secretory cell types. Biochemical approaches suffer from sensitivity problems and

fi*om the availability o f specific antibodies. Sensitivity is a more critical issue, as is

sample purity when attempting to directly identify components o f this machinery in

granulocytes by mass spectrometry methodologies, even though this is a less biased

approach. This study has however validated the RBL-2H3 cell line as a model system

for studying putative components o f the granule docking and fusion machinery and

eluded to the involvement o f Rab3a in granulocyte exocytosis. To gain further insight

into this process in haematopoeitic cells, more sensitive and possibly less stringent

4. IDENTIFICATION AND LOCALISATION OF COMPONENTS OF THE