Croquis De Ubicación De La Muestra

The work described in this chapter was aimed at establishing methodology suitable for investigating membrane traffic of newly synthesised regulated secretory organelle proteins. Empirically the biotinylation reaction described in section 3.3.1. would appear to be simple and easy to perform on PC I2 cells (table 3.1.) and the application to specific analysis of newly synthesised synaptophysin at the surface of PC I2 cells and CHO-38 cells has provided qualitative information (figures 3.4., 3.5. and 3.6.): Newly

synthesised synaptophysin arrives at the cell surface of both these cell types, internalises and undergoes further rounds of recycling in a manner similar to newly synthesised TR. These experiments also indicate that Trans^^S labelled synaptophysin is essentially behaving in the same manner as the [^^SJsulphate labelled synaptophysin in the initial study on constitutive appearance at the PC 12 cell surface by Régnier-Vigoroux et al.

(figure 4; 1991). Synaptophysin that has been labelled at the TGN is therefore behaving in essentially the same manner as that labelled at the HR, accumulated in the TGN during a 20°C incubation and able to leave the TGN at 37°C. This was important to establish, as studies on the sulphation of D^H have revealed that six percent of the D fH in PC 12 is suphated and constitutively secreted, whereas D fH stored intracellularly remains unsulphated (McHugh et at., 1985).

The large variation observed between synaptophysin time of arrival at the cell surface and the percentage of labelled molecules at the cell surface at any one time (figures 3.4. and 3.5.), indicate that the biotinylation procedure is extrememly limited in providing quantitative information on synaptophysin trafficking. Analysis of the cell surface appearance of other newly synthesised regulated secretory organelle proteins was therefore not pursued.

Rather, an examination of newly synthesised synaptophysin passage through the early endosome was undertaken using a transferrin horseradish peroxidase conjugate. This revealed that newly synthesised synaptophysin in CHO-38 cells is in the same

compartment as newly synthesised TR one hour after labelling, while only a proportion of that in PC I2 cells is in a compartment accessible to newly synthesised TR (figure 3.7.). This initial experiment was performed in preparation for pulse-chase experiments to look at the passage of newly synthesised synaptophysin through early endosomal compartments of both P C I2 cells and CHO-38 cells. During this work, a report in the literature emerged that showed the fate of fluid phase HRP upon chase from early endosomes, compared with the traffic route of newly synthesised [^^S]sulphate-labelled synaptophysin (Bauerfeind et al., 1993): Synaptophysin that was pulse labelled with [^^SJsulphate and chased for thirty minutes comigrated on equilibrium gradients with HRP internalised into early endosomes for five minutes, followed by a seven minute chase. HRP internalised to the early endosomes could be chased, like synaptophysin, to SLMVs after 180 minutes. Although this is a long chase period (where the fluid phase HRP and synaptophysin would have had time to recycle back to the TGN and perhaps become integrated into synaptic vesicles from this organelle) this study clearly

demonstrates the accessibility of SLMVs to content and membrane from early

endosomes. The planned pulse-chase experiments with TfnHRP or fluid phase HRP were aimed at resolving this question and were consequently not undertaken. However, this methodolgy (with fluid phase HRP or TfnHRP) could be refined and exploited for analysis of protein transport through fluid phase marker-accessible compartments, such as early endosomal compartments.

The above study by Bauerfeind et at. supports the earlier finding that PC 12 SLMVs are accessible to fluid phase HRP after two hours (Clift-O'Grady et at., 1990). In addition, electron microscopy studies have revealed that fluid phase HRP fed to PC 12 cells for five minutes is found in small synaptophysin-containing vesicles which could be SLMVs, although these structures could be recycling endosomal vesicles (Lah and Burry, 1993a). It is somewhat surprising that SLMVs are accessible to a content marker such as HRP, as passage of SLMV proteins through the early endosome would presumably function to sort content and membrane proteins from one another for SLMV biogenesis. However, since the studies described in this thesis were undertaken, several other reports in the

literature have revealed a relationship between synaptophysin and early endosomal structures of both neuroendocrine and nonneuroendocrine cells. Reports on the trafficking of other synaptic vesicle membrane proteins (section 1.4.3.) have also shed light on synaptic vesicle biogenesis:

Lah and Burry (1993a) immunoisolated synaptophysin-containing structures from PC 12 cells and recovered fluid phase HRP that had been fed to PC I2 cells for five minutes and chased in the absence of HRP for up to fifteen minutes (but not for longer chase periods). This confirms the prescence of synaptophysin in the early endosomal structures of PC 12 cells and provides indirect evidence for the absence of synaptophysin from prelysosomal structures.

When synaptic vesicle membrane proteins were expressed in CHO fibroblasts,

synaptophysin colocalised with endosomal markers, synaptotagmin accumulated at the cell surface, and SV2 was targeted to small vesicles lacking endosomal markers. In addition, when coexpressed in CHO cells, these proteins maintained their distinct compartments and did not colocalise in any one organelle (Feany et. a l, 1993b).

However, synaptophysin expression in nonneuroendocrine hepatocellular and vulvar carcinoma cell lines has led to the discovery of a subpopulation of synaptophysin in small electron translucent vesicles of 30-90nm that lack constitutively secreted proteins, TR and fluid phase HRP (fed to cells at intervals from five minutes to two hours) (Leube et. a l,

1994). This is inconsistent with the above reports, where HRP fed to PC 12 cells was incoportated into SLMVs, and is in contradiction with studies on synaptophysin

expression in CHO and 3T3 fibroblasts and epithelial MDCK cells, where synaptophysin was in compartments indistinguishable from endosomal organelles (Cameron et al., 1991; Johnston et a l, 1989; Linstedt and Kelly, 1991a). Perhaps these hepatocellular and vulvar carcinoma cell lines contain sorting components necessary for synaptophysin segregation from early endosomal compartments, components that are present in neuroendocrine and neuronal cells but not within CHO, 3T3 and MDCK cells.

Finally, a morphological analysis of synaptic vesicle protein distributions in cultured hippocampal neurons was undertaken by (Mundigl et. a l, 1993), where these neurones had gained axonal and dendritic polarity but not yet formed synapses: Synaptophysin, synaptotagmin, synaptobrevin, p29, SV2 and rabSa were found throughout the axonal and dendritic areas of these cells while the TR was confined to dendrites and the cell body. During treatment of these cells with the organelle-disrupting fungal metabolite, brefeldin A (BFA) the distribution of synaptic vesicle proteins in the axon remained unaffected. However, BFA treatment did result in the tubulation of TR-containing structures, together with the cotubulation of synaptophysin structures in the cell body and dendrites. Synaptotagmin, p29, synaptobrevin, SV2 and rabSa-containing structures in these regions did not undergo this tubulation. These authors suggest that in these cells synaptic vesicle proteins are differentially sorted at the TGN and are coassembled at the cell periphery for transport to presynaptic nerve terminals.

The presence of synaptophysin in endosomes need not necessarily prove a role in synaptic vesicle biogenesis and it may be that synaptophysin targeting to endosomes simply reflects a default pathway in the absence of synaptic-like microvesicles. However, we found that between ten and twenty percent of the newly synthesised synaptophysin was accessible to fluid phase HRP one hour after labelling at the ER of PC 12 cells (figure 3.7.) and Linstedt and Kelly (1991a) have estimated that

approximately one third of the synaptophysin present in P C I2 cells is in LDLR-

containing structures at steady state. This significant proportion of synaptophysin in the early endosomes of PC 12 cells suggests that this protein localisation is not a result of misstargeting. In addition, when synaptophysin missing its C-terminal cytoplasmic domain is expressed in 3T3 fibroblasts, it is no longer targeted to | endosomes (Linstedt and Kelly, 1991b). Conversly, if this cytoplasmic region replaces the cytoplasmic tail of the LDL receptor, the chimaera efficiently recycles through early endosomes (Kelly and Grote, 1993). Synaptophysin must therefore have information for the targeting to endosomes in addition to targeting to synaptic vesicles. The fact that synaptophysin sorts

away from other synaptic vesicle membrane proteins in the dendrites of BFA treated cultured neurones

(Mundigl et. a l, 1993) indicates that synaptophysin has a functional role in the endosomes of regulated secretory organelle-containing cells.

Full characterisation of the functional role of synaptophysin (section 1.4.3.1.) would perhaps explain this association with TR-containing compartments that is not seen with other synaptic vesicle membrane proteins (Feany et a l, 1993b; Mundigl et a l, 1993). For example, synaptophysin could potentially act as a chaperone protein for the rapid removal of other synaptic vesicle membrane proteins out of endosomal structures to synaptic vesicles. Perhaps synaptophysin interactions ensure the correct size and curvature of budding synaptic vesicles and synaptophysin is therefore in abundance in sorting compartments such as endosomes. Maybe synaptophysin in synaptic vesicle- containing cells is involved in the fusion of recycling vesicles or SLMVs with endosomal membrane and the plasma membrane, respectively. In CHO cells, synaptotagmin

accumulates at the cell surface (Feany et. a l, 1993b). Perhaps in neurones and neuroendocrine cells, synaptotagmin is transported to the cell surface and awaits synaptophysin (and other elements absent from CHO cells) for removal to synaptic vesicles. All these potential roles account for synaptophysin's recycling ability between the cell surface and intracellular compartments (figures 3.4. and 3.5.).

The formation of synaptic vesicles is likely to involve the inherent tendency of synaptic vesicle membrane proteins to form multi-protein complexes (section 1.4.3.8. and table

1.3.) and cellular sorting machinery that recognises these complexes. If this is the case, targeting information for the incoporation of these complexes into synaptic vesicles need only be contained in one constituent protein. Indeed, a signal within a predicted

amphipathic a-helix has recently been identified for the targeting of synaptobrevin to SLMVs in PC 12 cells (Grote et a l, 1995). As other synaptic vesicles are not predicted to contain such a-helices, these authors propose that synaptobrevin is targeted to SLMVs

through interactions with other synaptic vesicle membrane proteins and disruption of the amphipathic a-helix induces a conformational change that inhibits these interactions.

The above evidence suggests that the site of this synaptic vesicle formation involves endosomal structures, a process that could potentially occur at the tips of endosomal tubular extentions, as is the case for endosomal recycling vesicles (sections 1.2.2. and

1.2.3.). The evolution of synaptic vesicle formation could therefore have taken

advantage of a pre-existing pathway of membrane recycling by placing vesicular transfer from endosomes to the cell surface under Ca^+ control.

C H A PTE R FO U R: P-SELECTIN .

4.1. ABSTRACT.

A morphological analysis of the distribution of the secretory granule membrane protein p- selectin and p-selectin deletion mutants was made in PC 12 cells in order to determine the location of a “granule targeting signal” within the p-selectin cytoplasmic tail. The

p-selectin and p-selectin mutants were also transfected into H.Ep.2 cells to see where they would be directed in the absence of secretory granules and to give an insight into the relationship between secretory granules and other cellular organelles. In addition, a p-selectin-horseradish peroxidase chimaera was transfected into PC 12 and H.Ep.2 cells for a detailed morphological analysis of p-selectin targeting by electron microscopy.