Materials and Methods
B. RIA o f rGH in WT and transfected cell lines shows no significant difference in amount o f endogenous rGH with the expression o f GH-GFP construct RIA o f eGFP content in hGH-eGFP transfected GC cells
(green bar) reveals a 1000 fold reduction in protein content compared to GH.
( iKipter 3:
A)
SB
g
In v i n o s l i u i ie s in a (jH c e l l lin e sicihlv i r a n s f e c ic U v i i h e ijI - P
GC controls GFP channel 0.1%
B)
GCGFP #1.2C)
GFP channel 29.3% GCGFP #3.2 GFP channel 49.5% 0 10 100 1000 10000 Fluorescence intensity3,6 FACS analysis of p48hGH-eGFP GC cell lines
(A) FACS analysis o f untransfected GC cells. Fluorescence intensity is plotted along the x-axis and cell
number is plotted along the y-axis. The broken line to the right o f the cell population represents an arbitrary cut o ff linefor auto-fluorescence (amount o f fluorescence 0.1%).
(B) GC-GFP 1.2 and (C) GC-GFP 3.2 are typical FACS analyses o f transfected hGH-eGFP cells,
illustrating a broad population o f intensely fluorescent cells (B=29.3%; C=49.5%).
( In/p ie r 3: fil viiro studies in a ( , / / cell line siah/v iransfecteci with eGFP 10» 1 0 ' 102 103 10° 1 0 ’ 102 103 1 0 * 1 0‘ R17 R 18 R 19 R8 1 0' - 10» 1 0' 1 0* 10' 102 1 0 ° 1 0 * 1 0* R 18 R 17 R 20 R 19 R 25 R8 1 0' - 1 0* 1 0' 247 1 8 5 - 123 10° 1 0 ’ 1 0 *
Figure 3.7 FACS sorting o f hG H -eG FP GC cells lines
This figure shows scatter plots (left panels) and distribution (right panels) o f fluorescent intensities in GH- eGFP GC cells subjected to FACS sorting and analysis. Top panels. Gates were set arbitrarily to define GFP-ve (R19=12%) and GFP+ve (R20=88%) populations. Within these quadrants, subpopulations were collected o f extremely low (R8=4.7%) and highly (R25=38%) fluorescent cells, cultured separately for 3
days and then re-analysed by FACS. M iddle panels show cells from the R25 culture. The vast majority
o f these continue to be GFP+ve (R20= 98.1%) and highly fluorescent (62.5% within the R25 gate). Lower
panels show cells from the R8 culture. Very few GFP+ve cells emerge during culture (R20=1.4%;
R25=0.7%).
( l u i p le r 3: hi vitro studies m a (iH cell line stably transfecicd n ifh eG h P
In order to investigate the differing intensities o f fluorescence within a single population o f transfected GC cells, 1 repeated a FACS experiment but collected the cells, under sterile conditions, and divided them into populations of “weakly” fluorescing cells and “strongly” fluorescing cells. This was achieved by setting electronic gates at either end o f the fluorescence scale to collect exclusively the “very bright” cells or the “very dim” cells. These sorted populations were then re-cultured for 3 days before FACS analysis (figure 3.7). 1 found that the cells enriched for intense fluorescence contained almost 100% fluorescing cells (98.96% GFP+ve, 1.04% GFP-ve). The cells that were collected and re-cultured from the opposite end of the scale, containing little or no fluorescence, recovered a small population o f cells (3.36% GFP+ve) which were brightly fluorescing.
3.6 Total Internal Reflection Fluorescence Microscopy (TIRE)
Total internal reflection fluorescence microscopy (TIRF) is particularly well suited for real-time motion analysis o f secretory vesicles in GC cells transfected with p48hGH- eGFP. As previously described, vesicles in these transfected GC cells contain endogenous GFP. In collaboration with J-B Manneville (Physical Biochemistry, NIMR) we have been able to analyse the GH granules in these cells.
The TIRF set up is described in chapter 2 (2.17). The cells imaged were stuck firmly to the surface o f the slide, only the first hundred nanometres were illuminated, as represented in the diagram below:
GH-GFP vesicles cell cytoplasm
cell plasma membrane
/(2 )= /o e x p (-z /ô )
with Ô ~ 100-500nm
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The TIRF images o f GH-GFP transfected GC cells show GFP fluorescence in vesicles, near the plasm a membrane o f the cells attached to the slide. Some spots fluoresced brightly, others more dimly. Five hundred frames could be taken w ithout significant photo-bleaching, allowing time-resolved studies at the level o f single granules. Because the intensity o f the evanescent wave rapidly declines with distance from the glass substrate, vesicles closest to the plasmalemma are expected to be the brightest, while those more than 300nm away are too dim to be resolved. Real-time imaging o f these GH-GFP granules showed that they moved and could be tracked. Some granules slowly appeared and faded from view, while they moved into and out o f the evanescent wave; some granules remained stationary. There were also a few granules that could be tracked successfully, becoming brighter, before stopping and disappearing abruptly. Perhaps these granules were docking and undergoing exocytosis and the GFP, thus released, would diffuse away.
Figure 3.8 shows TIRF images o f GH-GFP GC cells, which have been captured and analysed. The raw fluorescent TIRF image (fig 3.8a) was filtered to enhance its contrast (fig 3.8b). Each frame can be superimposed onto the previous one to visualise the path o f vesicles, which can be seen for 10 frames in figure 3.8c. In order to visualise long-range movem ent in GC cells, 500 frames were filtered and superim posed (fig 3.8e) and skeletonised (fig 3.8f) using custom written macros in imaging analysis software (J.B. Manneville, NIMR).
Analysis o f single granule trajectories determines w hether the motion is random or directed. This is achieved by computer assisted analysis routines based on the imaging software Optimas and the ATI (Analytical Language for Images) to quantify in real-time the two- and three-dimensional motion o f the vesicle. Figure 3.9 displays two types o f vesicle movement in GC cells, showing long-range directed motion in 3.9a and short- range diffusive motion in 3.9b. X and y plots display parallel, horizontal motion, while z plots vertical motion. The long-range directed motion o f vesicles signifies vesicular transport on the cytoskeleton. M ost o f the granules observed displayed short-range diffusive motion and were most likely bound to the plasma membrane or docked.
Figure 3.8 TIRF microscopy of GH granule motion in p48hGH-eGFP GC cells The raw fluorescent TIRF image of GH-eGFP cells (A) is FFT (Fast Fourier Transform) filtered (B) to enhance its contrast. Each frame can be superimposed onto the previous one to visualise the path of vesicles (C shows path of vesicles in 10 frames). Long-range movement can be visualised by filtering and superimposing 500 frames (E ) and skeletonising (F). 1 frame = 40ms; scale bar = 5p.m.
^ long-range % ^ V . k motion " # f ® V * v ■ . ^ > • * \ stow diffusion 4 # ' N -0 05 ■0 15 -02 -05 0 5 t i m e ( s e c ) -05 0 05 1 1 L o n g - r a n g e , d i r e c t e d m o t i o n
B 1
0 2 0 5 0 15 '-0 05 -0.5 20 t i m e ( s e c ) 1 -0.5 0 0.5 1 1 X (nm) S h o r t - r a n g e , d i f f u s i v e m o t i o nFigure 3.9 Analysis of single granule trajectories in ph48hGH-eGFP GC cells
Analysis o f single granule trajectories in TIRF microscopy determines whether their motion is random or directed. Two types o f vesicle movement in GC cells
are displayed above, showing long-range directed motion in A and short-range diffusive motion in B. and y ploys display parallel, horizontal motion, while z
plots vertical motion. The long-range directed motion o f vesicles (shown by the arrow in the top xy plot) signifies vesicular transport on the cytoskeleton. Most
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3.7 Discussion
GFP has been used widely in cell biology in order to visualise and study cellular processes in real time (Tsien, 1998). Most studies have used transfection to express GFP, fused to a variety o f different proteins in cell lines, resulting in a fusion protein that maintains normal functions and localisations o f the host protein but is also fluorescent. The transfection o f eGFP into rat GC cells was performed to achieve targeting o f eGFP to GH granules in a GH cell and to assess the viability o f the human GH construct (p48GH- eGFP) in a rodent cell line in vitro, before making a transgenic mouse.
W hen expressed alone or with minimal N-terminal peptide extensions, eGFP pervades throughout the cytoplasm. However, targeting signals may be fused to GFP that direct localisation o f a fluorescent fusion product to specific sub-cellular structures or molecules e.g. progesterone receptor (Lim et a l, 1999). In particular, GFP variants targeted to secretory vesicles have been used to follow the genesis, trafficking and regulated release from these organelles in endocrine cell lines (Steyer et a l , 1999; Kaether et a l , 1997; Lang et a l, 2000). The hGH signal peptide (Martial et a l, 1979) is sufficient to enable heterologous reporter sequences to be processed through the secretory pathway in cell cultures (Pecceu et a l, 1991; Blam et a l, 1988). For the purpose o f my studies, eGFP was fused with the signal peptide and an additional portion o f the N- term inus o f hGH (p48hGH-eGFP), which directed the fluorescent product to GH secretory vesicles in rat GC cells.
Since we w ished to avoid the possibility that the fusion protein would retain hGH bioactivity, we chose not to fuse GFP onto the end o f the intact hGH to make a full- length fusion protein. Although less o f a concern in cell lines, we intended to use the same construct in animals, where it would be a concern. Expression o f intact human growth hormone in pituitaries o f transgenic mice would cause local feedback at pituitary and hypothalamic level and disrupt normal endogenous GH production. One example o f local feedback disruption is the Transgenic Growth R etarded (TGR) rat, previously characterised in our lab (Flavell et a l, 1996) in which the hGH gene was targeted to GRF neurones in the hypothalamus, inducing dominant-dwarfism by feedback inhibition o f GRF.
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Although we included the N-terminal zinc binding sites o f GH, my data does not show whether these residues were in fact necessary for granule packaging o f eGFP or merely fortuitous. The aim o f this was not to identify the sequences necessary, merely to find a construct that did achieve this. A further series o f constructs, knocking out each o f the three relevant histidines in hGH (’^His, ^’His and ^^"^His) that are thought to be involved in packaging o f GH dimers, would be required to address this issue and this is discussed more thoroughly in chapter 6.
It could be that the N-term inal GH sequences in the eGFP fusion product partially interacted with rat GH sequences which facilitated co-packaging in GC cells. However, this cannot be the only explanation since the same construct also gave granular staining when expressed in other secretory cell types (P C I2 cells, unpublished results) and also in hypothalamic GHRH neurones (McGuinness et aL, 1999) that do not express endogenous GH (see figure 3.10). I cannot assume from co-localisation studies that GFP is ‘aggregated’ in GH granules. The p48hGH-eGFP fusion protein contains the first 48 amino acids o f GH. After the signal peptide is cleaved, prior to sorting and packaging into GH granules in the TGN, only 22 residues o f hGH from the hGH-eGFP fusion protein rem ain. It is unlikely that the hGH-eGFP fusion protein dimerises with endogenous rGH, supported by the reduced amount o f eGFP compared to rGH in RIA o f GC cells. In my view, eGFP is co-packaged with rGH because it contains the signal peptide directing protein sorting, and aggregates to some degree with GH; during zinc m ediated GH dim érisation, the hGH-eGFP fusion protein may bind to a few GH molecules. However, the large differences in amounts should suggest that the hGH-eGFP cannot fully participate in the aggregation/condensation that achieves a much higher concentration o f itself.
I found the GFP fluorescence to be highly variable in all o f the stably transfected hGH- eGFP cell lines, which may indicate non-clonal transfection. However, the heterogeneity o f reporter expression has been described in several studies (Frawley et aL, 1994; White
et aL, 1995; Takasuka et aL, 1998) and may demonstrate variability in gene expression, implying that some cells are transcriptionally silent at given times.
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I
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cGFP I hGH SPB)
Figure 3.10 Expression of eGFP in GHRH neurones from rGHRH-eGFF
transgenic mice
(A) The cosmid construct engineered to target eGFP to vesicles in GHRH neurones in transgenic mice. The 1.3kb p48hGH-eGFP fusion fragment was inserted, using Mlul linkers, into the first exon o f the rGHRH gene.
(B) Expression of eGFP in GHRH neurones appeared granular (1) and was transported along axon fibres (2) to the ME (3). Scale bar = 10pm.
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The transfection o f p48GH-GFP into rat GC cells confirmed that a human GH-eGFP fusion protein can target rat growth hormone secretory granules in vitro. I have shown that hG H -G FP growth horm one cells can be purified and enriched using FACS. Endocrine cells release hormones by exocytosis o f secretory vesicles or granules. To become available for exocytosis, granules must move from the cytosol to the plasma membrane and dock there. It is o f interest to us to observe single granules near the plasm a m em brane in live GH cells, w hich we have accom plished using TIRF microscopy. In hGH-eGFP transfected GC cells, we have been able to track GH granules beneath the plasma membrane in three-dimensions to analyse their motion. Previously this has been done in chromaffin cells (Steyer & Aimers, 1999) and PC 12 cells (Lang et aL, 1997). When hGH-eGFP cells were analysed on our TIRF set up, we observed two different types o f motion. Short-range diffusive m otion, w here granules wander randomly around a resting position and long-range directed motion, where a granule would move along a track, becoming increasingly bright, before disappearing abruptly. Short-range motion could be modelled quantitatively by assuming granules to be attached or tethered to the plasma membrane prior to docking and fusion. Electron microscopy studies in chromaffin cells have shown chromaffin granules tethered to the cytoskeleton, and a filamentous actin mesh work beneath the plasmalemma o f chromaffin cells (Nakata & Hirokawa, 1992). The long-range directed motion o f granules which I have observed in these cells is likely to be vesicular transport on the cytoskeleton, immediately prior to exocytosis, which might explain the sudden disappearance o f GFP. However, this could also be explained by a granule moving out o f the evanescent field and hence out o f view.
Successful targeting o f GFP to GH secretory granules was a significant, preliminary result for my subsequent studies in transgenic mice in vivo. If transfection o f the hGH- eGFP fusion gene failed to target GFP to rat GH granules due to species specificity, a double transfection o f hGH-GFP in hGH-GC cells and double transgenic hGH animals would have been necessary. These studies in cell lines encouraged me to proceed to target GFP to GH granules in mouse somatotrophs in our transgenic hGH-eGFP mouse for in vivo studies.
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