1
Louis-Jeantet Research Laboratories, University Medical Centre, 1 rue Michel
Servet, 1211 Geneva 4, Switzerland
2
G. W. Hooper Foundation, Department of Microbiology and Immunology, and
Departments of Pharmaceutical Sciences and Pharmaceutical Chemistry,
University of California, San Francisco, CA 94143, USA
*
Author for correspondence (e-mail:
philippe.halban{at}medecine.unige.ch
)
Accepted May 12, 2001
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SUMMARY |
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Key words: Clathrin, Proinsulin, Insulin, C-peptide, Trafficking, Regulated secretion
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INTRODUCTION |
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Although the precise role of clathrin in the regulated secretory pathway of
ß-cells, and indeed any other neuroendocrine cell type, remains obscure,
two possibilities merit consideration. Clathrin may be implicated in sorting
events within the trans-Golgi network (TGN), allowing proinsulin and/or other
proteins important in the regulated pathway (including the conversion
endoproteases) to be delivered to nascent granules, or it could have a role in
the formation of such granules. Alternatively, and possibly additionally,
clathrin may be implicated in the formation of vesicles budding from maturing
granules (Arvan and Castle,
1998). There are two variants
of clathrin light, LCa and LCb. There is a predominance of LCb in cells with a
regulated secretory pathway (Acton and Brodsky,
1990
). This suggests that
clathrin may play a specific and perhaps unique role in this pathway.
Clathrin forms triskelions, consisting of clathrin heavy and light chains
(Brodsky, 1988; Kirchhausen,
2000
). The Hub fragment
(comprising the C-terminal third of the heavy chain) mimics the central
portion of the triskelion and can bind light chains to form nonproductive
heteropolymers incapable of coating membranes (Liu et al.,
1995
). Expression of Hub in
living cells leads to depletion of clathrin light chain and disruption of
clathrin function (Liu et al.,
1998
). The Hub fragment can
thus be considered a dominant-negative mutant clathrin heavy chain, and its
expression in HeLa cells led to the inhibition of clathrin-mediated membrane
transport (Liu et al., 1998
).
Such a dominant-negative strategy for the study of clathrin function in
mammalian cells complements earlier studies in clathrin-deficient mutant
organisms including yeast (Payne et al.,
1987
; Payne and Schekman,
1985
; Payne and Schekman,
1989
; Seeger and Payne,
1992
; Silveira et al.,
1990
). Hub has now been
expressed at high levels in primary rat pancreatic ß-cells using a
recombinant adenovirus, in order to study the importance of clathrin for
proinsulin trafficking and processing, as well as the regulated secretion of
proinsulin and its conversion products, insulin and C-peptide.
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MATERIALS AND METHODS |
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Preparation of recombinant adenovirus
The HindIII-BglII cDNA fragment encoding the T7-tagged Hub peptide from
pCDM8T7Hub (Liu et al., 1998)
was subcloned into the HindIII-BglII sites of the pTrackCMV vector (He et al.,
1998
) to produce the
pTrack-T7Hub. The bacterial strain BJ5183 was cotransformed with 0.1 µg of
pTrack-T7Hub linearized by PmeI and with 5 µg of the adenovirus DNA. HEK293
cells were transfected with the recombinant adenovirus DNA linearized by PacI
to produce adenovirus expressing the T7-tagged Hub peptide (`Hub virus'). The
control adenovirus without insert (`empty virus') was produced the same
way.
Cell culture and infection
Islets of Langerhans were isolated by collagenase digestion of the pancreas
of adult male Sprague-Dawley OFA rats and the islets digested with trypsin to
obtain a suspension consisting predominantly of single cells according to
standard procedures (Rouiller et al.,
1990). To allow them to
recover from the isolation and digestion procedures, the cells were placed in
plastic Petri dishes (35 mm diameter; 5x105 cells/dish)
coated with a matrix secreted by 804G (rat bladder carcinoma cells from
Desmos, San Diego, CA) and maintained in culture (DMEM, 11.2 mM glucose, 10%
FCS) for 24 hours, during which time they adhered to the matrix and spread
(Bosco et al., 2000
). Cells
were then incubated for 3 hours at 37°C in complete medium (as above) with
recombinant adenovirus expressing either enhanced green fluorescent protein
(EGFP) alone (empty virus) or EGFP and Hub fragment (`Hub virus') and then
washed 2x in PBS before culture overnight in complete medium. The
quantity of virus used for infection was established empirically to allow for
maximum expression of EGFP without any detectable toxicity. The multiplicity
of infection (moi) was in all cases less than 100, a titre shown by us in
previous experiments to be nontoxic for ß-cells (Irminger et al.,
1996
; Meyer et al.,
1998
). The following day, the
cells were trypsinized and then purified by fluorescence-activated cell
sorting (FACS using a FACStar Plus, Becton and Dickinson, Sunnyvale, CA) on
the basis of their EGFP fluorescence. The sorted (fluorescent) cells were
allowed to recover from the procedure for 6 hours in culture in nonadherent
plastic dishes and were then plated in droplets (5x104
cells/75 µl droplet of complete medium) on plastic dishes covered with 804G
matrix. Pulse-chase or immunofluorescence experiments were performed the
following day.
Pulse-chase
Infected (sorted) cells were washed twice with a modified Krebs-Ringer
bicarbonate buffer (KRB-Hepes: 134 mM NaCl, 4.8 mM KCl, 1 mM CaCl2,
1.2 mM MgSO4, 1.2 mM KH2PO4, 5 mM
NaHCO3, 2.8 mM glucose, 0.25% BSA, 10 mM Hepes pH 7.4) then
incubated for 15 minutes at 37°C in KRB-Hepes containing 16.7 mM glucose,
followed by 15 minutes at 37°C in the same buffer containing 1 mCi/ml
[3H]leucine. The cells were washed twice with KRB-Hepes and
incubated for 150 minutes at 37°C with KRB-Hepes (basal secretion),
followed by 1 hour at 37°C with KRB-Hepes supplemented with 16.7 mM
glucose, 10 mM leucine, 10 mM glutamine, 1 mM IBMX, 10 µM forskolin and 0.1
µM PMA (stimulated secretion). The two secretion media (basal and
stimulated) were centrifuged at 2000 rpm for 5 minutes to remove any cells
that may have detached from the dish during incubations. The cells were
extracted in 1 M acetic acid, 0.1% BSA, freeze-thawed twice to ensure complete
extraction of cellular proteins, and centrifuged to remove debris (10 minutes;
12,000 rpm, microfuge).
Samples were analysed by reverse-phase HPLC using a well-established method
allowing for separation and quantification of radiolabelled proinsulin,
conversion intermediates, insulin, C-peptide and truncated (des-(27-31)-)
C-peptide as described previously (Irminger et al.,
1996; Verchere et al.,
1996
).
Immunofluorescence
Cells were washed twice in Dulbecco's PBS (DPBS), then fixed in 4%
paraformaldehyde for 15 minutes, washed again twice with DPBS and
permeabilized for 10 minutes in DPBS plus 0.2% Triton X-100, 0.1% BSA. They
were blocked for 1 hour in DPBS containing 5% BSA before incubation for 1 hour
at room temperature with monoclonal anti-clathrin light chain (CON.1; BAbCO,
Richmond, CA) diluted 1/200 (in DPBS 1% BSA), monoclonal anti-proinsulin (GS
4G9, the generous gift of O. Madsen, Hagedorn Research Institute, Gentofte,
Denmark; this antibody recognizes proinsulin but not insulin) diluted 1/50 or
polyclonal anti-rat cathepsin B (Upstate biotechnology, Lake Placid, NY)
diluted 1/100. The cells were then washed twice with DPBS and incubated with a
goat anti-mouse or anti-rabbit TRITC conjugated antiserum diluted 1/1000 in
DPBS 1% BSA for 1 hour at room temperature in the dark, washed and mounted
using Vectashield (Vector Laboratories, Burlingame, CA). Immunofluorescence
and direct fluorescence (EGFP) images were taken using a confocal microscope
(Zeiss).
Presentation of data and statistical analysis
Data are presented as the mean±s.e.m. for `n' independent
experiments. Significance of differences between groups was evaluated using
Student's two-tailed t-test for unpaired groups (P<0.05 considered
significant).
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RESULTS |
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Use of FACS to sort cells expressing high levels of Hub on the basis
of their EGFP fluorescence
The effect of Hub on clathrin LC disposition was most evident in cells
expressing high levels of the peptide (for example, the cell in
Fig. 1A, top panel, indicated
by the arrow). In order to limit the studies to such a population of cells,
they were first sorted by FACS. The Hub peptide expressed in the recombinant
adenovirus carried an N-terminal T7 epitope allowing for quantification of Hub
expression using anti-T7 antibodies. Given that the adenovirus co-expressed
EGFP (not, it must be stressed, as a fusion protein) it follows that
expression of EGFP should correlate with that of T7 and thus of Hub itself.
This was seen to be the case when infected cells were examined by direct
fluorescence (for EGFP) in combination with immunofluorescence (using anti-T7
and rhodamine-labelled second antibody; data not shown). Cells expressing high
levels of Hub could thus be sorted by FACS on the basis of their EGFP
fluorescence. Analysis of fluorescence by FACS confirmed the expected
heterogeneity of EGFP expression. The sorting windows were set such as to
recover only the 20% most fluorescent cells. All biochemical experiments were
performed on such sorted cells. When examined for clathrin light chain
immunofluorescence, they all displayed the expected phenotype, with the barely
discernible diffuse staining typical for cells expressing high levels of Hub
as described above. For controls, cells were infected with the adenovirus
expressing EGFP alone and sorted using the same sorting parameters as for the
Hub-infected cells. Cells were sorted 24 hours after infection with adenovirus
and then maintained in tissue culture for a further 24 hours. This allowed
cells to recover from the sorting procedure and more importantly provided
sufficient time for Hub to exert its dominant-negative effect on clathrin
function.
Pulse-chase experiments and HPLC analysis of radioactive
products
Cells were infected and sorted as above. They were then pulse-labelled (10
minutes [3H]leucine). The fate of newly synthesized (radioactive)
proinsulin was then followed during a subsequent chase. The first 150 minutes
of chase were performed under basal conditions in order to keep secretion from
secretory granules of the regulated pathway to a minimum. During this time
period, any incorrectly sorted proinsulin would be secreted by the
constitutive pathway. The medium was changed, and the cells were incubated for
a further 60 minutes of chase in presence of a cocktail of secretagogues in
order to stimulate (regulated) secretion of products stored in secretory
granules. At the end of the second chase incubation, cells were extracted.
Both the chase media and cell extracts were analyzed by reverse-phase HPLC in
order to quantify radioactive proinsulin, insulin and C-peptide. A
representative elution profile is shown in
Fig. 2. Note that two
nonallelic proinsulin genes are expressed in the rat. The HPLC protocol allows
for separation of the two insulins and C-peptides but not of proinsulin 1 and
2. The minor peaks eluting before C-peptide have been shown to consist of the
two C-peptide molecules lacking the five C-terminal residues (truncated,
des-(27-31)-C-peptide) (see below).
|
Hub-expression does not perturb proinsulin sorting to the regulated
secretory pathway, its conversion to insulin or the regulated secretion of
proinsulin/insulin
The percentage of labelled proinsulin released by cells during the 150
minute basal chase (and considered to represent constitutive secretion) was
less than 1% of total labelled proinsulin + insulin (basal and stimulated
media + cell extracts). This indicates that both for cells expressing high
levels of Hub and for controls, >99% of all newly synthesized proinsulin
molecules were retained within the cell during this chase
(Fig. 3). Clearly, the mere
retention of proinsulin within cells does not constitute in itself any proof
that these molecules were correctly sorted to and stored within secretory
granules, although (along with the immunofluorescence staining pattern for
proinsulin seen in Fig. 1) it
is perfectly in keeping with this. It was thus necessary to investigate the
conversion of proinsulin to insulin and C-peptide, an event known to arise
within secretory granules and to quantify the proportion of proinsulin and/or
insulin released from cells in response to secretagogues.
|
Proinsulin conversion was also found to be unaffected by Hub expression. Thus, the amount of fully processed insulin (expressed as a percentage of insulin + conversion intermediates + proinsulin) in basal medium, stimulated medium and cell extracts was not significantly different in Hub- vs control-infected cells (Fig. 4). The percentage of proinsulin/insulin released in response to secretagogues was similarly unaffected by Hub (Fig. 5). Thus, neither the very low levels of secretion under basal conditions, nor the marked stimulation of secretion by secretagogues (hallmarks of the regulated secretory pathway and typical of primary ß-cells) were affected by expression of the Hub fragment.
|
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It is concluded that sorting of proinsulin to granules and its subsequent conversion to insulin is unimpaired in the absence of clathrin-coating activity and that exocytosis of granules is similarly normal.
Hub expression increases both the percentage of C-peptide released in
the form of truncated des-(27-31)-C-peptide during 150 minutes of chase under
basal conditions and more extensive C-peptide degradation thereafter
It has been shown previously that C-peptide, which is produced in equimolar
amounts with insulin by the conversion of proinsulin, can be truncated. This
truncation event results in loss of the end five (C-terminal) residues to give
rise to des-(27-31)-C-peptide (Verchere et al.,
1996). Such truncation arises
within granules by an as yet unidentified protease. There is further and more
extensive degradation of C-peptide, which occurs either in granules or
subsequent to its vesicular transfer to lysosomes. Insulin, by contrast, is
stable within granules. Thus, together, truncation and degradation of
C-peptide lead to insulin:C-peptide ratios greater than unity.
The relative amounts of intact and truncated C-peptide and the ratio of insulin to total (intact + truncated) C-peptide was examined in the media. In the 150 minute basal medium, the percentage of C-peptide in the form of des-(27-31)-C-peptide was doubled (from 5 to 10%) in cells expressing Hub (Fig. 6). There was no such difference in the stimulated medium. By contrast, the ratio of insulin to total detectable C-peptide in the basal medium was not affected by Hub expression, whereas it was significantly elevated in the stimulated medium (Fig. 7A).
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It is believed that truncation is the earliest proteolytic event to which
C-peptide is subject in granules, followed by more extensive degradation to
products no longer identified by the HPLC method used for analysis. Given the
known stability of insulin within granules (Halban et al.,
1987; Neerman-Arbez and
Halban, 1993
), if C-peptide is
only truncated but not subject to more extensive proteolysis, the ratio of
insulin to total detectable (intact + truncated) C-peptide will be unity.
Further degradation will result in an increased ratio. The ratio of
radiolabelled insulin:total C-peptide of 1.28 in the stimulated medium from
cells expressing Hub (Fig. 7A)
indicates that 21±2.3% of C-peptide had been degraded. We have shown
that such C-peptide degradation occurs within cells and not in the medium
following release from primary islet cells (Neerman-Arbez and Halban,
1993
). Taken together, and
given the sequential nature of the experimental protocol (150 minute basal
secretion followed by 60 minutes of stimulation), the data presented in Figs
6 and
7A thus suggest that expression
of Hub leads to both an increase in C-peptide truncation (as an early event
picked up in the basal medium) and degradation (a later event impacting on
insulin:total C-peptide ratio in stimulated medium).
It was predicted from the above data that the ratio of insulin to C-peptide
in the steady-state should be elevated in cells expressing Hub. Unfortunately,
there is no reliable analytical method available for direct measurement of rat
C-peptide. We therefore resorted to an indirect method for measuring the
insulin:C-peptide levels in ß-cells. Cell extracts were analyzed by HPLC
exactly as described for pulse-chase experiments. The absorbance of the HPLC
column effluent was continuously monitored at 213 nm and the peak areas for
insulin I and C-peptide I measured (note that steady-state levels of truncated
C-peptide were too low to be routinely measurable by U.V. absorbance). The
relative extinction coefficient of rat C-peptide vs insulin is not known.
Given that we have shown only very limited C-peptide degradation in primary
rat ß-cells (Neerman-Arbez and Halban,
1993), the results
(Fig. 7B) were normalized for
an insulin:C-peptide ratio of 1:1 in control cells. The ratio was increased by
58% in cells expressing Hub, confirming increased degradation of C-peptide
relative to that of insulin in such cells.
Taken together, these data suggest that clathrin is normally implicated in
maintaining C-peptide truncation and degradation at the low levels seen in
primary ß-cells (Neerman-Arbez and Halban,
1993; Verchere et al.,
1996
), presumably by virtue of
its involvement in purging granules of unwanted proteases. In an attempt to
document this, cathepsin B was examined by immunofluorescence. The enzyme has
been shown by others to be found in granules as well as in lysosomes (Kuliawat
et al., 1997
) and
co-localization of the enzyme with insulin was confirmed here (not shown).
There was, however, no evident impact of the expression of Hub on either
cathepsin B localization or levels (not shown). This negative result does not
necessarily indicate that Hub was without effect on the trafficking of this or
any other enzyme for the following reasons: (1) immunofluorescence only
provides an indication of steady-state levels and not of fluxes; (2) the
resolving power of light microscopy is not adequate for discrimination between
lysosomes and granules. This is a confounding factor in the analysis given
that insulin itself is known to reside in lysosomes in addition to granules
(Orci et al., 1984c
); (3)
cathepsin B may not be a relevant marker as it has not been implicated in the
truncation or degradation of C-peptide. More detailed analysis of the kinetics
of passage of candidate enzymes through granules on their way to
endosomes/lysosomes would thus be necessary to address this question.
Hub expression does not affect the amount of C-peptide released via
the postgranular constitutive pathway
Secretion of C-peptide during the 150 minute basal chase period can be
attributed to the combination of true basal exocytosis of large dense-core
granules of the regulated pathway and so-called `postgranular constitutive' or
`constitutive-like' secretion (Arvan and Castle,
1992; Arvan and Castle,
1998
). This latter secretory
pathway, first postulated for ß-cells by Arvan and colleagues (Kuliawat
and Arvan, 1992
), involves the
budding of vesicles from granules, taking with them soluble granular
components including C-peptide, followed by constitutive exocytosis of such
vesicles. Based on the assumption that insulin should be excluded from such
vesicles, it is possible to estimate the amount of C-peptide released uniquely
via the postgranular constitutive pathway during the 150 minute basal chase
(Neerman-Arbez and Halban,
1993
). The percentage of total
labelled C-peptide released in this way was vanishingly small, and there was
no significant effect of Hub (0.05±0.03 vs 0.19±0.12%/150
minutes, Hub vs control).
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DISCUSSION |
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The fact that immature granules carry only a discontinuous, rather than a
continuous, coat of clathrin is in itself suggestive that they are in a class
of their own and distinct from bona fide clathrin-coated vesicles (CCVs). One
cannot conclude from this observation alone that clathrin is irrelevant to
granule formation. It could, on purely theoretical grounds, be implicated in
two steps: sorting of granule proteins in the TGN and budding/scission of
granules per se (Arvan and Castle,
1998; Halban and Irminger,
1994
). The present study
clearly excludes such roles. The sorting of proinsulin to the regulated
secretory pathway was remarkably efficient (as reported by us previously;
Rhodes and Halban, 1987
) in
these primary rat ß-cells, with >99% of all newly synthesized
proinsulin being correctly sorted in both Hub-expressing and control cells.
This does not in itself preclude a role for clathrin in the sorting of other
granule constituents, however unlikely. The fact that the kinetics of
proinsulin conversion were similarly unaffected, however, certainly indicates
that the conversion enzymes (PC2, PC3 and carboxypeptidase E) were correctly
packaged in granules and that the granular ATP-dependent proton pump needed
for acidification and in turn for activity of the conversion endoproteases
(Orci et al., 1994
; Rhodes et
al., 1987
) was similarly
present and active in the granule membrane. Clathrin is thus not implicated in
these key events in granule maturation and function. Finally, the data show
clearly that clathrin is not needed for regulated exocytosis of either
immature (proinsulin) or mature (insulin) granules.
If clathrin is not needed for the sorting of granular constituents, the
formation of granules or their function as the proinsulin conversion
compartment and exocytotic vehicle, what purpose could it serve in the
regulated secretory pathway? It has been suggested that CCVs bud from maturing
granules (see Arvan and Castle,
1998, for review). Such
formation of CCVs was first proposed on the basis of morphological studies
some 20 years ago (Orci,
1982
). Subsequently, it has
been proposed that some C-peptide, as a soluble constituent of the granule, is
captured in CCVs as they form from maturing granules (Arvan et al.,
1991
; Kuliawat and Arvan,
1992
; Kuliawat and Arvan,
1994
). This may be the case in
less well differentiated transformed insulin-secretory cell lines, but in the
primary ß-cell <1% of C-peptide is secreted in this fashion
(Neerman-Arbez and Halban,
1993
). We confirm this in the
present study. Given these very low values, it was not surprising that no
significant inhibitory effect of clathrin inactivation was discernible at this
level. Certainly it was not stimulated. By contrast, the hypothesis that CCVs
may transport unwanted proteases away from granules to an endosomal
intermediate (Turner and Arvan,
2000
) is captivating, well
documented (albeit indirectly given that the vesicles themselves have yet to
be isolated) (Klumperman et al.,
1998
; Kuliawat et al.,
1997
) and of potential
physiological significance. Such passage of an enzyme into and then out of
granules has also been documented for the proprotein convertase furin (Dittie
et al., 1997
). A predicted
consequence of the inhibition of this vesicular pathway would be the elevation
of proteolytic activity in granules. C-peptide provides a useful marker
substrate for such activity. In particular, we have shown that it can be
selectively truncated to lose its five C-terminal residues (Verchere et al.,
1996
). Significantly, such
truncation has been shown to arise in granules and not subsequent to shunting
of C-peptide to another degradative compartment (Verchere et al.,
1996
). The observation of a
doubling of C-peptide truncation in the basal medium of cells expressing Hub
is quite in keeping with a role of clathrin in the removal of the (as yet to
be identified) truncation protease(s) from granules. Such truncation is
followed by more extensive degradation of C-peptide and this was also elevated
in cells expressing Hub.
In conclusion, clathrin is not essential for sorting of proinsulin within the TGN, for the formation of ß-cell secretory granules, for proinsulin conversion or for the regulated exocytosis of either proinsulin or insulin. Clathrin does appear, however, to be important for purging granules of proteases as they mature.
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ACKNOWLEDGMENTS |
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