Role of clathrin in the regulated secretory pathway of pancreatic ß-cells
Miguel Molinete1,
Stéphane Dupuis1,
Frances M. Brodsky2 and
Philippe A. Halban1,*
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
)
View larger version (41K):
[in a new window]
|
Fig. 1. Effect of Hub expression on immunolocalization by confocal microscopy of
clathrin light chain and proinsulin. Rat islet cells were infected with
Adeno-Hub/EGFP (a recombinant adenovirus expressing both the Hub peptide and
EGFP such that the latter is a marker for Hub expression) or with Adeno-EGFP
(only expressing EGFP) as indicated. The left-hand images show direct EGFP
fluorescence. Right-hand images show staining for (A) clathrin light chain;
(B) proinsulin. Control cells infected with Adeno-EGFP show a strong
perinuclear and punctate staining for clathrin light chain regardless of the
level of EGFP expression (A: lower). Expression of Hub leads to the
disappearance of the perinuclear and punctate staining pattern for clathrin
light chain in those cells with the highest level of expression of EGFP (and
thus Hub — see representative cell marked by arrow in A: upper), whereas
cells in the same cluster but expressing low or nondetectable levels of EGFP
(and thus Hub) present normal clathrin light chain staining (see
representative cell marked by arrowhead in A: upper). Proinsulin
immunostaining (B) was unaffected by expression of Hub, with all cells
presenting the same staining pattern regardless of the level of EGFP
expression. Bar, 10 µm.
|
|
View larger version (17K):
[in a new window]
|
Fig. 2. Reverse phase HPLC analysis of radioactive products in islet cells. Cells
were pulse-labelled and then chased for 150 minutes under basal conditions
before stimulation with a cocktail of secretagogues for 60 minutes. The
representative profile is for radioactive products released during this 60
minute period with elution times for the two nonallelic rat I and II
proinsulins (PI) and their conversion products as indicated by the arrows.
*Elution times of the conversion intermediates des-31,32-split
proinsulin I and II, and des-64,65-split proinsulin I and II (reading from
left to right); tCP, truncated (des-(27-31)-) C-peptide.
|
|
View larger version (15K):
[in a new window]
|
Fig. 3. Efficiency of sorting of proinsulin to the regulated secretory pathway is
unaffected by Hub. Cells were infected with recombinant adenovirus expressing
only EGFP (control) or EGFP and Hub (Hub). Those cells expressing the highest
levels of EGFP (and thus Hub) were sorted by FACS and pulse-labelled for 10
minutes with [3H]leucine before a 150 minute chase under basal
conditions. Labelled proinsulin and insulin secreted during this time and that
remaining within in the cells at the end of the incubation was quantified by
HPLC (see Fig. 2). The
percentage of labelled proinsulin directed to the regulated secretory pathway
was calculated as 100—x, where x is the percentage of labelled
proinsulin released without conversion during the 150 minute chase (and
attributed to constitutive secretion) and is presented as mean±s.e.m.,
n=4. Note the interrupted scale. Sorting efficiency was >99%
regardless of whether Hub was expressed in the cells or not.
|
|
View larger version (18K):
[in a new window]
|
Fig. 4. Expression of Hub does not affect the conversion of proinsulin to insulin.
Cells were infected with adenovirus, sorted and labelled as described in the
legend to Fig. 3. After the 150
minute basal chase, cells were incubated for a further 60 minutes in the
presence of secretagogues. The basal and stimulated chase media and extracts
of cells at the end of the two chase periods were analysed by HPLC as
described in Fig. 2. Proinsulin
conversion (mean±s.e.m., n=4) is presented as labelled
insulin/labelled proinsulin + intermediates + insulin x 100 (corrected
for the difference in the number of leucines in each).
|
|
View larger version (18K):
[in a new window]
|
Fig. 5. Expression of Hub does not affect the release of proinsulin/insulin. Cells
were infected with adenovirus, sorted and labelled as described in the legend
to Fig. 3. Samples of medium,
following a 150 minute incubation under basal conditions and a subsequent 60
minute period of stimulation, as well as cell extracts (after the stimulation)
were analysed by HPLC (see Fig.
2). Labelled proinsulin and insulin (corrected for the difference
in the number of leucines in each) is presented as a percentage of that found
in all samples (basal + stimulated medium + extracts). Data are
mean±s.e.m., n=4.
|
|
View larger version (16K):
[in a new window]
|
Fig. 6. Expression of Hub increases truncation of C-peptide. Cells were infected
with adenovirus, sorted and labelled as described in the legend to
Fig. 3. Radioactive truncated
(des-(27-31)-) and intact C-peptide were quantified by HPLC (see
Fig. 2). Truncated C-peptide is
expressed as a percentage of total detectable labelled C-peptide (intact +
truncated). Data are mean±s.e.m., n=4.
*P<0.01 for Hub vs control for products released during
the 150 minute basal chase.
|
|
View larger version (14K):
[in a new window]
|
Fig. 7. Expression of Hub increases C-peptide degradation (relative to insulin) at
later times of chase and in the steady state. Cells were infected with
adenovirus and sorted as described in the legend to
Fig. 3. (A) Pulse-chase. The
sorted cells were labelled (10 minutes [3H]leucine) and chased (150
minutes basal followed by 60 minutes stimulated). The two chase media were
analyzed by HPLC. The data (mean±s.e.m., n=4) are for the
ratio of labelled insulin to total (intact plus truncated) labelled C-peptide
(corrected for the difference in the number of leucines in each).
*P<0.02 for Hub vs control for products released during
the 60 minute chase with secretagogues (`stimulated'). (B) Steady-state. The
sorted cells were incubated for 150 minutes under basal conditions and then
for a further 60 minutes with secretagogues. In order to estimate the ratio of
(unlabelled) insulin:C-peptide, the 60 minute stimulated medium was analysed
by HPLC and the peaks monitored by an online spectrophotometer at 213 nm. Peak
areas for C-peptide I and insulin I were measured. The insulin:C-peptide ratio
for cells expressing Hub was normalized to that for control cells (for which
the measured value was 2.45±0.27). Data are presented as
mean±s.e.m., n=5 from four independent experiments.
*P<0.005.
|
|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
© The Company of Biologists Ltd 2001
