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First published online 19 August 2008
doi: 10.1242/jcs.031070

Journal of Cell Science 121, 3025-3034 (2008)
Published by The Company of Biologists 2008
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Efficient coupling of Sec23-Sec24 to Sec13-Sec31 drives COPII-dependent collagen secretion and is essential for normal craniofacial development

Anna K. Townley1, Yi Feng2, Katy Schmidt1, Deborah A. Carter2, Robert Porter3, Paul Verkade1,2 and David J. Stephens1,*

1 Cell Biology Laboratories, Department of Biochemistry, University of Bristol School of Medical Sciences, University Walk, Bristol BS8 1TD, UK
2 Department of Physiology and Pharmacology, and Wolfson Bioimaging Facility, University of Bristol School of Medical Sciences, University Walk, Bristol BS8 1TD, UK
3 School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UK


Figure 1
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  		Fig. 1. Depletion of Sec13 expression by siRNA causes concomitant loss of Sec31. HeLa cells were transfected with two independent siRNA duplexes (-1 and -2) targeting either (A) Sec13, (B) Sec31A or (C) Sec31B; 72 hours later, cells were lysed and immunoblotted as indicated. (D) A time course showing concomitant loss of Sec13 and Sec31A at 24, 48 and 72 hours. Lamin A/C was used as a control in all experiments and {alpha}-tubulin is included as a loading control. Molecular-mass markers are shown in kDa. The asterisk in A marks a non-specific band.

 

Figure 2
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  		Fig. 2. Depletion of Sec13 causes a loss of peripheral ERES labelling, and an accumulation of COPII and cargo in the juxtanuclear region of cells. (A) Cells depleted of Sec13 for 72 hours using one or both of the siRNA duplexes (-1 or -2) were fixed and immunolabelled for Sec31A, Sec24C, GM130, Sec16 or ERGIC-53 as indicated. (B) Cells transfected with siRNA duplexes targeting both Sec31A and Sec31B were immunolabelled for Sec16 and ERGIC-53. Note the juxtanuclear accumulation of COPII (Sec16) and cargo (ERGIC-53) in both cases (arrows). (C) Lysates of cells depleted of Sec13 were immunoblotted with antibodies specific for ERGIC-53 and {alpha}-tubulin. Molecular-mass markers are shown in kDa. (D) Localization of Sec16A and ERGIC-53 at sub-saturating pixel intensities in lamin-A/C- or Sec13-suppressed cells. (E) Suppression of Sec13 expression causes a reduction in COPI labelling of both Golgi and peripheral punctae compared with lamin-A/C-suppressed controls. Sec31A labelling is used to show effective suppression of Sec13/31 expression. Scale bars: 10 µm.

 

Figure 3
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  		Fig. 3. Depletion of Sec13 causes an increase in the half life and immobile fraction of Sec23/24 at ERES. (A) Half life of recovery of YFP-Sec23A during photobleaching shows a statistically significant increase following depletion of Sec13 (P=0.0072; n=45 ERES from three independent experiments). (B) The immobile fraction of YFP-Sec23A at ERES shows a statistically significant increase following depletion of Sec13 (P=0.0097; n=45 ERES from three independent experiments).

 

Figure 4
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  		Fig. 4. Secretory transport of tsO45-G-YFP is not significantly inhibited following Sec13 depletion. Cells depleted of Sec13 or lamin A/C were infected with an adenovirus expressing tsO45-G-YFP followed by incubation at the restrictive temperature of 39.5°C for 18 hours. (A) Transport of tsO45-G-YFP through the secretory pathway in cells transfected with either lamin-A/C or Sec13 siRNA. Upper panels: YFP (total tsO45-G) fluorescence 60 minutes after the shift to 32°C shows indistinguishable Golgi localization and plasma-membrane delivery in either experiment; note the enhanced juxtanuclear clustering of the Golgi in Sec13-suppressed cells. Lower panels: shows only plasma-membrane-localized tsO45-G-YFP 90 minutes after the shift to 32°C. Scale bars: 20 µm. (B) Quantification of arrival of tsO45-G-YFP at the plasma membrane (monitored by immunolabelling paraformaldehyde-fixed but non-permeabilized cells with an antibody against an extracellular epitope); error bars show standard deviation (n=100 cells from three independent experiments). (C,D) Arrival of protein at the Golgi following release of cargo from the ER at 32°C for the times indicated was determined by monitoring acquisition of EndoH resistance and immunoblotting for YFP. Samples were also immunoblotted to confirm the efficacy of Sec13 depletion and {alpha}-tubulin was included as a loading control. Molecular-mass markers are shown in kDa.

 

Figure 5
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  		Fig. 5. Ultrastructure of ER bud sites in Sec13-suppressed cells. Electron micrographs of high-pressure-frozen and freeze-substituted HeLa cells. (A) In control (lamin-A/C depleted) cells, small 50 nm vesicles are in close proximity to the Golgi apparatus (G). These vesicles are presumptive ER-to-Golgi transport vesicles but could potentially be either COPI- or COPII-coated. Occasionally, a coated (COPII) budding profile can be observed emanating from the ER (arrowhead). (B,C) In Sec13-depleted cells, the small 50 nm vesicles can still be observed (arrows) but, in addition, dilated ER with several budding profiles can frequently be seen (B, inset in C shows magnification of indicated area, large white arrowhead). These budding profiles (arrowheads in C) appear to be in different states of fission and contain an electron-dense coat. Scale bar: 500 nm (A,B), 350 nm (C).

 

Figure 6
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  		Fig. 6. Suppression of Sec13 in primary human fibroblasts results in defective collagen secretion and deposition. Primary human dermal fibroblasts were transfected with siRNA duplexes to suppress Sec13 expression for 72 hours. Cells were then processed for immunofluorescence using antibodies to detect type-I collagen and Sec31A. (A) Cells were labelled with antibodies to detect the C-terminal telopeptide of collagen I (LF-67) and Sec31A. Scale bars: 20 µm. (B) Quantification of collagen deposition from three independent experiments shows a significant reduction (P<0.001) in deposition of fibrillar collagen. Supplementary material Fig. S2 shows further detail and example images from this quantification. (C) Immunoblotting was used to verify suppression of Sec13 and concomitant loss of Sec31A in primary human dermal fibroblasts. Asterisks denote non-specific bands detected with the anti-Sec13 antibody; tubulin was included as a loading control. Molecular-mass markers are shown in kDa.

 

Figure 7
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  		Fig. 7. Suppression of Sec13 expression in D. rerio results in a phenotype similar to that of crusher mutant embryos. (A) Control and Sec13 morphant (using morpholino oligonucleotide MO1) zebrafish embryos at 5 dpf were photographed following paraformaldehyde fixation. (B) Live embryos were photographed at higher magnification to visualize craniofacial features (from experiments using MO2). Arrows indicate the facial features and arrowheads the pectoral fins that are present in controls but missing or dysmorphic in Sec13 morphants; note the absence of extension of the head beyond the eyes and kinked pectoral fin in Sec13 morphants. (C) Alcian-blue staining of fixed embryos at 4 and 5 dpf (following microinjection of MO1) shows defective deposition of proteoglycans and lack of formation of cartilage in multiple areas of the head. mc, Meckel's cartilage; pec fin, pectoral fin. Arrows indicate facial features and arrowheads the pectoral fins in controls that are missing or dysmorphic in Sec13 morphants (arrowheads). (D) Scanning electron micrographs showing the dorsal, lateral and ventral views of control and Sec13 morphant 5-dpf zebrafish embryos. Note the perturbation of development of the frontal region of the head (arrowhead), the pectoral fins (arrows) and the eyes in Sec13 morphants. Scale bars: 0.2 mm. (E) Immunoblotting of lysates from dechorionated control or Sec13 morphant embryos confirms suppression of Sec13 expression and concomitant loss of Sec31A. Immunoblotting of these lysates for {alpha}-tubulin is included as a loading control. Molecular-mass markers are shown in kDa.

 

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© The Company of Biologists Ltd 2008