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Porosome in Cystic Fibrosis.

Jena BP - Discoveries (Craiova) (2014 Jul-Sep)

Bottom Line: This understanding now provides a platform to address diseases that may result from secretory defects.Hence secretion of more viscous mucus prevents its proper transport, resulting in chronic and fatal airways disease such as cystic fibrosis (CF).The involvement of CFTR in porosome-mediated mucin secretion is hypothesized, and is currently being tested.

View Article: PubMed Central - PubMed

Affiliation: Wayne State University School of Medicine, Department of Physiology, Detroit, MI, USA.

ABSTRACT

Macromolecular structures embedded in the cell plasma membrane called 'porosomes', are involved in the regulated fractional release of intravesicular contents from cells during secretion. Porosomes range in size from 15 nm in neurons and astrocytes to 100-180 nm in the exocrine pancreas and neuroendocrine cells. Porosomes have been isolated from a number of cells, and their morphology, composition, and functional reconstitution well documented. The 3D contour map of the assembly of proteins within the porosome complex, and its native X-ray solution structure at sub-nm resolution has also advanced. This understanding now provides a platform to address diseases that may result from secretory defects. Water and ion binding to mucin impart hydration, critical for regulating viscosity of the mucus in the airways epithelia. Appropriate viscosity is required for the movement of mucus by the underlying cilia. Hence secretion of more viscous mucus prevents its proper transport, resulting in chronic and fatal airways disease such as cystic fibrosis (CF). CF is caused by the malfunction of CF transmembrane conductance regulator (CFTR), a chloride channel transporter, resulting in viscous mucus in the airways. Studies in mice lacking functional CFTR secrete highly viscous mucous that adhered to the epithelium. Since CFTR is known to interact with the t-SNARE protein syntaxin-1A, and with the chloride channel CLC-3, which are also components of the porosome complex, the interactions between CFTR and the porosome complex in the mucin-secreting human airway epithelial cell line Calu-3 was hypothesized and tested. Results from the study demonstrate the presence of approximately 100 nm in size porosome complex composed of 34 proteins at the cell plasma membrane in Calu-3 cells, and the association of CFTR with the complex. In comparison, the nuclear pore complex measures 120 nm and is comprised of over 500 protein molecules. The involvement of CFTR in porosome-mediated mucin secretion is hypothesized, and is currently being tested.

No MeSH data available.


Related in: MedlinePlus

Representative electron micrographs of Calu-3 cells in culture demonstrating the presence of microvilli (MV) and porosomes (P) at the cell plasma membrane [11]. (A) Calu-3 cells demonstrate the presence of dense microvilli and scattered porosomes at the cell plasma membrane. (B–D) Note the flask-shaped porosomes measuring nearly 100 nm in diameter (E) and from 200–300 nm in depth, with openings to the cell surface (red arrowhead). Mucus (C), is found at the opening of the porosome to the cell exterior. Of the two porosomes shown in (D), the one to the center appears to be sectioned right through the center of the organelle, where as the porosome to the left, has been sectioned at its base. (E) Similar to the AFM images in Figure 1, the microvilli measure on average 92 nm in diameter [11]. ©Bhanu Jena.
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Figure 2: Representative electron micrographs of Calu-3 cells in culture demonstrating the presence of microvilli (MV) and porosomes (P) at the cell plasma membrane [11]. (A) Calu-3 cells demonstrate the presence of dense microvilli and scattered porosomes at the cell plasma membrane. (B–D) Note the flask-shaped porosomes measuring nearly 100 nm in diameter (E) and from 200–300 nm in depth, with openings to the cell surface (red arrowhead). Mucus (C), is found at the opening of the porosome to the cell exterior. Of the two porosomes shown in (D), the one to the center appears to be sectioned right through the center of the organelle, where as the porosome to the left, has been sectioned at its base. (E) Similar to the AFM images in Figure 1, the microvilli measure on average 92 nm in diameter [11]. ©Bhanu Jena.

Mentions: It is well established that cup-shaped macromolecular lipoprotein structures called porosomes are secretory portals embedded in the cell plasma membrane in cells, where membrane-bound secretory vesicles transiently dock and fuse to expel intravesicular contents during secretion1–10. Porosomes have been isolated from a number of cells, including the exocrine pancreas5,6 (Figure 1), neurons3 (Figure 2), and in the mucin-secreting human airway epithelial cell line Calu-3 (Figure 3,4)11. The morphology, composition, and reconstitution of porosomes in the exocrine pancreas (Figure 5–7) and in neurons are well documented2–12, and the 3D contour map of the assembly of proteins within the structure has also been determined in great detail10. This new understanding of the secretory machinery in cells now provides a platform to address diseases resulting from secretory defects. The structure, and composition of the porosome complex in Calu-3 cells expressing cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been determined for the first time11, with promise to help better understand cystic fibrosis. CFTR is a plasma membrane chloride selective cyclic AMP-activated ion channel, localized at the apical membrane of secretory epithelial cells, including the conducting airways13. Besides mediating the secretion of Cl-, CFTR also regulates several other transport proteins, including K+ channels, aquaporin water channels, anion exchangers, the membrane fusion protein syntaxin-1A, and sodium bicarbonate transporters14–26. Accordingly, studies show that CFTR and its associated proteins are present in large macromolecular signaling complexes via scaffolding proteins containing PDZ domains13,25,27. The C-terminus of CFTR in humans contains the sequence Asp-Thr-Arg-Leu, that mediate binding to several PDZ domain proteins13. For example, ezrin and moesin present in the Calu-3 porosome complex11 are also known CFTR-PDZ binding protein13. In addition, CFTR has several other regions that mediate protein-protein interactions, such as a domain at its N-terminus that binds to syntaxin-1A and SNAP-2315,23. CFTR also contains a protein phosphatase-2A (PP2A)-binding, and an AMP kinase (AMPK)-binding domain12. Similarly, CFTR has a regulatory domain that is a substrate to both protein kinase A (PKA) and C (PKC)28. These interactions facilitate CFTR to form large CFTR-associated macromolecular signaling complexes at the plasma membrane. CF as a disease was first identified as cysts observed in the pancreas and the highly viscous mucus found in the lung of patients. However, since discovery that these observed defects are a result of a dysfunction of the CFTR chloride channel29,30, there has been little progress in our understanding of the link between CFTR dysfunction and the secretion of such highly viscous mucin in the lung of CF patients31. The surface of the airways is coated with a thin film of mucous composed of essentially mucin, salt, proteases, antioxidants, and antibodies31,32. Mucin lubricates, trap foreign particles and pathogens, and assists in the clearance of foreign particles from the airways via ciliary transport31,32. A key property of mucus is its appropriate viscosity that enables its movement by the underlying cilia. Secretion of more viscous mucus disallows its proper transport, resulting in chronic and fatal airways disease such as CF32. Similar to other secretory cells that undergo secretory vesicle volume increase during secretion35–45, goblet cells of the airways epithelia that store mucin in a dehydrated state within membrane-bound secretory granules are no exception. Since vesicle swelling is a requirement for cell secretion41, and both ion channels and water channels or aquaporins regulate this process42,44, altered chloride transport would impair secretory vesicle hydration and optimal release. Furthermore, recent studies in mice lacking functional CFTR31 showed that these animals secrete highly viscous mucous that adhered to the epithelium. Since CFTR is known to interact with syntaxin-1A, chloride channel CLC-3, and aquaporins14–26, which are components of the porosome complex1,2,5–7,46, the possible interactions between CFTR and the porosome in goblet cells was hypothesized and tested in a recent study11. Results from the study demonstrate the presence of approximately 100 nm in size porosomes and microvilli at the surface of the plasma membrane in Calu-3 cells (Figure 1,2)11. The t-SNARE SNAP-25 specific antibody conjugated to protein A-sepharose® has been utilized to isolate the porosome complex from Calu-3 cells11. For each immunoisolation, 1 mg of Triton-Lubrol-solubilized Calu-3 cells was used. The Triton/Lubrol solubilization buffer contained 0.5% Lubrol, 0.5% Triton X-100, 1 mM benzamidine, 5 mM Mg-ATP, and 5 mM EDTA in PBS at pH 7.5, supplemented with protease inhibitor mix (Sigma, St. Louis, MO). Ten micrograms of SNAP-25 antibody conjugated to the protein A-sepharose® were incubated with the 1 mg of the solubilized cells for 1 h at room temperature followed by three washes of 10 volumes of wash buffer (500 mM NaCl, 10 mM Tris, 2 mM EDTA, pH 7.5). The immunoprecipitated porosome attached to the immunosepharose beads was eluted using low pH buffer (pH 3.5) to dissociate the complex from the antibody bound to the bead, and the eluted sample immediately returned to neutral pH and stored at −80 degrees11. A combination of proteomics, Western blot analysis, and immunocytochemistry, were all used to determine the composition and distribution of the porosome complex in Calu-3 cells11. Proteomic analysis of isolated Calu-3 porosomes using mass spectrometry demonstrate the presence of CFTR as well as several proteins found in the neuronal porosome complex, including Syntaxin-1A, actin, rabs, heterotrimeric G-protein, and the GTPase activating protein GAP (Table I)11. Immunoblot analysis (Figure 3) of the isolated Calu-3 porosome complex, and immunocytochemistry (Figure 4) further confirms CFTR association with the porosome complex, reflecting important implication of CFTR in both normal mucus secretion in the airway epithelium in health, and in the impaired state in CF disease. In the past two decades, employing a combination of approaches including AFM, biochemistry, molecular biology, electrophysiology, EM, mass spectrometry, SAXS analysis, and database searches such as STRING 9.1 of known functional and predicted protein-protein interactions, further structural details of the porosome complex have been determined1–12. Although great progress have been made in our understanding of the porosome, of Ca+2 and SNARE-mediated membrane fusion47–76, and on secretory vesicle volume regulation required for regulated fractional release of intravesicular contents35–45 during cell secretion, a molecular level understanding of porosome-mediated secretion in mucin-secreting cells remains to be determined. Therefore, a clear understanding of the porosome in mucin secreting in Calu-3 cells, and the role of CFTR in porosome-mediated mucin secretion is critical in revealing how mucin secretion is precisely regulated.


Porosome in Cystic Fibrosis.

Jena BP - Discoveries (Craiova) (2014 Jul-Sep)

Representative electron micrographs of Calu-3 cells in culture demonstrating the presence of microvilli (MV) and porosomes (P) at the cell plasma membrane [11]. (A) Calu-3 cells demonstrate the presence of dense microvilli and scattered porosomes at the cell plasma membrane. (B–D) Note the flask-shaped porosomes measuring nearly 100 nm in diameter (E) and from 200–300 nm in depth, with openings to the cell surface (red arrowhead). Mucus (C), is found at the opening of the porosome to the cell exterior. Of the two porosomes shown in (D), the one to the center appears to be sectioned right through the center of the organelle, where as the porosome to the left, has been sectioned at its base. (E) Similar to the AFM images in Figure 1, the microvilli measure on average 92 nm in diameter [11]. ©Bhanu Jena.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581455&req=5

Figure 2: Representative electron micrographs of Calu-3 cells in culture demonstrating the presence of microvilli (MV) and porosomes (P) at the cell plasma membrane [11]. (A) Calu-3 cells demonstrate the presence of dense microvilli and scattered porosomes at the cell plasma membrane. (B–D) Note the flask-shaped porosomes measuring nearly 100 nm in diameter (E) and from 200–300 nm in depth, with openings to the cell surface (red arrowhead). Mucus (C), is found at the opening of the porosome to the cell exterior. Of the two porosomes shown in (D), the one to the center appears to be sectioned right through the center of the organelle, where as the porosome to the left, has been sectioned at its base. (E) Similar to the AFM images in Figure 1, the microvilli measure on average 92 nm in diameter [11]. ©Bhanu Jena.
Mentions: It is well established that cup-shaped macromolecular lipoprotein structures called porosomes are secretory portals embedded in the cell plasma membrane in cells, where membrane-bound secretory vesicles transiently dock and fuse to expel intravesicular contents during secretion1–10. Porosomes have been isolated from a number of cells, including the exocrine pancreas5,6 (Figure 1), neurons3 (Figure 2), and in the mucin-secreting human airway epithelial cell line Calu-3 (Figure 3,4)11. The morphology, composition, and reconstitution of porosomes in the exocrine pancreas (Figure 5–7) and in neurons are well documented2–12, and the 3D contour map of the assembly of proteins within the structure has also been determined in great detail10. This new understanding of the secretory machinery in cells now provides a platform to address diseases resulting from secretory defects. The structure, and composition of the porosome complex in Calu-3 cells expressing cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been determined for the first time11, with promise to help better understand cystic fibrosis. CFTR is a plasma membrane chloride selective cyclic AMP-activated ion channel, localized at the apical membrane of secretory epithelial cells, including the conducting airways13. Besides mediating the secretion of Cl-, CFTR also regulates several other transport proteins, including K+ channels, aquaporin water channels, anion exchangers, the membrane fusion protein syntaxin-1A, and sodium bicarbonate transporters14–26. Accordingly, studies show that CFTR and its associated proteins are present in large macromolecular signaling complexes via scaffolding proteins containing PDZ domains13,25,27. The C-terminus of CFTR in humans contains the sequence Asp-Thr-Arg-Leu, that mediate binding to several PDZ domain proteins13. For example, ezrin and moesin present in the Calu-3 porosome complex11 are also known CFTR-PDZ binding protein13. In addition, CFTR has several other regions that mediate protein-protein interactions, such as a domain at its N-terminus that binds to syntaxin-1A and SNAP-2315,23. CFTR also contains a protein phosphatase-2A (PP2A)-binding, and an AMP kinase (AMPK)-binding domain12. Similarly, CFTR has a regulatory domain that is a substrate to both protein kinase A (PKA) and C (PKC)28. These interactions facilitate CFTR to form large CFTR-associated macromolecular signaling complexes at the plasma membrane. CF as a disease was first identified as cysts observed in the pancreas and the highly viscous mucus found in the lung of patients. However, since discovery that these observed defects are a result of a dysfunction of the CFTR chloride channel29,30, there has been little progress in our understanding of the link between CFTR dysfunction and the secretion of such highly viscous mucin in the lung of CF patients31. The surface of the airways is coated with a thin film of mucous composed of essentially mucin, salt, proteases, antioxidants, and antibodies31,32. Mucin lubricates, trap foreign particles and pathogens, and assists in the clearance of foreign particles from the airways via ciliary transport31,32. A key property of mucus is its appropriate viscosity that enables its movement by the underlying cilia. Secretion of more viscous mucus disallows its proper transport, resulting in chronic and fatal airways disease such as CF32. Similar to other secretory cells that undergo secretory vesicle volume increase during secretion35–45, goblet cells of the airways epithelia that store mucin in a dehydrated state within membrane-bound secretory granules are no exception. Since vesicle swelling is a requirement for cell secretion41, and both ion channels and water channels or aquaporins regulate this process42,44, altered chloride transport would impair secretory vesicle hydration and optimal release. Furthermore, recent studies in mice lacking functional CFTR31 showed that these animals secrete highly viscous mucous that adhered to the epithelium. Since CFTR is known to interact with syntaxin-1A, chloride channel CLC-3, and aquaporins14–26, which are components of the porosome complex1,2,5–7,46, the possible interactions between CFTR and the porosome in goblet cells was hypothesized and tested in a recent study11. Results from the study demonstrate the presence of approximately 100 nm in size porosomes and microvilli at the surface of the plasma membrane in Calu-3 cells (Figure 1,2)11. The t-SNARE SNAP-25 specific antibody conjugated to protein A-sepharose® has been utilized to isolate the porosome complex from Calu-3 cells11. For each immunoisolation, 1 mg of Triton-Lubrol-solubilized Calu-3 cells was used. The Triton/Lubrol solubilization buffer contained 0.5% Lubrol, 0.5% Triton X-100, 1 mM benzamidine, 5 mM Mg-ATP, and 5 mM EDTA in PBS at pH 7.5, supplemented with protease inhibitor mix (Sigma, St. Louis, MO). Ten micrograms of SNAP-25 antibody conjugated to the protein A-sepharose® were incubated with the 1 mg of the solubilized cells for 1 h at room temperature followed by three washes of 10 volumes of wash buffer (500 mM NaCl, 10 mM Tris, 2 mM EDTA, pH 7.5). The immunoprecipitated porosome attached to the immunosepharose beads was eluted using low pH buffer (pH 3.5) to dissociate the complex from the antibody bound to the bead, and the eluted sample immediately returned to neutral pH and stored at −80 degrees11. A combination of proteomics, Western blot analysis, and immunocytochemistry, were all used to determine the composition and distribution of the porosome complex in Calu-3 cells11. Proteomic analysis of isolated Calu-3 porosomes using mass spectrometry demonstrate the presence of CFTR as well as several proteins found in the neuronal porosome complex, including Syntaxin-1A, actin, rabs, heterotrimeric G-protein, and the GTPase activating protein GAP (Table I)11. Immunoblot analysis (Figure 3) of the isolated Calu-3 porosome complex, and immunocytochemistry (Figure 4) further confirms CFTR association with the porosome complex, reflecting important implication of CFTR in both normal mucus secretion in the airway epithelium in health, and in the impaired state in CF disease. In the past two decades, employing a combination of approaches including AFM, biochemistry, molecular biology, electrophysiology, EM, mass spectrometry, SAXS analysis, and database searches such as STRING 9.1 of known functional and predicted protein-protein interactions, further structural details of the porosome complex have been determined1–12. Although great progress have been made in our understanding of the porosome, of Ca+2 and SNARE-mediated membrane fusion47–76, and on secretory vesicle volume regulation required for regulated fractional release of intravesicular contents35–45 during cell secretion, a molecular level understanding of porosome-mediated secretion in mucin-secreting cells remains to be determined. Therefore, a clear understanding of the porosome in mucin secreting in Calu-3 cells, and the role of CFTR in porosome-mediated mucin secretion is critical in revealing how mucin secretion is precisely regulated.

Bottom Line: This understanding now provides a platform to address diseases that may result from secretory defects.Hence secretion of more viscous mucus prevents its proper transport, resulting in chronic and fatal airways disease such as cystic fibrosis (CF).The involvement of CFTR in porosome-mediated mucin secretion is hypothesized, and is currently being tested.

View Article: PubMed Central - PubMed

Affiliation: Wayne State University School of Medicine, Department of Physiology, Detroit, MI, USA.

ABSTRACT

Macromolecular structures embedded in the cell plasma membrane called 'porosomes', are involved in the regulated fractional release of intravesicular contents from cells during secretion. Porosomes range in size from 15 nm in neurons and astrocytes to 100-180 nm in the exocrine pancreas and neuroendocrine cells. Porosomes have been isolated from a number of cells, and their morphology, composition, and functional reconstitution well documented. The 3D contour map of the assembly of proteins within the porosome complex, and its native X-ray solution structure at sub-nm resolution has also advanced. This understanding now provides a platform to address diseases that may result from secretory defects. Water and ion binding to mucin impart hydration, critical for regulating viscosity of the mucus in the airways epithelia. Appropriate viscosity is required for the movement of mucus by the underlying cilia. Hence secretion of more viscous mucus prevents its proper transport, resulting in chronic and fatal airways disease such as cystic fibrosis (CF). CF is caused by the malfunction of CF transmembrane conductance regulator (CFTR), a chloride channel transporter, resulting in viscous mucus in the airways. Studies in mice lacking functional CFTR secrete highly viscous mucous that adhered to the epithelium. Since CFTR is known to interact with the t-SNARE protein syntaxin-1A, and with the chloride channel CLC-3, which are also components of the porosome complex, the interactions between CFTR and the porosome complex in the mucin-secreting human airway epithelial cell line Calu-3 was hypothesized and tested. Results from the study demonstrate the presence of approximately 100 nm in size porosome complex composed of 34 proteins at the cell plasma membrane in Calu-3 cells, and the association of CFTR with the complex. In comparison, the nuclear pore complex measures 120 nm and is comprised of over 500 protein molecules. The involvement of CFTR in porosome-mediated mucin secretion is hypothesized, and is currently being tested.

No MeSH data available.


Related in: MedlinePlus