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CX3CL1, a chemokine finely tuned to adhesion: critical roles of the stalk glycosylation and the membrane domain.

Ostuni MA, Guellec J, Hermand P, Durand P, Combadière C, Pincet F, Deterre P - Biol Open (2014)

Bottom Line: The multi-domain CX3CL1 transmembrane chemokine triggers leukocyte adherence without rolling and migration by presenting its chemokine domain (CD) to its receptor CX3CR1.Our results indicate that the CX3CL1 molecular structure is finely adapted to capture CX3CR1 in circulating cells and that each domain has a specific purpose: the mucin stalk is stiffened by its high glycosylation to present the CD away from the membrane, the transmembrane domain generates the permanent aggregation of an adequate amount of monomers to guarantee adhesion and prevent rolling, and the cytosolic domain ensures adhesive robustness by interacting with the cytoskeleton.We propose a model in which quasi-immobile CX3CL1 bundles are organized to quickly generate adhesive patches with sufficiently high strength to capture CX3CR1+ leukocytes but with sufficiently low strength to allow their patrolling behavior.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U 1135, Centre d'Immunologie et des Maladies Infectieuses, F-75013, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMRS CR7, Centre d'Immunologie et des Maladies Infectieuses, F-75013, Paris, France CNRS, ERL 8255, Centre d'Immunologie et des Maladies Infectieuses, F-75013, Paris, France Present address: INSERM, U 1134, Biologie Intégrée du Globule Rouge; Université Paris Diderot; Institut National de la Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015, Paris, France.

No MeSH data available.


Related in: MedlinePlus

Lateral diffusion rate of CX3CL1-EYFP and its truncated, deglycosylated, or TM mutants.(A) Diffusion rate assessed by FRAP in COS7 cells of native CX3CL1-EYFP or CX3CL1-EYFP lacking its chemokine domain (named “w/o CD CX3CL1”), its cytosolic tail (named “w/o cyto CX3CL1”), its extracellular part (named “cyto-TM”), or both of its extracellular and intracellular regions (named “TM”) or deglycosylated (named “dgCX3CL1”); * p<0.05 and *** p<0.001 compared with native CX3CL1-EYFP. (B) Diffusion rate of CX3CL1-EYFP and various mutants as assessed by FRAP in CHOldlD cells cultured either with 10% FCS (empty bars) or 0.1% FCS (filled bars) medium. *** p<0.001 compared with the respective 10% FCS cultured CX3CL1-EYFP-expressing cells. (C) Immunoblotting of CX3CL1 chimera in CHOldlD. Thirty micrograms of membrane lysates from CHOldlD transfected with CX3CL1-EYFP and cultivated under normal (right) or deprived (left) serum conditions were loaded onto a 10% polyacrylamide gel under reducing conditions, transferred to a nitrocellulose membrane, and incubated overnight at 4°C under agitation with anti-human CX3CL1 primary antibody. The immune complexes were visualized with secondary peroxidase-conjugated antibodies using a chemiluminescent kit. (D) Lateral diffusion measured by FRAP in COS7 cells expressing the native CX3CL1-EYFP chimera or different constructions in which residues 321 to 325 were mutated to ALA (named “CX3CL1-5ALA”), residues 326 to 332 were mutated to ALA (“CX3CL1-7ALA”), or residues 321 to 332 were mutated to ALA (“CX3CL1-12ALA”). The lateral diffusion of CX3CL1-12ALA and of this mutated chemokine without its extracellular domain (“cyto-TM-12ALA”) or without its extra and intracellular domains (“TM-12ALA”) is also reported. *** p<0.001 compared with native CX3CL1-EYFP.
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f04: Lateral diffusion rate of CX3CL1-EYFP and its truncated, deglycosylated, or TM mutants.(A) Diffusion rate assessed by FRAP in COS7 cells of native CX3CL1-EYFP or CX3CL1-EYFP lacking its chemokine domain (named “w/o CD CX3CL1”), its cytosolic tail (named “w/o cyto CX3CL1”), its extracellular part (named “cyto-TM”), or both of its extracellular and intracellular regions (named “TM”) or deglycosylated (named “dgCX3CL1”); * p<0.05 and *** p<0.001 compared with native CX3CL1-EYFP. (B) Diffusion rate of CX3CL1-EYFP and various mutants as assessed by FRAP in CHOldlD cells cultured either with 10% FCS (empty bars) or 0.1% FCS (filled bars) medium. *** p<0.001 compared with the respective 10% FCS cultured CX3CL1-EYFP-expressing cells. (C) Immunoblotting of CX3CL1 chimera in CHOldlD. Thirty micrograms of membrane lysates from CHOldlD transfected with CX3CL1-EYFP and cultivated under normal (right) or deprived (left) serum conditions were loaded onto a 10% polyacrylamide gel under reducing conditions, transferred to a nitrocellulose membrane, and incubated overnight at 4°C under agitation with anti-human CX3CL1 primary antibody. The immune complexes were visualized with secondary peroxidase-conjugated antibodies using a chemiluminescent kit. (D) Lateral diffusion measured by FRAP in COS7 cells expressing the native CX3CL1-EYFP chimera or different constructions in which residues 321 to 325 were mutated to ALA (named “CX3CL1-5ALA”), residues 326 to 332 were mutated to ALA (“CX3CL1-7ALA”), or residues 321 to 332 were mutated to ALA (“CX3CL1-12ALA”). The lateral diffusion of CX3CL1-12ALA and of this mutated chemokine without its extracellular domain (“cyto-TM-12ALA”) or without its extra and intracellular domains (“TM-12ALA”) is also reported. *** p<0.001 compared with native CX3CL1-EYFP.

Mentions: We also assayed the effect of the deletion of the different parts of CX3CL1 on its diffusion rate in the COS7 cell line (Fig. 4A). The truncation of the CD (“w/o CD CX3CL1”) had no effect on the lateral diffusion of the molecule, whereas the truncation of the cytosolic tail (“w/o cyto CX3CL1”) resulted only in a modest 1.2-fold increase in the diffusion rate. In contrast, the truncation of the extracellular region (“cyto-TM”) yielded a net 2-fold increase. Moreover, the truncation of both the intracellular and extracellular regions of CX3CL1 without changing the TM domain resulted in a molecule that was almost 3-fold more mobile than the whole molecule. Thus, it appears that the extracellular part of the chemokine is largely involved in the restraining of its diffusion rate.


CX3CL1, a chemokine finely tuned to adhesion: critical roles of the stalk glycosylation and the membrane domain.

Ostuni MA, Guellec J, Hermand P, Durand P, Combadière C, Pincet F, Deterre P - Biol Open (2014)

Lateral diffusion rate of CX3CL1-EYFP and its truncated, deglycosylated, or TM mutants.(A) Diffusion rate assessed by FRAP in COS7 cells of native CX3CL1-EYFP or CX3CL1-EYFP lacking its chemokine domain (named “w/o CD CX3CL1”), its cytosolic tail (named “w/o cyto CX3CL1”), its extracellular part (named “cyto-TM”), or both of its extracellular and intracellular regions (named “TM”) or deglycosylated (named “dgCX3CL1”); * p<0.05 and *** p<0.001 compared with native CX3CL1-EYFP. (B) Diffusion rate of CX3CL1-EYFP and various mutants as assessed by FRAP in CHOldlD cells cultured either with 10% FCS (empty bars) or 0.1% FCS (filled bars) medium. *** p<0.001 compared with the respective 10% FCS cultured CX3CL1-EYFP-expressing cells. (C) Immunoblotting of CX3CL1 chimera in CHOldlD. Thirty micrograms of membrane lysates from CHOldlD transfected with CX3CL1-EYFP and cultivated under normal (right) or deprived (left) serum conditions were loaded onto a 10% polyacrylamide gel under reducing conditions, transferred to a nitrocellulose membrane, and incubated overnight at 4°C under agitation with anti-human CX3CL1 primary antibody. The immune complexes were visualized with secondary peroxidase-conjugated antibodies using a chemiluminescent kit. (D) Lateral diffusion measured by FRAP in COS7 cells expressing the native CX3CL1-EYFP chimera or different constructions in which residues 321 to 325 were mutated to ALA (named “CX3CL1-5ALA”), residues 326 to 332 were mutated to ALA (“CX3CL1-7ALA”), or residues 321 to 332 were mutated to ALA (“CX3CL1-12ALA”). The lateral diffusion of CX3CL1-12ALA and of this mutated chemokine without its extracellular domain (“cyto-TM-12ALA”) or without its extra and intracellular domains (“TM-12ALA”) is also reported. *** p<0.001 compared with native CX3CL1-EYFP.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4265755&req=5

f04: Lateral diffusion rate of CX3CL1-EYFP and its truncated, deglycosylated, or TM mutants.(A) Diffusion rate assessed by FRAP in COS7 cells of native CX3CL1-EYFP or CX3CL1-EYFP lacking its chemokine domain (named “w/o CD CX3CL1”), its cytosolic tail (named “w/o cyto CX3CL1”), its extracellular part (named “cyto-TM”), or both of its extracellular and intracellular regions (named “TM”) or deglycosylated (named “dgCX3CL1”); * p<0.05 and *** p<0.001 compared with native CX3CL1-EYFP. (B) Diffusion rate of CX3CL1-EYFP and various mutants as assessed by FRAP in CHOldlD cells cultured either with 10% FCS (empty bars) or 0.1% FCS (filled bars) medium. *** p<0.001 compared with the respective 10% FCS cultured CX3CL1-EYFP-expressing cells. (C) Immunoblotting of CX3CL1 chimera in CHOldlD. Thirty micrograms of membrane lysates from CHOldlD transfected with CX3CL1-EYFP and cultivated under normal (right) or deprived (left) serum conditions were loaded onto a 10% polyacrylamide gel under reducing conditions, transferred to a nitrocellulose membrane, and incubated overnight at 4°C under agitation with anti-human CX3CL1 primary antibody. The immune complexes were visualized with secondary peroxidase-conjugated antibodies using a chemiluminescent kit. (D) Lateral diffusion measured by FRAP in COS7 cells expressing the native CX3CL1-EYFP chimera or different constructions in which residues 321 to 325 were mutated to ALA (named “CX3CL1-5ALA”), residues 326 to 332 were mutated to ALA (“CX3CL1-7ALA”), or residues 321 to 332 were mutated to ALA (“CX3CL1-12ALA”). The lateral diffusion of CX3CL1-12ALA and of this mutated chemokine without its extracellular domain (“cyto-TM-12ALA”) or without its extra and intracellular domains (“TM-12ALA”) is also reported. *** p<0.001 compared with native CX3CL1-EYFP.
Mentions: We also assayed the effect of the deletion of the different parts of CX3CL1 on its diffusion rate in the COS7 cell line (Fig. 4A). The truncation of the CD (“w/o CD CX3CL1”) had no effect on the lateral diffusion of the molecule, whereas the truncation of the cytosolic tail (“w/o cyto CX3CL1”) resulted only in a modest 1.2-fold increase in the diffusion rate. In contrast, the truncation of the extracellular region (“cyto-TM”) yielded a net 2-fold increase. Moreover, the truncation of both the intracellular and extracellular regions of CX3CL1 without changing the TM domain resulted in a molecule that was almost 3-fold more mobile than the whole molecule. Thus, it appears that the extracellular part of the chemokine is largely involved in the restraining of its diffusion rate.

Bottom Line: The multi-domain CX3CL1 transmembrane chemokine triggers leukocyte adherence without rolling and migration by presenting its chemokine domain (CD) to its receptor CX3CR1.Our results indicate that the CX3CL1 molecular structure is finely adapted to capture CX3CR1 in circulating cells and that each domain has a specific purpose: the mucin stalk is stiffened by its high glycosylation to present the CD away from the membrane, the transmembrane domain generates the permanent aggregation of an adequate amount of monomers to guarantee adhesion and prevent rolling, and the cytosolic domain ensures adhesive robustness by interacting with the cytoskeleton.We propose a model in which quasi-immobile CX3CL1 bundles are organized to quickly generate adhesive patches with sufficiently high strength to capture CX3CR1+ leukocytes but with sufficiently low strength to allow their patrolling behavior.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U 1135, Centre d'Immunologie et des Maladies Infectieuses, F-75013, Paris, France Sorbonne Universités, UPMC Université Paris 06, UMRS CR7, Centre d'Immunologie et des Maladies Infectieuses, F-75013, Paris, France CNRS, ERL 8255, Centre d'Immunologie et des Maladies Infectieuses, F-75013, Paris, France Present address: INSERM, U 1134, Biologie Intégrée du Globule Rouge; Université Paris Diderot; Institut National de la Transfusion Sanguine, 6 rue Alexandre Cabanel, 75015, Paris, France.

No MeSH data available.


Related in: MedlinePlus