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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

Adhesive potency of the truncated and deglycosylated CX3CL1 mutants compared to native CX3CL1.(A) Immunoblotting of the different CX3CL1-EYFP chimeras expressed in the COS7 cell line with anti-CX3CL1 mAb. Thirty micrograms of membrane lysates from COS7 cells expressing CX3CL1 chimeras were loaded on 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. (B) Number of CX3CR1+ CHO cells adhering to a monolayer of COS7 cells expressing various forms of CX3CL1-EYFP chimera. The results were obtained using a flow adherence assay. ** p<0.01 and *** p<0.001 compared with native CX3CL1-EYFP. (C) Membrane localization of CX3CL1 and its variants. Representative images of COS7 cells expressing EYFP chimeras of CX3CL1, CX3CL1 w/o cyt, shCX3CL1, or dgCX3CL1. The images were taken with a Leica SP5 confocal microscope (63× dry objective). The bars represent 10 µm. (D) The membrane expression of the various constructs was evaluated by confocal microscopy as a ratio of membrane to total fluorescence. (E) Dissociation force, assessed by the two pipette assay, between CX3CR1+ CHO-cells and COS7 cells expressing the indicated CX3CL1-EYFP chimera. (F) Percentage of positive adherent cell doublets calculated for each CX3CL1-EYFP chimera. * p<0.05 and ** p<0.01 compared with native CX3CL1-EYFP.
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f01: Adhesive potency of the truncated and deglycosylated CX3CL1 mutants compared to native CX3CL1.(A) Immunoblotting of the different CX3CL1-EYFP chimeras expressed in the COS7 cell line with anti-CX3CL1 mAb. Thirty micrograms of membrane lysates from COS7 cells expressing CX3CL1 chimeras were loaded on 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. (B) Number of CX3CR1+ CHO cells adhering to a monolayer of COS7 cells expressing various forms of CX3CL1-EYFP chimera. The results were obtained using a flow adherence assay. ** p<0.01 and *** p<0.001 compared with native CX3CL1-EYFP. (C) Membrane localization of CX3CL1 and its variants. Representative images of COS7 cells expressing EYFP chimeras of CX3CL1, CX3CL1 w/o cyt, shCX3CL1, or dgCX3CL1. The images were taken with a Leica SP5 confocal microscope (63× dry objective). The bars represent 10 µm. (D) The membrane expression of the various constructs was evaluated by confocal microscopy as a ratio of membrane to total fluorescence. (E) Dissociation force, assessed by the two pipette assay, between CX3CR1+ CHO-cells and COS7 cells expressing the indicated CX3CL1-EYFP chimera. (F) Percentage of positive adherent cell doublets calculated for each CX3CL1-EYFP chimera. * p<0.05 and ** p<0.01 compared with native CX3CL1-EYFP.

Mentions: We first tested the contribution of different features of the CX3CL1 molecule to its adhesiveness, i.e., the presence of its cytosolic tail, the length of its mucin stalk, and its glycosylation. To this end, we constructed three CX3CL1 mutated analogues: one without the cytosolic tail (termed “w/o cyto CX3CL1”), one with half of the mucin stalk (termed “shCX3CL1”), and one with a fully deglycosylated stalk (termed “dgCX3CL1”), which was obtained by mutating the 52 serine or threonine residues of the mucin stalk to alanine, i.e., all of the potential sites for O-glycosylation. To follow the cellular expression of CX3CL1, these molecules were linked to EYFP at the C terminus, whose presence was indeed previously shown to not impair the function of the whole protein (Liu et al., 2005; Hermand et al., 2008; Huang et al., 2009). After expression in the COS7 cell line, the molecular weight of the CX3CL1-EYFP chimera leveled at 120 kDa, i.e., the sum of the complete CX3CL1 (90 kDa) and of the EYFP protein (30 kDa). Consistently, the w/o cyto CX3CL1 exhibited a molecular weight of 110 kDa, whereas the shCX3CL1 mutant had a weight of approximately 70 kDa (i.e., the sum of the 40 kDa short CX3CL1 plus the EYFP protein). The weight of the dgCX3CL1 mutant was 80 kDa, i.e., the sum of the polypeptide of CX3CL1 (50 kDa) plus the EYFP protein (Fig. 1A). The existence of some minor bands of lower molecular weight as seen in the w/o cyto CX3CL1 well is probably due to some intracellular forms of CX3CL1 that are not well processed as previously analyzed in detail (Garton et al., 2001).


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)

Adhesive potency of the truncated and deglycosylated CX3CL1 mutants compared to native CX3CL1.(A) Immunoblotting of the different CX3CL1-EYFP chimeras expressed in the COS7 cell line with anti-CX3CL1 mAb. Thirty micrograms of membrane lysates from COS7 cells expressing CX3CL1 chimeras were loaded on 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. (B) Number of CX3CR1+ CHO cells adhering to a monolayer of COS7 cells expressing various forms of CX3CL1-EYFP chimera. The results were obtained using a flow adherence assay. ** p<0.01 and *** p<0.001 compared with native CX3CL1-EYFP. (C) Membrane localization of CX3CL1 and its variants. Representative images of COS7 cells expressing EYFP chimeras of CX3CL1, CX3CL1 w/o cyt, shCX3CL1, or dgCX3CL1. The images were taken with a Leica SP5 confocal microscope (63× dry objective). The bars represent 10 µm. (D) The membrane expression of the various constructs was evaluated by confocal microscopy as a ratio of membrane to total fluorescence. (E) Dissociation force, assessed by the two pipette assay, between CX3CR1+ CHO-cells and COS7 cells expressing the indicated CX3CL1-EYFP chimera. (F) Percentage of positive adherent cell doublets calculated for each CX3CL1-EYFP chimera. * p<0.05 and ** p<0.01 compared with native CX3CL1-EYFP.
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Related In: Results  -  Collection

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f01: Adhesive potency of the truncated and deglycosylated CX3CL1 mutants compared to native CX3CL1.(A) Immunoblotting of the different CX3CL1-EYFP chimeras expressed in the COS7 cell line with anti-CX3CL1 mAb. Thirty micrograms of membrane lysates from COS7 cells expressing CX3CL1 chimeras were loaded on 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. (B) Number of CX3CR1+ CHO cells adhering to a monolayer of COS7 cells expressing various forms of CX3CL1-EYFP chimera. The results were obtained using a flow adherence assay. ** p<0.01 and *** p<0.001 compared with native CX3CL1-EYFP. (C) Membrane localization of CX3CL1 and its variants. Representative images of COS7 cells expressing EYFP chimeras of CX3CL1, CX3CL1 w/o cyt, shCX3CL1, or dgCX3CL1. The images were taken with a Leica SP5 confocal microscope (63× dry objective). The bars represent 10 µm. (D) The membrane expression of the various constructs was evaluated by confocal microscopy as a ratio of membrane to total fluorescence. (E) Dissociation force, assessed by the two pipette assay, between CX3CR1+ CHO-cells and COS7 cells expressing the indicated CX3CL1-EYFP chimera. (F) Percentage of positive adherent cell doublets calculated for each CX3CL1-EYFP chimera. * p<0.05 and ** p<0.01 compared with native CX3CL1-EYFP.
Mentions: We first tested the contribution of different features of the CX3CL1 molecule to its adhesiveness, i.e., the presence of its cytosolic tail, the length of its mucin stalk, and its glycosylation. To this end, we constructed three CX3CL1 mutated analogues: one without the cytosolic tail (termed “w/o cyto CX3CL1”), one with half of the mucin stalk (termed “shCX3CL1”), and one with a fully deglycosylated stalk (termed “dgCX3CL1”), which was obtained by mutating the 52 serine or threonine residues of the mucin stalk to alanine, i.e., all of the potential sites for O-glycosylation. To follow the cellular expression of CX3CL1, these molecules were linked to EYFP at the C terminus, whose presence was indeed previously shown to not impair the function of the whole protein (Liu et al., 2005; Hermand et al., 2008; Huang et al., 2009). After expression in the COS7 cell line, the molecular weight of the CX3CL1-EYFP chimera leveled at 120 kDa, i.e., the sum of the complete CX3CL1 (90 kDa) and of the EYFP protein (30 kDa). Consistently, the w/o cyto CX3CL1 exhibited a molecular weight of 110 kDa, whereas the shCX3CL1 mutant had a weight of approximately 70 kDa (i.e., the sum of the 40 kDa short CX3CL1 plus the EYFP protein). The weight of the dgCX3CL1 mutant was 80 kDa, i.e., the sum of the polypeptide of CX3CL1 (50 kDa) plus the EYFP protein (Fig. 1A). The existence of some minor bands of lower molecular weight as seen in the w/o cyto CX3CL1 well is probably due to some intracellular forms of CX3CL1 that are not well processed as previously analyzed in detail (Garton et al., 2001).

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