CX3CL1 homo-oligomerization drives cell-to-cell adherence

Abstract

During inflammatory response, blood leukocytes adhere to the endothelium. This process involves numerous adhesion molecules, including a transmembrane chemokine, CX3CL1, which behaves as a molecular cluster. How this cluster assembles and whether this association has a functional role remain unknown. The analysis of CX3CL1 clusters using native electrophoresis and single molecule fluorescence kinetics shows that CX3CL1 is a homo-oligomer of 3 to 7 monomers. Fluorescence recovery after photobleaching assays reveal that the CX3CL1-transmembrane domain peptide self-associates in both cellular and acellular lipid environments, while its random counterpart (i.e. peptide with the same residues in a different order) does not. This strongly indicates that CX3CL1 oligomerization is driven by its intrinsic properties. According to the molecular modeling, CX3CL1 does not associate in compact bundles but rather with monomers linearly assembled side by side. Finally, the CX3CL1 transmembrane peptide inhibits both the CX3CL1 oligomerization and the adhesive function, while its random counterpart does not. This demonstrates that CX3CL1 oligomerization is mandatory for its adhesive potency. Our results provide a new direction to control CX3CL1-dependent cellular adherence in key immune processes. The migration of blood leukocytes to damaged tissues is the first step of the inflammation process and involves a sequence of coordinated interactions between leukocytes and endothelial cells 1-3. The chemotactic cytokines called chemokines that primarily attract leukocytes, are central to the physiological and pathological inflammatory processes 4-6. Chemokines trigger leukocyte activation and their firm adhesion to the inflamed endothelium, mainly through integrins 7-9. Two members of the chemokine family are exceptions: CXCL16 and CX3CL1. In addition to their chemokine domain (CD), these two chemokines possess three domains: a mucin-like stalk, a transmembrane (TM) domain, and a cytosolic tail 10,11. When interacting with their cognate receptors (CXCR6 and CX3CR1, respectively), these chemokines induce cell-cell adhesion 12. CXCL16 and CX3CL1 can also be cleaved by metalloproteinases, such as ADAM10 and ADAM17 13-15 , to produce a soluble form with chemotactic functions. The CX3CL1 chemokine, with its unique CX3CR1 receptor 16 , is involved in adherence to the endothelium of the inflammatory monocyte population (CD14 hi CD16-CX3CR1 + CCR2 + in humans, Ly6C hi CX3CR1 + CCR2 + in mice) 12,17-20 likely through interaction with platelets 21,22. This chemokine is also involved in the recruitment of NK lymphocytes 23,24 and in lymphocyte survival as in allergic diseases 25 , as well as in monocytic 26,27 and neuronal survival 28-31. An additional function of the CX3CR1-CX3CL1 pair is the regulation of the patrolling behavior and the margination of monocytes in blood vessels 32,33 or their adherence to the bone marrow 34. The CX3CL1 chemokine is also involved in cytoadhesion of red blood cells infected with the malaria parasite Plasmodium

High resolution analysis of the cytosolic Ca2+events in beta cell collectives in situ

“…Fast and long-term recording of [Ca 2+ ] c oscillations was limited due to relatively weak fluorescence signal of the classical fluorophores, their high affinity Ca 2+ binding that yielded phase lags, slow sampling rates, and substantial bleaching [21,63]. However, novel fluorescent Ca 2+ -sensing dyes that overcome most of the aforementioned technical issues [47], used together with enhanced data analysis, have enabled quantitation of changes in cytosolic Ca 2+ concentrations with millisecond temporal and high spatial resolution in many cells at the same time enabling repeated stimulations. With these tools at hand, we conducted studies to revisit glucose-dependent beta cell activation and to test the contribution of IP 3 and RYR intracellular Ca 2+ release channels during this activation.…”

SMAD4 and TGFβ are architects of inverse genetic programs during fate determination of antiviral CTLs

“…Since very few SMAD4-deficient CTLs expressed KLRG1 during infection with LM-OVA, we investigated whether these cells were located in the vasculature or peripheral tissues. Fractalkine (CX 3 CL 1 ) is a chemoattractant with adhesive properties ( Ostuni et al, 2020 ) that alters CD8 T cell migration. Prior studies indicate that CTLs in the vasculature express the fractalkine receptor (CX 3 CR1) at high levels ( Nishimura et al, 2002 ).…”

Polymerization of misfolded Z alpha-1 antitrypsin protein lowers CX3CR1 expression in human PBMCs

“…Probably, specific conformational properties and/or molecular sizes of Z-AAT polymers are required for their interaction with CX3CR1. For example, cell surfaces express CX3CL1 as a constitutive oligomer (three to seven molecules), which is essential for efficient interaction with CX3CR1 ( Hermand et al, 2008 ; Ostuni et al, 2020 ). Numerous chemokines tend to self-associate that determines their activity ( Proudfoot et al, 2003 ), and therefore certain Z-AAT polymers may resemble chemokine structures competing for the same receptor(s).…”