The cortical actin network regulates avidity-dependent binding of hyaluronan by the lymphatic vessel endothelial receptor LYVE-1

Abstract

Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) mediates the docking and entry of dendritic cells to lymphatic vessels through selective adhesion to its ligand hyaluronan in the leukocyte surface glycocalyx. To bind hyaluronan efficiently, LYVE-1 must undergo surface clustering, a process that is induced efficiently by the large cross-linked assemblages of glycosaminoglycan present within leukocyte pericellular matrices but is induced poorly by the shorter polymer alone. These properties suggested that LYVE-1 may have limited mobility in the endothelial plasma membrane, but no biophysical investigation of these parameters has been carried out to date. Here, using super-resolution fluorescence microscopy and spectroscopy combined with biochemical analyses of the receptor in primary lymphatic endothelial cells, we provide the first evidence that LYVE-1 dynamics are indeed restricted by the submembranous actin network. We show that actin disruption not only increases LYVE-1 lateral diffusion but also enhances hyaluronan-binding activity. However, unlike the related leukocyte HA receptor CD44, which uses ERM and ankyrin motifs within its cytoplasmic tail to bind actin, LYVE-1 displays little if any direct interaction with actin, as determined by co-immunoprecipitation. Instead, as shown by super-resolution stimulated emission depletion microscopy in combination with fluorescence correlation spectroscopy, LYVE-1 diffusion is restricted by transient entrapment within submembranous actin corrals. These results point to an actin-mediated constraint on LYVE-1 clustering in lymphatic endothelium that tunes the receptor for selective engagement with hyaluronan assemblages in the glycocalyx that are large enough to cross-bridge the corral-bound LYVE-1 molecules and thereby facilitate leukocyte adhesion and transmigration.

Hyaluronan and Its Receptors as Regulatory Molecules of the Endothelial Interface

“…91 Recent studies demonstrate that cortical actin networks regulate LYVE-1 lateral diffusion and receptor clustering in the lymphatic endothelium, supporting the observation that selective engagement of LYVE-1 with HA within the glycocalyx facilitates leukocyte adhesion and transmigration. 94 In contrast to LYVE-1 which does not interact with the cytoskeleton but rather appears constrained by it, CD44 binds to the actin cytoskeleton via actin adapter proteins to promote CD44 clustering. 41,95 Understanding cues that promote in vivo dynamic clustering which allows LYVE-1 to discriminate HA present on the surface of leukocytes from soluble HA requires further study.…”

Hyaluronan and Its Receptors: Key Mediators of Immune Cell Entry and Trafficking in the Lymphatic System

“…Even so, homodimerisation is not sufficient in itself to enable constitutive binding of free HMW HA by the native receptor in LECs [148], and such binding requires further high-density clustering of LYVE-1 or prior cross-linking of HA in the form of multimolecular complexes with CD44 and/or one or more of its HA-binding partners [49]. This constraint is imposed by the limited lateral mobility of LYVE-1, owing to its corralling within the sub-membrane actin meshwork by means of the cytoplasmic tail, as evidenced by super-resolution microscopy [149,150]. As outlined earlier, the necessary conditions for HA-binding are met during DC transit by the redistribution of LYVE-1 from its normally punctate pattern in the plasma membrane [149] into receptor-dense lymphatic transmigratory cups at the leucocyte:endothelial interface, and by recruitment of the multivalent CD44 • HA glycocalyx to the pro-adhesive DC uropod [22,114].…”

“…As outlined earlier, the necessary conditions for HA-binding are met during DC transit by the redistribution of LYVE-1 from its normally punctate pattern in the plasma membrane [149] into receptor-dense lymphatic transmigratory cups at the leucocyte:endothelial interface, and by recruitment of the multivalent CD44 • HA glycocalyx to the pro-adhesive DC uropod [22,114]. Both these processes involve regulated assembly/disassembly of the sub-membrane actin cytoskeleton that restricts the membrane mobility of both LYVE-1 and CD44 [124,125,150]. Overall, it is likely that these features allow lymphatic vessels to discriminate between free HA present in interstitial fluid and cell (i.e., glycocalyx) bound HA and to respond appropriately to each.…”

Simple methods for quantifying super-resolved cortical actin

“…There is significant evidence that actin can regulate organisation of certain receptors within the membrane (e.g. serotonin 1a receptor (Shrivastava et al, 2020), α2Aadrenergic receptor (Sungkaworn et al, 2017), and the LYVE-1 receptor (Stanly et al, 2020)) but the precise mechanism by which this occurs has yet to be fully visualised. It would therefore be beneficial to image and analyse these actin corrals at high resolution.…”

“…Stimulated Emission Depletion (STED) microscopy and Structured Illumination Microscopy (SIM) techniques have a lesser resolving power than SMLM, but still provide at minimum a doubling of standard widefield resolution, and are therefore a powerful tool for actin imaging studies. As a recent example, Stanly et al (2020) used STED microscopy to image actin corrals in conjunction with the LYVE-1 receptor, reporting corrals with an average size of 100 nm -1.5 µm, each containing heterogeneously distributed receptor clusters. 1) direct physical impediment of receptor movement by the actin filaments, 2) alteration to lipid packing around picketing proteins, and 3) direct interaction with actin filaments, with or without adaptor proteins.…”