Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I ...cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.
Cholesterol (Chol) is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. Thus, it is of utmost interest to develop tools for visualization of Chol ...organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently labeled Chol analogs. Unfortunately, the introduction of the label may influence the characteristics of the analog, such as its localization, interaction, and trafficking in cells; hence, it is important to get knowledge of such bias. In this report, we compared different fluorescent lipid analogs for their performance in cellular assays: 1) plasma membrane incorporation, specifically the preference for more ordered membrane environments in phase-separated giant unilamellar vesicles and giant plasma membrane vesicles; 2) cellular trafficking, specifically subcellular localization in Niemann-Pick type C disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS)-based and super-resolution stimulated emission depletion-FCS-based measurements of membrane diffusion dynamics. The analogs exhibited strong differences, with some indicating positive performance in the membrane-based experiments and others in the intracellular trafficking assay. However, none showed positive performance in all assays. Our results constitute a concise guide for the careful use of fluorescent Chol analogs in visualizing cellular Chol dynamics.
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.
Platelets are specialized anucleate cells that play a major role in hemostasis following vessel injury. More recently, platelets have also been implicated in innate immunity and inflammation by ...directly interacting with immune cells and releasing proinflammatory signals. It is likely therefore that in certain pathologies, such as chronic parasitic infections and myeloid malignancies, platelets can act as mediators for hemostatic and proinflammatory responses. Fortunately, murine platelet function
is highly analogous to human, providing a robust model for functional comparison. However, traditional methods of studying platelet phenotype, function and activation status often rely on using large numbers of whole isolated platelet populations, which severely limits the number and type of assays that can be performed with mouse blood. Here, using cutting edge 3D quantitative phase imaging, holotomography, that uses optical diffraction tomography (ODT), we were able to identify and quantify differences in single unlabeled, live platelets with minimal experimental interference. We analyzed platelets directly isolated from whole blood of mice with either a JAK2V617F-positive myeloproliferative neoplasm (MPN) or
infection. Image analysis of the platelets indicates previously uncharacterized differences in platelet morphology, including altered cell volume and sphericity, as well as changes in biophysical parameters such as refractive index (RI) and dry mass. Together, these data indicate that, by using holotomography, we were able to identify clear disparities in activation status and potential functional ability in disease states compared to control at the level of single platelets.
Receptor clustering is known to trigger signalling events that contribute to critical changes in cellular functions. Faithful imaging of such clusters by means of fluorescence microscopy relies on ...the application of adequate cell fixation methods prior to immunolabelling in order to avoid artefactual redistribution by the antibodies themselves. Previous work has highlighted the inadequacy of fixation with paraformaldehyde (PFA) alone for efficient immobilisation of membrane-associated molecules, and the advantages of fixation with PFA in combination with glutaraldehyde (GA). Using fluorescence microscopy, we here highlight how inadequate fixation can lead to the formation of artefactual clustering of receptors in lymphatic endothelial cells, focussing on the transmembrane hyaluronan receptors LYVE-1 and CD44, and the homotypic adhesion molecule CD31, each of which displays their native diffuse surface distribution pattern only when visualised with the right fixation techniques, i.e. PFA/GA in combination. Fluorescence recovery after photobleaching (FRAP) confirms that the artefactual receptor clusters are indeed introduced by residual mobility. In contrast, we observed full immobilisation of membrane proteins in cells that were fixed and then subsequently permeabilised, irrespective of whether the fixative was PFA or PFA/GA in combination. Our study underlines the importance of choosing appropriate sample preparation protocols for preserving authentic receptor organisation in advanced fluorescence microscopy.
Janus kinase (JAK2)V617F is the most common mutation found in patients with Philadelphia chromosome negative myeloproliferative neoplasms (Ph- MPNs). The discovery of this mutation over 15 years ago ...revolutionised MPN diagnosis and inspired the development of JAK inhibitors as new therapeutic interventions. However, despite extensive structural and biophysical studies using JAK2 domains in isolation, the exact molecular mechanisms of JAK2V617F activation remains elusive. We have previously demonstrated that expression of the thrombopoietin (TPO) receptor, MPL, which interacts directly with JAK2, is essential for disease development in a mouse model of a JAK2V617F-positiveMPN (Blood 2014 124:3956-3963).
Using total internal reflection fluorescence (TIRF) microscopy, we visualized MPL interaction dynamics in live cells on single molecule level. Effective cell surface MPL fluorescence labelling and dual-color imaging allowed us to determine the level of MPL dimerization under various experimental conditions. Using this assay, we clearly established that MPL is monomeric at physiologically relevant receptor densities. However, TPO stimulation results in significant dimerization of MPL (>50%) and an equilibrium between monomers and dimers. This counters the current dogma that MPL exists at the membrane as a pre-formed dimer. Strikingly, we found that JAK2V617F shifts this monomer-dimer equilibrium leading to significant TPO-independent MPL dimerization providing a novel mechanistic model of oncogenic JAK2 activation.
To highlight the role of ligand-independent receptor dimerization in JAK2 activation, we compared three groups of autoactivating mutations in the PK domain covering the FERM-SH2 (FS2)-PK linker region (Group I), residues in the proximity of the αC helix (Group II) and at the autoinhibitory PK-TK interface (Group III). Consistent MPL dimerization was only observed for mutations in groups I and II. Mutations in these groups both localize to a potential homomeric PK/PK interface that has been implicated as a switch of JAK activation.
Using MD simulations, we also found that the FERM domain of JAK2 strongly interacts with the inner leaflet of the lipid bilayer of the plasma membrane via a single hydrophobic residue (L224) surrounded by several positively charged residues that allows the region to act as a membrane anchor. This tight coupling to the membrane enforces an appropriate orientation between the JAKs within the receptor dimers required for optimal intermolecular PK/PK interaction that is critical for receptor dimerization. To interfere with membrane anchoring, we introduced a negative charge in this position (L224E). Strikingly, ligand-independent MPL dimerization and activation by JAK2V617F was dramatically reduced upon introducing L224E, supporting the vital importance of L224 for orienting JAK2 at the membrane to allow productive PK-PK interactions.
Here, we demonstrate that JAK2V617F mutation acts by altering and strengthening the intermolecular interactions involving the PK/PK dimerization interface. In essence, these mutations drive cytoplasmic stabilization of receptor-JAK dimers, bypassing extracellular stabilization of dimers via cytokine binding. These results provide critical and entirely novel mechanistic insights into signal initiation in MPNs and readdress the roles of receptor-associated proteins.
Hubbard:Ajax Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Co-Founder.
To investigate whether monocytes contribute to matrix deposition in systemic sclerosis (SSc) by production of tissue-inhibitor of metalloproteinase-1 (TIMP-1).
Matrix metalloproteinase-1 (MMP-1) and ...TIMP-1 expression and secretion were measured by qRT-PCR and ELISA in circulating monocytes from patients with SSc, patients with rheumatoid arthritis (RA) and healthy controls (HC) and in healthy monocytes cultured in the presence of SSc or HC serum samples. Production of TIMP-1 was determined in response to a panel of Toll-like receptor (TLR) agonists and MyD88 inhibitory peptide. The functional effect of conditioned media from SSc and HC serum samples or TLR8-stimulated monocytes was studied in an MMP-1 activity assay.
TIMP-1 production by monocytes was upregulated in patients with SSc compared with patients with RA and HC. Incubation of HC monocytes with SSc serum samples resulted in functionally active TIMP-1 production. However, pretreatment with MyD88 inhibitor, but not control peptide, decreased TIMP-1 secretion. TIMP-1 production was significantly stronger when SSc and HC monocytes were stimulated with TLR8 (ssRNA) agonist, but the response was more pronounced in SSc monocytes. TIMP-1 production after TLR stimulation was also strongly reduced in the presence of MyD88 inhibitory peptide or in the monocytes isolated from a patient with a genetic TLR signalling defect. MMP-1 activity was significantly inhibited in media from serum samples or TLR8-stimulated monocytes indicative of functional TIMP activity.
This study demonstrates profibrotic properties of circulating monocytes from patients with SSc and a key role for TLR signalling, particularly TLR8, in TIMP-1 secretion and matrix remodelling.
The lymphatic endothelial receptor LYVE-1 investigated here is a weak binder to its ligand hyaluronan (HA) and uses receptor clustering to achieve the avidity required for stable interaction to HA. ...These clustering based interactions are highly dynamic and are often controlled by several molecular players that contribute to receptor functional changes. One such contributor is the cortical actin cytoskeleton. As the actin cytoskeleton lies immediately below the membrane surface, it creates compartments on the membrane that control the receptor organisation, dynamics and allows physical interaction of the receptors with actin, which either hinder or enhance their functions. In this study, I investigate the role of the actin cytoskeleton in regulating the dynamics and function of LYVE-1. At first, the native organisation of LYVE-1 and cross-linked clustered state of LYVE-1 (that are known to cause avidity induced increase in HA binding), on primary human dermal lymphatic endothelial cells (HDLECs), were identified by confocal and super-resolution STED microscopy. Following which the effects of actin disassembly on LYVE-1:HA binding function and cross-linked clustering of LYVE-1 were investigated using FACS and STED microscopy. The dynamics of LYVE-1 during actin steady state or disassembly was studied using diffusion measurements on primary and cytoplasmic tail truncated hLYVE-1 transfected HDLECs. The results from the diffusion experiments suggest that LYVE-1 mobility decreases after actin depletion and correlates with the increase in HA interaction as determined by FACS during actin disassembly, although there was no change in the surface organisation of LYVE-1. Further diffusion measurements using STED-FCS showed that LYVE-1 was 'trapped' or confined on the membrane surface of HDLECs in native conditions. This data along with biochemical assays show a hitherto unknown direct link of the actin cytoskeleton regulating LYVE-1:HA binding and dynamics, giving an indication of how LYVE-1 in vivo may modulate binding to HA.
The lymphatic endothelial receptor LYVE-1 investigated here is a weak binder to its ligand hyaluronan (HA) and uses receptor clustering to achieve the avidity required for stable interaction to HA. ...These clustering based interactions are highly dynamic and are often controlled by several molecular players that contribute to receptor functional changes. One such contributor is the cortical actin cytoskeleton. As the actin cytoskeleton lies immediately below the membrane surface, it creates compartments on the membrane that control the receptor organisation, dynamics and allows physical interaction of the receptors with actin, which either hinder or enhance their functions. In this study, I investigate the role of the actin cytoskeleton in regulating the dynamics and function of LYVE-1. At first, the native organisation of LYVE-1 and cross-linked clustered state of LYVE-1 (that are known to cause avidity induced increase in HA binding), on primary human dermal lymphatic endothelial cells (HDLECs), were identified by confocal and super-resolution STED microscopy. Following which the effects of actin disassembly on LYVE-1:HA binding function and cross-linked clustering of LYVE-1 were investigated using FACS and STED microscopy. The dynamics of LYVE-1 during actin steady state or disassembly was studied using diffusion measurements on primary and cytoplasmic tail truncated hLYVE-1 transfected HDLECs. The results from the diffusion experiments suggest that LYVE-1 mobility decreases after actin depletion and correlates with the increase in HA interaction as determined by FACS during actin disassembly, although there was no change in the surface organisation of LYVE-1. Further diffusion measurements using STED-FCS showed that LYVE-1 was 'trapped' or confined on the membrane surface of HDLECs in native conditions. This data along with biochemical assays show a hitherto unknown direct link of the actin cytoskeleton regulating LYVE-1:HA binding and dynamics, giving an indication of how LYVE-1 in vivo may modulate binding to HA.
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