Megan Joseph: Quantitative single molecule analysis of podoplanin clustering in fibroblastic reticular cells uncovers CD44 function
CLEC-2 binds to the membrane glycoprotein podoplanin (PDPN) on FRCs, inhibiting actomyosin contractility through the FRC network and permitting lymph node expansion. The hyaluronic acid receptor CD44 is known to be required for FRCs to respond to DCs but the mechanism of action is not fully elucidated. Here, we use DNA-PAINT, a quantitative single molecule super-resolution technique, to visualize and quantify how PDPN clustering is regulated in the plasma membrane of FRCs. Our results indicate that CLEC-2 interaction leads to the formation of large PDPN clusters (i.e. more than 12 proteins per cluster) in a CD44-dependent manner. These results suggest that CD44 expression is required to stabilize large pools of PDPN at the membrane of FRCs upon CLEC-2 interaction, revealing the molecular mechanism through which CD44 facilitates cellular crosstalk between FRCs and DCs.
Lymph nodes are highly organized tissues that contain and compartmentalize immune cell types to orchestrate adaptive immune responses. Lymph node tissue architecture is determined by stromal cell structures; fibroblastic reticular cells (FRCs) that establish cellular networks linking lymph and blood vasculature which provide trafficking routes for lymphocytes and myeloid cells and generate growth factor and survival factors for the immune cell populations they support . Further, the fibroblastic reticular network is the key mechanically sensitive component of the lymph node capable of determining the physical properties of the tissue in steady state and adapting to permit lymph node expansion. Podoplanin (PDPN) has been determined as a mechanical sensor in FRCs, and mice with conditional genetic deletion of PDPN in fibroblastic stroma, PdgfraΔPdpn mice, exhibit attenuated lymph node expansion and altered immune activation [2–4]. PDPN overexpression has also been noted in inflammatory diseases, tissue damage and a wide range of cancers, and is directly correlated with disease outcomes, but the downstream signalling pathways and mechanisms of action of PDPN are still not fully understood. PDPN overexpression has been linked to cell migration, cell adhesion and cytoskeletal contractility in cancer cells [5–7]. For example, in non-motile tissue structures, such as FRCs in lymph nodes and on lymphatic endothelial cells, PDPN can act as a ligand to promote the migration of dendritic cells along stromal cell scaffolds through the direct binding of the C-type lectin-like receptor CLEC-2 . PDPN can also interact with platelets through CLEC-2 which is a required interaction for the physiological separation of blood and lymphatic vasculature during development . The same interaction with platelets also plays an important role in the function of high endothelial venues (HEVs) in lymph nodes, acting to prevent blood from leaking into the tissues .
It is known that PDPN has only a very short cytoplasmic tail of just 10 amino acids . Therefore, it has been difficult to determine how PDPN is required for so many diverse functions in such a range of cell types and tissue contexts. It has been reported that PDPN can directly bind to ERM proteins (ezrin, radixin and moesin) to regulate RhoA GTPase and actomyosin contractility [2,5]. The transmembrane domain of PDPN may in fact be a key regulator of PDPN function, allowing PDPN molecules to rearrange within different regions of the plasma membrane and to permit the interactions between PDPN and other membrane binding partners . We have recently reported that CD44, a non-kinase transmembrane glycoprotein and receptor for hyaluronic acid, is a key PDPN binding partner required for the response of FRCs to CLEC-2+ dendritic cells . PDPN and CD44 interactions are mediated through their transmembrane domains and are also dependent on cholesterol levels in the plasma membrane [2,14]. Interestingly, Pdpn and Cd44 mRNA expression are also coregulated, and knockdown of Cd44 results in lower expression levels of PDPN . Furthermore, overexpression of CD44 can attenuate PDPN-driven actomyosin contractility [2,13]. CD44 and PDPN colocalize at the plasma membrane of FRCs and their interaction is increased when PDPN binds to CLEC-2, leading us to hypothesize that CD44 controls PDPN activity through the spatial organization and clustering of PDPN molecules within the membrane. However, we have lacked the technical capability to quantify PDPN clustering at a molecular scale to formally test this hypothesis.
Here we use a quantitative single-molecule localization microscopy (SMLM) technique, known as DNA-PAINT (point accumulation for imaging in nanoscale topography) , to study how the spatial organization of PDPN proteins in the plasma membrane of wild-type (WT) and CD44 knock-out (CD44 KO) FRCs responds to binding of CLEC-2. DNA-PAINT relies on the binding and unbinding of two types of short single-stranded DNA sequences, one chemically coupled to the antibody targeting the protein of interest, known as the ‘docking’ strand, and another one fluorescently labelled and freely diffusing in solution, known as the ‘imager’ strand. The transient, yet repetitive binding between imager and docking DNA strands creates the characteristic blinking effect needed for super resolution fluorescence microscopy, as the fluorescent signal is preferentially detected during binding events. The continuous replenishment of imager strands makes DNA-PAINT immune to photobleaching, thus the same target protein can be detected multiple times with virtually unlimited number of photons outperforming the localization precision obtained with more conventional SMLM methods such as PALM (photoactivated localization microscopy)  and STORM (stochastic optical reconstruction microscopy) . The high level of nanometre accuracy attained by DNA-PAINT (i.e. it can routinely obtain sub-10 nm localization precision) has proven capable of imaging individual molecular targets on synthetic samples that simulates biomolecular nanoclusters , as well as protein clusters in fixed cells [19–21]. Furthermore, due to the predictable binding kinetics between imager and docking strands, it is possible to correlate the frequency of single-molecule events with the underlying number of labelled molecular targets  overcoming ‘overcounting’ artefacts observed with other SMLM techniques. During the last years, examples of the use of DNA-PAINT to quantify protein clustering in different biological systems started to emerge  and here we use it to shed light into the role of CLEC-2 and CD44 on PDPN clustering.
2.1. CLEC-2 drives PDPN and CD44 co-localization in FRCs and cell spreading
Confocal imaging of FRCs in steady state confirms that PDPN and CD44 colocalize with each other on FRC membranes (figure 1a), as previously published . We studied PDPN-CD44 co-localization in a CLEC-2-Fc expressing FRC cell line , modelling prolonged CLEC-2 stimulation to mimic a crosstalk between dendritic cells and fibroblastic stroma during an adaptive immune response in vivo. We observed increased changes in the co-localization of PDPN-CD44 on FRC membranes (figure 1a), which is in line with our previous observations that PDPN-CD44 co-localization is significantly increased upon CLEC-2 stimulation . However, due to the diffraction-limited resolution of confocal imaging, it is not possible to accurately uncover the interaction between PDPN and CD44. To improve our understanding of the molecular interactions between PDPN and CD44, and the effect of CLEC-2 stimulation, we here investigated PDPN clustering with single-molecule localization microscopy (SMLM) under total internal reflection fluorescence (TIRF) excitation in WT and CD44 KO FRC cell lines. TIRF illumination limits the excitation of fluorophores to a very thin optical section of approximately 150 nm above the glass/specimen interface which is ideal for imaging PDPN on cell membranes. Accordingly, FRCs were stimulated with CLEC-2-Fc or control coated glass slides. To assess that this method of CLEC-2 stimulation was functional, we quantified the cell morphology index of unstimulated and stimulated FRCs. Previous work has shown that there is an increase in the morphology index between FRCs co-cultured with and without CLEC-2+ DCs  because of inhibition of FRC contractility, as measured by reduced F-actin+ stress fibres allowing cell spreading . Our results showed a similar increase in the morphology index when FRCs were cultured on CLEC-2 coated glass slides (figure 1b) compared to CLEC-2 expressing FRCs or DC interaction  which suggests that immobilized CLEC-2 on coated glass slides induces a CLEC-2 mediated response in FRCs. As such, we used this method to study in detail PDPN-CD44 interactions on the cell membrane of FRCs in absence or presence of CLEC-2-Fc.