Super-resolved trajectory-derived nanoclustering analysis using spatiotemporal indexing.

Single-molecule localization microscopy techniques are emerging as vital tools to unravel the nanoscale world of living cells by understanding the spatiotemporal organization of protein clusters at the nanometer scale. Current analyses define spatial nanoclusters based on detections but neglect important temporal information such as cluster lifetime and recurrence in "hotspots" on the plasma membrane. Spatial indexing is widely used in video games to detect interactions between moving geometric objects. Here, we use the R-tree spatial indexing algorithm to determine the overlap of the bounding boxes of individual molecular trajectories to establish membership in nanoclusters. Extending the spatial indexing into the time dimension allows the resolution of spatial nanoclusters into multiple spatiotemporal clusters. Using spatiotemporal indexing, we found that syntaxin1a and Munc18-1 molecules transiently cluster in hotspots, offering insights into the dynamics of neuroexocytosis. Nanoscale spatiotemporal indexing clustering (NASTIC) has been implemented as a free and open-source Python graphic user interface.

Non-beta blocker enantiomers of propranolol and atenolol inhibit vasculogenesis in infantile hemangioma.

Propranolol and atenolol, current therapies for problematic infantile hemangioma (IH), are composed of R(+) and S(-) enantiomers: the R(+) enantiomer is largely devoid of beta blocker activity. We investigated the effect of R(+) enantiomers of propranolol and atenolol on the formation of IH-like blood vessels from hemangioma stem cells (HemSCs) in a murine xenograft model. Both R(+) enantiomers inhibited HemSC vessel formation in vivo. In vitro, similar to R(+) propranolol, both atenolol and its R(+) enantiomer inhibited HemSC to endothelial cell differentiation. As our previous work implicated the transcription factor sex-determining region Y (SRY) box transcription factor 18 (SOX18) in propranolol-mediated inhibition of HemSC to endothelial differentiation, we tested in parallel a known SOX18 small-molecule inhibitor (Sm4) and show that this compound inhibited HemSC vessel formation in vivo with efficacy similar to that seen with the R(+) enantiomers. We next examined how R(+) propranolol alters SOX18 transcriptional activity. Using a suite of biochemical, biophysical, and quantitative molecular imaging assays, we show that R(+) propranolol directly interfered with SOX18 target gene trans-activation, disrupted SOX18-chromatin binding dynamics, and reduced SOX18 dimer formation. We propose that the R(+) enantiomers of widely used beta blockers could be repurposed to increase the efficiency of current IH treatment and lower adverse associated side effects.

A dominant-negative SOX18 mutant disrupts multiple regulatory layers essential to transcription factor activity.

Few genetically dominant mutations involved in human disease have been fully explained at the molecular level. In cases where the mutant gene encodes a transcription factor, the dominant-negative mode of action of the mutant protein is particularly poorly understood. Here, we studied the genome-wide mechanism underlying a dominant-negative form of the SOX18 transcription factor (SOX18RaOp) responsible for both the classical mouse mutant Ragged Opossum and the human genetic disorder Hypotrichosis-lymphedema-telangiectasia-renal defect syndrome. Combining three single-molecule imaging assays in living cells together with genomics and proteomics analysis, we found that SOX18RaOp disrupts the system through an accumulation of molecular interferences which impair several functional properties of the wild-type SOX18 protein, including its target gene selection process. The dominant-negative effect is further amplified by poisoning the interactome of its wild-type counterpart, which perturbs regulatory nodes such as SOX7 and MEF2C. Our findings explain in unprecedented detail the multi-layered process that underpins the molecular aetiology of dominant-negative transcription factor function.

Homodimerization regulates an endothelial specific signature of the SOX18 transcription factor.

During embryogenesis, vascular development relies on a handful of transcription factors that instruct cell fate in a distinct sub-population of the endothelium (1). The SOXF proteins that comprise SOX7, 17 and 18, are molecular switches modulating arterio-venous and lymphatic endothelial differentiation (2,3). Here, we show that, in the SOX-F family, only SOX18 has the ability to switch between a monomeric and a dimeric form. We characterized the SOX18 dimer in binding assays in vitro, and using a split-GFP reporter assay in a zebrafish model system in vivo. We show that SOX18 dimerization is driven by a novel motif located in the vicinity of the C-terminus of the DNA binding region. Insertion of this motif in a SOX7 monomer forced its assembly into a dimer. Genome-wide analysis of SOX18 binding locations on the chromatin revealed enrichment for a SOX dimer binding motif, correlating with genes with a strong endothelial signature. Using a SOX18 small molecule inhibitor that disrupts dimerization, we revealed that dimerization is important for transcription. Overall, we show that dimerization is a specific feature of SOX18 that enables the recruitment of key endothelial transcription factors, and refines the selectivity of the binding to discrete genomic locations assigned to endothelial specific genes.