BMP2-induced chemotaxis requires PI3K p55γ/p110α-dependent phosphatidylinositol (3,4,5)-triphosphate production and LL5β recruitment at the cytocortex

Background: BMP-induced chemotaxis of mesenchymal progenitors is fundamental for vertebrate development, disease and tissue repair. BMP2 induces Smad and non-Smad signalling. Whereas signal transduction via Smads lead to transcriptional responses, non-Smad signalling induces both, transcriptional and immediate/early non-transcriptional responses. However, the molecular mechanisms by which BMP2 facilitates planar cell polarity, cortical actin rearrangements, lamellipodia formation and chemotaxis of mesenchymal progenitors are poorly understood. Our aim was to uncover the molecular mechanism by which BMP2 facilitates chemotaxis via the BMP2-dependent activation of PI3K and spatiotemporal control of PIP3 production important for actin rearrangements at the mesenchymal cell cytocortex. Results: We unveiled the molecular mechanism by which BMP2 induces non-Smad signalling by PI3K and the role of the second messenger PIP3 in BMP2-induced planar cell polarity, cortical actin reorganisation and lamellipodia formation. By using protein interaction studies, we identified the class Ia PI3K regulatory subunit p55γ to act as a specific and non-redundant binding partner for BMP receptor type II (BMPRII) in concert with the catalytic subunit p110α. We mapped the PI3K interaction to a region within the BMPRII kinase. Either BMP2 stimulation or increasing amounts of BMPRI facilitated p55γ association with BMPRII, but BMPRII kinase activity was not required for the interaction. We visualised BMP2-dependent PIP3 production via PI3K p55γ/p110α and were able to localise PIP3 to the leading edge of intact cells during the process of BMP2-induced planar cell polarity and actin dependent lamellipodia formation. Using mass spectrometry, we found the highly PIP3-sensitive PH-domain protein LL5β to act as a novel BMP2 effector in orchestrating cortical actin rearrangements. By use of live cell imaging we found that knock-down of p55γ or LL5β or pharmacological inhibition of PI3K impaired BMP2-induced migratory responses. Conclusions: Our results provide evidence for an important contribution of the BMP2-PI3K (p55γ/p110α)- PIP3-LL5β signalling axis in mesenchymal progenitor cell chemotaxis. We demonstrate molecular insights into BMP2-induced PI3K signalling on the level of actin reorganisation at the leading edge cytocortex. These findings are important to better understand BMP2–induced cytoskeletal reorganisation and chemotaxis of mesenchymal progenitors in different physiological or pathophysiological contexts

Zfp281 orchestrates interconversion of pluripotent states by engaging Ehmt1 and Zic2.

Developmental cell fate specification is a unidirectional process that can be reverted in response to injury or experimental reprogramming. Whether differentiation and de-differentiation trajectories intersect mechanistically is unclear. Here, we performed comparative screening in lineage-related mouse naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), and identified the constitutively expressed zinc finger transcription factor (TF) Zfp281 as a bidirectional regulator of cell state interconversion. We showed that subtle chromatin binding changes in differentiated cells translate into activation of the histone H3 lysine 9 (H3K9) methyltransferase Ehmt1 and stabilization of the zinc finger TF Zic2 at enhancers and promoters. Genetic gain-of-function and loss-of-function experiments confirmed a critical role of Ehmt1 and Zic2 downstream of Zfp281 both in driving exit from the ESC state and in restricting reprogramming of EpiSCs. Our study reveals that cell type-invariant chromatin association of Zfp281 provides an interaction platform for remodeling the cis-regulatory network underlying cellular plasticity

Phenotypic landscape of intestinal organoid development

Intestinal organoids are an ex vivo culture system of the intestinal epithelium that recapitulate many key aspects of the parent tissue: cell type diversity, spatial organization, but also the ability to regenerate and return to homeostatic conditions following damage. Intestinal organoids can develop from single cells, forming an emergent and self-organizing system undergoing temporally controlled cell fate transitions and spatial rearrangements. This complicated process is orchestrated by crosstalk of many pathways that together form a complex network of functional interactions. Although some components of this densely interconnected network have been resolved and described in detail, a systematic approach to mapping all of the involved players and their interdependence has not been attempted. To address this complex question, I developed an image-based screen in intestinal organoids cultured from single cells using an annotated compound library. I designed a novel approach generating multivariate feature profiles for hundreds of thousands of individual organoids to quantitatively describe phenotypes observed in each of the screened conditions. These were used to identify stable phenotypic outcomes of intestinal organoid development in a data-driven manner. The relative abundance of each of the detected phenotypes produced a unique phenotypic fingerprint for every screened condition, quantitatively describing the phenotypic landscape of organoid development. I used the generated phenotypic fingerprints to find conditions with significant and reproducible effects, identifying 230 target genes. Subsequently, I used this multivariate dataset to infer the functional genetic interactions of identified genes generating the first map of interactions that govern intestinal organoid formation. This allowed me to discover modules of genes that regulate cell identity transitions and maintain the balance between regeneration and homeostasis. With network analysis I confirmed known players involved in key steps of organoid development but also revealed several novel potential upstream regulators. In the second part of this study, I focused on conditions identified by the screen to enrich for a regenerative phenotype characterized by absence of differentiated cells of both absorptive and secretory lineage. Abundance of this phenotype marked conditions that potentially improved the regeneration potential of the intestinal epithelium. Among these I discovered two key components of the retinoic acid signaling pathway: RXR and RAR. Follow-up studies allowed me to describe novel roles for nuclear retinoic acid receptors and retinol metabolism in intestinal damage response and homeostasis. By combining quantitative imaging with RNA sequencing I confirmed the role of endogenous retinoic acid signaling and metabolism for initiating transcriptional programs that guide intestinal epithelium cell fate transitions. I also observed that RXR inhibition not only suppressed differentiation, but induced a regenerative fetal-like transcriptional identity. To validate the physiological relevance of this finding, together with our collaborators we designed an in vivo study using a mouse model of cycling cell ablation to induce acute damage in the intestine, treating mice with the compound over the course of recovery. The mouse assay corroborated the findings from the organoid studies, showing that a small molecule inhibitor of RXR identified in the organoid screen improved intestinal regeneration in vivo. Taken together, this study presents a novel approach for data-driven phenotypic discovery suitable for large image-based screens. This approach can be used on any other arrayed screen with single object resolution offering means to robustly identify phenotypic effects also in a complicated landscape characterized by pleiotropic phenotypes. Furthermore, it establishes a novel paradigm in genetic interaction screening applied to an emergent self-organized system that was ultimately instrumental to identify a small molecule that improved regeneration of the intestinal epithelium in vivo

Nanoscale Coupling of Endocytic Pit Growth and Stability

Clathrin-mediated endocytosis, an essential process for plasma membrane homeostasis and cell signaling, is characterized by stunning heterogeneity in the size and lifetime of clathrin-coated endocytic pits (CCPs). If and how CCP growth and lifetime are coupled and how this relates to their physiological function are unknown. We combine computational modeling, automated tracking of CCP dynamics, electron microscopy, and functional rescue experiments to demonstrate that CCP growth and lifetime are closely correlated and mechanistically linked by the early-acting endocytic F-BAR protein FCHo2. FCHo2 assembles at the rim of CCPs to control CCP growth and lifetime by coupling the invagination of early endocytic intermediates to clathrin lattice assembly. Our data suggest a mechanism for the nanoscale control of CCP growth and stability that may similarly apply to other metastable structures in cells

ER-Golgi-localized proteins TMED2 and TMED10 control the formation of plasma membrane lipid nanodomains

To promote infections, pathogens exploit host cell machineries such as structural elements of the plasma membrane. Studying these interactions and identifying molecular players are ideal for gaining insights into the fundamental biology of the host cell. Here, we used the anthrax toxin to screen a library of 1,500 regulatory, cell-surface, and membrane trafficking genes for their involvement in the intoxication process. We found that endoplasmic reticulum (ER)-Golgi-localized proteins TMED2 and TMED10 are required for toxin oligomerization at the plasma membrane of human cells, an essential step dependent on localization to cholesterol-rich lipid nanodomains. Biochemical, morphological, and mechanistic analyses showed that TMED2 and TMED10 are essential components of a supercomplex that operates the exchange of both cholesterol and ceramides at ER-Golgi membrane contact sites. Overall, this study of anthrax intoxication led to the discovery that lipid compositional remodeling at ER-Golgi interfaces fully controls the formation of functional membrane nanodomains at the cell surface