Flow cytometry is a powerful method, which is widely used for high-throughput quantitative and qualitative analysis of cells. However, its straightforward applicability for extracellular vesicles (EVs) and mainly exosomes is hampered by several challenges, reflecting mostly the small size of these vesicles (exosomes: ~80–200 nm, microvesicles: ~200–1,000 nm), their polydispersity, and low refractive index. The current best and most widely used protocol for beads-free flow cytometry of exosomes uses ultracentrifugation (UC) coupled with floatation in sucrose gradient for their isolation, labeling with lipophilic dye PKH67 and antibodies, and an optimized version of commercial high-end cytometer for analysis. However, this approach requires an experienced flow cytometer operator capable of manual hardware adjustments and calibration of the cytometer. Here, we provide a novel and fast approach for quantification and characterization of both exosomes and microvesicles isolated from cell culture media as well as from more complex human samples (ascites of ovarian cancer patients) suitable for multiuser labs by using a flow cytometer especially designed for small particles, which can be used without adjustments prior to data acquisition. EVs can be fluorescently labeled with protein-(Carboxyfluoresceinsuccinimidyl ester, CFSE) and/or lipid- (FM) specific dyes, without the necessity of removing the unbound fluorescent dye by UC, which further facilitates and speeds up the characterization of microvesicles and exosomes using flow cytometry. In addition, double labeling with protein- and lipid-specific dyes enables separation of EVs from common contaminants of EV preparations, such as protein aggregates or micelles formed by unbound lipophilic styryl dyes, thus not leading to overestimation of EV numbers. Moreover, our protocol is compatible with antibody labeling using fluorescently conjugated primary antibodies. The presented methodology opens the possibility for routine quantification and characterization of EVs from various sources. Finally, it has the potential to bring a desired level of control into routine experiments and non-specialized labs, thanks to its simple bead-based standardization.

Extracellular vesicles (EVs) function as important conveyers of information between cells and thus can be exploited as drug delivery systems or disease biomarkers. Transmission electron microscopy (TEM) remains the gold standard method for visualisation of EVs, however the analysis of individual EVs in TEM images is time-consuming if performed manually. Therefore, we present here a software tool for computer-assisted evaluation of EVs in TEM images. TEM ExosomeAnalyzer detects EVs based on their shape and edge contrast criteria and subsequently analyses their size and roundness. The software tool is compatible with common negative staining protocols and isolation methods used in the field of EV research; even with challenging TEM images (EVs both lighter and darker than the background, images containing artefacts or precipitated stain, etc.). If the fully-automatic analysis fails to produce correct results, users can promptly adjust the detected seeds of EVs as well as their boundaries manually. The performance of our tool was evaluated for three different modes with variable levels of human interaction, using two datasets with various heterogeneity. The semi-automatic mode analyses EVs with high success rate in the homogenous dataset (F1 score 0.9094, Jaccard coefficient 0.8218) as well as in the highly heterogeneous dataset containing EVs isolated from cell culture medium and patient samples (F1 score 0.7619, Jaccard coefficient 0.7553). Moreover, the extracted size distribution profiles of EVs isolated from malignant ascites of ovarian cancer patients overlap with those derived by cryo-EM and are comparable to NTA- and TRPS-derived data. In summary, TEM ExosomeAnalyzer is an easy-to-use software tool for evaluation of many types of vesicular microparticles and is available at http://cbia.fi.muni.cz/exosome-analyzer free of charge for non-commercial and research purposes. The web page contains also detailed description how to use the software tool including a video tutorial.

Dishevelled (DVL) is the key component of the Wnt signaling pathway. Currently, DVL conformational dynamics under native conditions is unknown. To overcome this limitation, we develop the Fluorescein Arsenical Hairpin Binder- (FlAsH-) based FRET in vivo approach to study DVL conformation in living cells. Using this single-cell FRET approach, we demonstrate that (i) Wnt ligands induce open DVL conformation, (ii) DVL variants that are predominantly open, show more even subcellular localization and more efficient membrane recruitment by Frizzled (FZD) and (iii) Casein kinase 1 ɛ (CK1ɛ) has a key regulatory function in DVL conformational dynamics. In silico modeling and in vitro biophysical methods explain how CK1ɛ-specific phosphorylation events control DVL conformations via modulation of the PDZ domain and its interaction with DVL C-terminus. In summary, our study describes an experimental tool for DVL conformational sampling in living cells and elucidates the essential regulatory role of CK1ɛ in DVL conformational dynamics.

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Mammalian limb development is driven by the integrative input from several signaling pathways; a failure to receive or a misinterpretation of these signals results in skeletal defects. The brachydactylies, a group of overlapping inherited human hand malformation syndromes, are mainly caused by mutations in BMP signaling pathway components. Two closely related forms, Brachydactyly type B2 (BDB2) and BDB1 are caused by mutations in the BMP antagonist Noggin (NOG) and the atypical receptor tyrosine kinase ROR2 that acts as a receptor in the non-canonical Wnt pathway. Genetic analysis of Nog and Ror2 functional interaction via crossing Noggin and Ror2 mutant mice revealed a widening of skeletal elements in compound but not in any of the single mutants, thus indicating genetic interaction. Since ROR2 is a non-canonical Wnt co-receptor specific for Wnt-5a we speculated that this phenotype might be a result of deregulated Wnt-5a signaling activation, which is known to be essential for limb skeletal elements growth and patterning. We show that Noggin potentiates activation of the Wnt-5a-Ror2-Disheveled (Dvl) pathway in mouse embryonic fibroblast (MEF) cells in a Ror2-dependent fashion. Rat chondrosarcoma chondrocytes (RCS), however, are not able to respond to Noggin in this fashion unless growth arrest is induced by FGF2. In summary, our data demonstrate genetic interaction between Noggin and Ror2 and show that Noggin can sensitize cells to Wnt-5a/Ror2-mediated non-canonical Wnt signaling, a feature that in cartilage may depend on the presence of active FGF signaling. These findings indicate an unappreciated function of Noggin that will help to understand BMP and Wnt/PCP signaling pathway interactions.

during cancer treatment. neural stem cells and is a selective target for neuroprotection HCN channel activity balances quiescence and proliferation in Updated version

Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are malignancies characterized by the dependence on B-cell receptor (BCR) signaling and by the high expression of ROR1, the cell surface receptor for Wnt-5a. Both, BCR and ROR1 are therapeutic targets in these diseases and the understanding of their mutual cross talk is thus of direct therapeutic relevance. In this study we analyzed the role of Lyn, a kinase from the Src family participating in BCR signaling, as a mediator of the BCR-ROR1 crosstalk. We confirm the functional interaction between Lyn and ROR1 and demonstrate that Lyn kinase efficiently phosphorylates ROR1 in its kinase domain and aids the recruitment of the E3 ligase c-CBL. We show that ROR1 surface dynamics in migrating primary CLL cells as well as chemotactic properties of CLL cells were inhibited by Lyn inhibitor dasatinib. Our data establish Lyn-mediated phosphorylation of ROR1 as a point of crosstalk between BCR and ROR1 signaling pathways.

word count: 168 words 26 Main text word count: 4183 words 27 Number of Figures: 6 28 Number of tables: 2 29 Running title: Lyn controls chemotaxis and motility of CLL cells via phosphorylation of ROR1.30 Abstract 33 Chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) are malignancies 34 characterized by the dependence on B-cell receptor (BCR) signaling and by the high 35 expression of the cell surface receptor ROR1. Both, BCR and ROR1 are therapeutic targets 36 in these diseases and the understanding of their mutual cross talk is thus of direct 37 therapeutic relevance. In this study we analyzed the role of Lyn, a kinase from the Src 38 family, as a mediator of the BCR-ROR1 crosstalk. We confirm the functional interaction 39 between Lyn and ROR1 and demonstrate that Lyn kinase efficiently phosphorylates ROR1 40 in its kinase domain and aids the recruitment of an E3 ligase c-CBL. The absence of Lyn in 41 Lyn KO Maver-1 cells produced by CRISPR-Cas9 resulted in the increased ROR1 cell 42 surface levels and deregulated migratory properties. Similar correlations between ROR1 43 surface dynamics, levels of active Lyn and chemotactic properties were confirmed in primary 44 CLL samples. Our data establish Lyn-mediated phosphorylation of ROR1 as a point of 45 crosstalk between BCR and ROR1 signaling pathways. 46 47 48 49 50 51 52 53 54 55 56 57 58 59 3 Introduction 60The ROR protein family comprises of ROR1 and ROR2, which are both type 1 61 transmembrane receptors. Upon discovery, ROR proteins were referred to as orphan 62 receptors on account of the lack of identity of their ligands. However, subsequent studies 63 identified their ligands to be the Wnt proteins, mostly Wnt-5a protein 1,2 . Wnt-5a/ROR 64 pathway is an essential signaling pathway that controls cell polarity and migration during 65 embryonic development and tissue homeostasis 3,4 . During embryonic development, RORs 66 are highly and uniformly expressed, most prominently in the skeletal and neural tissues, but 67 postnatally their expression becomes highly restricted 5 . 68Interestingly, ROR1 or ROR2 upregulation has been observed in many cancers: 69ROR1 is upregulated in solid tumors or hematologic malignancies while ROR2 is 70 overexpressed in osteosarcomas or renal cell carcinomas 6 . High expression of ROR1 is 71 typical for some B-cell lymphomas such as mantle cell lymphoma (MCL) 7 and chronic 72 lymphocytic leukemia(CLL) 8,9 . CLL is a form of hematologic cancer which is manifested as a 73 steady accumulation of mature CD5 + B-cells in the bone marrow, lymphoid tissues and 74 peripheral blood. It is the most common form of adult leukemia in the western hemisphere, 75with an incidence of 5 per 100 000 each year and an average median age of on-set around 76 70 years. Most of the CLL cases remain asymptomatic for a long time, in which case 77 therapeutic intervention is not necessary. However, part of the CLL cases progress rapidly, 78 require treatment and their overall life expectancy is decreased 10 . 79CLL cells are in most cases hig...