Spatiotemporal monitoring of hard tissue development reveals unknown features of tooth and bone development.

Mineralized tissues, such as bones or teeth, are essential structures of all vertebrates. They enable rapid movement, protection, and food processing, in addition to providing physiological functions. Although the development, regeneration, and pathogenesis of teeth and bones have been intensely studied, there is currently no tool to accurately follow the dynamics of growth and healing of these vital tissues in space and time. Here, we present the BEE-ST (Bones and tEEth Spatio-Temporal growth monitoring) approach, which allows precise quantification of development, regeneration, remodeling, and healing in any type of calcified tissue across different species. Using mouse teeth as model the turnover rate of continuously growing incisors was quantified, and role of hard/soft diet on molar root growth was shown. Furthermore, the dynamics of bones and teeth growth in lizards, frogs, birds, and zebrafish was uncovered. This approach represents an effective, highly reproducible, and versatile tool that opens up diverse possibilities in developmental biology, bone and tooth healing, tissue engineering, and disease modeling.

Role of casein kinase 1 in the amoeboid migration of B-cell leukemic and lymphoma cells: A quantitative live imaging in the confined environment.

The migratory properties of leukemic cells are commonly associated with their pathological potential and can significantly affect the disease progression. While the research in immunopathology mostly employed powerful indirect methods such as flow cytometry, these cells were rarely observed directly using live imaging microscopy. This is especially true for the malignant cells of the B-cell lineage, such as those originating from chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In this study, we employed open-source image analysis tools to automatically and quantitatively describe the amoeboid migration of four B-cell leukemic and lymphoma cell lines and primary CLL cells. To avoid the effect of the shear stress of the medium on these usually non-adherent cells, we have confined the cells using a modified under-agarose assay. Surprisingly, the behavior of tested cell lines differed substantially in terms of basal motility or response to chemokines and VCAM1 stimulation. Since casein kinase 1 (CK1) was reported as a regulator of B-cell migration and a promoter of CLL, we looked at the effects of CK1 inhibition in more detail. Migration analysis revealed that CK1 inhibition induced rapid negative effects on the migratory polarity of these cells, which was quantitatively and morphologically distinct from the effect of ROCK inhibition. We have set up an assay that visualizes endocytic vesicles in the uropod and facilitates morphological analysis. This assay hints that the effect of CK1 inhibition might be connected to defects in polarized intracellular transport. In summary, 1) we introduce and validate a pipeline for the imaging and quantitative assessment of the amoeboid migration of CLL/MCL cells, 2) we provide evidence that the assay is sensitive enough to mechanistically study migration defects identified by the transwell assay, and 3) we describe the polarity defects induced by inhibition or deletion of CK1ε.

Lyn Phosphorylates and Controls ROR1 Surface Dynamics During Chemotaxis of CLL Cells.

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.