The Nanomechanical Properties of CLL Cells Are Linked to the Actin Cytoskeleton and Are a Potential Target of BTK Inhibitors.

Chronic lymphocytic leukemia (CLL) is an incurable disease characterized by an intense trafficking of the leukemic cells between the peripheral blood and lymphoid tissues. It is known that the ability of lymphocytes to recirculate strongly depends on their capability to rapidly rearrange their cytoskeleton and adapt to external cues; however, little is known about the differences occurring between CLL and healthy B cells during these processes. To investigate this point, we applied a single-cell optical (super resolution microscopy) and nanomechanical approaches (atomic force microscopy, real-time deformability cytometry) to both CLL and healthy B lymphocytes and compared their behavior. We demonstrated that CLL cells have a specific actomyosin complex organization and altered mechanical properties in comparison to their healthy counterpart. To evaluate the clinical relevance of our findings, we treated the cells in vitro with the Bruton's tyrosine kinase inhibitors and we found for the first time that the drug restores the CLL cells mechanical properties to a healthy phenotype and activates the actomyosin complex. We further validated these results in vivo on CLL cells isolated from patients undergoing ibrutinib treatment. Our results suggest that CLL cells' mechanical properties are linked to their actin cytoskeleton organization and might be involved in novel mechanisms of drug resistance, thus becoming a new potential therapeutic target aiming at the normalization of the mechanical fingerprints of the leukemic cells.

3D-STED Super-Resolution Microscopy Reveals Distinct Nanoscale Organization of the Hematopoietic Cell-Specific Lyn Substrate-1 (HS1) in Normal and Leukemic B Cells.

HS1, the hematopoietic homolog of cortactin, acts as a versatile actin-binding protein in leucocytes. After phosphorylation, it is involved in GTPase and integrin activation, and in BCR, TCR, and CXCR4 downstream signaling. In normal and leukemic B cells, HS1 is a central cytoskeletal interactor and its phosphorylation and expression are prognostic factors in chronic lymphocytic leukemia (CLL) patients. We here introduce for the first time a super-resolution imaging study based on single-cell 3D-STED microscopy optimized for revealing and comparing the nanoscale distribution of endogenous HS1 in healthy B and CLL primary cells. Our study reveals that the endogenous HS1 forms heterogeneous nanoclusters, similar to those of YFP-HS1 overexpressed in the leukemic MEC1 cell line. HS1 nanoclusters in healthy and leukemic B cells form bulky assemblies at the basal sides, suggesting the recruitment of HS1 for cell adhesion. This observation agrees with a phasor-FLIM-FRET and STED colocalization analyses of the endogenous MEC1-HS1, indicating an increased interaction with Vimentin at the cell adhesion sites. In CLL cells isolated from patients with poor prognosis, we observed a larger accumulation of HS1 at the basal region and a higher density of HS1 nanoclusters in the central regions of the cells if compared to good-prognosis CLL and healthy B cells, suggesting a different role for the protein in the cell types analyzed. Our 3D-STED approach lays the ground for revealing tiny differences of HS1 distribution, its functionally active forms, and colocalization with protein partners.

Stem cell niche organization in the Drosophila ovary requires the ECM component Perlecan.

Stem cells reside in specialized microenvironments or niches that balance stem cell proliferation and differentiation.1,2 The extracellular matrix (ECM) is an essential component of most niches, because it controls niche homeostasis, provides physical support, and conveys extracellular signals.3-11 Basement membranes (BMs) are thin ECM sheets that are constituted mainly by Laminins, Perlecan, Collagen IV, and Entactin/Nidogen and surround epithelia and other tissues.12 Perlecans are secreted proteoglycans that interact with ECM proteins, ligands, receptors, and growth factors such as FGF, PDGF, VEGF, Hedgehog, and Wingless.13-18 Thus, Perlecans have structural and signaling functions through the binding, storage, or sequestering of specific ligands. We have used the Drosophila ovary to assess the importance of Perlecan in the functioning of a stem cell niche. Ovarioles in the adult ovary are enveloped by an ECM sheath and possess a tapered structure at their anterior apex termed the germarium. The anterior tip of the germarium hosts the germline niche, where two to four germline stem cells (GSCs) reside together with a few somatic cells: terminal filament cells (TFCs), cap cells (CpCs), and escort cells (ECs).19 We report that niche architecture in the developing gonad requires trol, that niche cells secrete an isoform-specific Perlecan-rich interstitial matrix, and that DE-cadherin-dependent stem cell-niche adhesion necessitates trol. Hence, we provide evidence to support a structural role for Perlecan in germline niche establishment during larval stages and in the maintenance of a normal pool of stem cells in the adult niche.

Laminin Levels Regulate Tissue Migration and Anterior-Posterior Polarity during Egg Morphogenesis in Drosophila.

Basement membranes (BMs) are specialized extracellular matrices required for tissue organization and organ formation. We study the role of laminin and its integrin receptor in the regulation of tissue migration during Drosophila oogenesis. Egg production in Drosophila involves the collective migration of follicle cells (FCs) over the BM to shape the mature egg. We show that laminin content in the BM increases with time, whereas integrin amounts in FCs do not vary significantly. Manipulation of integrin and laminin levels reveals that a dynamic balance of integrin-laminin amounts determines the onset and speed of FC migration. Thus, the interplay of ligand-receptor levels regulates tissue migration in vivo. Laminin depletion also affects the ultrastructure and biophysical properties of the BM and results in anterior-posterior misorientation of developing follicles. Laminin emerges as a key player in the regulation of collective cell migration, tissue stiffness, and the organization of anterior-posterior polarity in Drosophila.

ECM-Regulator timp Is Required for Stem Cell Niche Organization and Cyst Production in the Drosophila Ovary.

The extracellular matrix (ECM) is a pivotal component adult tissues and of many tissue-specific stem cell niches. It provides structural support and regulates niche signaling during tissue maintenance and regeneration. In many tissues, ECM remodeling depends on the regulation of MMP (matrix metalloproteinase) activity by inhibitory TIMP (tissue inhibitors of metalloproteinases) proteins. Here, we report that the only Drosophila timp gene is required for maintaining the normal organization and function of the germline stem cell niche in adult females. timp mutant ovaries show reduced levels of both Drosophila Collagen IV α chains. In addition, tissue stiffness and the cellular organization of the ovarian niche are affected in timp mutants. Finally, loss of timp impairs the ability of the germline stem cell niche to generate new cysts. Our results demonstrating a crucial role for timp in tissue organization and gamete production thus provide a link between the regulation of ECM metabolism and tissue homeostasis.