Guidelines for DC preparation and flow cytometry analysis of mouse nonlymphoid tissues.

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various nonlymphoid tissues. DC are sentinels of the immune system present in almost every mammalian organ. Since they represent a rare cell population, DC need to be extracted from organs with protocols that are specifically developed for each tissue. This article provides detailed protocols for the preparation of single-cell suspensions from various mouse nonlymphoid tissues, including skin, intestine, lung, kidney, mammary glands, oral mucosa and transplantable tumors. Furthermore, our guidelines include comprehensive protocols for multiplex flow cytometry analysis of DC subsets and feature top tricks for their proper discrimination from other myeloid cells. With this collection, we provide guidelines for in-depth analysis of DC subsets that will advance our understanding of their respective roles in healthy and diseased tissues. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all coauthors, making it an essential resource for basic and clinical DC immunologists.

LSP1-myosin1e bimolecular complex regulates focal adhesion dynamics and cell migration.

Several cytoskeleton-associated proteins and signaling pathways work in concert to regulate actin cytoskeleton remodeling, cell adhesion, and migration. Although the leukocyte-specific protein 1 (LSP1) has been shown to interact with the actin cytoskeleton, its function in the regulation of actin cytoskeleton dynamics is, as yet, not fully understood. We have recently demonstrated that the bimolecular complex between LSP1 and myosin1e controls actin cytoskeleton remodeling during phagocytosis. In this study, we show that LSP1 downregulation severely impairs cell migration, lamellipodia formation, and focal adhesion dynamics in macrophages. Inhibition of the interaction between LSP1 and myosin1e also impairs these processes resulting in poorly motile cells, which are characterized by few and small lamellipodia. Furthermore, cells in which LSP1-myosin1e interaction is inhibited are typically associated with inefficient focal adhesion turnover. Collectively, our findings show that the LSP1-myosin1e bimolecular complex plays a pivotal role in the regulation of actin cytoskeleton remodeling and focal adhesion dynamics required for cell migration.

Functionalized Cellulose Nanocrystals for Cellular Labeling and Bioimaging.

Cellulose nanocrystals (CNCs) are unique and promising natural nanomaterials that can be extracted from native cellulose fibers by acid hydrolysis. In this study, we developed chemically modified CNC derivatives by covalent tethering of PEGylated biotin and perylenediimide (PDI)-based near-infrared organic dye and evaluated their suitability for labeling and imaging of different cell lines including J774A.1 macrophages, NIH-3T3 fibroblasts, HeLa adenocarcinoma cells, and primary murine dendritic cells. PDI-labeled CNCs showed a superior photostability compared to similar commercially available dyes under long periods of constant and high-intensity illumination. All CNC derivatives displayed excellent cytocompatibility toward all cell types and efficiently labeled cells in a dose-dependent manner. Moreover, CNCs were effectively internalized and localized in the cytoplasm around perinuclear areas. Thus, our findings demonstrate the suitability of these new CNC derivatives for labeling, imaging, and long-time tracking of a variety of cell lines and primary cells.

Highly fluorescent hybrid nanofibers as potential nanofibrous scaffolds for studying cell-fiber interactions.

In this study, we report on the fabrication of hybrid nanofibers for labeling and bioimaging applications. Our approach is involved for developing highly fluorescent nanofibers using a blend of polylactic acid, polyethyleneglycol, and perylenediimide dyes, through the solution blow spinning technique. The nanofibers are exhibited diameters ranging from 330 nm to 420 nm. Nanofibers showed excellent red and near-infrared fluorescence emissive properties in fluorescent spectroscopy. Moreover, the strong two-photon absorption phenomenon was observed for nanofibers under confocal microscopy. To assess the applicability of these fluorescent nanofibers in bioimaging settings, we employ two types of mammalian cells B16F1 melanoma cells and J774.A1 macrophages. Both cell types exhibit negligible cytotoxicity after 24 h incubation with the nanofibers, indicating the suitability of nanofibers for cell-based experiments. We also observe strong interactions between the nanofibers and cells, as evidenced by two major events: a) the acquisition of an elongated cellular morphology with the major cellular axis parallel to the nanofibers and b) the accumulation of actin filaments along the points of contact of the cells with the fibers. Our findings demonstrate the suitability of these newly developed fluorescent nanofibers in cell-based applications for guiding cellular behavior. We expect that these fluorescent nanofibers have the potential to serve as scaffold materials for long-time tracking of cell-fiber interactions in fluorescence microscopy.

Met-Signaling Controls Dendritic Cell Migration in Skin by Regulating Podosome Formation and Function.

Signaling through the HGF receptor/Met in skin-resident Langerhans cells (LCs) and dermal dendritic cells (DCs) is essential for their emigration toward draining lymph nodes upon inflammation-induced activation. In this study, we addressed the role of Met signaling in distinct steps of LC/dermal DC emigration from the skin by employing a conditionally Met-deficient mouse model (Metflox/flox). We found that Met deficiency severely impaired podosome formation in DCs and concomitantly decreased the proteolytic degradation of gelatin. Accordingly, Met-deficient LCs failed to efficiently cross the extracellular matrix-rich basement membrane between the epidermis and the dermis. We further observed that HGF-dependent Met activation reduced the adhesion of bone marrow-derived LCs to various extracellular matrix factors and enhanced the motility of DCs in three-dimensional collagen matrices, which was not the case for Met-deficient LCs/DCs. We found no impact of Met signaling on the integrin-independent amoeboid migration of DCs in response to the CCR7 ligand CCL19. Collectively, our data show that the Met-signaling pathway regulates the migratory properties of DC in HGF-dependent and HGF-independent manners.

PLA/Hydroxyapatite scaffolds exhibit in vitro immunological inertness and promote robust osteogenic differentiation of human mesenchymal stem cells without osteogenic stimuli.

Bone defects stand out as one of the greatest challenges of reconstructive surgery. Fused deposition modelling (FDM) allows for the printing of 3D scaffolds tailored to the morphology and size of bone damage in a patient-specific and high-precision manner. However, FDM still suffers from the lack of materials capable of efficiently supporting osteogenesis. In this study, we developed 3D-printed porous scaffolds composed of polylactic acid/hydroxyapatite (PLA/HA) composites with high ceramic contents (above 20%, w/w) by FDM. The mechanical properties of the PLA/HA scaffolds were compatible with those of trabecular bone. In vitro degradation tests revealed that HA can neutralize the acidification effect caused by PLA degradation, while simultaneously releasing calcium and phosphate ions. Importantly, 3D-printed PLA/HA did not induce the upregulation of activation markers nor the expression of inflammatory cytokines in dendritic cells thus exhibiting no immune-stimulatory properties in vitro. Evaluations using human mesenchymal stem cells (MSC) showed that pure PLA scaffolds exerted an osteoconductive effect, whereas PLA/HA scaffolds efficiently induced osteogenic differentiation of MSC even in the absence of any classical osteogenic stimuli. Our findings indicate that 3D-printed PLA scaffolds loaded with high concentrations of HA are most suitable for future applications in bone tissue engineering.

Curauá-derived carbon dots: Fluorescent probes for effective Fe(III) ion detection, cellular labeling and bioimaging.

This study reports the generation of curauá-derived carbon dots (C-dots) and their suitability for Fe(III) detection, bioimaging and FACS analysis. C-dots were generated from curauá (Ananas erectifolius) fibers by a facile one-step hydrothermal approach. They exhibited graphite-like structure with a mean diameter of 2.4 nm, high water solubility, high levels of carboxyl and hydroxyl functional groups, excitation-dependent multicolor fluorescence emission (in the range 450 nm - 560 nm) and superior photostability. C-dots were highly selective and effective for the detection of ferric Fe(III) ion in an aqueous medium with a detection limit of 0.77 µM in the linear range of 0-30 µM, a value much lower than the guideline limits proposed by the World Health Organization (WHO). In biological cell systems, C-dots were very well tolerated by B16F1 mouse melanoma and J774.A1 mouse macrophages cell lines, both of which effectively internalized C-dots in their cytoplasmic compartment. Finally, C-dots were effective probes for long-term live cell imaging experiments and multi-channel flow cytometry analysis. Collectively, our findings demonstrate that curauá-derived C-dots serve as versatile and effective natural products for Fe(III) ion sensing, labeling and bioimaging of various cell types. This study adds novel C-dots to the library of carbon-based probes and paves the way towards a sustainable conversion of a most abundant biomass waste into value-added products.

Guiding cell adhesion and motility by modulating cross-linking and topographic properties of microgel arrays.

Biomaterial-driven modulation of cell adhesion and migration is a challenging aspect of tissue engineering. Here, we investigated the impact of surface-bound microgel arrays with variable geometry and adjustable cross-linking properties on cell adhesion and migration. We show that cell migration is inversely correlated with microgel array spacing, whereas directionality increases as array spacing increases. Focal adhesion dynamics is also modulated by microgel topography resulting in less dynamic focal adhesions on surface-bound microgels. Microgels also modulate the motility and adhesion of Sertoli cells used as a model for cell migration and adhesion. Both focal adhesion dynamics and speed are reduced on microgels. Interestingly, Gas2L1, a component of the cytoskeleton that mediates the interaction between microtubules and microfilaments, is dispensable for the regulation of cell adhesion and migration on microgels. Finally, increasing microgel cross-linking causes a clear reduction of focal adhesion turnover in Sertoli cells. These findings not only show that spacing and rigidity of surface-grafted microgels arrays can be effectively used to modulate cell adhesion and motility of diverse cellular systems, but they also form the basis for future developments in the fields of medicine and tissue engineering.

Surface Topography Guides Morphology and Spatial Patterning of Induced Pluripotent Stem Cell Colonies.

The relevance of topographic cues for commitment of induced pluripotent stem cells (iPSCs) is largely unknown. In this study, we demonstrate that groove-ridge structures with a periodicity in the submicrometer range induce elongation of iPSC colonies, guide the orientation of apical actin fibers, and direct the polarity of cell division. Elongation of iPSC colonies impacts also on their intrinsic molecular patterning, which seems to be orchestrated from the rim of the colonies. BMP4-induced differentiation is enhanced in elongated colonies, and the submicron grooves impact on the spatial modulation of YAP activity upon induction with this morphogen. Interestingly, TAZ, a YAP paralog, shows distinct cytoskeletal localization in iPSCs. These findings demonstrate that topography can guide orientation and organization of iPSC colonies, which may affect the interaction between mechanosensors and mechanotransducers in iPSCs.

GAR22ß regulates cell migration, sperm motility, and axoneme structure.

Spatiotemporal cytoskeleton remodeling is pivotal for cell adhesion and migration. Here we investigated the function of Gas2-related protein on chromosome 22 (GAR22ß), a poorly characterized protein that interacts with actin and microtubules. Primary and immortalized GAR22ß(-/-) Sertoli cells moved faster than wild-type cells. In addition, GAR22ß(-/-) cells showed a more prominent focal adhesion turnover. GAR22ß overexpression or its reexpression in GAR22ß(-/-) cells reduced cell motility and focal adhesion turnover. GAR22ß-actin interaction was stronger than GAR22ß-microtubule interaction, resulting in GAR22ß localization and dynamics that mirrored those of the actin cytoskeleton. Mechanistically, GAR22ß interacted with the regulator of microtubule dynamics end-binding protein 1 (EB1) via a novel noncanonical amino acid sequence, and this GAR22ß-EB1 interaction was required for the ability of GAR22ß to modulate cell motility. We found that GAR22ß is highly expressed in mouse testes, and its absence resulted in reduced spermatozoa generation, lower actin levels in testes, and impaired motility and ultrastructural disorganization of spermatozoa. Collectively our findings identify GAR22ß as a novel regulator of cell adhesion and migration and provide a foundation for understanding the molecular basis of diverse cytoskeleton-dependent processes.

Crucial role for the LSP1-myosin1e bimolecular complex in the regulation of Fcγ receptor-driven phagocytosis.

Actin cytoskeleton remodeling is fundamental for Fcγ receptor-driven phagocytosis. In this study, we find that the leukocyte-specific protein 1 (LSP1) localizes to nascent phagocytic cups during Fcγ receptor-mediated phagocytosis, where it displays the same spatial and temporal distribution as the actin cytoskeleton. Down-regulation of LSP1 severely reduces the phagocytic activity of macrophages, clearly demonstrating a crucial role for this protein in Fcγ receptor-mediated phagocytosis. We also find that LSP1 binds to the class I molecular motor myosin1e. LSP1 interacts with the SH3 domain of myosin1e, and the localization and dynamics of both proteins in nascent phagocytic cups mirror those of actin. Furthermore, inhibition of LSP1-myosin1e and LSP1-actin interactions profoundly impairs pseudopodial formation around opsonized targets and their subsequent internalization. Thus the LSP1-myosin1e bimolecular complex plays a pivotal role in the regulation of actin cytoskeleton remodeling during Fcγ receptor-driven phagocytosis.

Expression of liver plasma membrane transporters in gallstone-susceptible and gallstone-resistant mice.

We tested the hypothesis that differential expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates between gallstone-susceptible C57L/J and gallstone-resistant AKR/J mice. Plasma membrane fractions and total RNA isolated from livers of mice fed with a control or lithogenic (15% fat/1.25% cholesterol/0.5% cholic acid) diet were used for measurements of steady-state gene expression of hepatobiliary transport systems for bile salts (Ntcp1/Slc10a1, Oatp1/Slc21a1 and Bsep/Abcb11), phospholipids (Mdr2/Abcb4), organic anions (Mrp2/Abcc2) and organic cations (Oct1/Slc22a1). Irrespective of the diet, the steady-state gene expression of hepatobiliary transporters did not differ significantly between the two strains. Despite a higher basal bile flow and bile-salt secretion in C57L mice, Mrp2 (Abcc2) and Bsep (Abcb11) expression did not differ between the two strains. Elevated biliary phospholipid secretion in response to the lithogenic diet was linked to increased Mdr2 (Abcb4) protein expression, whereas the induction of Oct1 (Slc22a1) might reflect an enhanced uptake of choline for augmented phospholipid synthesis. In response to the lithogenic diet, Bsep (Abcb11) protein expression was up-regulated only marginally and bile salt secretion did not increase. The down-regulation of Ntcp1 (Slc10a1) protein expression might protect hepatocytes from high intracellular bile-salt loads. We conclude that variations in protein function rather than in the gene expression of liver plasma membrane transporters might account for variations in biliary lipid secretion rates. Our findings support the concept that the formation of lithogenic bile is caused by the hypersecretion of bile salts as a result of augmented availability of canalicular membrane cholesterol, possibly amplified by bile-salt-phospholipid uncoupling due to the increased bile flow.