α4 integrins are used by leukocytes and neural crest derivatives for adhesion and migration during embryogenesis, immune responses and tumour invasion. The pro-migratory activity of α4 integrin is mediated in part through the direct binding of the cytoplasmic domain to paxillin. Here, using intermolecular FRET and biochemical analyses, we report a novel interaction of the α4 integrin cytoplasmic domain with 14-3-3ζ. This interaction depends on serine phosphorylation of α4 integrin at a site (S978) distinct from that which regulates paxillin binding (S988). Using a combination of metabolic labelling and targeted mass spectrometry by multiple reaction monitoring we demonstrate the low stoichiometry phosphorylation of S978. The interaction between α4 integrin and 14-3-3ζ is enhanced by the direct association between 14-3-3ζ and paxillin, resulting in the formation of a ternary complex that stabilises the recruitment of each component. Although pair-wise interaction between α4 integrin and paxillin is sufficient for normal Rac1 regulation, the integrity of the ternary complex is essential for focused Cdc42 activity at the lamellipodial leading edge and directed cell movement. Taken together, these data identify a key signalling nexus mediating α4 integrin-dependent migration.

Intercellular communication between cancer cells, especially between cancer and stromal cells, plays an important role in disease progression. We examined the intercellular transfer of organelles and proteins in vitro and in vivo and the role of tunneling nanotubes (TNTs) in this process. TNTs are membrane bridges that facilitate intercellular transfer of organelles of unclear origin. Using 3-dimensional quantitative and qualitative confocal microscopy, we showed that TNTs contain green fluorescent protein (GFP)-early endosome antigen (EEA) 1, GFP Rab5, GFP Rab11, GFP Rab8, transferrin (Tf), and Tf receptor (Tf-R) fused to mCherry (Tf-RmCherry). Tf-RmCherry was transferred between cancer cells by a contactdependent but secretion-independent mechanism. Live cell imaging showed TNT formation preceding the transfer of Tf-RmCherry and involving the function of the small guanosine triphosphatase (GTPase) Rab8, which colocalized with Tf-RmCherry in the TNTs and was cotransferred to acceptor cells. Tf-RmCherry was transferred from cancer cells to fibroblasts, a noteworthy finding that suggests that this process occurs between tumor and stromal cells in vivo. We strengthened this hypothesis in a xenograft model of breast cancer using enhanced (e)GFP-expressing mice. Tf-RmCherry transferred from tumor to stromal cells and this process correlated with an increased opposite transfer of eGFP from stromal to tumor cells, together pointing toward complex intercellular communication at the tumor

Human exposure to titanium dioxide nanoparticles (nano-TiO ) is increasing. An internal source of nano-TiO is represented by titanium-based orthopedic and dental implants can release nanoparticles (NPs) upon abrasion. Little is known about how the size of NPs influences their interaction with cytoskeletal protein networks and the functional/homeostatic consequences that might follow at the implant-bone interface with regard to osteoblasts. We investigated the effects of size of anatase nano-TiO on SaOS-2 human osteoblast-like cells exposed to clinically relevant concentrations (0.05, 0.5, 5 mg/L) of 5 and 40 nm spherical nano-TiO . Cell viability and proliferation, adhesion, spread and migration were assessed, as well as the orientation of actin and microtubule cytoskeletal networks. The phosphorylation of focal adhesion kinase (p-FAK ) and the expression of vinculin in response to nano-TiO were also assessed. Treatment with nano-TiO disrupted the actin and microtubule cytoskeletal networks leading to morphological modifications of SaOS-2 cells. The phosphorylation of p-FAK and the expression of vinculin were also modified depending on the particle size, which affected cell adhesion. Consequently, the cell migration was significantly impaired in the 5 nm-exposed cells compared to unexposed cells. The present work shows that the orientation of cytoskeletal networks and the focal adhesion proteins and subsequently the adhesion, spread and migration of SaOS-2 cells were affected by the selected nano-TiO in a size dependent manner. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2582-2593, 2018.

The development of reliable and cost-efficient methods to assess the toxicity of nanomaterials (NMs) is critical for the proper identification of their impact on human health and for ensuring a safe progress of nanotechnology. In this study, we investigated the reliability and applicability of label-free impedance flow cytometry (IFC) for in vitro nanotoxicity screening, which avoids time-consuming labelling steps and minimizes possible NM-induced interferences. U937 human lymphoma cells were exposed for 24 h to eight different nanomaterials at five concentrations (2, 10, 20, 50, and 100 μg/mL). The NMs’ effect on viability was measured using IFC and the results were compared to those obtained by trypan blue (TB) dye exclusion and conventional flow cytometry (FC). To discriminate viable from necrotic cells, the IFC measurement settings regarding signal trigger level and frequency, as well as the buffer composition, were optimised. A clear discrimination between viable and necrotic cells was obtained at 6 MHz in a sucrose-based measurement buffer. Nanomaterial-induced interferences were not detected for IFC. The IFC and TB assay results were in accordance for all NMs. The IFC was found to be robust, reliable and less prone to interferences due to the advantage of being label-free.

Cellular organelles have been shown to shuttle between cells in co-culture. We hereby show that titanium dioxide (TiO2) nanoparticles (NPs) can be transferred in such a manner, between cells in direct contact, along with endosomes and lysosomes. A co-culture system was employed for this purpose and the NP transfer was observed in mammalian cells including normal rat kidney (NRK) and HeLa cells. We found that the small GTPase Arf6 facilitates the intercellular transfer of smaller NPs and agglomerates. Spherical, anatase nano-TiO2 with sizes of 5 (Ti5) and 40 nm (Ti40) were used in this study. Humans are increasingly exposed to TiO2 NPs from external sources such as constituents of foods, cosmetics, and pharmaceuticals, or from internal sources represented by Ti-based implants, which release NPs upon abrasion. Exposure to 5 mg/l of Ti5 and Ti40 for 24 h did not affect cellular viability but modified their ability to communicate with surrounding cells. Altogether, our results have important implications for the design of nanomedicines, drug delivery and toxicity.

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Introduction: PH is a disorder characterized by remodeling of the pulmonary vasculature which causes increased pulmonary vascular resistance and ultimately right ventricular (RV) failure leading to death. MetS is a recognized risk factor for PH, particularly in patients suffering from PH associated with left-sided heart failure (WHO Group 2 PH) for which there are no approved therapies. Activins and growth differentiation factors (GDFs) are implicated in both PH pathology as well as body composition and metabolism by genetic disease association, preclinical, translational, and clinical evidence. HS135 is a rationally designed first-in-class activin-receptor (ActR)-based fusion protein that addresses both vascular remodeling and MetS in PH without affecting hematological parameters; hematological effects limit the dosing of other ActR-based agents. Methods: HS135’s ability to counteract vascular remodeling and heart failure in PH was assessed using a rat monocrotaline (MCT) model: a single dose of 60 mg/kg MCT was administered on Day 0 followed by 4 weeks of twice weekly treatment with either HS135 (1, 4, or 16 mg/kg) or vehicle. PH efficacy was assessed at end of study by echocardiography and histological readouts. HS135’s ability to improve body composition and metabolism was assessed in wild type mice by administering HS135 (1, 3, 10, 20, or 50 mg/kg) or vehicle twice weekly for 3 weeks. The effect of HS135 on hematological parameters was assessed by whole blood cell count in non-human primates (NHPs) 2 weeks following a single administration of HS135 at 3 or 30 mg/kg. Results: HS135 at 3 or 30 mg/kg did not lead to changes in hematological parameters in NHPs. Compared to vehicle, HS135 was effective at counteracting the MCT-induced PH phenotype in rats as evidenced by significant reductions in Fulton index and improved echocardiography parameters. In wild type mice, HS135 led to significant improvements in body composition. Conclusions: Contrary to other ActR-based therapies, HS135 did not lead to increases in hematocrit which would limit its dosing. HS135 was efficacious at treating PH while simultaneously improving body composition in preclinical models. Clinical trials with HS135 are expected to commence within 2023.

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