Highlights d ZO proteins self-organize membrane-attached compartments via phase separation d Multivalent protein interactions and phosphorylation control the phase transition d Phase separation of ZO1 is sufficient to sequester key tightjunction proteins d ZO1 phase separation is required for the assembly of functional tight junctions in tissue
Design, synthesis, characterisation and in vitro studies of hydrophilic, colloidally stable, 64 Cu(II)-labelled, ultrasmall iron oxide nanoparticles in a range of human cell lines †The application of ultra-small super-paramagnetic iron oxide nanoparticles (USPIONs) as versatile diagnostic probes for multimodal imaging in biomedicine, including via magnetic resonance imaging (MRI) and positron emission tomography (PET), requires hydrophilic and biocompatible surface coatings. Herein, we describe the development of USPIONs stabilised by octylamine-modified polyacrylic acid (OPA) and the subsequent conjugation of a 64 Cu(II) chelator, N-(4-aminophenyl)-2-[4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan-1-yl]-acetamide (amino-dmptacn), for radioactivity-based detection. Transmission electron microscopic analysis and dynamic light scattering measurements confirmed the monodispersity and stability of the OPAUSPIONs in aqueous media and revealed a hydrodynamic size of ca. 15 nm. Furthermore, the biocompatibility and cellular uptake efficiency of the functionalised USPIONs was investigated in a range of normal and tumour cell lines. The results clearly show a cell type-as well as time-dependent internalisation of the OPA-USPIONs via active energy-dependent pathways. Biocompatibility of OPAUSPIONs in the concentration range of 10-50 mg mL À1 was demonstrated, while impairment of cellular viability was observed for human umbilical vein endothelial cells at 100 mg mL À1 . Upon exposure to human serum, several biomolecules cover the negatively-charged surface of the nanoparticles and a biomolecular corona is formed. Nonetheless, the nanoparticles represent a promising platform for the future development of a bimodal PET-MRI tumour-imaging agent. ; Fax: +61 3 9903 9582; Tel: +61 3 9903 9706 † Electronic supplementary information (ESI) available. See
We report the synthesis and detailed in vitro evaluation of zwitterionic ultrasmall superparamagnetic iron oxide NPs (USPIONs) comprising oleic acid/oleyl alcohol‐stabilized magnetite particles (5 nm core diameter) coated with an amphiphilic zwitterionic polymer, poly(maleic anhydride‐alt‐1‐decene) substituted with 3‐(dimethylamino)propylamine (PMAL). These particles display a near‐neutral zeta potential at pH≥7 and possess high colloidal stability, maintaining a hydrodynamic diameter of ca. 15–20 nm over a wide range of pHs (4–10) and ionic strength (up to 1 m NaCl). They exhibit very low levels of nonspecific protein binding upon exposure to serum, and negligible uptake by phagocytic and non‐phagocytic hepatocarcinoma cells, which suggests that they may be able to resist rapid accumulation in the liver and spleen, a common in vivo fate for NPs. The PMAL‐USPIONs exhibit very low cytotoxicity and do not elicit an inflammatory response or display hemolytic activity in vitro. Minimal nonspecific uptake by either cancerous or non‐cancerous cell lines was observed, an important precondition to achieve highly selective targeting upon further functionalization with an active targeting agent (e.g., antibody or peptide). Overall, this study establishes the considerable potential of PMAL‐USPIONs as a platform for the future development of “stealth” NP‐based imaging and/or therapeutic agents.
Tight junctions are cell adhesion complexes that seal tissues and are involved in cell polarity and signalling. Supra-molecular assembly and positioning of tight junctions as continuous networks of adhesion strands is dependent on the two membrane associated scaffolding proteins ZO1 and ZO2. To understand how ZO proteins organize junction assembly, we performed quantitative cell biology and in vitro reconstitution experiments. We discovered that ZO proteins self-organize membrane attached compartments via phase separation. We identified the multivalent interactions of the conserved PDZ-SH3-GuK supra-domain as the driver of phase separation. These interactions are regulated by phosphorylation and intra-molecular binding. Formation of condensed ZO protein compartments is sufficient to specifically enrich and localize tight junction proteins including adhesion receptors, cytoskeletal adapters and transcription factors. Our results suggest that an active phase transition of ZO proteins into a condensed membrane bound compartment drives claudin polymerization and coalescence of a continuous tight junction belt.Introduction:
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