The late endosomal adaptor protein LAMTOR2/p14 is essential for tissue homeostasis by controlling MAPK and mTOR signaling, which in turn regulate cell growth and proliferation, migration and spreading.

Electrospun nanofibres are an excellent cell culture substrate, enabling the fast and non-disruptive harvest and transfer of adherent cells for microscopical and biochemical analyses. Metabolic activity and cellular structures are maintained during the only half a minute-long harvest and transfer process. We show here that such samples can be optimally processed by means of cryofixation combined either with freeze-substitution, sample rehydration and cryosection-immunolabelling or with freeze-fracture replica-immunolabelling. Moreover, electrospun fibre substrates are equally suitable for complementary approaches, such as biochemistry, fluorescence microscopy and cytochemistry. In vitro grown vertebrate cells are an indispensable tool for cell biological research, and are widely used for the subcellular localisation of macromolecules at the level of electron microscopy (EM). Yet, the fast and efficient harvest of living, adherent cells without affecting cellular morphology and physiology is still a not satisfactorily solved problem. Thus, the potential of recent improvements (1-3) of cryo-immuno-EM (cryo-IEM) has so far not been fully exploited.To begin with, immunogold-labelling of cryosections according to Tokuyasu (4) has been successfully combined with rapid cryofixation (2,3). The new 'hybrid' approach avoids artefacts resulting from conventional chemical fixation. It is based on cryofixation of native, unfixed specimens, followed by chemical stabilisation of cellular ultrastructure and antigenicity at around −90• C by means of freeze-substitution (FS) and, finally, sample rehydration and postfixation prior to cryosectioning (2,3). So far, this modified Tokuyasu-technique was mainly used for tissues and suspension cultures (2,3,5), but not regularly for adherent cell cultures, with very few exceptions, when relatively large, voluminous cells (HepG2) were cultured on gelatine beads (Cytodex™, Sigma: Ø ∼100 μm; refs. (2,6)). Yet, in our previous studies we found that commercially available beads are less suitable for applying this advanced IEMtechnique to flat and/or small cells, such as mouse embryonic fibroblasts (MEF) with a maximal height of ∼4 μm (ref.(7) and our unpublished data). Considering the adverse ratio of bead-diameter versus cell-height it is evident that most of the area of an average 400 × 400 μm-cryosection is occupied by section profiles of the carrier beads, but not by the cells under investigation. The analysis of a fair amount of cells, however, is mandatory for unbiased stereology (8). Furthermore, sampling of cells grown on beads prior to cryofixation requires time-consuming intermediate enrichment-steps, possibly leading to unwanted physiological and ultrastructural alterations.The second cutting-edge IEM-technique to mention here is sodium dodecyl sulphate-digested freeze-fracture replica labelling (SDS-FRL (1)). So far, SDS-FRL was predominantly applied to chemically fixed tissues e.g. (9); see also (10) for review. The few SDS-FRL studies on natively cryofixed monolayers relayed on...

We present a new synthesis protocol for a multivalent, multimodality, nucleophilic nanoparticle ideal for in vivo imaging. Stability requirements necessitated covalent cross-linking of the carbohydrate cage, easy functionalization the introduction of sterically accessible amine groups. The new protocol aimed at more uniform particle size, less clustering and superior magnetic properties compared with commercial nanoparticles. Particles were precipitated from Fe(2+) and Fe(3+) in the presence of 10 kDa dextran monodispersed from the aerosol phase. Cross-linking was achieved with epichlorhydrin, nuclophilication with NH3, purification with ultrafiltration and dialysis. Particles and a commercial product (Rienso®, Takeda Pharma) underwent physicochemical characterizations. Biocompatibility was assessed by Resazurin on LLC-PK1 cells; the internalization rate was measured for three cell lines (HAEC, HASMC, HT29). Core size was 5.61 ± 1.25 nm; hydrodynamic size was 49.56 ± 11.73 nm. The number of sterically accessible amine groups averaged 9.9. The cores showed cubic magnetite structure. Values of r1 and r2 were 10.9 and 148.17 mM(-1) s(-1). Cellular viability was unchanged after incubation. Introduction of aerosol phase dextran resulted in a reduction of the overall hydrodynamic diameter and a narrower size distribution of the synthesized particles. Electron tomography visualized for the first time the postulated 'hairy layer' of the dextran coating and enabled the measurement of the overall diameter of 100.2 ± 7.92 nm. The resulting nanoparticle is biocompatible, functionalizable and detectable at nanomolar concentrations with MRI and optical imaging. It can potentially serve as a platform for multimodal molecular imaging and targeted therapy approaches.

By combination of diamond thin films with a polydimethylsiloxane (PDMS) microfluidic channel, we realize a system for electronic and optical monitoring of cells under in vivo-like conditions of culturing medium exchange with no direct contact to ambient air. We make use of surface conductivity and high optical transparency of hydrogen-terminated nanocrystalline diamond films (thickness 400 nm) to create a microscopic impedance electrode or transistor channel. We show that the diamond-PDMS system exhibits stable adhesion at least up to 5 mL min À1 flow and electrical sensitivity to adsorption of bovine serum albumin. Next, HeLa cells were cultured in the diamond-PDMS channel while electric cell-substrate impedance sensing and optical microscopy were used for long-term (168 h) cell culture monitoring. We observe correlation of the cell culture morphology and impedance characteristics as a function of time. These results confirm the applicability of the diamond-PDMS microfluidic system.