Combining high-pressure freezing with pre-embedding immunogold electron microscopy and tomography.

Immunogold labeling of permeabilized whole-mount cells or thin-sectioned material is widely used for the subcellular localization of biomolecules at the high spatial resolution of electron microscopy (EM). Those approaches are well compatible with either 3-dimensional (3D) reconstruction of organelle morphology and antigen distribution or with rapid cryofixation-but not easily with both at once. We describe here a specimen preparation and labeling protocol for animal cell cultures, which represents a novel blend of specifically adapted versions of established techniques. It combines the virtues of reliably preserved organelle ultrastructure, as trapped by rapid freezing within milliseconds followed by freeze-substitution and specimen rehydration, with the advantages of robust labeling of intracellular constituents in 3D through means of pre-embedding NANOGOLD-silver immunocytochemistry. So obtained thin and semi-thick epoxy resin sections are suitable for transmission EM imaging, as well as tomographic reconstruction and modeling of labeling patterns in the 3D cellular context.

Ultrastructural Morphometry Points to a New Role for LAMTOR2 in Regulating the Endo/Lysosomal System.

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. Moreover, LAMTOR2 critically controls architecture and function of the endocytic system, including epidermal growth factor receptor (EGFR) degradation in lysosomes, positioning of late endosomes and defense against intracellular pathogens. Here we describe the multifaceted ultrastructural phenotype of the endo/lysosomal system of LAMTOR2-deficient mouse embryonic fibroblasts. Quantitative (immuno-)electron microscopy of cryo-fixed samples revealed significantly reduced numbers of recycling tubules emanating from maturing multivesicular bodies (MVB). Instead, a distinct halo of vesicles surrounded MVB, tentatively interpreted as detached, jammed recycling tubules. These morphological changes in LAMTOR2-deficient cells correlated with the presence of growth factors (e.g. EGF), but were similarly induced in control cells by inactivating mTOR. Furthermore, proper transferrin receptor trafficking and recycling were apparently dependent on an intact LAMTOR complex. Finally, a severe imbalance in the relative proportions of endo/lysosomes was found in LAMTOR2-deficient cells, resulting from increased amounts of mature MVB and (autophago)lysosomes. These observations suggest that the LAMTOR/Ragulator complex is required not only for maintaining the homeostasis of endo/lysosomal subpopulations but also contributes to the proper formation of MVB-recycling tubules, and regulation of membrane/cargo recycling from MVB.

Cryo-immunoelectron microscopy of adherent cells improved by the use of electrospun cell culture substrates.

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.

Stability of the endosomal scaffold protein LAMTOR3 depends on heterodimer assembly and proteasomal degradation.

LAMTOR3 (MP1) and LAMTOR2 (p14) form a heterodimer as part of the larger Ragulator complex that is required for MAPK and mTOR1 signaling from late endosomes/lysosomes. Here, we show that loss of LAMTOR2 (p14) results in an unstable cytosolic monomeric pool of LAMTOR3 (MP1). Monomeric cytoplasmic LAMTOR3 is rapidly degraded in a proteasome-dependent but lysosome-independent manner. Mutational analyses indicated that the turnover of the protein is dependent on ubiquitination of several lysine residues. Similarly, other Ragulator subunits, LAMTOR1 (p18), LAMTOR4 (c7orf59), and LAMTOR5 (HBXIP), are degraded as well upon the loss of LAMTOR2. Thus the assembly of the Ragulator complex is monitored by cellular quality control systems, most likely to prevent aberrant signaling at the convergence of mTOR and MAPK caused by a defective Ragulator complex.

Proteomic analysis of endosomes from genetically modified p14/MP1 mouse embryonic fibroblasts.

The p14/MP1 scaffold complex binds MEK1 and ERK1/2 on late endosomes, thus regulating the strength, duration and intracellular location of MAPK signaling. By organelle proteomics we have compared the protein composition of endosomes purified from genetically modified p14⁻/⁻, p14+/⁻ and p14(rev) mouse embryonic fibroblasts. The latter ones were reconstituted retrovirally from p14⁻/⁻ mouse embryonic fibroblasts by reexpression of pEGFP-p14 at equimolar ratios with its physiological binding partner MP1, as shown here by absolute quantification of MP1 and p14 proteins on endosomes by quantitative MS using the Equimolarity through Equalizer Peptide strategy. A combination of subcellular fractionation, 2-D DIGE and MALDI-TOF/TOF MS revealed 31 proteins differentially regulated in p14⁻/⁻ organelles, which were rescued by reexpression of pEGFP-p14 in p14⁻/⁻ endosomes. Regulated proteins are known to be involved in actin remodeling, endosomal signal transduction and trafficking. Identified proteins and their in silico interaction networks suggested that endosomal signaling might regulate such major cellular functions such as proliferation, differentiation, migration and survival.

Loss-of-function of MYO5B is the main cause of microvillus inclusion disease: 15 novel mutations and a CaCo-2 RNAi cell model.

Autosomal recessive microvillus inclusion disease (MVID) is characterized by an intractable diarrhea starting within the first few weeks of life. The hallmarks of MVID are a lack of microvilli on the surface of villous enterocytes, occurrence of intracellular vacuoles lined by microvilli (microvillus inclusions), and the cytoplasmic accumulation of periodic acid-Schiff (PAS)-positive vesicles in enterocytes. Recently, we identified mutations in MYO5B, encoding the unconventional type Vb myosin motor protein, in a first cohort of nine MVID patients. In this study, we identified 15 novel nonsense and missense mutations in MYO5B in 11 unrelated MVID patients. Fluorescence microscopy, Western blotting, and electron microscopy were applied to analyze the effects of MYO5B siRNA knock-down in polarized, brush border possessing CaCo-2 cells. Loss of surface microvilli, increased formation of microvillus inclusions, and subapical enrichment of PAS-positive endomembrane compartments were induced in polarized, filter-grown CaCo-2 cells, following MYO5B knock-down. Our data indicate that MYO5B mutations are a major cause of microvillus inclusion disease and that MYO5B knock-down recapitulates most of the cellular phenotype in vitro, thus independently showing loss of MYO5B function as the cause of microvillus inclusion disease.

Extracellular signal regulated MAP-kinase signalling in osmotically stressed trout hepatocytes.

Activation of the extracellular signal-regulated MAP-kinase (ERK) by anisoosmotic conditions, the underlying signalling pathways, and the role of protein kinases in cell volume regulation were investigated in trout hepatocytes. While hyperosmolarity left phosphorylated ERK (pERK) levels unaffected, hypoosmolarity caused a significant increase of pERK within 2 min which peaked at around 30 min. Chelating extracellular Ca2+ to prevent the influx of Ca2+ associated with swelling reduced iso- and abolished hypoosmotic ERK activation. Similarly, inhibiting the ERK activator MEK, tyrosine kinases, or PKC inhibited the increase of pERK. In contrast, exposing cells to chelerytrine or staurosporine, PKC inhibitors of little specificity, increased pERK independently from osmotic conditions. Blocking PI3 kinase, application of 8-Br-cAMP, exposure to a P-receptor antagonist, and inhibition of p38 MAP-kinase had no effect on ERK activity. A significant reduction of regulatory volume decrease (RVD) after hypoosmotic swelling caused by MEK-inhibition and an even more pronounced reduction due to p38 inhibition indicates a role for MAP-kinases in volume regulation, but a lack of correlation between the impact of protein kinase inhibitors on pERK levels and on RVD suggests that ERK may merely modulate volume recovery. Immunocytochemical detection of pERK indicated cytoplasmic activation, but no nuclear accumulation within 30 min, supporting the notion that ERK exerts non-genomic effects. Overall, our data underscore the complexity of hypoosmotic ERK signalling and suggest a role of ERK and p38 in acute cell volume regulation.

Therapeutic potential of adipose-derived stromal cells in age-related osteoporosis.

Adipose-derived stromal cells (ASCs) are increasingly being used for orthopedic-based tissue engineering approaches due to their ability to readily undergo osteogenic differentiation. In the present study, we used in vitro and in vivo approaches to evaluate the use of ASCs as a treatment strategy for age-related osteoporosis. Molecular, histological and micro-computed tomography (micro-CT) based approaches confirmed that ASCs isolated from 18-week-old osteoporotic senescence-accelerated mice (SAMP6) were capable of undergoing osteogenesis when cultured in either silk fibroin (SF) scaffolds or scaffold-free microtissues (ASC-MT). A single intratibial injection of CM-Dil-labeled isogeneic ASCs or ASC-MT into SAMP6 recipients significantly improved trabecular bone quality after 6 weeks in comparison to untreated contralateral bones, as determined by micro-CT. Injected ASCs could be observed in paraffin wax bone sections at 24 h and 6 weeks post treatment and induced a significant increase in several molecular markers of bone turnover. Furthermore, a significant improvement in the osteogenic potential of osteoporotic patient-derived human bone marrow stromal cells (BMSCs) was observed when differentiated in conditioned culture media harvested from osteoporotic patient-derived human ASCs. These findings therefore support the use of ASCs as an autologous cell-based approach for the treatment of osteoporosis.

The late endosomal p14-MP1 (LAMTOR2/3) complex regulates focal adhesion dynamics during cell migration.

Cell migration is mediated by the dynamic remodeling of focal adhesions (FAs). Recently, an important role of endosomal signaling in regulation of cell migration was recognized. Here, we show an essential function for late endosomes carrying the p14-MP1 (LAMTOR2/3) complex in FA dynamics. p14-MP1-positive endosomes move to the cell periphery along microtubules (MTs) in a kinesin1- and Arl8b-dependent manner. There they specifically target FAs to regulate FA turnover, which is required for cell migration. Using genetically modified fibroblasts from p14-deficient mice and Arl8b-depleted cells, we demonstrate that MT plus end-directed traffic of p14-MP1-positive endosomes triggered IQGAP1 disassociation from FAs. The release of IQGAP was required for FA dynamics. Taken together, our results suggest that late endosomes contribute to the regulation of cell migration by transporting the p14-MP1 scaffold complex to the vicinity of FAs.

Apoptosis and necroptosis are induced in rainbow trout cell lines exposed to cadmium.

Cadmium is an important environmental toxicant that can kill cells. A number of studies have implicated apoptosis as well as necrosis and, most recently, a form of programmed necrosis termed necroptosis in the process of cadmium-mediated toxicity, but the exact mechanism remains ill-defined and may depend on the affected cell type. This study investigated which mode of cell death may be responsible for cell death induction in cadmium-exposed trout cell lines from gill and liver and if this cell death was sensitive to inhibitors of necroptosis or apoptosis, respectively. It was observed that intermediate levels of cadmium that killed approximately 50% of the cells over 96-120h of exposure caused cell death that morphologically resembled apoptosis and was associated with an increase of apoptotic markers such as the number of cells with diminished DNA content (sub-G1 cells), condensed or fragmented nuclei, and elevation of caspase-3 activity. At the same time, however, cells also lost plasma membrane integrity, as indicated by uptake of propidium iodide, showed a decrease of ATP levels and mitochondrial membrane potential, and displayed cell swelling, signs associated with secondary necrosis, or equally possible, necroptotic cell death. Importantly, many of these alterations were at least partly inhibited by the necroptosis inhibitor necrostatin-1 and were to a lesser extent also sensitive to the pan-caspase inhibitor zVAD-fmk, indicating that multiple modes of cell death are concurrently induced in cadmium-exposed trout cells, including necroptosis and apoptosis. Cell death appeared to lack concurrent radical formation, consistent with genetically regulated necroptotic cell death, but was characterized by the rapid induction of DNA damage markers, and the early onset of disintegration of the Golgi complex. Comparative experiments evaluating copper-toxicity indicated that in comparison to cadmium much higher concentrations of this metal were required to induce cell death and that neither necrostatin-1 nor a pan-caspase inhibitor conferred protection, suggesting that additional modes of cell death can be triggered in response to poisoning with heavy metals.

3D versus 2D cell culture implications for electron microscopy.

Cell culture systems are indispensable tools for basic research and a wide range of clinical in vitro studies. However, conventional 2D cell cultures poorly mimic the conditions in the living organism. This limitation may seriously compromise the reliability and significance of data obtained from such approaches. Therefore, we present here a comparative study on selected 3D and 2D cell cultures of U87-MG human glioblastoma cells that were processed by means of high-pressure freezing and freeze-substitution as well as by conventional chemical fixation and Tokuyasu cryo-section immuno-labeling. Three-dimensional cultures comprised pseudo-vascularized cultures, fiber and bead scaffold cultures, and spheroid cultures. Cell cultures in dishes and on coverslips were the static 2D culture systems used as reference models. We will discuss morphological and immuno-cytochemical observations with respect to the feasibility of the cell culture systems investigated for the state-of-the-art electron microscopy.

MYO5B mutations cause microvillus inclusion disease and disrupt epithelial cell polarity.

Following homozygosity mapping in a single kindred, we identified nonsense and missense mutations in MYO5B, encoding type Vb myosin motor protein, in individuals with microvillus inclusion disease (MVID). MVID is characterized by lack of microvilli on the surface of enterocytes and occurrence of intracellular vacuolar structures containing microvilli. In addition, mislocalization of transferrin receptor in MVID enterocytes suggests that MYO5B deficiency causes defective trafficking of apical and basolateral proteins in MVID.

Activation and nuclear translocation of ERK in response to ligand-dependent and -independent stimuli in liver and gill cells from rainbow trout.

The mitogen-activated protein kinase ERK is an important signalling molecule involved in the control of cell proliferation, differentiation and cell death, targeting molecules at the cell membrane, in the cytosol, and in the nucleus. This study investigated the activation pattern and subcellular distribution of ERK in liver and gill cells of rainbow trout upon hypo-osmotic shock, addition of epidermal growth factor (EGF) and copper treatment. It further set out to characterize the hypothetical role of nuclear-export signal (NES)-dependent relocation of ERK after nuclear entry and the potential involvement of the ERK activator MEK. Although, in primary hepatocytes, ERK was activated in all conditions in a stimulus-specific manner, it did not accumulate in the nucleus, irrespective of the absence or presence of the inhibitor of NES-dependent export leptomycin B (LB). Similarly, in trout hepatoma cells, where pERK levels increased upon osmotic and mitotic stimulation, but not after toxic insult, no significant nuclear translocation was observed. In a gill cell line, levels of pERK increased after osmotic and mitotic stimulation and showed a decrease during incubation with a toxicant. Again, none of these conditions triggered nuclear accumulation of pERK in the gill cells in the absence of LB, but in contrast to the observation in liver cells, both osmotic and mitotic stimulation caused nuclear accumulation in the presence of the inhibitor. The ERK activator MEK, which possesses a NES-sequence, was apparently not involved in nuclear export, as it did not seem to enter the nucleus. Altogether, ERK is activated in trout cells in a stimulus- and cell type-specific manner, and our data suggest that it acutely acts primarily on cytoplasmic or membrane-situated targets in liver cells, whereas it presumably triggers rapid transcriptional activities in gill cells.

Importance of cytoskeletal elements in volume regulatory responses of trout hepatocytes.

The role of cytoskeletal elements in volume regulation was studied in trout hepatocytes by investigating changes in F-actin distribution during anisotonic exposure and assessing the impact of cytoskeleton disruption on volume regulatory responses. Hypotonic challenge caused a significant decrease in the ratio of cortical to cytoplasmic F-actin, whereas this ratio was unaffected in hypertonic saline. Disruption of microfilaments with cytochalasin B (CB) or cytochalasin D significantly slowed volume recovery following hypo- and hypertonic exposure in both attached and suspended cells. The decrease of net proton release and the intracellular acidification elicited by hypotonicity were unaltered by CB, whereas the increase of proton release in hypertonic saline was dramatically reduced. Because amiloride almost completely blocked the hypertonic increase of proton release and cytoskeleton disruption diminished the associated increase of intracellular pH (pH(i)), we suggest that F-actin disruption affected Na(+)/H(+) exchanger activity. In line with this, pH(i) recovery after an ammonium prepulse was significantly inhibited in CB-treated cells. The increase of cytosolic Na(+) under hypertonic conditions was not diminished but, rather, enhanced by F-actin disruption, presumably due to inhibited Na(+)-K(+)-ATPase activity and stimulated Na(+) channel activity. The elevation of cytosolic Ca(2+) in hypertonic medium was significantly reduced by CB. Altogether, our results indicate that the F-actin network is of crucial importance in the cellular responses to anisotonic conditions, possibly via interaction with the activity of ion transporters and with signalling cascades responsible for their activation. Disruption of microtubules with colchicine had no effect on any of the parameters investigated.