Hannes L. Ebner

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.

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. Hum Mutat 31:1–8, 2010.

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

Late endosomes locally regulate cell migration by transporting the p14–MP1 scaffold complex to the vicinity of focal adhesions.

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.

Copper, but not cadmium, is acutely toxic for trout hepatocytes: short-term effects on energetics and ion homeostasis

The toxic effects of cadmium (Cd) and copper (Cu) on cellular energy metabolism and ion homeostasis were investigated in hepatocytes from the rainbow trout, Oncorhynchus mykiss. The metal content of cells did not increase during incubation with Cu, whereas a dose-dependent increase was seen with Cd. Cell viability was unaffected in the presence of 100 microM Cd and 10 microM Cu but was significantly reduced after 30 min of exposure to 100 microM Cu, both in the presence and absence of extracellular calcium. Oxygen consumption (VO(2)) was not affected by 100 microM Cd or 10 microM Cu, whereas 100 microM Cu caused a significant and calcium-dependent increase of VO(2). Lactate production and basal glucose release were not altered by either of the metals. However, the epinephrine-stimulated rate of glucose release was significantly reduced after 2 h of incubation with 100 microM Cu. Hepatocytes exposed to Cd showed only a marginal increase of intracellular free calcium (Ca(i)(2+)), whereas with Cu a pronounced and dose-dependent increase of Ca(i)(2+) was induced after a delay of 10 to 15 min, the calcium being of extracellular origin. Intracellular pH was not altered by Cd but decreased significantly in the presence of Cu. Overall our data demonstrate that Cu, but not Cd, is acutely toxic for trout hepatocytes. Since Cu does not enter the cells in the short term it appears to exert its acutely toxic effects at the cell membrane. Although Cu toxicity is associated with an uptake of calcium from extracellular space, leading to an elevation of cellular respiration, cytotoxicity does not appear to be dependent on the presence of extracellular calcium.

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.

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

SUMMARY 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.)

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.

Stability of the Endosomal Scaffold Protein LAMTOR3 Depends on Heterodimer Assembly and Proteasomal Degradation

Background: LAMTOR3 is an endosomal MAPK scaffold recently shown also to modulate the mTORC1 pathway. Results: Association to LAMTOR2 and recruitment to the endosomal membrane stabilize LAMTOR3 and prevent the premature degradation of the protein by the proteasome. Conclusion: The ubiquitin-proteasome pathway and LAMTOR complex assembly tightly control LAMTOR3 abundance. Significance: Cells may modulate the dual ERK/mTORC1 activity of LAMTOR3 by tightly controlling its intracellular protein level. 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 p14rev 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.