Molecular mechanism to recruit galectin-3 into multivesicular bodies for polarized exosomal secretion.

The beta-galactoside binding lectin galectin-3 (Gal3) is found intracellularly and in the extracellular space. Secretion of this lectin is mediated independently of the secretory pathway by a not yet defined nonclassical mechanism. Here, we found Gal3 in the lumen of exosomes. Superresolution and electron microscopy studies visualized Gal3 recruitment and sorting into intraluminal vesicles. Exosomal Gal3 release depends on the endosomal sorting complex required for transport I (ESCRT-I) component Tsg101 and functional Vps4a. Either Tsg101 knockdown or expression of dominant-negative Vps4aE228Q causes an intracellular Gal3 accumulation at multivesicular body formation sites. In addition, we identified a highly conserved tetrapeptide P(S/T)AP motif in the amino terminus of Gal3 that mediates a direct interaction with Tsg101. Mutation of the P(S/T)AP motif results in a loss of interaction and a dramatic decrease in exosomal Gal3 secretion. We conclude that Gal3 is a member of endogenous non-ESCRT proteins which are P(S/T)AP tagged for exosomal release.

DYRK1B regulates Hedgehog-induced microtubule acetylation.

The posttranslational modification (PTM) of tubulin subunits is important for the physiological functions of the microtubule (MT) cytoskeleton. Although major advances have been made in the identification of enzymes carrying out MT-PTMs, little knowledge is available on how intercellular signaling molecules and their associated pathways regulate MT-PTM-dependent processes inside signal-receiving cells. Here we show that Hedgehog (Hh) signaling, a paradigmatic intercellular signaling system, affects the MT acetylation state in mammalian cells. Mechanistically, Hh pathway activity increases the levels of the MT-associated DYRK1B kinase, resulting in the inhibition of GSK3ß through phosphorylation of Serine 9 and the subsequent suppression of HDAC6 enzyme activity. Since HDAC6 represents a major tubulin deacetylase, its inhibition increases the levels of acetylated MTs. Through the activation of DYRK1B, Hh signaling facilitates MT-dependent processes such as intracellular mitochondrial transport, mesenchymal cell polarization or directed cell migration. Taken together, we provide evidence that intercellular communication through Hh signals can regulate the MT cytoskeleton and contribute to MT-dependent processes by affecting the level of tubulin acetylation.

Miz1 Controls Schwann Cell Proliferation via H3K36me2 Demethylase Kdm8 to Prevent Peripheral Nerve Demyelination.

Schwann cell differentiation and myelination depends on chromatin remodeling, histone acetylation, and methylation, which all affect Schwann cell proliferation. We previously reported that the deletion of the POZ (POxvirus and Zinc finger) domain of the transcription factor Miz1 (Myc-interacting zinc finger protein; encoded by Zbtb17) in mouse Schwann cells (Miz1ΔPOZ) causes a neuropathy at 90 d after birth [postnatal day (P) 90], with a subsequent spontaneous regeneration. Here we show that RNA sequencing from Miz1ΔPOZ and control animals at P30 revealed a set of upregulated genes with a strong correlation to cell-cycle regulation. Consistently, a subset of Schwann cells did not exit the cell cycle as observed in control animals and the growth fraction increased over time. From the RNAseq gene list, two direct Miz1 target genes were identified, one of which encodes the histone H3K36me2 demethylase Kdm8. We show that the expression of Kdm8 is repressed by Miz1 and that its release in Miz1ΔPOZ cells induces a decrease of H3K36me2, especially in deregulated cell-cycle-related genes. The linkage between elevated Kdm8 expression, hypomethylation of H3K36 at cell-cycle-relevant genes, and the subsequent re-entering of adult Schwann cells into the cell cycle suggests that the release of Kdm8 repression in the absence of a functional Miz1 is a central issue in the development of the Miz1ΔPOZ phenotype.SIGNIFICANCE STATEMENT The deletion of the Miz1 (Myc-interacting zinc finger protein 1) POZ (POxvirus and Zinc finger) domain in Schwann cells causes a neuropathy. Here we report sustained Schwann cell proliferation caused by an increased expression of the direct Miz1 target gene Kdm8, encoding a H3K36me2 demethylase. Hence, the demethylation of H3K36 is linked to the pathogenesis of a neuropathy.

Resistance to apoptosis and autophagy leads to enhanced survival in Sertoli cells.

STUDY QUESTION: What is the underlying mechanism of Sertoli cell (SC) resistance to cell death? SUMMARY ANSWER: High expression of prosurvival B-cell lymphoma-2 (BCL2) proteins and inhibition of apoptosis and autophagy prolongs SC survival upon exposure to stress stimuli. WHAT IS KNOWN ALREADY: In human and in experimental models of orchitis, tolerogenic SC survive stress conditions, while germ cells undergo massive apoptosis. In general, non-dividing highly differentiated cells tend to resist stress conditions for a longer time by favoring activation of prosurvival mechanisms and inhibition of cell death pathways. STUDY DESIGN, SIZE, DURATION: In this cross sectional study, conditions stimulating apoptosis and autophagy were used to induce cell death in primary rat SC. Primary rat peritubular cells (PTC) and immortalized rat 93RS2 SC were used as controls. Each cell isolation was counted as one experiment (n = 1), and each experiment was repeated three to six times. PARTICIPANTS/MATERIALS, SETTING, METHODS: Testis biopsy samples from infertile or subfertile patients and testis samples from rats with experimental autoimmune orchitis were used for immunohistological analysis. Primary SC were isolated from 19-day-old male Wistar rats. To maintain cell purity, cells were cultured in serum-free medium for apoptosis experiments and in medium supplemented with 1% serum for autophagy analyses. To induce apoptosis, cells were stimulated with staurosporine, borrelidin, cisplatin and etoposide for 4 or 24 h. Caspase three activation was examined by immunoblotting and enzymatic activity assay. Mitochondrial membrane potential was measured using tetramethylrhodamine methyl ester followed by flow cytometric analysis. Cytochrome c release was monitored by immunofluorescence. Cell viability was determined using the methylthiazole tetrazolium assay. To monitor autophagy flux, cells were deprived of nutrients using Hank's balanced salt solution for 1, 2 and 3 h. Formation of autophagosomes was analyzed by using immunoblotting, immunofluorescence labeling and ultrastructural analyses. Relative mRNA levels of genes involved in the regulation of apoptosis and autophagy were evaluated. Extracellular high mobility group box protein one was measured as a marker of necrosis using ELISA. MAIN RESULTS AND THE ROLE OF CHANCE: SC survive the inflammatory conditions in vivo in human testis and in experimental autoimmune orchitis. Treatment with apoptosis inducing chemotherapeutics did not cause caspase three activation in isolated rat SC. Moreover, mitochondrial membrane potential and mitochondrial localization of cytochrome c were not changed by treatment with staurosporine, suggesting a premitochondrial blockade of apoptosis in SC. Expression levels of prosurvival BCL2 family members were significantly higher in SC compared to PTC at both mRNA and protein levels. Furthermore, after nutrient starvation, autophagy signaling was initiated in SC as observed by decreased levels of phosphorylated UNC- 51-like kinase -1 (ULK1). However, levels of light chain 3 II (LC3 II) and sequestosome1 (SQSTM1) remained unchanged, indicating blockade of the autophagy flux. Lysosomal activity was intact in SC as shown by accumulation of LC3 II following administration of lysosomal protease inhibitors, indicating that inhibition of autophagy flux occurs at a preceding stage. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, we have used primary SC from prepubertal rats. Caution should be taken when translating our results to adult animals, where crosstalk with other testicular cells and hormonal factors may also play a role in regulating survival of SC. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that inhibition of autophagy and apoptosis following exposure to extrinsic stress stimuli promotes SC survival, and is a possible mechanism to explain the robustness of SC in response to stress. Cell death resistance in SC is crucial for the recovery of spermatogenesis after chemotherapy treatment in cancer patients. Additionally, understanding the molecular mechanisms of SC survival unravels valuable target proteins, such as BCL2, that may be manipulated therapeutically to control cell viability depending on the context of the disease. STUDY FUNDING AND COMPETING INTEREST(S): This study was funded by the Deutsche Forschungsgemeinschaft (DFG) Grant BH93/1-1, and by the International Research Training Group between Justus Liebig University of Giessen and Monash University, Melbourne (GRK 1871/1) funded by the DFG and Monash University. The support of the Medical Faculty of Justus-Liebig University of Giessen is gratefully acknowledged. The authors declare no conflict of interest.

Late onset neuropathy with spontaneous clinical remission in mice lacking the POZ domain of the transcription factor Myc-interacting zinc finger protein 1 (Miz1) in Schwann cells.

The transcription factor Miz1 (Myc-interacting zinc finger 1) is a known regulator of the cell cycle but also has cell cycle-independent functions. Here we analyzed the role of Miz1 in the peripheral nervous system, using an early embryonic conditional knock-out model in which the Miz1 POZ domain is ablated in Schwann cells. Although the development of myelinated nerve fibers was not impaired, Miz1ΔPOZ mice acquired behavioral signs of a peripheral neuropathy at the age of 3 months. At this time, ultrastructural analysis of the sciatic nerve showed de- and dysmyelination of fibers, with massive outfoldings and a focal infiltration of macrophages. Although the expression of genes encoding structural myelin proteins, such as periaxin, myelin basic protein, and myelin protein zero, was decreased, genes associated with a negative regulation of myelination, including c-Jun, Sox2, and Id2, were up-regulated in Miz1ΔPOZ mice compared with controls. In animals older than 4 months, the motor disabilities vanished, and the ultrastructure of the sciatic nerve exhibited numerous tomacula and remyelinated fibers, as indicated by thinner myelin. No second acute attack was observed up to the age of 1 year. Thus, the deletion of the Miz1 POZ domain in Schwann cells induces an acute neuropathy with a subsequent regeneration in which there is ongoing balancing between de- and remyelination. Miz1ΔPOZ mice are impaired in the maintenance of myelinated fibers and are a promising model for studying remyelination in adult peripheral nerves.

Altered glycogen metabolism causes hepatomegaly following an Atg7 deletion.

Autophagy is a lysosomal degradation process involved in the turnover of organelles or other cell constituents, in providing sources for energy production under starving conditions and in cell metabolism. A key protein in the macroautophagic machinery is the autophagy-related protein (Atg) 7. Constitutive deletion of Atg7 is lethal at birth. A conditional deletion of Atg7 in hepatocytes leads to hepatomegaly and in aged animals to liver tumors. With this study, we aim at analyzing the hepatomegaly development in more detail. The 3- to 4-fold enlargement of the liver takes place between days 25 and 35 after birth (P25-P35) and persists at least until P90. This is accompanied by a change in the expression of enzymes involved in the glycogen/glucose metabolism. While glycogen synthesis is inhibited, glucose is preferentially kept as glucose-6-phosphate inside the cells, inducing a swelling of the cells caused by hyperosmolarity. An increase of lipogenic enzymes suggests that glucose-6-phosphate is delivered to lipogenic pathways, which is supported by the occurrence of a steatosis around P30. The development of hepatomegaly is accompanied by a polyploidisation of hepatocytes, an enhanced expression of genes related to inflammatory processes and an infiltration of macrophages and granulocytes. Our data provide evidence that the attenuation of macroautophagy in hepatocytes leads to a glucose retention that causes cell swelling. The resulting hepatomegaly, which develops in a time interval of about 10 days, perturbs liver perfusion and induces an inflammatory reaction together with polyploidisation.

Alterations in the secretory pattern of dermal dendritic cells following melanin uptake.

Under a variety of circumstances, melanin occurs in the dermal compartment of the skin, being mostly observed in cells that have been termed melanophages, some of which have been identified as dermal dendritic cells. We analysed changes in the expression and secretion pattern of cytokines by dendritic cells after the uptake of melanin from various sources. Dendritic cells were derived from human primary blood monocytes or from the human monocytic cell line THP-1. Melanin uptake increased the secretion of the chemokines MIP-1ß (CCL4) and MCP-1 (CCL2). The higher MIP-1ß secretion was accompanied by higher MIP-1ß gene expression. Elevation of MIP-1ß secretion was dependent on the uptake of melanin but could not be induced by the phagocytosis of latex beads, indicating that the phagocytic process itself was not sufficient to increase the secretion of this cytokine. The data thus show that the uptake of melanin changes the cytokine expression and secretion pattern of dendritic-like cells.

Humans possess two mitochondrial ferredoxins, Fdx1 and Fdx2, with distinct roles in steroidogenesis, heme, and Fe/S cluster biosynthesis.

Mammalian adrenodoxin (ferredoxin 1; Fdx1) is essential for the synthesis of various steroid hormones in adrenal glands. As a member of the [2Fe-2S] cluster-containing ferredoxin family, Fdx1 reduces mitochondrial cytochrome P450 enzymes, which then catalyze; e.g., the conversion of cholesterol to pregnenolone, aldosterone, and cortisol. The high protein sequence similarity between Fdx1 and its yeast adrenodoxin homologue (Yah1) suggested that Fdx1, like Yah1, may be involved in the biosynthesis of heme A and Fe/S clusters, two versatile and essential protein cofactors. Our study, employing RNAi technology to deplete human Fdx1, did not confirm this expectation. Instead, we identified a Fdx1-related mitochondrial protein, designated ferredoxin 2 (Fdx2) and found it to be essential for heme A and Fe/S protein biosynthesis. Unlike Fdx1, Fdx2 was unable to efficiently reduce mitochondrial cytochromes P450 and convert steroids, indicating that the two ferredoxin isoforms are highly specific for their substrates in distinct biochemical pathways. Moreover, Fdx2 deficiency had a severe impact, via impaired Fe/S protein biogenesis, on cellular iron homeostasis, leading to increased cellular iron uptake and iron accumulation in mitochondria. We conclude that mammals depend on two distinct mitochondrial ferredoxins for the specific production of either steroid hormones or heme A and Fe/S proteins.

Redox atlas of the mouse. Immunohistochemical detection of glutaredoxin-, peroxiredoxin-, and thioredoxin-family proteins in various tissues of the laboratory mouse.

BACKGROUND: Oxidoreductases of the thioredoxin family of proteins have been thoroughly studied in numerous cellular and animal models mimicking human diseases. Despite of their well documented role in various disease conditions, no systematic information on the presence of these proteins is available. METHODS: Here, we have systematically analyzed the presence of some of the major constituents of the glutaredoxin (Grx)-, peroxiredoxin (Prx)-, and thioredoxin (Trx)-systems, i.e. Grx1, Grx2, Grx3 (TXNL-2/PICOT), Grx5, nucleoredoxin (Nrx), Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, Trx1, thioredoxin reductase 1 (TrxR1), Trx2, TrxR2, and γ-glutamyl cysteine synthetase (γ-GCS) in various tissues of the mouse using immunohistochemistry. RESULTS: The identification of the Trx family proteins in the central nervous system, sensory organs, digestive system, lymphatic system, reproductive system, urinary system, respiratory system, endocrine system, skin, heart, and muscle revealed a number of significant differences between these proteins with respect to their distribution in these tissues. CONCLUSION: Our results imply more specific functions and interactions between the proteins of this family than previously assumed. GENERAL SIGNIFICANCE: Crucial functions of Trx family proteins have been demonstrated in various disease conditions. A detailed overview on their distribution in various tissues will be helpful to fully comprehend their potential role and the interactions of these proteins in the most thoroughly studied model for human diseases-the laboratory mouse. This article is part of a Special Issue entitled Human and Murine Redox Protein Atlases.

Replacement of Rbpj with Rbpjl in the PTF1 complex controls the final maturation of pancreatic acinar cells.

BACKGROUND & AIMS: The mature pancreatic acinar cell is dedicated to the production of very large amounts of digestive enzymes. The early stages of pancreatic development require the Rbpj form of the trimeric Pancreas Transcription Factor 1 complex (PTF1-J). As acinar development commences, Rbpjl gradually replaces Rbpj; in the mature pancreas, PTF1 contains Rbpjl (PTF1-L). We investigated whether PTF1-L controls the expression of genes that complete the final stage of acinar differentiation. METHODS: We analyzed acinar development and transcription in mice with disrupted Rbpjl (Rbpjl(ko/ko) mice). We performed comprehensive analyses of the messenger RNA population and PTF1 target genes in pancreatic acinar cells from these and wild-type mice. RESULTS: In Rbpjl(ko/ko) mice, acinar differentiation was incomplete and characterized by decreased expression (as much as 99%) of genes that encode digestive enzymes or proteins of regulated exocytosis and mitochondrial metabolism. Whereas PTF1-L bound regulatory sites of genes in normal adult pancreatic cells, the embryonic form (PTF1-J) persisted in the absence of Rbpjl and replaced PTF1-L; the extent of replacement determined gene expression levels. Loss of PTF1-L reduced expression (>2-fold) of only about 50 genes, 90% of which were direct targets of PTF1-L. The magnitude of the effects on individual digestive enzyme genes correlated with the developmental timing of gene activation. Absence of Rbpjl increased pancreatic expression of liver-restricted messenger RNA. CONCLUSIONS: Replacement of Rbpj by Rbpjl in the PTF1 complex drives acinar differentiation by maximizing secretory protein synthesis, stimulating mitochondrial metabolism and cytoplasmic creatine-phosphate energy stores, completing the packaging and secretory apparatus, and maintaining acinar-cell homeostasis.

Myc regulates keratinocyte adhesion and differentiation via complex formation with Miz1.

Myc plays a key role in homeostasis of the skin. We show that Miz1, which mediates Myc repression of gene expression, is expressed in the epidermal basal layer. A large percentage of genes regulated by the Myc-Miz1 complex in keratinocytes encode proteins involved in cell adhesion, and some, including the alpha6 and beta1 integrins, are directly bound by Myc and Miz1 in vivo. Using a Myc mutant deficient in Miz1 binding (MycV394D), we show that Miz1 is required for the effects of Myc on keratinocyte responsiveness to TGF-beta. Myc, but not MycV394D, decreases keratinocyte adhesion and spreading. In reconstituted epidermis, Myc induces differentiation and loss of cell polarization in a Miz1-dependent manner. In vivo, overexpression of beta1 integrins restores basal layer polarity and prevents Myc-induced premature differentiation. Our data show that regulation of cell adhesion is a major function of the Myc-Miz1 complex and suggest that it may contribute to Myc-induced exit from the epidermal stem cell compartment.

Disruption of the Gardos channel (KCa3.1) in mice causes subtle erythrocyte macrocytosis and progressive splenomegaly.

Gardos channel, the erythrocyte Ca(2+)-activated K(+) channel (K(Ca)3.1), is considered a major regulator of red blood cell (RBC) volume by mediating efflux of potassium and thus cell dehydration and shrinkage. However, the functional importance of K(Ca)3.1 in RBC in vivo is incompletely understood. Here, we used K(Ca)3.1(-/-)-mice to investigate the consequences of K(Ca)3.1 deficiency for RBC indices, functions, and sequestration. RBCs of K(Ca)3.1(-/-)-mice of all ages were mildly macrocytic but their biconcave appearance being preserved. RBC number, total hemoglobin, and hematocrit were unchanged in the adult K(Ca)3.1(-/-)-mice and increased in the premature K(Ca)3.1(-/-)-mice. Filterability, Ca(2+)-dependent volume decrease and osmotic tolerance of RBCs lacking K(Ca)3.1 were noticeably reduced when compared to RBC of wild-type littermates. Deformability to increasing shear stress was unchanged. Strikingly, K(Ca)3.1(-/-)-mice developed progressive splenomegaly which was considerable ( approximately 200% of controls) in the >6-month-old mice and was paralleled by increased iron deposition in the aged mice presumably as a consequence of enhanced RBC sequestration. Daily injections of the K(Ca)3.1-blocker TRAM-34 (120 mg/kg) also produced mild splenomegaly in wild-type mice. We conclude that genetic deficit of erythroid K(Ca)3.1 causes mild RBC macrocytosis, presumably leading to reduced filterability, and impairs volume regulation. These RBC defects result in mild but progressive splenomegaly.

Role of human mitochondrial Nfs1 in cytosolic iron-sulfur protein biogenesis and iron regulation.

The biogenesis of iron-sulfur (Fe/S) proteins in eukaryotes is a complex process involving more than 20 components. So far, functional investigations have mainly been performed in Saccharomyces cerevisiae. Here, we have analyzed the role of the human cysteine desulfurase Nfs1 (huNfs1), which serves as a sulfur donor in biogenesis. The protein is located predominantly in mitochondria, but small amounts are present in the cytosol/nucleus. huNfs1 was depleted efficiently in HeLa cells by a small interfering RNA (siRNA) approach, resulting in a drastic growth retardation and striking morphological changes of mitochondria. The activities of both mitochondrial and cytosolic Fe/S proteins were strongly impaired, demonstrating that huNfs1 performs an essential function in Fe/S protein biogenesis in human cells. Expression of murine Nfs1 (muNfs1) in huNfs1-depleted cells restored both growth and Fe/S protein activities to wild-type levels, indicating the specificity of the siRNA depletion approach. No complementation of the growth retardation was observed, when muNfs1 was synthesized without its mitochondrial presequence. This extramitochondrial muNfs1 did not support maintenance of Fe/S protein activities, neither in the cytosol nor in mitochondria. In conclusion, our study shows that the essential huNfs1 is required inside mitochondria for efficient maturation of cellular Fe/S proteins. The results have implications for the regulation of iron homeostasis by cytosolic iron regulatory protein 1.

Schwann cell Myc-interacting zinc-finger protein 1 without pox virus and zinc finger: epigenetic implications in a peripheral neuropathy.

Functionality of adult peripheral nerves essentially relies on differentiation of Schwann cells during postnatal development, as well as fine-tuned re- and transdifferentiation in response to peripheral nerve injury. Epigenetic histone modifications play a major role during the differentiation of embryonic stem cells and diverse organ specific progenitor cells, yet only little is known about the epigenetic regulation of Schwann cells. Just recently, Fuhrmann et al. reported how the transcription factor Myc-interacting zinc-finger protein 1 (Miz1) might contribute to Schwann cell differentiation through repression of the histone demethylase Kdm8. Here, we discuss the potential novel role of Miz1 in Schwann cell differentiation and give a short overview about previously reported histone modifications underlying peripheral nerve development and response to injury.

Mechanisms of iron-sulfur protein maturation in mitochondria, cytosol and nucleus of eukaryotes.

Iron-sulfur (Fe/S) clusters are important cofactors of numerous proteins involved in electron transfer, metabolic and regulatory processes. In eukaryotic cells, known Fe/S proteins are located within mitochondria, the nucleus and the cytosol. Over the past years the molecular basis of Fe/S cluster synthesis and incorporation into apoproteins in a living cell has started to become elucidated. Biogenesis of these simple inorganic cofactors is surprisingly complex and, in eukaryotes such as Saccharomyces cerevisiae, is accomplished by three distinct proteinaceous machineries. The "iron-sulfur cluster (ISC) assembly machinery" of mitochondria was inherited from the bacterial ancestor of mitochondria. ISC components are conserved in eukaryotes from yeast to man. The key principle of biosynthesis is the assembly of the Fe/S cluster on a scaffold protein before it is transferred to target apoproteins. Cytosolic and nuclear Fe/S protein maturation also requires the function of the mitochondrial ISC assembly system. It is believed that mitochondria contribute a still unknown compound to biogenesis outside the organelle. This compound is exported by the mitochondrial "ISC export machinery" and utilised by the "cytosolic iron-sulfur protein assembly (CIA) machinery". Components of these two latter systems are also highly conserved in eukaryotes. Defects in the mitochondrial ISC assembly and export systems, but not in the CIA machinery have a strong impact on cellular iron uptake and intracellular iron distribution showing that mitochondria are crucial for both cellular Fe/S protein assembly and iron homeostasis.

Characterisation of lipofuscin-like lysosomal inclusion bodies from human placenta.

A structural hallmark of lysosomes is heterogeneity of their contents. We describe a method for isolation of particulate materials from human placental lysosomes. After a methionine methyl ester-induced disruption of lysosomes and two density gradient centrifugations we obtained a homogeneous membrane fraction and another one enriched in particulate inclusions. The latter exhibited a yellow-brown coloration and contained bodies lacking a delimiting membrane, which were characterised by a granular pattern and high electron density. The lipofuscin-like inclusion materials were rich in tripeptidyl peptidase I, beta-glucuronidase, acid ceramidase and apolipoprotein D and contained proteins originating from diverse subcellular localisations. Here we show that human term placenta contains lipofuscin-like lysosomal inclusions, a phenomenon usually associated with senescence in postmitotic cells. These findings imply that a simple pelleting of a lysosomal lysate is not appropriate for the isolation of lysosomal membranes, as the inclusions tend to be sedimented with the membranes.

Miz1 is required for early embryonic development during gastrulation.

Miz1 is a member of the POZ domain/zinc finger transcription factor family. In vivo, Miz1 forms a complex with the Myc oncoprotein and recruits Myc to core promoter elements. Myc represses transcription through Miz1 binding sites. We now show that the Miz1 gene is ubiquitously expressed during mouse embryogenesis. In order to elucidate the physiological function of Miz1, we have deleted the mouse Miz1 gene by homologous recombination. Miz1(+/-) mice are indistinguishable from wild-type animals; in contrast, Miz1(-/-) embryos are not viable. They are severely retarded in early embryonic development and do not undergo normal gastrulation. Expression of Goosecoid and Brachyury is detectable in Miz1(-/-) embryos, suggesting that Miz1 is not required for signal transduction by Nodal. Expression of p21Cip1, a target gene of Miz1 is unaltered; in contrast, expression of p57Kip2, another target gene of Miz1 is absent in Miz1(-/-) embryos. Miz1(-/-) embryos succumb to massive apoptosis of ectodermal cells around day 7.5 of embryonic development. Our results show that Miz1 is required for early embryonic development during gastrulation.

Glutathione-conjugating and membrane-remodeling activity of GDAP1 relies on amphipathic C-terminal domain.

Mutations in the ganglioside-induced differentiation associated protein 1 (GDAP1) cause severe peripheral motor and sensory neuropathies called Charcot-Marie-Tooth disease. GDAP1 expression induces fission of mitochondria and peroxisomes by a currently elusive mechanism, while disease causing mutations in GDAP1 impede the protein's role in mitochondrial dynamics. In silico analysis reveals sequence similarities of GDAP1 to glutathione S-transferases (GSTs). However, a proof of GST activity and its possible impact on membrane dynamics are lacking to date. Using recombinant protein, we demonstrate for the first time theta-class-like GST activity for GDAP1, and it's activity being regulated by the C-terminal hydrophobic domain 1 (HD1) of GDAP1 in an autoinhibitory manner. Moreover, we show that the HD1 amphipathic pattern is required to induce membrane dynamics by GDAP1. As both, fission and GST activities of GDAP1, are critically dependent on HD1, we propose that GDAP1 undergoes a molecular switch, turning from a pro-fission active to an auto-inhibited inactive conformation.

Isolation of Sertoli Cells and Peritubular Cells from Rat Testes.

The testis, and in particular the male gamete, challenges the immune system in a unique way because differentiated sperm first appear at the time of puberty - more than ten years after the establishment of systemic immune tolerance. Spermatogenic cells express a number of proteins that may be seen as non-self by the immune system. The testis must then be able to establish tolerance to these neo-antigens on the one hand but still be able to protect itself from infections and tumor development on the other hand. Therefore the testis is one of a few immune privileged sites in the body that tolerate foreign antigens without evoking a detrimental inflammatory immune response. Sertoli cells play a key role for the maintenance of this immune privileged environment of the testis and also prolong survival of cotransplanted cells in a foreign environment. Therefore primary Sertoli cells are an important tool for studying the immune privilege of the testis that cannot be easily replaced by established cell lines or other cellular models. Here we present a detailed and comprehensive protocol for the isolation of Sertoli cells - and peritubular cells if desired - from rat testes within a single day.