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Kozjak-Pavlovic V.,Max Planck Institute for Infection Biology | Kozjak-Pavlovic V.,University of Wurzburg | Ross K.,Max Planck Institute for Infection Biology | Gotz M.,University of Wurzburg | And 3 more authors.
Journal of Molecular Biology | Year: 2010

β-Barrel proteins are found in the outer membranes of bacteria, chloroplasts and mitochondria. The evolutionary conserved sorting and assembly machinery (SAM complex) assembles mitochondrial β-barrel proteins, such as voltage-dependent anion-selective channel 1 (VDAC1), into complexes in the outer membrane by recognizing a sorting β-signal in the carboxy-terminal part of the protein. Here we show that in mammalian mitochondria, masking of the C-terminus of β-barrel proteins by a tag leads to accumulation of soluble misassembled protein in the intermembrane space, which causes mitochondrial fragmentation and loss of membrane potential. A similar phenotype is observed if the β-signal is shortened, removed or when the conserved hydrophobic residues in the β-signal are mutated. The length of the tag at the C-terminus is critical for the assembly of VDAC1, as well as the amino acid residues at positions 130, 222, 225 and 251 of the protein. We propose that if the recognition of the β-signal or the folding of the β-barrel proteins is inhibited, the nonassembled protein will accumulate in the intermembrane space, aggregate and damage mitochondria. This effect offers easy tools for studying the requirements for the membrane assembly of β-barrel proteins, but also advises caution when interpreting the outcome of the β-barrel protein overexpression experiments. © 2010 Elsevier Ltd. All rights reserved.

Guerfal M.,Unit for Medical Biotechnology | Guerfal M.,Ghent University | Claes K.,Unit for Medical Biotechnology | Claes K.,Ghent University | And 6 more authors.
Microbial Cell Factories | Year: 2013

Background: Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved. Results: We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the {increment}pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the {increment}pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER. Conclusions: We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of biotechnological interest, such as insect cells and mammalian cells. © 2013 Guerfal et al.; licensee BioMed Central Ltd.

News Article
Site: www.scientificcomputing.com

This 40x photograph shows 3-D reconstruction of mouse brown adipose (fat) tissue. It received an honorable mention in the 2015 Nikon Small World Photomicrophotography Competition, which recognizes excellence in photography with the optical microscope and was taken by Dr. Daniela Malide of the National Institutes of Health (NIH) National Heart, Lung and Blood Institute, Light Microscopy Core Facility, in Bethesda, MD, using third harmonic generation microscopy.

All zebrafish work was performed according to standard protocols approved by The University of Chicago (ACUP #72074). No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. In situ hybridization for the hox13, Cre, and1 and shha genes were performed according to standard protocols29 after fixation in 4% paraformaldehyde overnight at 4 °C. Probes for hox13 and shha were as previously described18. Primers to clone Cre and and1 into vectors can be found in Extended Data Tables 1 and 2. Specimens were visualized on a Leica M205FA microscope. In order to create a destination vector for lineage tracing, we first designed a random sequence of 298 bp that contained a SmaI site to be used in downstream cloning. This sequence was ordered as a gBlocks fragment (IDT) and ligated into the pCR8/GW/TOPO TA cloning vector (Invitrogen). We then performed a Gateway LR reaction according to the manufacturers specifications between this entry vector and pXIG–cFos–GFP, which abolished an NcoI site present in the gateway cassette and introduced a SmaI site. We then removed the GFP gene with NcoI and BglII of the destination vector and ligated in Cre with (primers in Extended Data Table 1), using the ‘pCR8GW–Cre–pA–FRT–kan–FRT’ (kind gift of M. L. Suster, Sars International Center for Marine Molecular Biology, University of Bergen, Bergen, Norway) as a template for Cre PCR and Platinum Taq DNA polymerase High Fidelity (Invitrogen). In order to add a late phase enhancer to this vector, we first ordered four identical oligos (IDT gBlocks) of the core e16 sequence from gar, each flanked by different restriction sites. Each oligo was then ligated into pCR8/GW/TOPO, and sequentially cloned via restriction sites into a single pCR8/GW/TOPO vector. This entry vector was used a template to PCR the final Lo-e16x4 sequence and ligate it into the Cre destination vector using XhoI and SmaI, creating Lo-e16x4–Cre. The early phase enhancer Dr-CNS65x3 was cloned into the destination vector using the same strategy. Final vectors were confirmed by sequencing. A full list of sequences and primers used can be found in Extended Data Table 1. *AB zebrafish embryos were collected from natural spawning and injected according to the Tol2 system as described previously21. Transposase RNA was synthesized from the pCS2-zT2TP vector using the mMessage mMachine SP6 kit (Ambion)21. All injected embryos were raised to sexual maturity according to standard protocols. Adult F0 fish were outcrossed to wild-type *AB, and the total F1 clutch was lysed and DNA isolated at 24 hpf for genotyping (see Extended Data Table 1 for primers) to confirm germline transmission of Cre plasmids in the F0 founders. Multiple founders were identified and tested for the strongest and most consistent expression via antibody staining and in situ hybridization. One founder fish was identified as best, and all subsequent experiments were performed using offspring of this individual fish. Founder Lo-e16x4–Cre and Dr-CNS65x3–Cre fish were crossed to the Tg(ubi:Switch) line (kind gift from L. I. Zon). Briefly, this line contains a construct in which a constitutively active promoter (ubiquitin) drives expression of a loxP flanked GFP protein in all cells of the fish assayed. When Cre is introduced, the GFP gene is removed and the ubiquitin promoter is exposed to mCherry, thus permanently labelling the cell. We crossed our founder Cre fish to Tg(ubi:Switch) and fixed progeny at different time points to track cell fate. In order to detect the mCherry signal, embryos or adults were fixed overnight in 4% paraformaldehyde and subsequently processed for whole-mount antibody staining according to standard protocols30 using the following antibodies and dilutions: 1st rabbit anti-mCherry/DsRed (Clontech #632496) at 1:250, 1st mouse anti-Zns-5 (Zebrafish International Resource Center, USA) at 1:200, 2nd goat anti-rabbit Alexa Fluor 546 (Invitrogen #A11071) at 1:400, 2nd goat anti-mouse Alexa 647 (Invitrogen #A21235) at 1:400. Stained zebrafish were mounted under a glass slide and visualized using an LSM 710 confocal microscope (Organismal Biology and Anatomy, the University of Chicago). Antibody stains on adult zebrafish (90 dpf) fins were imaged on a Leica SP5 II tandem scanner AOBS Laser Scanning Confocal (the University of Chicago Integrated Light Microscopy Core Facility). Two mutations were simultaneously introduced into the first exon of each hox13 gene by CRISPR/Cas9 system as previously described in Xenopus tropicalis31. Briefly, two gRNAs that match the sequence of exon 1 of each hox13 gene were designed by ZiFiT (http://zifit.partners.org/ZiFiT/). To synthesize gRNAs, forward and reverse oligonucleotides that are unique for individual target sequences were synthesized by Integrated DNA Technologies, Inc. (IDT). Each oligonucleotide sequence can be found in Extended Data Table 2. Subsequently, each forward and reverse oligonucleotide were hybridized, and double stranded products were individually amplified by PCR with primers that include a T7 RNA promoter sequence, followed by purification by NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel). Each gRNA was synthesized from the purified PCR products by in vitro transcription with the MEGAscript T7 Transcription kit (Ambion). Cas9 mRNA was synthesized by mMESSAGE mMACHINE SP6 Transcription Kit according to the manufacturer’s instructions (Ambion). Two gRNAs targeting exon 1 of each hox gene were injected with Cas9 mRNA into zebrafish eggs at the one-cell stage. We injected ~2 nl of the injection solution (5 μl solution containing 1,000 ng of each gRNA and 500 ng Cas9 diluted in nuclease-free water) into the single cell of the embryo. Injected embryos were raised to adulthood, and at three months were genotyped by extracting DNA from tail clips. Briefly, zebrafish were anaesthetized by Tricaine (0.004%) and tips of the tail fin (2–3 mm2) were removed and placed in an Eppendorf tube. The tissue was lysed in standard lysis buffer (10 mM Tris pH 8.2, 10 mM EDTA, 200 mM NaCl, 0.5% SDS, 200 μg/ml proteinase K) and DNA recovered by ethanol precipitation. Approximately 800-1,100 bp of exon 1 from each gene was amplified by PCR using the primers described in Extended Data Table 2. To determine whether mutations were present, PCR products were subjected to T7E1 (T7 endonuclease1) assay as previously reported32. After identification of mutant fish by T7E1 assay, detailed analysis of mutation patterns were performed by sequencing at the Genomics Core at the University of Chicago. Identified mutant fish were outcrossed to wild type to select frameshift mutations from mosaic mutational patterns and establish single heterozygous lines. Obtained embryos were raised to adults (~3 months), then analysed by T7E1 assay and sequenced. Among a variety of mutational patterns, fish that have frameshift mutations were used for assays as single heterozygous fish. We obtained several independent heterozygous mutant lines for each hox13 gene to compare the phenotype among different frameshift mutations. To obtain hoxa13a+/−, hoxa13b+/− double heterozygous mutant fish, each single heterozygous mutant line was crossed with the other mutant line. Offspring were analysed by T7E1 assay and sequenced after three months, and double heterozygous mutant fish were selected. To generate double homozygous hoxa13 mutant embryos and adult fish (hoxa13a−/−, hoxa13b−/−), double heterozygous fish (hoxa13a+/−, hoxa13b+/−) were crossed with each other. The ratio of each genotype from crossing heterozygous fish is summarized in Extended Data Table 4. After mutant lines were established, single (hoxa13a or hoxa13b) or double (hoxa13a, hoxa13b) mutant embryos and adult fish were genotyped by PCR for each analysis. Primer sequences for PCR are listed in Extended Data Table 2. To identify an 8 bp deletion in exon 1 of hoxa13a, the PCR product was treated by Ava1 at 37 °C for 2 h, because the 8 bp deletion produces a new Ava1 site in the PCR product (‘zebra hoxa13a_8 bp del’ primers, wild type; 231 bp, mutant; 111 bp and 119 bp). Final product size was confirmed by 3% agarose gel electrophoresis. To identify a 29 bp deletion in exon 1 of hoxa13a, the PCR product was confirmed by gel electrophoresis (‘zebra hoxa13a_29 bp del’ primers, wild type; 110 bp, mutant; 81 bp). To identify a 14 bp insertion in exon 1 of hoxa13b, the PCR product was treated by Bcc1 at 37°C for 2 h, because the 14 bp insertion produces a new Bcc1 site in the PCR product (‘zebra hoxa13b_14 bp ins’ primers, wild type; 98 bp, mutant; 53 bp + 57 bp). The final product size was confirmed by 3% agarose gel electrophoresis. The details of the mutant sequence are summarized in Extended Data Table 3a–c. Two gRNAs targeting exon 1 of hoxa13b and two gRNAs targeting exon 1 of hoxd13a were injected with Cas9 mRNA into zebrafish one-cell eggs that were obtained from crossing hoxa13a+/− and hoxa13a+/−, hoxa13b+/−, hoxd13a+/− (gRNAs were same as that were used to establish single hox13 knockout fishes and found in Extended Data Table 2). Injected eggs were raised to adult fish and genotyped by extracting DNA from tail fins. PCR products of each hox13 gene were cloned into PCRIITOPO (Invitrogen) and deep sequencing was performed (Genomic Core, the University of Chicago). At four months old, skeletal staining and CT scanning were performed to analyse the effect of triple gene deletions. The knockout ratios of each hox13 allele were calculated from the results of deep sequencing. Embryos were obtained by crossing hoxa13a+/−, hoxa13b+/− to each other and raised to 72 hpf or 96 hpf. After fixation by 4% PFA for 15 h, caudal halves were used for PCR genotyping. Pectoral fins of wild type and hoxa13a−/−, hoxa13b−/− were detached from the embryonic body and placed horizontally on glass slides. The fins were photographed with a Leica M205FA microscope, and the fin fold length along the proximodistal axis at the centre of the fin was measured using ImageJ. The resulting data were analysed by t-test comparing the means. Embryos were obtained by crossing hoxa13a+/−, hoxa13b+/− to each other and raised to 96 hpf. After fixation by 4% PFA for 15 h, caudal halves were used for PCR genotyping. Wild type and hoxa13a−/−, hoxa13b−/− embryos were stained by DAPI (1:4,000 in PBS-0.1% Triton) for 3 h and washed for 3 h by PBS−0.1% Triton. Pectoral fins were detached from the embryonic body, placed on glass slides and covered by a coverslip. The DAPI signal was detected by Zeiss LSM 710 (Organismal Biology and Anatomy, the University of Chicago). Individual nuclei were manually marked using Adobe Illustrator and the number of nuclei was counted. The data were analysed by t-test comparing the means. Skeletal staining was performed as previously described33. Briefly, fish were fixed in 10% neutral-buffered formalin overnight. After washing with milli-Q water, solutions were substituted by 70% EtOH in a stepwise fashion and then by 30% acetic acid/70% EtOH. Cartilage was stained with 0.02% alcian blue in 30% acetic acid/70% EtOH overnight. After washingwith milli-Q water, the solution was changed to a 30% saturated sodium borate solution and incubated overnight. Subsequently, specimens were immersed in 1% trypsin/30% saturated sodium borate and incubated at room temperature overnight. Following a milli-Q water wash, specimens were transferred into a 1% KOH solution containing 0.005% Alzarin Red S. The next day, specimens were washed with milli-Q water and subjected to glycerol substitution. Three replicates for each genotype were investigated. After skeletal staining, girdles and pectoral fins were manually separated from the body. Girdles and fins were stained with 0.5% PMA (phosphomolybdic acid) in milli-Q water for 16 h and followed by washes with milli-Q water. Specimens were placed into 1.5 ml Eppendorf tubes with water and kept overnight to settle in the tubes. The next day, tubes containing specimens were set and scanned with the UChicago PaleoCT scanner (GE Phoenix v/tome/x 240kv/180kv scanner) (http://luo-lab.uchicago.edu/paleoCT.html), at 50 kVp, 160 μA, no filtration, 5×-averaging, exposure timing of 500 ms per image, and a resolution of 8 μm per slice (512 μm3 per voxel). Scanned images were analysed and segmented using Amira 3D Software 6.0 (FEI). Three replicates for single and double homozygotes and five for mosaic triple knockout were investigated.

Remijsen Q.,Catholic University of Leuven | Remijsen Q.,Molecular Signaling and Cell Death Unit | Remijsen Q.,Ghent University | Berghe T.V.,Molecular Signaling and Cell Death Unit | And 12 more authors.
Cell Research | Year: 2011

Neutrophil extracellular traps (NETs) are extracellular chromatin structures that can trap and degrade microbes. They arise from neutrophils that have activated a cell death program called NET cell death, or NETosis. Activation of NETosis has been shown to involve NADPH oxidase activity, disintegration of the nuclear envelope and most granule membranes, decondensation of nuclear chromatin and formation of NETs. We report that in phorbol myristate acetate (PMA)-stimulated neutrophils, intracellular chromatin decondensation and NET formation follow autophagy and superoxide production, both of which are required to mediate PMA-induced NETosis and occur independently of each other. Neutrophils from patients with chronic granulomatous disease, which lack NADPH oxidase activity, still exhibit PMA-induced autophagy. Conversely, PMA-induced NADPH oxidase activity is not affected by pharmacological inhibition of autophagy. Interestingly, inhibition of either autophagy or NADPH oxidase prevents intracellular chromatin decondensation, which is essential for NETosis and NET formation, and results in cell death characterized by hallmarks of apoptosis. These results indicate that apoptosis might function as a backup program for NETosis when autophagy or NADPH oxidase activity is prevented. © 2011 IBCB, SIBS, CAS All rights reserved.

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