No abstract

Assembly of the β-barrel outer membrane proteins (OMPs) is an essential cellular process in Gram negative bacteria and in the mitochondria and chloroplasts of eukaryotes—two organelles of bacterial origin. Central to this process is the conserved β-barrel OMP that belongs to the Omp85 superfamily. In Escherichia coli, BamA is the core β-barrel OMP, and together with four outer membrane lipoproteins, BamBCDE, constitute the β-barrel assembly machine (BAM). In this paper, we investigated the roles of BamD, an essential lipoprotein, and BamB in BamA biogenesis. Depletion of BamD caused impairment in BamA biogenesis and cessation of cell growth. These defects of BamD depletion were partly reversed by single amino acid substitutions mapping within the β-barrel domain of BamA. However, in the absence of BamB, the positive effects of the β-barrel substitutions on BamA biogenesis under BamD depletion conditions were nullified. By employing a BamA protein bearing one such substitution, F494L, it was demonstrated that the mutant BamA protein could not only assemble without BamD, but it could also facilitate the assembly of wild-type BamA expressed in trans. Based on these data, we propose a model in which the Bam lipoproteins, which are localized to the outer membrane by the BAM-independent Lol pathway, aid in the creation of new BAM complexes by serving as outer membrane receptors and folding factors for nascent BamA molecules. The newly assembled BAM holocomplex then catalyzes the assembly of substrate OMPs and BamA. These in vivo findings are corroborated by recently published in vitro data.

Spino-Cerebellar-Ataxia type 38 (SCA38) is caused by missense mutations in the very long chain fatty acid elongase 5 gene, ELOVL5. The main clinical findings in this disease are ataxia, hyposmia and cerebellar atrophy. Mice in which Elovl5 has been knocked out represent a model of the loss of function hypothesis of SCA38. In agreement with this hypothesis, Elovl5 knock out mice reproduced the main symptoms of patients, motor deficits at the beam balance test and hyposmia. The cerebellar cortex of Elovl5 knock out mice showed a reduction of thickness of the molecular layer, already detectable at 6 months of age, confirmed at 12 and 18 months. The total perimeter length of the Purkinje cell (PC) layer was also reduced in Elovl5 knock out mice. Since Elovl5 transcripts are expressed by PCs, whose dendrites are a major component of the molecular layer, we hypothesized that an alteration of their dendrites might be responsible for the reduced thickness of this layer. Reconstruction of the dendritic tree of biocytin-filled PCs, followed by Sholl analysis, showed that the distribution of distal dendrites was significantly reduced in Elovl5 knock out mice. Dendritic spine density was conserved. These results suggest that Elovl5 knock out mice recapitulate SCA38 symptoms and that their cerebellar atrophy is due, at least in part, to a reduced extension of PC dendritic arborization.

Abstract Polymorphisms associated with BIN1 (bridging integrator 1) confer the second greatest risk for developing late-onset Alzheimer’s disease. The biological consequences of this genetic variation are not fully understood; however, BIN1 is a binding partner for tau. Tau is normally a highly soluble cytoplasmic protein, but in Alzheimer’s disease, tau is abnormally phosphorylated and accumulates at synapses to exert synaptotoxicity. The purpose of this study was to determine whether alterations in BIN1 and tau in Alzheimer’s disease promote the damaging redistribution of tau to synapses, as a mechanism by which BIN1 polymorphisms may increase the risk of developing Alzheimer’s disease. We show that BIN1 is lost from the cytoplasmic fraction of Alzheimer’s disease cortex, and this is accompanied by the progressive mislocalization of phosphorylated tau to synapses. We confirmed proline 216 in tau as critical for tau interaction with the BIN1-SH3 domain and showed that the phosphorylation of tau disrupts this binding, suggesting that tau phosphorylation in Alzheimer’s disease disrupts tau–BIN1 associations. Moreover, we show that BIN1 knockdown in rat primary neurons to mimic BIN1 loss in Alzheimer’s disease brain causes the damaging accumulation of phosphorylated tau at synapses and alterations in dendritic spine morphology. We also observed reduced release of tau from neurons upon BIN1 silencing, suggesting that BIN1 loss disrupts the function of extracellular tau. Together, these data indicate that polymorphisms associated with BIN1 that reduce BIN1 protein levels in the brain likely act synergistically with increased tau phosphorylation to increase the risk of Alzheimer’s disease by disrupting cytoplasmic tau–BIN1 interactions, promoting the damaging mis-sorting of phosphorylated tau to synapses to alter synapse structure and reducing the release of physiological forms of tau to disrupt tau function.

Human induced pluripotent stem cells (iPSCs) are a powerful tool for studying development and disease. However, different iPSC lines show considerable phenotypic variation. The lack of common well-characterized cell lines that are used widely frustrates efforts to integrate data across research groups or replicate key findings. Inspired by model organism communities who addressed this issue by establishing a limited number of widely accepted strains, we characterised candidate iPSC lines in unprecedented detail to select a well-performing line to underpin collaborative studies. Specifically, we characterised the morphology, growth rates, and single-cell transcriptomes of iPSC lines in the pluripotent state and assessed their genomic integrity using karyotyping, DNA microarrays, whole genome sequencing, and functional assays for p53 activity. We further tested their ability to be edited by CRISPR/Cas9 and used single-cell RNA sequencing to compare the efficiency with which they could be differentiated into multiple lineages. We found that there was significant variability in the performance of lines across the tested assays that enabled the rational selection of a lead line, KOLF2.1J, which is a gene-corrected derivative of a publicly available line from the Human Induced Pluripotent Stem Cells Initiative (HipSci) resource. We are now using this line in an initiative from the NIH Center for Alzheimer’s and Related Dementias to derive hundreds of gene-edited and functionalized sub-clones to be distributed widely throughout the research community along with associated datasets, with the aim of promoting the standardisation required for large-scale collaborative science in the stem cell field.SummaryThe authors of this collaborative science study describe a deep characterization of widely available induced pluripotent stem cell (iPSC) lines to rationally select a line that performs well in multiple experimental approaches. Analysis of transcriptional patterns in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and differentiation efficiency towards multiple lineages identified KOLF2.1J as a well-performing cell line. The widespread distribution and use of this line makes it an attractive cell line for comparative and collaborative efforts in the stem cell field.HighlightsDeep genotyping and phenotyping reveals KOLF2.1J as well-performing cell line that is readily distributed and could serve as common reference lineDespite rare copy-neutral loss of heterozygosity (CN-LOH) events, iPSC lines retain genomic fidelity after CRISPR/Cas9-based gene editingOur multifactorial pipeline serves as a blueprint for future efforts to identify other lead iPSC linesGraphical abstract