Chromosome Translocation Inflates Bacillus Forespores and Impacts Cellular Morphology.

The means by which the physicochemical properties of different cellular components together determine bacterial cell shape remain poorly understood. Here, we investigate a programmed cell-shape change during Bacillus subtilis sporulation, when a rod-shaped vegetative cell is transformed to an ovoid spore. Asymmetric cell division generates a bigger mother cell and a smaller, hemispherical forespore. The septum traps the forespore chromosome, which is translocated to the forespore by SpoIIIE. Simultaneously, forespore size increases as it is reshaped into an ovoid. Using genetics, timelapse microscopy, cryo-electron tomography, and mathematical modeling, we demonstrate that forespore growth relies on membrane synthesis and SpoIIIE-mediated chromosome translocation, but not on peptidoglycan or protein synthesis. Our data suggest that the hydrated nucleoid swells and inflates the forespore, displacing ribosomes to the cell periphery, stretching septal peptidoglycan, and reshaping the forespore. Our results illustrate how simple biophysical interactions between core cellular components contribute to cellular morphology.

Shaping an Endospore: Architectural Transformations During Bacillus subtilis Sporulation.

Endospore formation in Bacillus subtilis provides an ideal model system for studying development in bacteria. Sporulation studies have contributed a wealth of information about the mechanisms of cell-specific gene expression, chromosome dynamics, protein localization, and membrane remodeling, while helping to dispel the early view that bacteria lack internal organization and interesting cell biological phenomena. In this review, we focus on the architectural transformations that lead to a profound reorganization of the cellular landscape during sporulation, from two cells that lie side by side to the endospore, the unique cell within a cell structure that is a hallmark of sporulation in B. subtilis and other spore-forming Firmicutes. We discuss new insights into the mechanisms that drive morphogenesis, with special emphasis on polar septation, chromosome translocation, and the phagocytosis-like process of engulfment, and also the key experimental advances that have proven valuable in revealing the inner workings of bacterial cells.

Stable Isotope Labeling of Amino Acids in Flies (SILAF) Reveals Differential Phosphorylation of Mitochondrial Proteins Upon Loss of OXPHOS Subunits.

Drosophila melanogaster has been a workhorse of genetics and cell biology for more than a century. However, proteomic-based methods have been limited due to the complexity and dynamic range of the fly proteome and the lack of efficient labeling methods. Here, we advanced a chemically defined food source into direct stable-isotope labeling of amino acids in flies (SILAF). It allows for the rapid and cost-efficient generation of a large number of larvae or flies, with full incorporation of lysine-[13C6] after six labeling days. SILAF followed by fractionation and enrichment gave proteomic insights at a depth of 7196 proteins and 8451 phosphorylation sites, which substantiated metabolic regulation on enzymatic level. We applied SILAF to quantify the mitochondrial phosphoproteome of an early-stage leucine-rich PPR motif-containing protein (LRPPRC)-knockdown fly model of mitochondrial disease that almost exclusively affects protein levels of the oxidative phosphorylation (OXPHOS) system. While the mitochondrial compartment was hypo-phosphorylated, two conserved phosphosites on OXPHOS subunits NDUFB10 and NDUFA4 were significantly upregulated upon impaired OXPHOS function. The ease and versatility of the method actuate the fruit fly as an appealing model in proteomic and posttranslational modification studies, and it enlarges potential metabolic applications based on heavy amino acid diets.

Thioredoxin-80 protects against amyloid-beta pathology through autophagic-lysosomal pathway regulation.

Aggregation and accumulation of amyloid beta (Aß) are believed to play a key role in the pathogenesis of Alzheimer's disease (AD). We previously reported that Thioredoxin-80 (Trx80), a truncated form of Thioredoxin-1, prevents the toxic effects of Aß and inhibits its aggregation in vitro. Trx80 levels were found to be dramatically reduced both in the human brain and cerebrospinal fluid of AD patients. In this study, we investigated the effect of Trx80 expression using in vivo and in vitro models of Aß pathology. We developed Drosophila melanogaster models overexpressing either human Trx80, human Aß42, or both Aß42/Trx80 in the central nervous system. We found that Trx80 expression prevents Aß42 accumulation in the brain and rescues the reduction in life span and locomotor impairments seen in Aß42 expressing flies. Also, we show that Trx80 induces autophagosome formation and reverses the inhibition of Atg4b-Atg8a/b autophagosome formation pathway caused by Aß42. These effects were also confirmed in human neuroblastoma cells. These results give insight into Trx80 function in vivo, suggesting its role in the autophagosome biogenesis and thus in Aß42 degradation. Our findings put Trx80 on the spotlight as an endogenous agent against Aß42-induced toxicity in the brain suggesting that strategies to enhance Trx80 levels in neurons could potentially be beneficial against AD pathology in humans.

Defects of mitochondrial RNA turnover lead to the accumulation of double-stranded RNA in vivo.

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology.

Severe congenital lactic acidosis and hypertrophic cardiomyopathy caused by an intronic variant in NDUFB7.

Mutations in structural subunits and assembly factors of complex I of the oxidative phosphorylation system constitute the most common cause of mitochondrial respiratory chain defects. Such mutations can present a wide range of clinical manifestations, varying from mild deficiencies to severe, lethal disorders. We describe a patient presenting intrauterine growth restriction and anemia, which displayed postpartum hypertrophic cardiomyopathy, lactic acidosis, encephalopathy, and a severe complex I defect with fatal outcome. Whole genome sequencing revealed an intronic biallelic mutation in the NDUFB7 gene (c.113-10C>G) and splicing pattern alterations in NDUFB7 messenger RNA were confirmed by RNA Sequencing. The detected variant resulted in a significant reduction of the NDUFB7 protein and reduced complex I activity. Complementation studies with expression of wild-type NDUFB7 in patient fibroblasts normalized complex I function. Here we report a case with a primary complex I defect due to a homozygous mutation in an intron region of the NDUFB7 gene.

Metabolic regulation of neurodifferentiation in the adult brain.

Understanding the mechanisms behind neurodifferentiation in adults will be an important milestone in our quest to identify treatment strategies for cognitive disorders observed during our natural ageing or disease. It is now clear that the maturation of neural stem cells to neurones, fully integrated into neuronal circuits requires a complete remodelling of cellular metabolism, including switching the cellular energy source. Mitochondria are central for this transition and are increasingly seen as the regulatory hub in defining neural stem cell fate and neurodevelopment. This review explores our current knowledge of metabolism during adult neurodifferentiation.

Spatiotemporally regulated proteolysis to dissect the role of vegetative proteins during Bacillus subtilis sporulation: cell-specific requirement of σH and σA.

Sporulation in Bacillus subtilis is a paradigm of bacterial development, which involves the interaction between a larger mother cell and a smaller forespore. The mother cell and the forespore activate different genetic programs, leading to the production of sporulation-specific proteins. A critical gap in our understanding of sporulation is how vegetative proteins, made before sporulation initiation, contribute to spore formation. Here we present a system, spatiotemporally regulated proteolysis (STRP), which enables the rapid, developmentally regulated degradation of target proteins, thereby providing a suitable method to dissect the cell- and developmental stage-specific role of vegetative proteins. STRP has been used to dissect the role of two major vegetative sigma factors, σH and σA , during sporulation. The results suggest that σH is only required in predivisional cells, where it is essential for sporulation initiation, but that it is dispensable during subsequent steps of spore formation. However, evidence has been provided that σA plays different roles in the mother cell, where it replenishes housekeeping functions, and in the forespore, where it plays an unexpected role in promoting spore germination and outgrowth. Altogether, the results demonstrate that STRP has the potential to provide a comprehensive molecular dissection of every stage of sporulation, germination and outgrowth.

Absence of the Autophagy Adaptor SQSTM1/p62 Causes Childhood-Onset Neurodegeneration with Ataxia, Dystonia, and Gaze Palsy.

SQSTM1 (sequestosome 1; also known as p62) encodes a multidomain scaffolding protein involved in various key cellular processes, including the removal of damaged mitochondria by its function as a selective autophagy receptor. Heterozygous variants in SQSTM1 have been associated with Paget disease of the bone and might contribute to neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Using exome sequencing, we identified three different biallelic loss-of-function variants in SQSTM1 in nine affected individuals from four families with a childhood- or adolescence-onset neurodegenerative disorder characterized by gait abnormalities, ataxia, dysarthria, dystonia, vertical gaze palsy, and cognitive decline. We confirmed absence of the SQSTM1/p62 protein in affected individuals' fibroblasts and found evidence of a defect in the early response to mitochondrial depolarization and autophagosome formation. Our findings expand the SQSTM1-associated phenotypic spectrum and lend further support to the concept of disturbed selective autophagy pathways in neurodegenerative diseases.

Exposure Assessment For Air-To-Skin Uptake of Semivolatile Organic Compounds (SVOCs) Indoors.

Semivolatile organic compounds (SVOCs) are ubiquitous in the indoor environment and a priority for exposure assessment because of the environmental health concerns that they pose. Direct air-to-skin dermal uptake has been shown to be comparable to the inhalation intake for compounds with certain chemical properties. In this study, we aim to further understand the transport of these types of chemicals through the skin, specifically through the stratum corneum (SC). Our assessment is based on collecting three sequential forehead skin wipes, each hypothesized to remove pollutants from successively deeper skin layers, and using these wipe analyses to determine the skin concentration profiles. The removal of SVOCs with repeated wipes reveals the concentration profiles with depth and provides a way to characterize penetration efficiency and potential transfer to blood circulation. We used a diffusion model applied to surface skin to simulate concentration profiles of SVOCs and compared them with the measured values. We found that two phthalates, dimethyl and diethyl phthalates, penetrate deeper into skin with similar exposure compared to other phthalates and targeted SVOCs, an observation supported by the model results as well. We also report the presence of statistically significant declining patterns with skin depth for most SVOCs, indicating that their diffusion through the SC is relevant and eventually can reach the blood vessels in the vascularized dermis. Finally, using a nontarget approach, we identified skin oxidation products, linked to respiratory irritation symptoms, formed from the reaction between ozone and squalene.

Mitochondrial Polyadenylation Is a One-Step Process Required for mRNA Integrity and tRNA Maturation.

Polyadenylation has well characterised roles in RNA turnover and translation in a variety of biological systems. While polyadenylation on mitochondrial transcripts has been suggested to be a two-step process required to complete translational stop codons, its involvement in mitochondrial RNA turnover is less well understood. We studied knockdown and knockout models of the mitochondrial poly(A) polymerase (MTPAP) in Drosophila melanogaster and demonstrate that polyadenylation of mitochondrial mRNAs is exclusively performed by MTPAP. Further, our results show that mitochondrial polyadenylation does not regulate mRNA stability but protects the 3' terminal integrity, and that despite a lack of functioning 3' ends, these trimmed transcripts are translated, suggesting that polyadenylation is not required for mitochondrial translation. Additionally, loss of MTPAP leads to reduced steady-state levels and disturbed maturation of tRNACys, indicating that polyadenylation in mitochondria might be important for the stability and maturation of specific tRNAs.

Phosphorylation of spore coat proteins by a family of atypical protein kinases.

The modification of proteins by phosphorylation occurs in all life forms and is catalyzed by a large superfamily of enzymes known as protein kinases. We recently discovered a family of secretory pathway kinases that phosphorylate extracellular proteins. One member, family with sequence similarity 20C (Fam20C), is the physiological Golgi casein kinase. While examining distantly related protein sequences, we observed low levels of identity between the spore coat protein H (CotH), and the Fam20C-related secretory pathway kinases. CotH is a component of the spore in many bacterial and eukaryotic species, and is required for efficient germination of spores in Bacillus subtilis; however, the mechanism by which CotH affects germination is unclear. Here, we show that CotH is a protein kinase. The crystal structure of CotH reveals an atypical protein kinase-like fold with a unique mode of ATP binding. Examination of the genes neighboring cotH in B. subtilis led us to identify two spore coat proteins, CotB and CotG, as CotH substrates. Furthermore, we show that CotH-dependent phosphorylation of CotB and CotG is required for the efficient germination of B. subtilis spores. Collectively, our results define a family of atypical protein kinases and reveal an unexpected role for protein phosphorylation in spore biology.

Multirobot Heterogeneous Control Considering Secondary Objectives.

Cooperative robotics has considered tasks that are executed frequently, maintaining the shape and orientation of robotic systems when they fulfill a common objective, without taking advantage of the redundancy that the robotic group could present. This paper presents a proposal for controlling a group of terrestrial robots with heterogeneous characteristics, considering primary and secondary tasks thus that the group complies with the following of a path while modifying its shape and orientation at any time. The development of the proposal is achieved through the use of controllers based on linear algebra, propounding a low computational cost and high scalability algorithm. Likewise, the stability of the controller is analyzed to know the required features that have to be met by the control constants, that is, the correct values. Finally, experimental results are shown with different configurations and heterogeneous robots, where the graphics corroborate the expected operation of the proposal.

Vmp1 regulates PtdIns3P signaling during autophagosome formation in Dictyostelium discoideum.

Generation and turnover of phosphatidylinositol 3-phosphate (PtdIns3P) signaling is essential for autophagosome formation and other membrane traffic processes. In both Dictyostelium discoideum and mammalian cells, autophagosomes are formed from specialized regions of the endoplasmic reticulum (ER), called omegasomes, which are enriched in the signaling lipid PtdIns3P. Vacuole membrane protein 1 (Vmp1) is a multispanning membrane protein localized at the ER that is required for autophagosome formation. There are conflicting reports in the literature as to whether Vmp1 is strictly required or not for autophagy-related PtdIns3P signaling and its hierarchical relationship with Atg1 and PI3K. We have now addressed these questions in the Dictyostelium model. We show that Dictyostelium cells lacking Vmp1 have elevated and aberrant PtdIns3P signaling on the ER, resulting in an increased and persistent recruitment of Atg18 and other autophagic proteins. This indicates that Vmp1 is not strictly essential for the generation of PtdIns3P signaling but rather suggests a role in the correct turnover or modulation of this signaling. Of interest, these PtdIns3P-enriched regions of the ER surround ubiquitinated protein aggregates but are unable to form functional autophagosomes. vmp1 null cells also have additional defects in macropinocytosis and growth, which are not shared by other autophagy mutants. Remarkably, we show that these defects and also the aberrant PtdIns3P distribution are largely suppressed by the concomitant loss of Atg1, indicating that aberrant autophagic signaling on the ER inhibits macropinocytosis. These results suggest that Atg1 functions upstream of Vmp1 in this signaling pathway and demonstrates a previously unappreciated link between abnormal autophagy signaling and macropinocytosis.

Whole-exome DNA sequence analysis of Brca2- and Trp53-deficient mouse mammary gland tumours.

Germline mutations in the tumour suppressor BRCA2 predispose to breast, ovarian and a number of other human cancers. Brca2-deficient mouse models are used for preclinical studies but the pattern of genomic alterations in these tumours has not yet been described in detail. We have performed whole-exome DNA sequencing analysis of mouse mammary tumours from Blg-Cre Brca2(f/f) Trp53(f/f) animals, a model of BRCA2-deficient human cancer. We also used the sequencing data to estimate DNA copy number alterations in these tumours and identified a recurrent copy number gain in Met, which has been found amplified in other mouse mammary cancer models. Through a comparative genomic analysis, we identified several mouse Blg-Cre Brca2(f/f) Trp53(f/f) mammary tumour somatic mutations in genes that are also mutated in human cancer, but few of these genes have been found frequently mutated in human breast cancer. A more detailed analysis of these somatic mutations revealed a set of genes that are mutated in human BRCA2 mutant breast and ovarian tumours and that are also mutated in mouse Brca2-null, Trp53-null mammary tumours. Finally, a DNA deletion surrounded by microhomology signature found in human BRCA1/2-deficient cancers was not common in the genome of these mouse tumours. Although a useful model, there are some differences in the genomic landscape of tumours arising in Blg-Cre Brca2(f/f) Trp53(f/f) mice compared to human BRCA-mutated breast cancers. Therefore, this needs to be taken into account in the use of this model.

A eukaryotic-like 3' untranslated region in Salmonella enterica hilD mRNA.

Long 3' untranslated regions (3'UTRs) are common in eukaryotic mRNAs. In contrast, long 3'UTRs are rare in bacteria, and have not been characterized in detail. We describe a 3'UTR of 310 nucleotides in hilD mRNA, a transcript that encodes a transcriptional activator of Salmonella enterica pathogenicity island 1 (SPI-1). Deletion of the hilD 3'UTR increases the hilD mRNA level, suggesting that the hilD 3'UTR may play a role in hilD mRNA turnover. Cloning of the hilD 3'UTR downstream of the green fluorescent protein (gfp) gene decreases green fluorescent protein (GFP) activity in both Escherichia coli and S. enterica, indicating that the hilD 3'UTR can act as an independent module. S. enterica mutants lacking either ribonuclease E or polynucleotide phosphorylase contain similar amounts of hilD and hilD Δ3'UTR mRNAs, suggesting that the hilD 3'UTR is a target for hilD mRNA degradation by the degradosome. The hilD 3'UTR is also necessary for modulation of hilD and SPI-1 expression by the RNA chaperone Hfq. Overexpression of SPI-1 in the absence of the hilD 3'UTR retards Salmonella growth and causes uncontrolled invasion of epithelial cells. Based on these observations, we propose that the S. enterica hilD 3'UTR is a cis-acting element that contributes to cellular homeostasis by promoting hilD mRNA turnover.

Bistable forespore engulfment in Bacillus subtilis by a zipper mechanism in absence of the cell wall.

To survive starvation, the bacterium Bacillus subtilis forms durable spores. The initial step of sporulation is asymmetric cell division, leading to a large mother-cell and a small forespore compartment. After division is completed and the dividing septum is thinned, the mother cell engulfs the forespore in a slow process based on cell-wall degradation and synthesis. However, recently a new cell-wall independent mechanism was shown to significantly contribute, which can even lead to fast engulfment in [Formula: see text] 60 [Formula: see text] of the cases when the cell wall is completely removed. In this backup mechanism, strong ligand-receptor binding between mother-cell protein SpoIIIAH and forespore-protein SpoIIQ leads to zipper-like engulfment, but quantitative understanding is missing. In our work, we combined fluorescence image analysis and stochastic Langevin simulations of the fluctuating membrane to investigate the origin of fast bistable engulfment in absence of the cell wall. Our cell morphologies compare favorably with experimental time-lapse microscopy, with engulfment sensitive to the number of SpoIIQ-SpoIIIAH bonds in a threshold-like manner. By systematic exploration of model parameters, we predict regions of osmotic pressure and membrane-surface tension that produce successful engulfment. Indeed, decreasing the medium osmolarity in experiments prevents engulfment in line with our predictions. Forespore engulfment may thus not only be an ideal model system to study decision-making in single cells, but its biophysical principles are likely applicable to engulfment in other cell types, e.g. during phagocytosis in eukaryotes.

Sample preparation strategies for improving the identification of membrane proteins by mass spectrometry.

Despite enormous advances in the mass spectrometry and proteomics fields during the last two decades, the analysis of membrane proteins still remains a challenge for the proteomic community. Membrane proteins play a wide number of key roles in several cellular events, making them relevant target molecules to study in a significant variety of investigations (e.g., cellular signaling, immune surveillance, drug targets, vaccine candidates, etc.). Here, we critically review the several attempts that have been carried out on the different steps of the sample preparation procedure to improve and modify existing conventional proteomic strategies in order to make them suitable for the study of membrane proteins. We also revise novel techniques that have been designed to tackle the difficult but relevant task of identifying and characterizing membrane proteins.

Peptidoglycan transformations during Bacillus subtilis sporulation.

While vegetative Bacillus subtilis cells and mature spores are both surrounded by a thick layer of peptidoglycan (PG, a polymer of glycan strands cross-linked by peptide bridges), it has remained unclear whether PG surrounds prespores during engulfment. To clarify this issue, we generated a slender ΔponA mutant that enabled high-resolution electron cryotomographic imaging. Three-dimensional reconstructions of whole cells in near-native states revealed a thin PG-like layer extending from the lateral cell wall around the prespore throughout engulfment. Cryotomography of purified sacculi and fluorescent labelling of PG in live cells confirmed that PG surrounds the prespore. The presence of PG throughout engulfment suggests new roles for PG in sporulation, including a new model for how PG synthesis might drive engulfment, and obviates the need to synthesize a PG layer de novo during cortex formation. In addition, it reveals that B. subtilis can synthesize thin, Gram-negative-like PG layers as well as its thick, archetypal Gram-positive cell wall. The continuous transformations from thick to thin and back to thick during sporulation suggest that both forms of PG have the same basic architecture (circumferential). Endopeptidase activity may be the main switch that governs whether a thin or a thick PG layer is assembled.