Biology subjects

Johansson, M.

Publications and source records attributed to Johansson, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

A complex between IF2 and NusA links transcription and translation

The main function of translation initiation factors is to assist ribosomes in selecting the correct reading frame on an mRNA. This process has been extensively studied with the help of reconstituted in vitro systems, but the dynamics in living cells have not been characterized. In this study, we performed single-molecule tracking of the bacterial initiation factors IF2, IF3, as well as the initiator fMet-tRNAfMet directly in growing Escherichia coli cells. Our results reveal the kinetics of factor association with the ribosome and, among other things, highlight the respective antagonistic roles of IF2 and IF3 in the process. Importantly, our comparisons of in vivo binding kinetics of the two naturally occurring isoforms of IF2 reveal that the longer IF2 isoform directly interacts with the transcriptional factor NusA, a finding further corroborated by pull-down and cross-linking experiments. We further show that cells with compromised binding between IF2 and NusA display slow adaptation to new growth conditions. Taken together, these results suggest a novel coupling mechanism between transcription and translation initiation, important for the rewiring of the transcriptome during a shift of growth media.

molecular biology↗

bioRxiv2024

Imaging single particle profiler to study nanoscale bioparticles using conventional confocal microscopy

Single particle profiler is a unique methodology to study nanoscale bioparticles such as liposomes, lipid nanoparticles, extracellular vesicles and lipoproteins in single particle and high throughput manner. The original version requires the single photon counting modules for data acquisition. Here, we present imaging-based SPP (iSPP) which can be performed by imaging a spot over time in common imaging mode with photomultiplier tubes. We also provide a user-friendly software with graphical user interface to facilitate the application of this technique. We demonstrate that iSPP can be used to decipher lipid-protein interactions, membrane modifications by drugs and the heterogeneity of extracellular vesicles isolated from cells lines and urine of human donors.

biophysics↗

bioRxiv2024

Anabolic sensitivity in healthy, lean, older men is associated with higher expression of amino acid sensors and mTORC1 activators

BackgroundSarcopenia is thought to be underlined by age-associated anabolic resistance and dysregulation of intracellular signalling pathways. However, it is unclear whether these phenomena are driven by ageing per se or other confounding factors. MethodsLean and healthy young (n=10, 22 {+/-} 3 yrs, BMI; 23.4 {+/-} 0.8 kg/m2) and old men (n=10, 70 {+/-} 3 yrs, BMI; 22.7 {+/-} 1.3 kg/m2) performed unilateral resistance exercise followed by intake of essential amino acids (EAA). Muscle biopsies were collected from the rested and the exercised leg before, immediately after, as well as 60 and 180 minutes after EAA intake. Muscle samples were analyzed for amino acid concentrations, muscle protein synthesis (MPS) and associated anabolic signaling. ResultsFollowing exercise, peak plasma levels of EAA and leucine were similar between groups, but the area under the curve was [~]11% and [~]28% lower in Young (p<0.01). Absolute levels of muscle EAA and leucine peaked 60 min after exercise, with [~]15 and [~]21 % higher concentrations in the exercising leg (p<0.01) but with no difference between groups. MPS increased in both the resting ([~] 0.035%{middle dot}h-1 to 0.056%{middle dot}h-1, p<0.05) and exercising leg ([~] 0.035%{middle dot}h-1 to 0.083%{middle dot}h-1, p<0.05) with no difference between groups. Phosphorylation of S6K1Thr389 increased to a similar extent in the exercising leg in both groups but was 2.8-fold higher in the resting leg of Old at the 60 min timepoint (p<0.001). Phosphorylation of 4E-BP1Ser65 increased following EAA intake and exercise, but differences between legs were statistically different only at 180 min (p<0.001). However, phosphorylation of this site was on average 78% greater across all timepoints in Old (p<0.01). Phosphorylation of eEF2Thr56 was reduced ([~] 66 and 39%) in the exercising leg at both timepoints after EAA intake and exercise, with no group differences (p<0.05). However, phosphorylation at this site was reduced by [~] 27% also in the resting leg at 60 min, an effect that was only seen in Old (p<0.01). Total levels of Rheb ([~] 45%), LAT1 ([~] 31%) and Rag B ([~] 31%) were higher in Old (p<0.001). ConclusionLean and healthy old men do not manifest AR as evidenced by potent increases in MPS and mTORC1 signalling following EAA intake and exercise. Maintained anabolic sensitivity with age appears to be a function of a compensatory increase in basal levels of proteins involved in anabolic signalling. Therefore, our results suggest that age per se does not appear to cause AR in human skeletal muscle.

physiology↗

bioRxiv2024

A microfluidic platform for in situ studies of bacteria electroporation

Electroporation of dye-labelled bio-molecules has proven to be a valuable alternative to fluorescent protein fusion for single-molecule tracking in living cells. However, control over cell viability, electroporation efficiency and environment conditions before, during and after electroporation is difficult to achieve in bulk experiments. Here, we present a microfluidic platform capable of single-cell electroporation with in situ microscopy and demonstrate delivery of DNA into bacteria. Via real time observation of the electroporation process, we find that the effect of electrophoresis plays an important role when performing electroporation in a miniaturized platform and show that its undesired action can be balanced by using bipolar electrical pulses. We suggest that a low temperature of the sample during electroporation is important for cell viability due to temperature-dependant viscoelastic properties of the cell membrane. We further found that the presence of low conductive liquid between cells and the electrodes leads to a voltage divider effect which strongly influences the success of on-chip electroporation. Finally, we conclude that electroporation is intrinsically a highly stochastic process that is difficult to fully control via external parameters and envision that the microfluidic system presented here, capable of single-cell read-out, can be used for further fundamental studies to increase our understanding of the electroporation process.

bioengineering↗

bioRxiv2024

Dynamic binding of the bacterial chaperone Trigger factor to translating ribosomes

The bacterial chaperone Trigger factor (TF) binds to ribosome-nascent chain complexes (RNCs) and co-translationally aids the folding of proteins in bacteria. Decades of studies have given a broad, but often conflicting, description of the substrate specificity of TF, its RNC-binding dynamics, and competition with other RNC-binding factors, such as the Signal Recognition Particle (SRP). Previous RNC-binding kinetics experiments were conducted on stalled RNCs in reconstituted systems, and consequently, may not represent the interaction of TF with ribosomes translating mRNA in the cytoplasm of the cell. Here, we used single-particle tracking (SPT) to measure TF binding to actively translating ribosomes inside living Escherichia coli. In cells, TF displays two distinct binding modes -- long (ca 1 s) target-specific RNC binding, and shorter (ca 50 ms) sampling of non-target RNCs. RNC binding events are interrupted only by transient excursions to a freely diffusing state (ca 40 ms). We also show that TF competes with SRP for RNC binding in vivo, and in doing so, tunes the binding selectivity of SRP.

biophysics↗

bioRxiv2022

Real-time single-molecule 3D tracking in E. coli based on cross-entropy minimization

Sub-ms 3D tracking of individual molecules in living cells is an important goal for microscopy since it will enable measurements at the scale of diffusion limited macromolecular interactions. Here, we present a 3D tracking principle based on the true excitation point spread function and cross-entropy minimization for position localization of moving fluorescent reporters that approaches the relevant regime. When tested on beads moved on a stage, we reached 67nm lateral and 109nm axial precision with a time resolution of 0.84 ms at a photon count rate of 60kHz, coming close to the theoretical and simulated predictions. A critical step in the implementation was a new method for microsecond 3D PSF positioning that combines 3D holographic beam shaping and electro-optical deflection. For the analysis of tracking data, a new point estimator for diffusion was derived and evaluated by a detailed simulation of the 3D tracking principle applied to a fictive reaction-diffusion process in an E. coli-like geometry. Finally, we successfully applied these methods to track the Trigger Factor protein in living bacterial cells. Overall our results show that it is possible to reach sub-millisecond live-cell single-molecule tracking, but that it is still hard to resolve state transitions based on diffusivity at this time scale.

biophysics↗

bioRxiv2022

Episodic memory formation in naturalistic viewing

The brain systems of episodic memory and oculomotor control are tightly linked, suggesting a crucial role of eye movements in memory. But little is known about the neural mechanisms of memory formation across eye movements in unrestricted viewing behavior. Here, we leverage simultaneous eye tracking and EEG recording to examine episodic memory formation in free viewing. Participants memorized multi-element events while their EEG and eye movements were concurrently recorded. Each event comprised elements from three categories (face, object, place), with two exemplars from each category, in different locations on the screen. A subsequent associative memory test assessed participants memory for the between-category associations that specified each event. We used a deconvolution approach to overcome the problem of overlapping EEG responses to sequential saccades in free viewing. Brain activity was time-locked to the fixation onsets, and we examined EEG power in the theta and alpha frequency bands, the putative oscillatory correlates of episodic encoding mechanisms. Three modulations of fixation-related EEG predicted high subsequent memory performance: 1) theta increase at fixations after between-category gaze transitions, 2) theta and alpha increase at fixations after within-element gaze transitions, 3) alpha decrease at fixations after between-exemplar gaze transitions. Thus, event encoding with unrestricted viewing behavior was characterized by three neural mechanisms, manifested in fixation-locked theta and alpha EEG activity that rapidly turned on and off during the unfolding eye movement sequences. These three distinct neural mechanisms may be the essential building blocks that subserve the buildup of coherent episodic memories during unrestricted viewing behavior.

neuroscience↗

bioRxiv2021

Genotype-phenotype correlation analysis and therapeutic development using a patient stem cell-derived disease model of Wolfram syndrome

Wolfram syndrome is a rare genetic disorder largely caused by pathogenic variants in the WFS1 gene and manifested by diabetes mellitus, optic nerve atrophy, and progressive neurodegeneration. Recent genetic and clinical findings have revealed Wolfram syndrome as a spectrum disorder. Therefore, a genotype-phenotype correlation analysis is needed for diagnosis and therapeutic development. Here, we focus on the WFS1 c.1672C>T, p.R558C variant which is highly prevalent in the Ashkenazi-Jewish population. Clinical investigation indicates that subjects carrying the homozygous WFS1 c.1672C>T, p.R558C variant show mild forms of Wolfram syndrome phenotypes. Expression of WFS1 p.R558C is more stable compared to the other known recessive pathogenic variants associated with Wolfram syndrome. Stem cell-derived islets (SC-islets) homozygous for WFS1 c.1672C>T variant recapitulates genotype-related Wolfram phenotypes, which are milder than those of SC-islets with compound heterozygous WFS1 c.1672C>T (p.R558C), c.2654C>T (p.P885L). Enhancing residual WFS1 function by a combination treatment of chemical chaperones, sodium 4-phenylbutyrate (4-PBA) and tauroursodeoxycholic acid (TUDCA), mitigates detrimental effects caused by the WFS1 c.1672C>T, p.R558C variant and restored SC-islet function. Thus, the WFS1 c.1672C>T, p.R558C variant causes a mild form of Wolfram syndrome phenotypes, which can be remitted with a combination treatment of chemical chaperones. We demonstrate that our patient stem cell-derived disease model provides a valuable platform for further genotype-phenotype analysis and therapeutic development for Wolfram syndrome. One sentence summaryDevelopment of personalized therapy for Wolfram syndrome using genetics and iPSC model.

genetics↗

bioRxiv2021

Role of the Topoisomerase IIα Chromatin Tether domain in Nucleosome Binding & Chromosome Segregation

Due to the intrinsic nature of DNA replication, replicated genomes retain catenated genomic loci that must be resolved to ensure faithful segregation of sister chromatids in mitosis. Type II DNA Topoisomerase (TopoII) decatenates the catenated genomic DNA through its unique Strand Passage Reaction (SPR). Loss of SPR activity results in anaphase chromosome bridges and formation of Polo-like Kinase Interacting Checkpoint Helicase (PICH)-coated ultra-fine DNA bridges (UFBs) whose timely resolution is required to prevent micronuclei formation. Vertebrates have two TopoII isoforms- TopoII and TopoII{beta}, that share a conserved catalytic core. However, the essential mitotic function of TopoII cannot be compensated by TopoII{beta}, due to differences in their catalytically inert C-terminal domains (CTDs). Using genome-edited human cells, we show that specific binding of TopoII to methylated histone, tri-methylated H3K27 (H3K27me3), via its Chromatin Tether (ChT) domain within the CTD contributes critically to avoid anaphase UFB formation. Reducing H3K27 methylation prior to mitosis increases UFBs, revealing a requirement for proper establishment of H3K27me3 after DNA replication to facilitate TopoII-ChT dependent UFB prevention. We propose that interaction of the TopoII-ChT with H3K27me3 is a key factor that ensures the complete resolution of catenated loci to permit faithful chromosome segregation in human cells. Summary StatementGenomic catenations originating from the DNA replication process must be resolved by DNA Topoisomerase II (TopoII) to permit sister chromatid disjunction. The results show that specific recognition of methylated histone containing chromatin by TopoII is critical for complete resolution of the genome.

cell biology↗

bioRxiv2021

A new pipeline for the normalization and pooling of metabolomics data

Pooling metabolomics data across studies is often desirable to increase the statistical power of the analysis. However, this can raise methodological challenges as several preanalytical and analytical factors could introduce differences in measured concentrations and variability between datasets. Specifically, different studies may use variable sample types (e.g., serum versus plasma) collected, treated and stored according to different protocols, and assayed in different laboratories using different instruments. To address these issues, a new pipeline was developed to normalize and pool metabolomics data through a set of sequential steps: (i) exclusions of the least informative observations and metabolites and removal of outliers; imputation of missing data; (ii) identification of the main sources of variability through PC-PR2 analysis; (iii) application of linear mixed models to remove unwanted variability, including samples originating study and batch, and preserve biological variations while accounting for potential differences in the residual variances across studies. This pipeline was applied to targeted metabolomics data acquired using Biocrates AbsoluteIDQ kits in eight case-control studies nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Comprehensive examination of metabolomics measurements indicated that the pipeline improved the comparability of data across the studies. Our pipeline can be adapted to normalize other molecular data, including biomarkers as well as proteomics data, and could be used for pooling molecular datasets, for example in international consortia, to limit biases introduced by inter-study variability. This versatility of the pipeline makes our work of potential interest to molecular epidemiologists.

bioinformatics↗

bioRxiv2020

Direct measurements of mRNA translation kinetics in living cells

Ribosome mediated mRNA translation is central to life as we know it. The cycle of translation has, however, not been characterized in a living cell. Here we have developed a live-cell ribosome-labeling method, which allows us to characterize the whole processes of finding an mRNA and translating it, using single-molecule tracking techniques. We find that more than 90% of both bacterial ribosomal subunits are engaged in elongation at any particular time, and that neither of the subunits, in general, continues translation from one open reading frame to the next on a poly-cistronic mRNA. Furthermore, we find that a variety of previously published orthogonal ribosomes, with altered anti-Shine-Dalgarno sequences, show significant binding to endogenous mRNAs, with the rate of translation initiation only modestly affected. Hence, our results suggest that other mRNA elements than the SD sequence play major roles in directing the ribosome to the correct translation start sites.

molecular biology↗

bioRxiv2020

Integration of polygenic risk scores with modifiable risk factors improves risk prediction: results from a pan-cancer analysis

Cancer risk is determined by a complex interplay of environmental and heritable factors. Polygenic risk scores (PRS) provide a personalized genetic susceptibility profile that may be leveraged for disease prediction. Using data from the UK Biobank (413,753 individuals; 22,755 incident cancer cases), we quantify the added predictive value of integrating cancer-specific PRS with family history and modifiable risk factors for 16 cancers. We show that incorporating PRS measurably improves prediction accuracy for most cancers, but the magnitude of this improvement varies substantially. We also demonstrate that stratifying on levels of PRS identifies significantly divergent 5-year risk trajectories after accounting for family history and modifiable risk factors. At the population level, the top 20% of the PRS distribution accounts for 4.0% to 30.3% of incident cancer cases, exceeding the impact of many lifestyle-related factors. In summary, this study illustrates the potential for improving cancer risk assessment by integrating genetic risk scores.

genetics↗

bioRxiv2020

Mechanism of DNA surface exploration and operator bypassing during target search

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific DNA1-5. Here we combine resonance energy transfer and fluorescence correlation measurements to characterize how individual lac repressor (LacI) molecules explore DNA during the 1D phase of target search. To track the rotation of sliding LacI molecules on the microsecond time scale during DNA surface search, we use real-time single-molecule confocal laser tracking combined with fluorescence correlation spectroscopy (SMCT-FCS). The fluorescence signal fluctuations are accurately described by rotation-coupled sliding, where LacI traverses ~40 base pairs (bp) per revolution. This distance substantially exceeds the 10.5-bp helical pitch of DNA, suggesting that the sliding protein frequently hops out of the DNA groove, which would result in frequent bypassing of target sequences. Indeed, we directly observe such bypassing by single-molecule fluorescence resonance energy transfer (smFRET). A combined analysis of the smFRET and SMCT-FCS data shows that LacI at most hops one to two grooves (10-20 bp) every 250 s. Overall, our data suggest a speed-accuracy trade-off during sliding; the weak nature of non-specific protein-DNA interactions underlies operator bypassing but also facilitates rapid sliding. We anticipate that our SMCT-FCS method to monitor rotational diffusion on the microsecond time scale while tracking individual molecules with millisecond time resolution will be applicable to the real-time investigation of many other biological interactions and effectively extends the accessible time regime by two orders of magnitude.

biophysics↗

bioRxiv2019

Immune-mediated genetic pathways resulting in pulmonary function impairment increase lung cancer susceptibility

Impaired lung function is often caused by cigarette smoking, making it challenging to disentangle its role in lung cancer susceptibility. Investigation of the shared genetic basis of these phenotypes in the UK Biobank and International Lung Cancer Consortium (29,266 cases, 56,450 controls) shows that lung cancer is genetically correlated with reduced forced expiratory volume in one second (FEV1: rg=0.098, p=2.3x10-8) and the ratio of FEV1 to forced vital capacity (FEV1/FVC: rg=0.137, p=2.0x10-12). Mendelian randomization analyses demonstrate that reduced FEV1 increases squamous cell carcinoma risk (odds ratio (OR)=1.51, 95% confidence intervals: 1.21-1.88), while reduced FEV1/FVC increases the risk of adenocarcinoma (OR=1.17, 1.01-1.35) and lung cancer in never smokers (OR=1.56, 1.05-2.30). These findings support a causal role of pulmonary impairment in lung cancer etiology. Integrative analyses reveal that pulmonary function instruments, including 73 novel variants, influence lung tissue gene expression and implicate immune-related pathways in mediating the observed effects on lung carcinogenesis.

genetics↗

bioRxiv2019

SSD1 suppresses phenotypes induced by the lack of Elongator-dependent tRNA modifications

The Elongator complex promotes formation of 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) side-chains on uridines at the wobble position of cytosolic eukaryotic tRNAs. In all eukaryotic organisms tested to date, the inactivation of Elongator not only leads to the lack of mcm5/ncm5 groups in tRNAs, but also a wide variety of phenotypes. Although the phenotypes are most likely caused by a translational defect induced by reduced functionality of the hypomodified tRNAs, the mechanism(s) underlying individual phenotypes are poorly understood. In this study, we show that the genetic background modulates the phenotypes induced by the lack of mcm5/ncm5 groups in Saccharomyces cerevisiae. We show that the stress-induced growth defects of Elongator mutants are stronger in the W303 than in the closely related S288C genetic background and that the phenotypic differences are caused by the known polymorphism at the locus for the mRNA binding protein Ssd1. Moreover, the mutant ssd1 allele found in W303 cells is required for the reported histone H3 acetylation and telomeric gene silencing defects of Elongator mutants. The difference at the SSD1 locus also partially explains why the simultaneous lack of mcm5 and 2-thio groups at wobble uridines is lethal in the W303 but not in the S288C background. Collectively, our results demonstrate that the SSD1 locus modulates phenotypes induced by the lack of Elongator-dependent tRNA modifications. Author SummaryModified nucleosides in the anticodon region of tRNAs are important for the efficiency and fidelity of translation. The Elongator complex promotes formation of several related modified uridine residues at the wobble position of eukaryotic tRNAs. In yeast, plants, worms, mice and humans, mutations in genes for Elongator subunits lead to a wide variety of different phenotypes. Here, we show that the genetic background modulates the phenotypic consequences of the inactivation of budding yeast Elongator. This background effect is largely a consequence of a polymorphism at the SSD1 locus, encoding a RNA binding protein that influences translation, stability and/or localization of mRNAs. We show that several phenotypes reported for yeast Elongator mutants are either significantly stronger or only detectable in strains harboring a mutant ssd1 allele. Thus, SSD1 is a suppressor of the phenotypes induced by the hypomodification of tRNAs.

genetics↗

bioRxiv2019

A Collaborative Multi-Model Ensemble for Real-Time Influenza Season Forecasting in the U.S.

Seasonal influenza results in substantial annual morbidity and mortality in the United States and worldwide. Accurate forecasts of key features of influenza epidemics, such as the timing and severity of the peak incidence in a given season, can inform public health response to outbreaks. As part of ongoing efforts to incorporate data and advanced analytical methods into public health decision-making, the United States Centers for Disease Control and Prevention (CDC) has organized seasonal influenza forecasting challenges since the 2013/2014 season. In the 2017/2018 season, 22 teams participated. A subset of four teams created a research consortium called the FluSight Network in early 2017. During the 2017/2018 season they worked together to produce a collaborative multi-model ensemble that combined 21 separate component models into a single model using a machine learning technique called stacking. This approach creates a weighted average of predictive densities where the weight for each component is based on that components forecast accuracy in past seasons. In the 2017/2018 influenza season, one of the largest seasonal outbreaks in the last 15 years, this multi-model ensemble performed better on average than all individual component models and placed second overall in the CDC challenge. It also outperformed the baseline multi-model ensemble created by the CDC that took a simple average of all models submitted to the forecasting challenge. This project shows that collaborative efforts between research teams to develop ensemble forecasting approaches can bring measurable improvements in forecast accuracy and important reductions in the variability of performance from year to year. Efforts such as this, that emphasize real-time testing and evaluation of forecasting models and facilitate the close collaboration between public health officials and modeling researchers, are essential to improving our understanding of how best to use forecasts to improve public health response to seasonal and emerging epidemic threats.

epidemiology↗

bioRxiv2018

Elimination of ribosome inactivating factors improves the efficiency of Bacillus subtilis and Saccharomyces cerevisiae cell-free translational systems

Cell-free translational systems based on cellular lysates optimized for in vitro protein synthesis have multiple applications both in basic and applied science, ranging from studies of translation regulation to cell-free production of proteins and ribosome-nascent chain complexes. In order to achieve both high activity and reproducibility in a translational system, it is essential that the ribosomes in the cellular lysate are enzymatically active. Here we demonstrate that genomic disruption of genes encoding ribosome inactivating factors - HPF in Bacillus subtilis and Stm1 in Saccharomyces cerevisiae - robustly improve the activities of bacterial and yeast translational systems. A possible next step in developing strains for production of even more efficient cell-free translation systems could be achieved by combining a genomic disruption of the ribosome hibernation machinery with inactivation of the genes responsible for proteolysis and RNA degradation.

biochemistry↗

bioRxiv2018

Direct observation of rotation-coupled protein diffusion along DNA on the microsecond timescale

Many proteins that bind specific DNA sequences search the genome by combining three dimensional (3D) diffusion in the cytoplasm with one dimensional (1D) sliding on non-specific regions of the DNA1-5. It is however not known how sliding proteins are oriented with respect to DNA in order to recognize specific sequences. Here we measure the polarization of fluorescence emission from single fluorescently labeled lac repressor (LacI) molecules sliding on stretched DNA. Real-time feedback-coupled confocal single-particle tracking allows us to measure fluorescence correlation of the sliding molecules. We find that the fluctuations in the fluorescence signal on the s timescale are accurately described by rotation-coupled sliding on DNA. On average, LacI moves [~]50 base pairs per revolution, which is significantly longer than the 10.5 bp helical periodicity of DNA. Our data support a facilitated diffusion model1 where the transcription factor (TF) scans the DNA grooves for hydrogen bonding opportunities in a pre-aligned orientation with occasional slippage out of the groove.

biophysics↗