Biology subjects

Loose, M.

Publications and source records attributed to Loose, M..

29 records · Page 2Linked to original sources

bioRxiv2019

Computational analysis of filament polymerization dynamics in cytoskeletal networks

The polymerization-depolymerization dynamics of cytoskeletal proteins play essential roles in the self-organization of cytoskeletal structures, in eukaryotic as well as prokaryotic cells. While advances in fluorescence microscopy and in vitro reconstitution experiments have helped to study the dynamic properties of these complex systems, methods that allow to collect and analyze large quantitative datasets of the underlying polymer dynamics are still missing. Here, we present a novel image analysis workflow to study polymerization dynamics of active filaments in a non-biased, highly automated manner. Using treadmilling filaments of the bacterial tubulin FtsZ as an example, we demonstrate that our method is able to specifically detect, track and analyze growth and shrinkage of polymers, even in dense networks of filaments. We believe that this automated method can facilitate the analysis of a large variety of dynamic cytoskeletal systems, using standard time-lapse movies obtained from experiments in vitro as well as in the living cell. Moreover, we provide scripts implementing this method as supplementary material.

biochemistry↗

bioRxiv2019

Stochastic activation and bistability in a Rab GTPase regulatory network.

Rab GTPases are the central regulators of intracellular traffic. Their function relies on a conformational change triggered by nucleotide exchange and hydrolysis. While this switch is well understood for an individual protein, how Rab GTPases collectively transition between states to generate a biochemical signal in space and time is unclear. Here, we combine in vitro reconstitution experiments with theoretical modeling to study a minimal Rab5 activation network. We find that positive feedback in this network gives rise to bistable switching of Rab5 activation and provide evidence that controlling the inactive population of Rab5 on the membrane can shape the network response. Together, our findings reveal new insights into the non-equilibrium properties and general principles of biochemical signaling networks underlying the spatiotemporal organization of the cell.

systems biology↗

bioRxiv2019

Telomere-to-telomere assembly of a complete human X chromosome

After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38 2, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3, we reconstructed the [~]2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.

bioinformatics↗

bioRxiv2019

ZapA stabilizes FtsZ filament bundles without slowing down treadmilling dynamics

For bacterial cell division, treadmilling filaments of FtsZ organize into a ring-like structure at the center of the cell. What governs the architecture and stability of this dynamic Z-ring is currently unknown, but FtsZ-associated proteins have been suggested to play an important role. Here, we used an in vitro reconstitution approach combined with fluorescence microscopy to study the influence of the well-conserved protein ZapA on the organization and dynamics of FtsZ filaments recruited to a supported membrane. We found that ZapA increases the spatial order and stabilizes the steady-state architecture of the FtsZ filament network in a highly cooperative manner. Despite its strong influence on their large-scale organization, ZapA binds only transiently to FtsZ filaments and has no effect on their treadmilling velocity. Together, our data explains how FtsZ-associated proteins can contribute to the precision and stability of the Z-ring without compromising treadmilling dynamics.

biophysics↗

bioRxiv2018

FtsZ assembles the bacterial cell division machinery by a diffusion-and-capture mechanism

The mechanism of bacterial cell division is largely unknown. The protein machinery performing cell division is organized by FtsZ, a tubulin-homolog that forms treadmilling filaments at the cell division site. Treadmilling is thought to actively move proteins around the cell thereby distributing peptidoglycan synthesis to make two new cell poles. To understand this process, we reconstituted part of the bacterial cell division machinery using the purified components FtsZ, FtsA and truncated transmembrane proteins essential for cell division. We found that membrane-bound cytosolic peptides of FtsN and FtsQ co-migrated with treadmilling FtsZ-FtsA filaments. Remarkably, rather than moving in a directed fashion, individual peptides followed FtsZ filaments by a diffusion-and-capture mechanism. Our work provides a mechanism for how the Z-ring dynamically recruits divisome proteins and highlights the importance of transient interactions for the self-organization of complex biological structures. We propose that this mechanism is used more widely to organize and transmit spatiotemporal information in living cells.\n\nOne Sentence SummaryFtsZ treadmilling assembles bacterial division machinery by diffusion-and-capture mechanism.

biochemistry↗

bioRxiv2018

Lineage segregation, pluripotency and X-chromosome inactivation in the pig pre-gastrulation embryo

High-resolution molecular programs delineating the cellular foundations of mammalian embryogenesis have emerged recently. Similar analysis of human embryos is limited to pre-implantation stages, since early post-implantation embryos are inaccessible. Notwithstanding, we previously suggested conserved principles of pig and human early development. For further insight on pluripotent states and lineage delineation, we analysed pig embryos at single cell resolution. Here we show progressive segregation of inner cell mass and trophectoderm in early blastocysts, and then of epiblast and hypoblast in late blastocysts. We detected distinct pluripotent states, first as a short naive state followed by a protracted primed state. Dosage compensation with respect to the X-chromosome in females is attained via X-inactivation in late epiblasts. Detailed human-pig comparison is a basis towards comprehending early human development and a foundation for further studies of human pluripotent stem cell differentiation in pig interspecies chimeras.

developmental biology↗

bioRxiv2018

Whale watching with BulkVis: A graphical viewer for Oxford Nanopore bulk fast5 files.

MotivationThe Oxford Nanopore Technologies (ONT) MinION is used for sequencing a wide variety of sample types with diverse methods of sample extraction. Nanopore sequencers output fast5 files containing signal data subsequently base called to fastq format. Optionally, ONT devices can collect data from all sequencing channels simultaneously in a bulk fast5 file enabling inspection of signal in any channel at any point. We sought to visualise this signal to inspect challenging or difficult to sequence samples, or where flow cell performance is modified by an external agent, such as Read Until.\n\nResultsThe BulkVis tool can load a bulk fast5 file and overlays MinKNOW classifications on the signal trace. Users can navigate to a channel and time or, given a fastq header from a read, jump to its specific position. BulkVis can export regions as Nanopore base caller compatible reads. Using BulkVis, we find long reads can be incorrectly divided by MinKNOW resulting in single DNA molecules being split into two or more reads. The longest seen to date is 2,272,580 bases in length and reported in eleven consecutive reads. We provide helper scripts that identify and reconstruct split reads given a sequencing summary file and alignment to a reference. We note that incorrect read splitting appears to vary according to input sample type and is more common in ultra long read preparations.\n\nAvailabilityThe software is available freely under an MIT license at https://github.com/LooseLab/bulkVis. The software requires python3 to run.

genomics↗

bioRxiv2017

Nanopore sequencing and assembly of a human genome with ultra-long reads

Nanopore sequencing is a promising technique for genome sequencing due to its portability, ability to sequence long reads from single molecules, and to simultaneously assay DNA methylation. However until recently nanopore sequencing has been mainly applied to small genomes, due to the limited output attainable. We present nanopore sequencing and assembly of the GM12878 Utah/Ceph human reference genome generated using the Oxford Nanopore MinION and R9.4 version chemistry. We generated 91.2 Gb of sequence data ([~]30x theoretical coverage) from 39 flowcells. De novo assembly yielded a highly complete and contiguous assembly (NG50 [~]3Mb). We observed considerable variability in homopolymeric tract resolution between different basecallers. The data permitted sensitive detection of both large structural variants and epigenetic modifications. Further we developed a new approach exploiting the long-read capability of this system and found that adding an additional 5x-coverage of ultra-long reads (read N50 of 99.7kb) more than doubled the assembly contiguity. Modelling the repeat structure of the human genome predicts extraordinarily contiguous assemblies may be possible using nanopore reads alone. Portable de novo sequencing of human genomes may be important for rapid point-of-care diagnosis of rare genetic diseases and cancer, and monitoring of cancer progression. The complete dataset including raw signal is available as an Amazon Web Services Open Dataset at: https://github.com/nanopore-wgs-consortium/NA12878.

genomics↗

bioRxiv2017

Epidemic establishment and cryptic transmission of Zika virus in Brazil and the Americas

Zika virus (ZIKV) transmission in the Americas was first confirmed in May 2015 in Northeast Brazil1. Brazil has the highest number of reported ZIKV cases worldwide (>200,000 by 24 Dec 20162) as well as the greatest number of cases associated with microcephaly and other birth defects (2,366 confirmed cases by 31 Dec 20162). Following the initial detection of ZIKV in Brazil, 47 countries and territories in the Americas have reported local ZIKV transmission, with 22 of these reporting ZIKV-associated severe disease3. Yet the origin and epidemic history of ZIKV in Brazil and the Americas remain poorly understood, despite the value of such information for interpreting past trends in reported microcephaly. To address this we generated 53 complete or partial ZIKV genomes, mostly from Brazil, including data generated by the ZiBRA project - a mobile genomics lab that travelled across Northeast Brazil in 2016. One sequence represents the earliest confirmed ZIKV infection in Brazil. Joint analyses of viral genomes with ecological and epidemiological data estimate that the ZIKV epidemic first became established in NE Brazil by March 2014 and likely disseminated from there, both nationally and internationally, before the first detection of ZIKV in the Americas. Estimated dates of the international spread of ZIKV from Brazil coincide with periods of high vector suitability in recipient regions and indicate the duration of pre-detection cryptic transmission in those regions. NE Brazils role in the establishment of ZIKV in the Americas is further supported by geographic analysis of ZIKV transmission potential and by estimates of the virus basic reproduction number.\n\nOne Sentence SummaryVirus genomes reveal the establishment of Zika virus in Northeast Brazil and the Americas, and provide an appropriate timeframe for baseline (pre-Zika) microcephaly in different regions.

genomics↗

bioRxiv2017

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples without isolation remains challenging for viruses such as Zika, where metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence complete genomes comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimised library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved starting with clinical samples in 1-2 days following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas.

genomics↗

bioRxiv2016

Same-day diagnostic and surveillance data for tuberculosis via whole genome sequencing of direct respiratory samples.

Routine full characterization of Mycobacterium tuberculosis (TB) is culture-based, taking many weeks. Whole-genome sequencing (WGS) can generate antibiotic susceptibility profiles to inform treatment, augmented with strain information for global surveillance; such data could be transformative if provided at or near point of care.\n\nWe demonstrate a low-cost DNA extraction method for TB WGS direct from patient samples. We initially evaluated the method using the Illumina MiSeq sequencer (40 smear-positive respiratory samples, obtained after routine clinical testing, and 27 matched liquid cultures). M. tuberculosis was identified in all 39 samples from which DNA was successfully extracted. Sufficient data for antibiotic susceptibility prediction was obtained from 24 (62%) samples; all results were concordant with reference laboratory phenotypes. Phylogenetic placement was concordant between direct and cultured samples. Using an Illumina MiSeq/MiniSeq the workflow from patient sample to results can be completed in 44/16 hours at a cost of {pound}96/{pound}198 per sample.\n\nWe then employed a non-specific PCR-based library preparation method for sequencing on an Oxford Nanopore Technologies MinION sequencer. We applied this to cultured Mycobacterium bovis BCG strain (BCG), and to combined culture-negative sputum DNA and BCG DNA. For the latest flowcell, the estimated turnaround time from patient to identification of BCG was 6 hours, with full susceptibility and surveillance results 2 hours later. Antibiotic susceptibility predictions were fully concordant. A critical advantage of the MinION is the ability to continue sequencing until sufficient coverage is obtained, providing a potential solution to the problem of variable amounts of M. tuberculosis in direct samples.

microbiology↗