Biology subjects

Kaminski Schierle, G. S.

Publications and source records attributed to Kaminski Schierle, G. S..

31 records · Page 2Linked to original sources

bioRxiv2021

The SARS-CoV-2 nucleocapsid protein associates with the replication organelles before viral assembly at the Golgi/ERGIC and lysosome-mediated egress

Despite being the target of extensive research efforts due to the COVID-19 pandemic, relatively little is known about the dynamics of SARS-CoV-2 replication within cells. We investigate and characterise the tightly orchestrated sequence of events during different stages of the infection cycle by visualising the spatiotemporal dynamics of the four structural proteins of SARS-CoV-2 at high resolution. The nucleoprotein is expressed first and accumulates around folded ER membranes in convoluted layers that connect to viral RNA replication foci. We find that of the three transmembrane proteins, the membrane protein appears at the Golgi apparatus/ERGIC before the spike and envelope proteins. Relocation of the lysosome marker LAMP1 towards the assembly compartment and its detection in transport vesicles of viral proteins confirm an important role of lysosomes in SARS-CoV-2 egress. These data provide new insights into the spatiotemporal regulation of SARS-CoV-2 assembly, and refine current understanding of SARS-CoV-2 replication.

microbiology↗

bioRxiv2021

The role of water mobility in protein misfolding

The propensity for intrinsically disordered proteins to aggregate is heavily influenced by their surrounding environment. Here, we show that the mobility of the surrounding water molecules directly influences the aggregation rate of -synuclein (aSyn), a protein associated with Parkinsons disease. We observe that the addition of NaCl reduces the mobility of water, while addition of CsI increases the mobility of water. In turn, this reduces and increases the mobility of aSyn, respectively, given the change in strength and lifetime of the intermolecular forces. The reduction of aSyn mobility in the presence of NaCl ions leads to increased aggregation rates, which may be due to aggregation-competent conformations being stable for longer, thereby increasing the likelihood of establishing interactions between two adjacent monomers. In contrast, aSyn is more mobile when CsI is dissolved in the aqueous phase which leads to a reduction of successful monomeric interactions. We thus highlight the importance of the surrounding environment and describe how ion content can influence water mobility and the misfolding rate of amyloidogenic proteins, such as aSyn. By modulating the cellular environment to increase water mobility or finding small molecules to increase protein dynamics, new therapeutic targets may be found.

biophysics↗

bioRxiv2020

Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

Misfolded -synuclein is a major component of Lewy bodies, which are a hallmark of Parkinsons disease. A large body of evidence shows that -synuclein can self-assemble into amyloid fibrils, but the relationship between amyloid formation and Lewy body formation still remains unclear. Here we show, both in vitro and in a C. elegans model of Parkinsons disease, that -synuclein undergoes liquid-liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel. This maturation process towards the amyloid state is delayed in the presence of model synaptic vesicles in vitro. Taken together, these results suggest that the formation of Lewy bodies is linked to the arrested maturation of -synuclein condensates in the presence of lipids and other cellular components.

molecular biology↗

bioRxiv2020

Satellite repeat transcripts modulate heterochromatin condensates and safeguard chromosome stability in mouse embryonic stem cells

Heterochromatin maintains genome integrity and function, and is organised into distinct nuclear domains. Some of these domains are proposed to form by phase separation through the accumulation of HP1. Mammalian heterochromatin contains noncoding major satellite repeats (MSR), which are highly transcribed in mouse embryonic stem cells (ESCs). Here, we report that MSR transcripts can drive the formation of HP1 droplets in vitro, and scaffold heterochromatin into dynamic condensates in ESCs, leading to the formation of large nuclear domains that are characteristic of pluripotent cells. Depleting MSR transcripts causes heterochromatin to transition into a more compact and static state. Unexpectedly, changing heterochromatins biophysical properties has severe consequences for ESCs, including chromosome instability and mitotic defects. These findings uncover an essential role for MSR transcripts in modulating the organisation and properties of heterochromatin to preserve genome stability. They also provide new insights into the processes that could regulate phase separation and the functional consequences of disrupting the properties of heterochromatin condensates.

molecular biology↗

bioRxiv2020

Purification of recombinant α-Synuclein: a comparison of commonly used protocols

The insoluble aggregated form of the protein alpha-synuclein (aSyn) is associated with synucleinopathies, such as Parkinsons Disease, therefore great effort is put into understanding why and how this initially soluble protein misfolds. The initial state of aSyn, e.g. presence of contaminants, adducts, oligomers or degradation products, can greatly influence the outcome of an assay, such as determining its aggregation kinetics. Here, we compare four commonly used protocols for the isolation of recombinant aSyn from E. coli by boiling, acid precipitation, ammonium sulphate precipitation and periplasmic lysis followed by ion exchange chromatography and gel filtration. We identified, using non-denaturing electrospray ionisation mass spectrometry of the differently extracted aSyn samples, that aSyn isolated by acid precipitation and periplasmic lysis yielded the highest percentage of monomer, 100% and 96.5% respectively. aSyn purity was again highest in samples isolated by acid precipitation and periplasmic lysis, yet aggregation assays displayed differences in the aggregation rate of aSyn isolated by all four methods. HighlightsO_LIA rapid protocol; expression day one, two step purification day two. C_LIO_LIThe periplasmic lysis-based protocol yielded 95% pure aSyn. C_LIO_LIAcid precipitation and periplasmic lysis-based protocols yielded the highest proportion of monomeric aSyn at 100% and 96.5%, respectively. C_LI

biophysics↗

bioRxiv2020

Fast purification of recombinant monomeric amyloid-β from E. coli and amyloid-β-mCherry aggregates from mammalian cells

The Alzheimers disease related peptide, Amyloid-beta (A{beta})1-40 and 1-42, has proven difficult to be purified as a recombinant monomeric protein due its expression in E. coli leading to the formation of insoluble inclusion bodies and its tendency to quickly form insoluble aggregates. A vast array of methods have been used so far, yet many have pitfalls, such as the use of tags for ease of A{beta} isolation, the formation of A{beta} multimers within the time frame of extraction or the need to reconstitute A{beta} from a freeze dried state. Here, we present a rapid protocol to produce highly pure and monomeric recombinant A{beta} using a one-step ion exchange purification method and to label the peptide using a maleimide dye. The solublisation and purification steps take only three hours. We also present a protocol for the isolation of A{beta}-mCherry from mammalian cells. HighlightsO_LIPurification of untagged, monomeric recombinant A{beta} from E. coli. C_LIO_LIA fast protocol; 6 hours for E. coli growth and A{beta} expression, 2 hours to clean inclusion bodies, 45 mins to solublise and purify the peptide. C_LIO_LINo freeze drying step that can lead to oligomer formation. C_LIO_LIPurification of fluorescent A{beta}-mCherry from mammalian cells. C_LI

biophysics↗

bioRxiv2020

Illuminating chromatin compaction in live cells and fixed tissues using SiR-DNA fluorescence lifetime

The global compaction state of chromatin in a nucleus is an important component of cell identity that has been difficult to measure. We have developed a quantitative method to measure the chromatin compaction state in both live and fixed cells, without the need for genetic modification, using the fluorescence lifetime of SiR-DNA dye. After optimising this method using live cancer cell lines treated to induce chromatin compaction or decompaction, we observed chromatin compaction in differentiating epithelial cells in fixed tissue sections, as well as local decompaction foci that may represent transcription factories. In addition, we shed new light on chromatin decompaction during embryonic stem cell transition out of their naive pluripotent state. This method will be useful to studies of nuclear architecture, and may be easy, cheap, and accessible enough to serve as a general assay of stem-ness.

cell biology↗

bioRxiv2020

Formal model of Parkinson's disease neurons unveils possible causality links in the pathophysiology of the disease

Parkinsons Disease is the second most common neurodegenerative disease after Alzheimers disease. Despite extensive research, the initial cause of the disease is still unknown, although substantial advances were made in understanding of its genetics and the cognate neurophysiological mechanisms. Determining the causality relationships and the chronological steps pertaining to Parkinsons Disease is essential for the discovery of novel drug targets. We developed a systematic in silico model based on available data, which puts the possible sequence of events occurring in a neuron during disease onset into light. This is the first ever attempt, to our knowledge, to model comprehensively the primary modifications in the molecular pathways that manifest in compromised neurons from the commencement of the disease to the consequences of its progression. We showed that our proposed disease pathway was relevant for unveiling yet incomplete knowledge on calcium homeostasis in mitochondria, ROS production and -synuclein misfolding. Graphical abstract HighlightsO_LIVarying calcium concentration in aging dopaminergic neurons triggers disease onset. C_LIO_LIROS production in the mitochondria potentially causes iron accumulation. C_LIO_LIIron homeostasis dysregulation is linked to -synuclein aggregation. C_LI

systems biology↗

bioRxiv2020

Intrinsic fluorescence in non-aromatic peptide structures is induced by collective vibrations, charge reorganisation and short hydrogen bonds, as shown in a new glutamine-related structure

Fluorescence in biological systems is usually associated with the presence of aromatic groups. Here, we show that specific hydrogen bonding networks can significantly affect fluorescence employing a combined experimental and computational approach. In particular, we reveal that the single amino acid L-glutamine, by undergoing a chemical transformation leading to the formation of a short hydrogen bond, displays optical properties that are significantly enhanced compared to L-glutamine itself. Ab initio molecular dynamics simulations highlight that these short hydrogen bonds prevent the appearance of a conical intersection between the excited and the ground states and thereby significantly decrease non-radiative transition probabilities. Our findings open the doors for the design of new photoactive materials with biophotonic applications. Significance statementIntrinsic fluorescence of non-aromatic amino acids is a puzzling phenomenon with an enormous potential for technological and biomedical applications. The physical origins of this effect, however, remain elusive. Herein, we demonstrate how specific hydrogen bond networks can modulate fluorescence. We highlight the key role played by short hydrogen bonds in the networks on the ensuing fluorescence and we provide a detailed molecular mechanism to explain this unusual non-aromatic optical properties. Our findings should benefit the design of novel optically active biomaterials for applications in biosensing and imaging.

biophysics↗

bioRxiv2019

Extent of N-terminus exposure by altered long-range interactions of monomeric alpha-synuclein determines its aggregation propensity

As an intrinsically disordered protein, monomeric alpha synuclein (aSyn) constantly reconfigures and probes the conformational space. Long-range interactions across the protein maintain its solubility and mediate this dynamic flexibility, but also provide residual structure. Certain conformations lead to aggregation prone and non-aggregation prone intermediates, but identifying these within the dynamic ensemble of monomeric conformations is difficult. Herein, we used the biologically relevant calcium ion to investigate the conformation of monomeric aSyn in relation to its aggregation propensity. By using calcium to perturb the conformational ensemble, we observe differences in structure and intra-molecular dynamics between two aSyn C-terminal variants, D121A and pS129, and the aSyn familial disease mutants, A30P, E46K, H50Q, G51D, A53T and A53E, compared to wild-type (WT) aSyn. We observe that the more exposed the N-terminus and the beginning of the NAC region are, the more aggregation prone monomeric aSyn conformations become. N-terminus exposure occurs upon release of C-terminus interactions when calcium binds, but the level of exposure is specific to the aSyn mutation present. There was no correlation between single charge alterations, calcium affinity, or the number of ions bound on aSyns aggregation propensity, indicating that sequence or post-translation modification (PTM)-specific conformational differences between the N- and C-termini and the specific local environment mediate aggregation propensity instead. Understanding aggregation prone conformations of monomeric aSyn and the environmental conditions they form under will allow us to design new therapeutics targeted to the monomeric protein, to stabilise aSyn in non-aggregation prone conformations, by either preserving long-range interactions between the N- and C-termini or by protecting the N-terminus from exposure.

biophysics↗

bioRxiv2019

Intra-mitochondrial protein degradationalleviates alpha-synuclein seeding and Abeta 42 aggregation

Mitochondria have long been implicated in Parkinsons disease (PD), however, it is not clear how mitochondrial impairment and alpha-synuclein pathology are coupled. We report here that intra-mitochondrial protein homeostasis plays a major role in alpha-synuclein fibril elongation, as interference with intra-mitochondrial proteases and mitochondrial protein import significantly aggravate alpha-synuclein aggregation. In contrast, direct inhibition of mitochondrial complex I, increase in intracellular calcium concentration or formation of reactive oxygen species (ROS), all of which have been associated with mitochondrial stress, did not affect alpha-synuclein pathology. We further demonstrate that similar mechanisms are involved in Amyloid {beta} 1-42 (A{beta}42) aggregation, suggesting that mitochondria are directly capable of influencing cytosolic protein homeostasis of aggregation-prone proteins.

neuroscience↗

bioRxiv2018

Intrinsically aggregation-prone proteins form amyloid-like aggregates and contribute to tissue aging in C. elegans

Reduced protein homeostasis and increased protein instability is a common feature of aging. Yet it remains unclear whether protein instability is a cause of aging. In neurodegenerative diseases and amyloidoses, specific proteins self-assemble into amyloid fibrils and accumulate as pathological solid aggregates in a variety of tissues. More recently, widespread protein aggregation has been described during normal aging, in the absence of disease processes. Until now, an extensive characterization of the nature of age-dependent protein aggregation and its consequences for aging has been lacking. Here, we show that age-dependent aggregates are rapidly formed by newly synthesized proteins and contain amyloid-like structures similar to disease-associated protein aggregates. Moreover, we demonstrate that age-dependent protein aggregation accelerates the functional decline of different tissues in C. elegans. Together, these finding reveal that the formation of amyloid aggregates is a generic problem of aging and likely to be an important target for strategies designed to maintain physiological functions in later stages of life.

cell biology↗

bioRxiv2018

Fast fluorescence lifetime imaging reveals the maturation process of α-synuclein aggregates in ageing Caenorhabditis elegans

The nematode worm Caenorhabditis elegans has emerged as an important model organism to study the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system, however, remains a challenge. To address this problem, we here present a fast time-gated fluorescence lifetime imaging (TG-FLIM) method and show that it provides functional insights into the process of protein aggregation in living animals by enabling the rapid characterisation of different types of aggregates. More specifically, in longitudinal studies of C. elegans models of Parkinsons and Huntingtons diseases, we observed marked differences in the aggregation kinetics and the nature of the protein inclusions formed by -synuclein and polyglutamine. In particular, we found that -synuclein inclusions do not display amyloid-like features until late in the life of the worms, whereas polyglutamine forms amyloid characteristics rapidly in early adulthood. Furthermore, we show that the TG-FLIM method is capable of imaging live and non-anaesthetised worms moving in specially designed agarose micro-chambers. Taken together, our results show that the TG-FLIM method enables high-throughput functional imaging of living C. elegans that can be used to study in vivo mechanisms of aggregation and that has the potential to aid the search for therapeutic modifiers of protein aggregation and toxicity.

biophysics↗