Biology subjects

Müller, M.

Publications and source records attributed to Müller, M..

42 records · Page 3Linked to original sources

bioRxiv2019

DMD-based super-resolution structured illumination microscopy visualizes live cell dynamics at high speed and low cost

Structured illumination microscopy (SIM) is among the most widely used super-resolution fluorescence microscopy techniques for visualizing the dynamics of cellular organelles, such as mitochondria, the endoplasmic reticulum, or the cytoskeleton. In its most wide-spread implementation, SIM relies on the creation of an interference pattern at the diffraction limit using the coherent addition of laser beams created by a diffraction pattern.\n\nSpatial light modulators based on liquid crystal displays allow SIM micro-scopes to run at image rates of up to hundreds of super-resolved images per second. Digital micromirror devices are another natural choice for creating interference-based SIM patterns, but are not used to their fullest potential because of the blazed grating effect. This effect arises due to the fixed angles between which the mirrors can be switched, creating a sawtooth arrangement of mirrors and thus leading to a change in the intensity distribution of the diffracted beams. This results in SIM patterns with varying modulation contrast which are prone to reconstruction artifacts.\n\nWe have carefully studied the blazed grating effect of DMDs by simulations, varying a range of parameters and compared the simulation results with experiments. This allowed us to identify settings which result in very high modulation contrast across all angles and phases required to generate 2-beam SIM pattern. The use of inexpensive industry-grade CMOS cameras as well as low-cost lasers enabled us to construct a cost-effective, high-speed SIM system. Reconstruction of the super-resolved SIM images is achieved on a recently demonstrated parallel-computing platform, which allowed us to visualize living cells with super-resolution at multiple reconstructed frames per second in real time. We demonstrate the versatility of this new platform by imaging cellular organelle dynamics based on live-cell fluorescent stains as well as with fluorescent protein stained samples.

biophysics↗

bioRxiv2019

Unraveling the Complexity of Amyloid Polymorphism Using Gold Nanoparticles and Cryo-EM

The misfolding and self-assembly of proteins into {beta}-sheet-rich amyloid fibrils of various structures and morphologies is a hallmark of several neurodegenerative and systemic diseases. Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicity and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the use of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) to label the edges of synthetic, recombinant and native amyloid fibrils to assess amyloid morphological polymorphism using cryogenic transmission electron microscopy (cryo-EM). The fibrils studied were derived from amyloid proteins involved in disorders of the central nervous system (amyloid-{beta}, tau, -synuclein) and in systemic amyloidosis (a fragment of an immunoglobulin {lambda} light chain). The labeling efficiency enabled imaging and characterization of amyloid fibrils of different morphologies under hydrated conditions using cryo-EM. These NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including TEM with use of the negative stain or cryo-EM imaging. We also demonstrate the use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of an Alzheimers disease patient, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain (AL) amyloidosis. Analysis of the cryo-EM images of amyloids decorated with NPs shows exceptional homogeneity across the fibrils derived from human tissue in comparison to fibrils aggregated in vitro. The use of these NPs enabled us to gain novel insight into the structural features that distinguish amyloid fibrils formed in vivo from those formed in cell-free in vitro systems. Our findings demonstrate that these NPs represent a powerful tool for rapid imaging and profiling of amyloid morphological polymorphism in different types of samples, including those derived from complex biological aggregates found in human tissue and animal models of amyloid diseases. These advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM but also pave the way to elucidate the structural basis of amyloid strains and toxicity and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

neuroscience↗

bioRxiv2019

High speed multi-plane super-resolution structured illumination microscopy of living cells using an image-splitting prism

Super-resolution structured illumination microscopy (SR-SIM) can be conducted at video-rate acquisition speeds when combined with high-speed spatial light modulators and sCMOS cameras, rendering it particularly suitable for live cell imaging. If, however, three-dimensional (3D) information is desired, the sequential acquisition of vertical image stacks employed by current setups significantly slows down the acquisition process. In this work we present a multi-plane approach to SR-SIM that overcomes this slowdown via the simultaneous acquisition of multiple object planes, employing a recently introduced multi-plane image splitting prism combined with high-speed SR-SIM illumination. This strategy requires only the introduction of a single optical element and the addition of a second camera to acquire a laterally super-resolved three-dimensional image stack. We demonstrate the performance of multi-plane SR-SIM by applying this instrument to the dynamics of live mitochondrial network.

biophysics↗

bioRxiv2019

Chemical genetics of AGC-kinases reveals shared targets of Ypk1, Protein Kinase A and Sch9

Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take centre stage in regulating this process in S. cerevisiae. To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated downstream of Ypk1. Signalling through Ypk1 is therefore more closely related to PKA- and Sch9-signalling than previously appreciated and may perform functions previously only attributed to the latter kinases.

cell biology↗

bioRxiv2018

Association between Apolipoprotein E genotype and the gut microbiome composition in humans and mice

Apolipoprotein E (APOE) genotype is the strongest prevalent genetic risk factor for Alzheimers disease. Numerous studies have provided insights into the pathological mechanisms. However, a comprehensive understanding of the impact of APOE genotype on microflora speciation and metabolism is completely lacking. The aim of this study was to investigate the association between APOE genotype and the gut microbiome composition in human and APOE-targeted replacement (TR, APOE3 and APOE4) transgenic mice. Faecal microbiota amplicon sequencing from matched individuals with different APOE genotypes revealed no significant differences in overall microbiota diversity (alpha or beta diversity) in group-aggregated human APOE genotypes. However, several bacterial taxa showed significantly different relative abundance between APOE genotypes. Notably, we detected an association of Prevotellaceae and Ruminococcaceae and several butyrate-producing genera abundances with APOE genotypes. These findings were confirmed by comparing the gut microbiota of APOE-TR mice. Furthermore, metabolomic analysis of faecal water from murine samples detected significant differences in microbe-associated amino acids and short-chain fatty acids between APOE genotypes. Together, the findings indicate that APOE genotype associated with specific gut microbiome profiles in both humans and in APOE-TR mice. This suggests that the gut microbiome is worth further investigation as a potential therapeutic target to mitigate the deleterious impact of the APOE4 allele on cognitive decline and the prevention and treatment of AD.

genomics↗

bioRxiv2018

Boundaries support specific long-distance interactions between enhancers and promoters in Drosophila Bithorax complex

Drosophila bithorax complex (BX-C) is one of the best model systems for studying the role of boundaries (insulators) in gene regulation. Expression of three homeotic genes, Ubx, abd-A, and Abd-B, is orchestrated by nine parasegment-specific regulatory domains. These domains are flanked by boundary elements, which function to block crosstalk between adjacent domains, ensuring that they can act autonomously. Paradoxically, seven of the BX-C regulatory domains are separated from their gene target by at least one boundary, and must \"jump over\" the intervening boundaries. To understand the jumping mechanism, the Mcp boundary was replaced with Fab-7 and Fab-8. Mcp is located between the iab-4 and iab-5 domains, and defines the border between the set of regulatory domains controlling abd-A and Abd-B. When Mcp is replaced by Fab-7 or Fab-8, they direct the iab-4 domain (which regulates abd-A) to inappropriately activate Abd-B in abdominal segment A4. For the Fab-8 replacement, ectopic induction was only observed when it was inserted in the same orientation as the endogenous Fab-8 boundary. A similar orientation dependence for bypass activity was observed when Fab-7 was replaced by Fab-8. Thus, boundaries perform two opposite functions in the context of BX-C - they block crosstalk between neighboring regulatory domains, but at the same time actively facilitate long distance communication between the regulatory domains and their respective target genes.\n\nAuthor SummaryDrosophila bithorax complex (BX-C) is one of a few examples demonstrating in vivo role of boundary/insulator elements in organization of independent chromatin domains. BX-C contains three HOX genes, whose parasegment-specific pattern is controlled by cis-regulatory domains flanked by boundary/insulator elements. Since the boundaries ensure autonomy of adjacent domains, the presence of these elements poses a paradox: how do the domains bypass the intervening boundaries and contact their proper regulatory targets? According to the textbook model, BX-C regulatory domains are able to bypass boundaries because they harbor special promoter targeting sequences. However, contrary to this model, we show here that the boundaries themselves play an active role in directing regulatory domains to their appropriate HOX gene promoter.

genetics↗