Biology subjects

Ries, J.

Publications and source records attributed to Ries, J..

At least 55 records · Page 3Linked to original sources

bioRxiv2020

General DNA methylation patterns and environmentally-induced differential methylation in the eastern oyster (Crassostrea virginica)

Epigenetic modification, specifically DNA methylation, is one possible mechanism for intergenerational plasticity. Before inheritance of methylation patterns can be characterized, we need a better understanding of how environmental change modifies the parental epigenome. To examine the influence of experimental ocean acidification on eastern oyster (Crassostrea virginica) gonad tissue, oysters were cultured in the laboratory under control (491 {+/-} 49 atm) or high (2550 {+/-} 211 atm) pCO2 conditions for four weeks. DNA from reproductive tissue was isolated from five oysters per treatment, then subjected to bisulfite treatment and DNA sequencing. Irrespective of treatment, DNA methylation was primarily found in gene bodies with approximately 22% of CpGs (2.7% of total cytosines) in the C. virginica genome predicted to be methylated. In response to elevated pCO2, we found 598 differentially methylated loci primarily overlapping with gene bodies. A majority of differentially methylated loci were in exons (61.5%) with less intron overlap (31.9%). While there was no evidence of a significant tendency for the genes with differentially methylated loci to be associated with distinct biological processes, the concentration of these loci in gene bodies, including genes involved in protein ubiquitination and biomineralization suggests DNA methylation may be important for transcriptional control in response to ocean acidification. Changes in gonad methylation also indicate potential for these methylation patterns to be inherited by offspring. Understanding how experimental ocean acidification conditions modify the oyster epigenome, and if these modifications are inherited, allows for a better understanding of how ecosystems will respond to environmental change.

genomics↗

bioRxiv2019

Three dimensional particle averaging for structural imaging of macromolecular complexes by localization microscopy

We present an approach for 3D particle fusion in localization microscopy which dramatically increases signal-to-noise ratio and resolution in single particle analysis. Our method does not require a structural template, and properly handles anisotropic localization uncertainties. We demonstrate 3D particle reconstructions of the Nup107 subcomplex of the nuclear pore complex (NPC), cross-validated using multiple localization microscopy techniques, as well as two-color 3D reconstructions of the NPC, and reconstructions of DNA-origami tetrahedrons.

molecular biology↗

bioRxiv2019

A cost-efficient open source laser engine for microscopy

Scientific-grade lasers are costly components of modern microscopes. For high-power applications, such as single-molecule localization microscopy, their price can become prohibitive. Here, we present an open-source high-power laser engine that can be built for a fraction of the cost. It uses affordable, yet powerful laser diodes at wavelengths of 405 nm, 488 nm and 640 nm and optionally a 561 nm diode-pumped solid-state laser. The light is delivered to the microscope via an agitated multimode fiber in order to suppress speckles. We provide the part lists, CAD files and detailed descriptions, allowing any research group to build their own laser engine.

biophysics↗

bioRxiv2019

MINFLUX nanoscopy delivers multicolor nanometer 3D-resolution in (living) cells

The ultimate goal of biological superresolution fluorescence microscopy is to provide three-dimensional resolution at the size scale of a fluorescent marker. Here, we show that, by localizing individual switchable fluorophores with a probing doughnut-shaped excitation beam, MINFLUX nanoscopy provides 1-3 nanometer resolution in fixed and living cells. This progress has been facilitated by approaching each fluorophore iteratively with the probing doughnut minimum, making the resolution essentially uniform and isotropic over scalable fields of view. MINFLUX imaging of nuclear pore complexes of a mammalian cell shows that this true nanometer scale resolution is obtained in three dimensions and in two color channels. Relying on fewer detected photons than popular camera-based localization, MINFLUX nanoscopy is poised to open a new chapter in the imaging of protein complexes and distributions in fixed and living cells.

biophysics↗

bioRxiv2019

Organotypic slice culture model demonstrates interneuronal spreading of alpha-synuclein aggregates

Here we describe the use of an organotypic hippocampal slice model for studying -synuclein aggregation and inter-neuronal spreading initiated by injection of preformed -synuclein filaments (PFFs). PFF injection at dentate gyrus templates the endogenous -synuclein to form aggregates in axons and cell bodies that spread to CA3 and CA1 regions. Aggregates were insoluble and phosphorylated at serine 129, recapitulating Lewy pathology features found in Parkinsons disease and other synucleinopathies. The spreading of the aggregates were favoring the anterograde direction in the slice model. The model allowed development of slices expressing only serine-129 phosphorylation-deficient human -synuclein (S129G) using adeno-associated viral (AAV) vector in -synuclein knockout slices. Processes of aggregation and spreading of -synuclein were thereby shown to be independent of phosphorylation at serine 129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated by using viral vectors allow testing of hypotheses on mechanisms involved in formation of -synuclein aggregates and their prion-like spreading.

neuroscience↗

bioRxiv2019

Photoactivation of silicon rhodamines via a light-induced protonation

We present a new type of photoactivatable fluorophore that forms a bright silicon rhodamine derivative through a light-dependent isomerization followed by protonation. In contrast to other photoactivatable fluorophores, no caging groups are required, nor are there any undesired side-products released. Using this photoactivatable fluorophore, we created probes for HaloTag and actin for live-cell single-molecule localization microscopy and single-particle tracking experiments. The unusual mechanism of photoactivation and the fluorophores outstanding spectroscopic properties make it a powerful tool for live-cell super-resolution microscopy.

biophysics↗

bioRxiv2019

Nanoscale subcellular architecture revealed by multicolor 3D salvaged fluorescence imaging

Combining the molecular specificity of fluorescent probes with three-dimensional (3D) imaging at nanoscale resolution is critical for investigating the spatial organization and interactions of cellular organelles and protein complexes. We present a super-resolution light microscope that enables simultaneous multicolor imaging of whole mammalian cells at ~20 nm 3D resolution. We show its power for cell biology research with fluorescence images that resolved the highly convoluted Golgi apparatus and the close contacts between the endoplasmic reticulum and the plasma membrane, structures that have traditionally been the imaging realm of electron microscopy.\n\nOne Sentence SummaryComplex cellular structures previously only resolved by electron microscopy can now be imaged in multiple colors by 4Pi-SMS.

biophysics↗

bioRxiv2019

Nuclear pores as versatile reference standards for quantitative superresolution microscopy

Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions.\n\nHere we exploit the stereotypic arrangement of proteins in the nuclear pore complex as in situ reference structures to characterize the performance of a variety of microscopy modalities. We created four genome edited cell lines in which we endogenously labeled the nucleoporin Nup96 with mEGFP, SNAP-tag or HaloTag or the photoconvertible fluorescent protein mMaple. We demonstrate their use a) as 3D resolution standards for calibration and quality control, b) to quantify absolute labeling efficiencies and c) as precise reference standards for molecular counting.\n\nThese cell lines will enable the broad community to assess the quality of their microscopes and labels, and to perform quantitative, absolute measurements.

biophysics↗

bioRxiv2019

Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT

The Nuclear Pore Complex (NPC) is one of the largest and most complex protein assemblies in the cell and - among other functions - serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and superresolution studies advancing our understanding of the NPC's complex architecture. However, the specific and direct visualization of single copies of NPC proteins and thus the ability to observe single-molecule heterogeneities of these complex structures is thus far elusive. Here, we combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. We employ the high localization precision in DNA-PAINT and molecular contrast of these protein tags to optically resolve single copies of nucleoporins in the human Y-complex in three dimensions with a precision of ~3 nm. This technological advancement now enables structural studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy.

cell biology↗

bioRxiv2019

Depth-dependent PSF calibration and aberration correction for 3D single-molecule localization

3D Single molecule localization microscopy relies on fitting of the individual molecules with a point spread function (PSF) model. The reconstructed images often show local squeezing or expansion in z. A common cause are depth-induced aberrations in conjunction with an imperfect PSF model calibrated from beads on a coverslip, resulting in a mismatch between measured PSF and real PSF. Here, we developed a strategy for accurate z-localization in which we use the imperfect PSF model for fitting, determine the fitting errors and correct for them in a post-processing step. We present an open-source software tool and a simple experimental calibration procedure that allow retrieving accurate z-positions in any PSF engineering approach or fitting modality, even at large imaging depths.

biophysics↗

bioRxiv2018

Type-I myosins promote actin polymerization to drive membrane bending in endocytosis

Clathrin-mediated endocytosis in budding yeast requires the formation of a dynamic actin network that produces the force to invaginate the plasma membrane against the intracellular turgor pressure. The type-I myosins Myo3 and Myo5 are important for endocytic membrane reshaping, but mechanistic details of their function remain scarce. Here, we studied the function of Myo3 and Myo5 during endocytosis using quantitative live-cell imaging and genetic perturbations. We show that the type-I myosins promote, in a dose-dependent way, the growth and expansion of the actin network, which controls the speed of membrane and coat internalization. We found that this myosin-activity is independent of the actin nucleation promoting activity of myosins, and cannot be compensated for by increasing actin nucleation. Our results suggest a new mechanism for type-I myosins to produce force by promoting actin filament polymerization.

cell biology↗

bioRxiv2018

Cytoskeleton mechanics determine resting size and activation dynamics of platelets.

Platelets are cell fragments of various size that help maintain hemostasis. The way platelets respond during a clotting process is known to depend on their size, with important physiological consequences. We characterized the cytoskeleton of platelets as a function of their size. In resting Human and Mice platelets, we find a quadradic law between the size of a platelet and the amount of microtubule polymer it contains. We further estimate the length and number of microtubules in the marginal band using Electron and Super-resolution microscopy. In platelets activated with ADP, the marginal band coils as a consequence of cortical contraction driven by actin. We observe that this elastic coiling response is accompanied by a reversible shortening of the marginal band. Moreover, larger platelets have a higher propensity to coil. These results establish the dynamic equilibrium that is responsible for platelet size and differential response on a more quantitative level.\n\nHighlightsO_LIPlatelet size scales consistently with amount of polymerized tubulin in both mouse and human.\nC_LIO_LIPolymerized actin is required for ADP-induced marginal band coiling.\nC_LIO_LIUpon activation, the marginal band exhibits a reversible visco-elastic response involving shortening.\nC_LIO_LILarger marginal bands have a higher propensity to coil than shorter ones.\nC_LI\n\nIn briefThe cytoskeleton is adapted to platelet size and its mechanical properties determine propensity of a platelet to undergo morphological changes in response to agonists.

cell biology↗

bioRxiv2018

Topological data analysis quantifies biological nano-structure from single molecule localization microscopy

The study of complex molecular organisation and nano-structure by localization based microscopy is limited by the available analysis tools. We present a segmentation protocol which, through the application of persistence based clustering, is capable of probing densely packed structures which vary in scale. An increase in segmentation performance over state-of-the-art methods is demonstrated. Moreover we employ persistent homology to move beyond clustering, and quantify the topological structure within data. This provides new information about the preserved shapes formed by molecular architecture. Our methods are flexible and we demonstrate this by applying them to receptor clustering in platelets, nuclear pore components and endocytic proteins. Both 2D and 3D implementations are provided within RSMLM, an R package for pointillist based analysis and batch processing of localization microscopy data.

bioinformatics↗

bioRxiv2018

Super-resolution fight club: A broad assessment of 2D & 3D single-molecule localization microscopy software

With the widespread uptake of 2D and 3D single molecule localization microscopy, a large set of different data analysis packages have been developed to generate super-resolution images. To guide researchers on the optimal analytical software for their experiments, we have designed, in a large community effort, a competition to extensively characterise and rank these options. We generated realistic simulated datasets for popular imaging modalities - 2D, astigmatic 3D, biplane 3D, and double helix 3D - and evaluated 36 participant packages against these data. This provides the first broad assessment of 3D single molecule localization microscopy software, provides a holistic view of how the latest 2D and 3D single molecule localization software perform in realistic conditions, and ultimately provides insight into the current limits of the field.

biophysics↗

bioRxiv2018

Nearshore corals on the Mesoamerican Barrier Reef System on pace to cease growing as soon as year 2110

Anthropogenic global change and local anthropogenic stressors are decreasing coral growth and survival globally, thus altering the structure and function of coral reef ecosystems. We show that skeletal extension rates of nearshore colonies of Siderastrea siderea and Pseudodiploria strigosa across the Belize Mesoamerican Barrier Reef System (MBRS) have declined at average rates of 0.01 and 0.08 mm/yr, respectively, over approximately the past century, while offshore conspecifics exhibited no significant trend in extension with time. This caused extension rates of nearshore colonies to converge with their historically slower-growing offshore conspecifics. Bleaching events negatively impacted extension rates in S. siderea but not in P. strigosa. The more negative trend in linear extension for nearshore versus offshore colonies may arise from ocean warming combined with stronger land-based anthropogenic stressors within nearshore environments. Extrapolating these trends in linear extension into the future suggests that nearshore P. strigosa and S. siderea will cease growing by years 2110 and 2370, respectively.

ecology↗

bioRxiv2017

Systematic analysis of the molecular architecture of endocytosis reveals a nanoscale actin nucleation template that drives efficient vesicle formation

Clathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. We found that proteins assemble by radially-ordered recruitment according to function. WASP family proteins form a circular nano-scale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template creates sufficient force for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.

cell biology↗

bioRxiv2017

Fast, robust and precise 3D localization for arbitrary point spread functions

We present a fitter for 3D single-molecule localization of arbitrary, experimental point spread functions (PSFs) that reaches minimum uncertainty for EMCCD and sCMOS cameras, and achieves more than 105 fits/s. We provide tools to robustly model experimental PSFs and correct for depth induced aberrations, which allowed us to achieve an unprecedented 3D resolution with engineered astigmatic PSFs, and acquire high quality 3D superresolution images even on standard microscopes without 3D optics.

biophysics↗

bioRxiv2017

A real-time compression library for microscopy images

Fluorescence imaging techniques such as single molecule localization microscopy, high-content screening and light-sheet microscopy are producing ever-larger datasets, which poses increasing challenges in data handling and data sharing. Here, we introduce a real-time compression library that allows for very fast (beyond 1 GB/s) compression and de-compression of microscopy datasets during acquisition. In addition to an efficient lossless mode, our algorithm also includes a lossy option, which limits pixel deviations to the intrinsic noise level of the image and yields compression ratio of up to 100-fold. We present a detailed performance analysis of the different compression modes for various biological samples and imaging modalities.

bioinformatics↗