Biology subjects

Shroff, H.

Publications and source records attributed to Shroff, H..

34 records · Page 2Linked to original sources

bioRxiv2022

Plasticity in structure and assembly of SARS-CoV-2 nucleocapsid protein

Worldwide SARS-CoV-2 sequencing efforts track emerging mutations in its spike protein, as well as characteristic mutations in other viral proteins. Besides their epidemiological importance, the observed SARS-CoV-2 sequences present an ensemble of viable protein variants, and thereby a source of information on viral protein structure and function. Charting the mutational landscape of the nucleocapsid (N) protein that facilitates viral assembly, we observe variability exceeding that of the spike protein, with more than 86% of residues that can be substituted, on average by 3-4 different amino acids. However, mutations exhibit an uneven distribution that tracks known structural features but also reveals highly protected stretches of unknown function. One of these conserved regions is in the central disordered linker proximal to the N-G215C mutation that has become dominant in the Delta variant, outcompeting G215 variants without further spike or N-protein substitutions. Structural models suggest that the G215C mutation stabilizes conserved transient helices in the disordered linker serving as protein-protein interaction interfaces. Comparing Delta variant N-protein to its ancestral version in biophysical experiments, we find a significantly more compact and less disordered structure. N-G215C exhibits substantially stronger self-association, shifting the unliganded protein from a dimeric to a tetrameric oligomeric state, which leads to enhanced co-assembly with nucleic acids. This suggests that the sequence variability of N-protein is mirrored by high plasticity of N-protein biophysical properties, which we hypothesize can be exploited by SARS-CoV-2 to achieve greater efficiency of viral assembly, and thereby enhanced infectivity.

biophysics↗

bioRxiv2021

Systematic analysis of C. elegans embryo behavior reveals a stereotyped developmental progression and a rhythmic pattern of behavioral quiescence

AO_SCPLOWBSTRACTC_SCPLOWSystematic analysis of rich behavioral recordings is being used to uncover how circuits encode complex behaviors. Here we apply this approach to embryos. What are the first embryonic behaviors and how do they evolve as early neurodevelopment ensues? To address these questions, we present a systematic description of behavioral maturation for Caenorhabditis elegans embryos. Posture libraries were built using a genetically encoded motion capture suit imaged with light-sheet microscopy and annotated using custom tracking software. Analysis of cell trajectories, postures, and behavioral motifs revealed a stereotyped developmental progression. Early movement is dominated by flipping between dorsal and ventral coiling, which gradually slows into a period of reduced motility. Late-stage embryos exhibit sinusoidal waves of dorsoventral bends, prolonged bouts of directed motion, and a rhythmic pattern of pausing, which we designate slow wave twitch (SWT). Synaptic transmission is required for late-stage motion but not for early flipping nor the intervening inactive phase. A high-throughput behavioral assay and calcium imaging revealed that SWT is elicited by the rhythmic activity of a quiescence-promoting neuron (RIS). Similar periodic quiescent states are seen prenatally in diverse animals and may play an important role in promoting normal developmental outcomes.

neuroscience↗

bioRxiv2021

Cytonemes coordinate asymmetric signaling and organization in the Drosophila muscle progenitor niche

Asymmetric signaling and organization in the stem-cell niche determine stem-cell fates. Here, we investigate the basis of asymmetric signaling and stem-cell organization using the Drosophila wing-disc that creates an adult muscle progenitor (AMP) niche. We show that AMPs extend polarized cytonemes to contact the disc epithelial junctions and adhere themselves to the disc/niche. Niche-adhering cytonemes localize FGF-receptor to selectively adhere to the FGF-producing disc and receive FGFs in a contact-dependent manner. Activation of FGF signaling in AMPs, in turn, reinforces disc-specific cytoneme polarity/adhesion, which maintains their disc-proximal positions. Loss of cytoneme-mediated adhesion promotes AMPs to lose niche occupancy and FGF signaling, occupy a disc-distal position, and acquire morphological hallmarks of differentiation. Discrete AMP organization and diversification patterns are determined by localized expression and presentation patterns of two different FGFs in the wing-disc and their polarized target-specific distribution through niche-adhering cytonemes. Thus, cytonemes are essential for asymmetric signaling and niche-specific AMP organization.

developmental biology↗

bioRxiv2021

Multiview super-resolution microscopy

We enhance the performance of confocal microscopy over imaging scales spanning tens of nanometers to millimeters in space and milliseconds to hours in time, improving volumetric resolution more than 10-fold while simultaneously reducing phototoxicity. We achieve these gains via an integrated, four-pronged approach: 1) developing compact line-scanners that enable sensitive, rapid, diffraction-limited imaging over large areas; 2) combining line-scanning with multiview imaging, developing reconstruction algorithms that improve resolution isotropy and recover signal otherwise lost to scattering; 3) adapting techniques from structured illumination microscopy, achieving super-resolution imaging in densely labeled, thick samples; 4) synergizing deep learning with these advances, further improving imaging speed, resolution and duration. We demonstrate these capabilities on more than twenty distinct fixed and live samples, including protein distributions in single cells; nuclei and developing neurons in Caenorhabditis elegans embryos, larvae, and adults; myoblasts in Drosophila wing imaginal disks; and mouse renal, esophageal, cardiac, and brain tissues.

bioengineering↗

bioRxiv2021

Behavior emerges from unstructured muscle activity in response to neuromodulation

Identifying neural substrates of behavior requires defining actions in terms that map onto brain activity. Brain and muscle activity naturally correlate via the output of motor neurons, but apart from simple movements it has been difficult to define behavior in terms of muscle contractions. By mapping the musculature of the pupal fruit fly and comprehensively imaging muscle activation at single cell resolution, we here describe a multiphasic behavioral sequence in Drosophila. Our characterization identifies a previously undescribed behavioral phase and permits extraction of major movements by a convolutional neural network. We deconstruct movements into a syllabary of co-active muscles and identify specific syllables that are sensitive to neuromodulatory manipulations. We find that muscle activity shows considerable variability, which reduces upon neuromodulation. Our work provides a platform for studying whole-animal behavior, quantifying its variability across multiple spatiotemporal scales, and analyzing its neuromodulatory regulation at cellular resolution.

neuroscience↗

bioRxiv2020

A polymer gel index-matched to water enables diverse applications in fluorescence microscopy

We demonstrate diffraction-limited and super-resolution imaging through thick layers (tens-hundreds of microns) of BIO-133, a biocompatible, UV-curable, commercially available polymer with a refractive index (RI) matched to water. We show that cells can be directly grown on BIO-133 substrates without the need for surface passivation and use this capability to perform extended time-lapse volumetric imaging of cellular dynamics 1) at isotropic resolution using dual-view light-sheet microscopy, and 2) at super-resolution using instant structured illumination microscopy. BIO-133 also enables immobilization of 1) Drosophila tissue, allowing us to track membrane puncta in pioneer neurons, and 2) Caenorhabditis elegans, which allows us to image and inspect fine neural structure and to track pan-neuronal calcium activity over hundreds of volumes. Finally, BIO-133 is compatible with other microfluidic materials, enabling optical and chemical perturbation of immobilized samples, as we demonstrate by performing drug and optogenetic stimulation on cells and C. elegans.

bioengineering↗

bioRxiv2020

A neurite-zippering mechanism, mediated by layer-specific expression of IgCAMs, regulates synaptic laminar specificity in the C. elegans nerve ring neuropil

During development, neurites and synapses segregate into specific neighborhoods or layers within nerve bundles. The developmental programs guiding placement of neurites in specific layers, and hence their incorporation into specific circuits, are not well understood. We implement novel imaging methods and quantitative models to document the embryonic development of the C. elegans brain neuropil, and discover that differential adhesion mechanisms control precise placement of single neurites onto specific layers. Differential adhesion is orchestrated via developmentally-regulated expression of the IgCAM SYG-1, and its partner ligand SYG-2. Changes in SYG-1 expression across neuropil layers result in changes in adhesive forces, which sort SYG-2-expressing neurons. Sorting to layers occurs, not via outgrowth from the neurite tip, but via an alternate mechanism of retrograde zippering, involving interactions between neurite shafts. Our study indicates that biophysical principles from differential adhesion govern neurite placement and synaptic specificity in vivo in developing neuropil bundles.

neuroscience↗

bioRxiv2020

Three-dimensional residual channel attention networks denoise and sharpen fluorescence microscopy image volumes

We demonstrate residual channel attention networks (RCAN) for restoring and enhancing volumetric time-lapse (4D) fluorescence microscopy data. First, we modify RCAN to handle image volumes, showing that our network enables denoising competitive with three other state-of-the-art neural networks. We use RCAN to restore noisy 4D super-resolution data, enabling image capture over tens of thousands of images (thousands of volumes) without apparent photobleaching. Second, using simulations we show that RCAN enables class-leading resolution enhancement, superior to other networks. Third, we exploit RCAN for denoising and resolution improvement in confocal microscopy, enabling [~]2.5-fold lateral resolution enhancement using stimulated emission depletion (STED) microscopy ground truth. Fourth, we develop methods to improve spatial resolution in structured illumination microscopy using expansion microscopy ground truth, achieving improvements of [~]1.4-fold laterally and [~]3.4-fold axially. Finally, we characterize the limits of denoising and resolution enhancement, suggesting practical benchmarks for evaluating and further enhancing network performance.

bioengineering↗

bioRxiv2020

Signaling through polymerization and degradation: Analysis and simulations of T cell activation mediated by Bcl10

The adaptive immune system serves as a potent and highly specific defense mechanism against pathogen infection. One component of this system, the effector T cell, facilitates pathogen clearance upon detection of specific antigens by the T cell receptor (TCR). A critical process in effector T cell activation is transmission of signals from the TCR to a key transcriptional regulator, NF-{kappa}B. The transmission of this signal involves a highly dynamic process in which helical filaments of Bcl10, a key protein constituent of the TCR signaling cascade, undergo competing processes of polymeric assembly and macroautophagy-dependent degradation. Through computational analysis of three-dimensional super-resolution microscopy data, we quantitatively characterized TCR-stimulated Bcl10 filament assembly and length dynamics, demonstrating that filaments become shorter over time. Additionally, we developed an image-based bootstrap-like resampling method to quantitatively demonstrate preferred association between autophagosomes and Bcl10-filament ends and punctate-Bcl10 structures, implying that autophagosome-driven macroautophagy is directly responsible for Bcl10 filament shortening. We probe Bcl10 polymerization-depolymerization dynamics with a stochastic Monte-Carlo simulation of nucleation-limited filament assembly and degradation, and we show that high probabilities of filament nucleation in response to TCR engagement could provide the observed robust, homogeneous, and tunable response dynamic. Furthermore, the speed of autophagic degradation of filaments preferentially at filament ends provides effective regulatory control. Taken together, these data suggest that Bcl10 filament growth and degradation act as an excitable system that provides a digital response mechanism and the reliable timing critical for T cell activation and regulatory processes. Author SummaryThe immune system serves to protect organisms against pathogen-mediated disease. While a strong immune response is needed to eliminate pathogens in host organisms, immune responses that are too robust or too persistent can trigger autoimmune disorders, cancer, and a variety of additional serious human pathologies. Thus, a careful balance of activating and inhibitory mechanisms are necessary to prevent detrimental health outcomes of immune responses. For example, activated effector T cells marshal the immune response and direct killing of pathogen-infected cells; however, effector T cells that are chronically activated can damage and destroy healthy tissue. Here, we study an important internal activation pathway in effector T cells that involves the growth and counterbalancing degradation (via a process called macroautophagy) of filamentous cytoplasmic signaling structures. We utilize image analysis of 3-D super-resolution images and Monte Carlo simulations to study a key signal-transduction protein, Bcl10. We found that the speed of filament degradation has the greatest effect on the magnitude and duration of the response, implying that pharmaceutical interventions aimed at macroautophagy may have substantial impact on effector T cell function. Given that filamentous structures are utilized in numerous immune signaling pathways, our analysis methods could have broad applicability in the signal transduction field.

biophysics↗

bioRxiv2020

Structural and developmental principles of neuropil assembly in C. elegans

Neuropil is a fundamental form of tissue organization within brains1. In neuropils, densely packed neurons synaptically interconnect into precise circuit architecture2,3, yet the structural and developmental principles governing nanoscale precision in bundled neuropil assembly remain largely unknown4-6. Here we use diffusion condensation, a coarse-graining clustering algorithm7, to identify nested circuit structures within the C. elegans cerebral neuropil (called the nerve ring). We determine that the nerve ring neuropil is organized into four tightly bundled strata composed of related behavioral circuits. We demonstrate that the stratified architecture of the neuropil is a geometrical representation of the functional segregation of sensory information and motor outputs, with specific sensory organs and muscle quadrants mapping onto particular neuropil strata. We identify groups of neurons with unique morphologies that integrate information across strata and that create a sophisticated honeycomb-shaped scaffold that encases the strata within the nerve ring. We resolve the developmental sequence leading to stratified neuropil organization through the integration of lineaging and cell tracking algorithms with high resolution light-sheet microscopy, and reveal principles of cell position, migration and hierarchical outgrowth that guide neuropil organization. Our results uncover conserved design principles underlying nerve ring neuropil architecture and function, and a pioneer neuron-based, temporal progression of outgrowth that guides the hierarchical development of the layered neuropil. Our findings provide a blueprint for using structural and developmental approaches to systematically understand neuropil organization within brains.

neuroscience↗

bioRxiv2020

Nanoscale pattern extraction from relative positions of sparse 3D localisations

We present a method for extracting high-resolution ordered features from localisation microscopy data by analysis of relative molecular positions in 2D or 3D. This approach allows pattern recognition at sub-1% protein detection efficiencies, in large and heterogeneous samples, and in 2D and 3D datasets. We used this method to infer ultrastructure of the nuclear pore, the cardiomyocyte Z-disk, DNA origami structures and the centriole.

cell biology↗

bioRxiv2019

Accelerating iterative deconvolution and multiview fusion by orders of magnitude

We describe theoretical and practical advances in algorithm and software design, resulting in ten to several thousand-fold faster deconvolution and multiview fusion than previous methods. First, we adapt methods from medical imaging, showing that an unmatched back projector accelerates Richardson-Lucy deconvolution by at least 10-fold, in most cases requiring only a single iteration. Second, we show that improvements in 3D image-based registration with GPU processing result in speedups of 10-100-fold over CPU processing. Third, we show that deep learning can provide further accelerations, particularly for deconvolution with a spatially varying point spread function. We illustrate the power of our methods from the subcellular to millimeter spatial scale, on diverse samples including single cells, nematode and zebrafish embryos, and cleared mouse tissue. Finally, we show that our methods facilitate the use of new microscopes that improve spatial resolution, including dual-view cleared tissue light-sheet microscopy and reflective lattice light-sheet microscopy.

bioengineering↗

bioRxiv2019

N-WASP regulates the mobility of the B cell receptor and co-receptors during signaling activation

Regulation of membrane receptor mobility is important in tuning the cells response to external signals. This is particularly relevant in the context of immune receptor signaling. The binding of B cell receptors (BCR) to antigen induces B cell receptor activation. While actin dynamics and BCR signaling are known to be linked, the role of actin dynamics in modulating receptor mobility is not well understood. Here, we use single molecule imaging to examine BCR movement during signaling activation and examine the role of actin dynamics on BCR mobility. We use a novel machine learning based method to classify BCR trajectories into distinct diffusive states and show that the actin regulatory protein N-WASP regulates receptor mobility. Constitutive loss or acute inhibition of N-WASP, which is associated with enhanced signaling, leads to a predominance of BCR trajectories with lower diffusivity and is correlated with a decrease in actin dynamics. Furthermore, loss of N-WASP reduces diffusivity of CD19, a stimulatory co-receptor of the BCR but not that of unstimulated Fc{gamma}RIIB, an inhibitory co-receptor. The effect of N-WASP is mirrored by inhibition of the Arp2/3 complex and formins. Our results implicate the dynamic actin network in fine-tuning receptor mobility and receptor-ligand interactions, thereby modulating B cell signaling.

biophysics↗

bioRxiv2017

Single-shot super-resolution total internal reflection fluorescence microscopy

We demonstrate a simple method for combining instant structured illumination microscopy (SIM) with total internal reflection fluorescence microscopy (TIRF), doubling the spatial resolution of TIRF (down to 115 +/-13 nm) and enabling imaging frame rates up to 100 Hz over hundreds of time points. We apply instant TIRF-SIM to multiple live samples, achieving rapid, high contrast super-resolution imaging in close proximity to the coverslip surface.

bioengineering↗

bioRxiv2017

A scheme for 3-dimensional morphological reconstruction and force inference in the early C. elegans embryo

In this study, we present novel schemes for the reconstruction of cellular morphology and the inference of forces in the early C. elegans embryo. We have developed and bench-marked a morphological reconstruction scheme that transforms live-imaging of cellular membranes into a point cloud of smoothed surface patches, which facilitates accurate estimation of membrane curvatures and the angles between membranes.\n\nAssuming an isotropic and homogeneous distribution of tensions along a membrane, we infer a pattern of forces that are 7% deviated from force balance at edges, and 10% deviated from the Young-Laplace relation at membrane faces. We have also demonstrated the stability of our scheme by sensitivity analysis of the coefficient matrices involved and the reproducibility of our image-analysis and force inference pipeline.

biophysics↗

bioRxiv2017

Reflective imaging improves resolution, speed, andcollection efficiency in light sheet microscopy

Light-sheet fluorescence microscopy (LSFM) enables high-speed, high-resolution, gentle imaging of live biological specimens over extended periods. Here we describe a technique that improves the spatiotemporal resolution and collection efficiency of LSFM without modifying the underlying microscope. By imaging samples on reflective coverslips, we enable simultaneous collection of multiple views, obtaining 4 complementary views in 250 ms, half the period it would otherwise take to collect only two views in symmetric dual-view selective plane illumination microscopy (diSPIM). We also report a modified deconvolution algorithm that removes the associated epifluorescence contamination and fuses all views for resolution recovery. Furthermore, we enhance spatial resolution (to < 300 nm in all three dimensions) by applying our method to a new asymmetric diSPIM, permitting simultaneous acquisition of two high-resolution views otherwise difficult to obtain due to steric constraints at high numerical aperture (NA). We demonstrate the broad applicability of our method in a variety of samples of moderate (< 50 m) thickness, studying mitochondrial, membrane, Golgi, and microtubule dynamics in single cells and calcium activity in nematode embryos.

bioengineering↗