Biology subjects

Dutka, P.

Publications and source records attributed to Dutka, P..

17 recordsLinked to original sources

bioRxiv2026

CryoSHIELDs: tunable amphipathic peptide additives for cryo-EM

While cryogenic electron microscopy (cryo-EM) has transformed structural biology, sample preparation remains a major determinant of success. Specifically, the air-water interface (AWI) can damage complexes, impose preferred orientation, and reduce particle density. Here, we introduce cryoSHIELDs, a chemically diverse library of peptide additives designed to passivate the AWI and improve cryo-EM grid preparation. Across 24 protein systems spanning GPCRs, ABC and SLC transporters, soluble and Fab-containing complexes, and enzymes, cryoSHIELDs increased the success rate of structure determination. Individual cryoSHIELDs improved particle density, angular sampling, complex stability, and, for routine targets, map quality and resolution. This toolkit enabled the structural characterization of important targets, including the ABCC6 transporter, revealing unique TMD0-mediated stabilization, and DAMGO-bound -opioid receptor-Gi complex using substantially lower protein concentration. CryoSHIELDs provide a simple, tunable, and broadly applicable platform for more reliable cryo-EM in discovery biology and structure-based drug development.

biophysics↗

bioRxiv2026

A scalable human neuron model of Alzheimer's disease relevant tauopathy reveals mechanisms linking Tau fibrillization to synaptic dysfunction

Tauopathies, including Alzheimer's disease, are driven by pathological aggregation of hyperphosphorylated Tau, which disrupts synaptic integrity, impairs neuronal communication, and contributes to cognitive decline. To dissect tauopathy pathogenesis and enable therapeutic discovery, reliable and scalable human iPSC-neuron models are essential. Here, we developed two complementary iPSC-derived neuron models: an endogenous Tau seeding model, in which neurons are challenged with pre-formed Tau fragments that form paired helical filament (PHF)-consistent structures, and a Tau-0N3R overexpression seeding model to accelerate pathology. Both models recapitulate hallmark features of tauopathy, including the progressive formation of intracellular, hyperphosphorylated, sarkosyl-insoluble, and conformationally altered Tau aggregates (AT8, MC1 positive), along with synaptic and neuronal dysfunction. Cryogenic electron tomography (cryo-ET) further revealed the morphology of Tau fibrils within cells, as well as the ultrastructure of Tau fibrils trapping synaptic vesicles in situ. Using this platform, we performed integrated phosphoproteomics, high-content screening, and functional validation to identify key pathways driving Tau aggregation. MARK2-mediated phosphorylation within Tau's microtubule-binding domain emerged as an early trigger of aggregation, confirmed by site-specific mutagenesis. In parallel, small molecules targeting the PI3K/mTOR/GSK3 pathway reduced aggregation and restored synaptic function, with GSK3 inhibition lowering phosphorylation at critical aggregation-driving sites on Tau. Together, these findings establish a physiologically relevant, scalable platform for therapeutic screening that connects Tau seed uptake, site-specific phosphorylation, fibril formation, and synaptic disruption, ultimately identifying mechanistically separable intervention points along the aggregation cascade.

cell biology↗

bioRxiv2025

Sono-uncaging for Spatiotemporal Control of Chemical Reactivity

Photo-uncaging - the use of light to reveal the active part of a chemical compound by photolysis of a protecting group - has long been used to study and actuate biochemical processes. However, light scattering limits the applications of photo-uncaging in opaque specimens or tissues. Here, we introduce sono-uncaging, a process in which a chemical functional group becomes exposed upon the application of ultrasound, which can be applied and focused in optically opaque materials. We engineered gas vesicles (GVs), air-filled protein nanostructures sensitive to ultrasound, to contain cysteines on their concealed inner surface, hypothesizing that the application of ultrasound would collapse the GV shell and reveal the cysteines. The resulting SonoCage construct reacted with monobromobimane (mBBr), a fluorogenic, thiol-reactive molecule, only after treatment with ultrasound, establishing the sono-uncaging proof of concept. We then demonstrated the spatial patterning capability of sono-uncaging by embedding the SonoCages in an mBBr-containing hydrogel and creating fluorescent patterns with phased array ultrasound. This patterning could be accomplished using a diagnostic imaging transducer. This work establishes sono-uncaging as a method for spatiotemporal control over chemical reactivity using widely available ultrasound technology.

bioengineering↗

bioRxiv2024

Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening

A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 {micro}m.1 Within the past decade, reporter genes for US have been introduced2,3 and engineered4,5 to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs).6 Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semi-automated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologs of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

bioengineering↗

bioRxiv2024

Structural analysis and conformational dynamics of a holo-adhesion GPCR reveal interplay between extracellular and transmembrane domains

Adhesion G Protein-Coupled Receptors (aGPCRs) are key cell-adhesion molecules involved in numerous physiological functions. aGPCRs have large multi-domain extracellular regions (ECR) containing a conserved GAIN domain that precedes their seven-pass transmembrane domain (7TM). Ligand binding and mechanical force applied on the ECR regulate receptor function. However, how the ECR communicates with the 7TM remains elusive, because the relative orientation and dynamics of the ECR and 7TM within a holoreceptor is unclear. Here, we describe the cryo-EM reconstruction of an aGPCR, Latrophilin3/ADGRL3, and reveal that the GAIN domain adopts a parallel orientation to the membrane and has constrained movement. Single-molecule FRET experiments unveil three slow-exchanging FRET states of the ECR relative to the 7TM within the holoreceptor. GAIN-targeted antibodies, and cancer-associated mutations at the GAIN-7TM interface, alter FRET states, cryo-EM conformations, and receptor signaling. Altogether, this data demonstrates conformational and functional coupling between the ECR and 7TM, suggesting an ECR-mediated mechanism of aGPCR activation.

biochemistry↗

bioRxiv2023

Bioorthogonal labeling enables in situ fluorescence imaging of expressed gas vesicle nanostructures

Gas vesicles (GVs) are proteinaceous nanostructures that, along with virus-like particles, encapsulins, nano-cages, and other macromolecular assemblies are being developed for potential biomedical applications. To facilitate such development, it would be valuable to characterize these nanostructures sub-cellular assembly and localization. However, traditional fluorescent protein fusions are not tolerated by GVs primary constituent protein, making optical microscopy a challenge. Here, we introduce a method for fluorescently visualizing intracellular GVs using the bioorthogonal label FlAsH, which becomes fluorescent upon binding the six-amino acid tetracysteine (TC) tag. We engineered the GV subunit protein, GvpA, to display the TC tag, and showed that GVs bearing TC-tagged GvpA can be successfully assembled and fluorescently visualized in HEK 293T cells. We used fluorescence images of the tagged GVs to study GV size and distance distributions within these cells. This bioorthogonal labeling approach will enable research to provide a greater understanding of GVs and could be adapted to similar proteinaceous nanostructures.

synthetic biology↗

bioRxiv2023

Ultrasonic reporters of calcium for deep tissue imaging of cellular signals

Calcium imaging has enabled major biological discoveries. However, the scattering of light by tissue limits the use of standard fluorescent calcium indicators in living animals. To address this limitation, we introduce the first genetically encoded ultrasonic reporter of calcium (URoC). Based on a unique class of air-filled protein nanostructures called gas vesicles, we engineered URoC to produce elevated nonlinear ultrasound signal upon binding to calcium ions. With URoC expressed in mammalian cells, we demonstrate noninvasive ultrasound imaging of calcium signaling in vivo during drug-induced receptor activation. URoC brings the depth and resolution advantages of ultrasound to the in vivo imaging of dynamic cellular function and paves the way for acoustic biosensing of a broader variety of biological signals.

bioengineering↗

bioRxiv2023

Truly tiny acoustic biomolecules for ultrasound imaging and therapy

Nanotechnology offers significant advantages for medical imaging and therapy, including enhanced contrast and precision targeting. However, integrating these benefits into ultrasonography has been challenging due to the size and stability constraints of conventional bubble-based agents. Here we describe bicones, truly tiny acoustic contrast agents based on gas vesicles, a unique class of air-filled protein nanostructures naturally produced in buoyant microbes. We show that these sub-80 nm particles can be effectively detected both in vitro and in vivo, infiltrate tumors via leaky vasculature, deliver potent mechanical effects through ultrasound-induced inertial cavitation, and are easily engineered for molecular targeting, prolonged circulation time, and payload conjugation.

synthetic biology↗

bioRxiv2023

Structure and Function of the Dot/Icm T4SS

The Legionella pneumophila Dot/Icm type IV secretion system (T4SS) delivers effector proteins into host cells during infection. Despite its significance as a potential drug target, our current understanding of its atomic structure is limited to isolated subcomplexes. In this study, we used subtomogram averaging and integrative modeling to construct a nearly-complete model of the Dot/Icm T4SS accounting for seventeen protein components. We locate and provide insights into the structure and function of six new components including DotI, DotJ, DotU, IcmF, IcmT, and IcmX. We find that the cytosolic N-terminal domain of IcmF, a key protein forming a central hollow cylinder, interacts with DotU, providing insight into previously uncharacterized density. Furthermore, our model, in combination with analyses of compositional heterogeneity, explains how the cytoplasmic ATPase DotO is connected to the periplasmic complex via interactions with membrane-bound DotI/DotJ proteins. Coupled with in situ infection data, our model offers new insights into the T4SS-mediated secretion mechanism.

biochemistry↗

bioRxiv2022

Structural remodeling of Coxiella burnetii during its biphasic developmental cycle revealedby cryo-electron tomography

Coxiella burnetii is an obligate zoonotic bacterium that targets macrophages to cause a disease known as Q fever. It has a biphasic developmental lifecycle where the extracellular and metabolically inactive small cell variant (SCV) transforms, under host acidic environment, into the vegetative large cell variant (LCV). However, the details about the morphological and structural changes that accompany this biphasic cycle are still lacking. Here, we used cryo-electron tomography to image the different cell variants of C. burnetii grown either under axenic conditions in different pH or purified directly from host cells revealing the major developmental, morphological and structural transitions. We show that SCVs are characterized by equidistant stacks of inner membrane that presumably allow a smooth transition to LCV, a transition coupled with the expression of the Dot/Icm type IVB secretion system (T4BSS). A class of T4BSS particles were associated with extracellular densities including a tubular structure possibly involved in host interaction or effector delivery. Also, SCVs and cells in the transition state contained spherical multilayered membrane structures of different sizes and locations suggesting that they are not related to a sporulation process as once assumed.

microbiology↗

bioRxiv2022

Isoform- and ligand-specific modulation of the adhesion GPCR ADGRL3/Latrophilin3 by a synthetic binder

Adhesion G protein-coupled receptors (aGPCRs) are cell-surface proteins with large extracellular regions that bind to multiple ligands to regulate key biological functions including neurodevelopment and organogenesis. Modulating a single function of a specific aGPCR isoform while affecting no other function and no other receptor is not trivial. Here, we engineered an antibody, termed LK30, that binds to the extracellular region of the aGPCR ADGRL3, and specifically acts as an agonist for ADGRL3 but not for its isoform, ADGRL1. The LK30/ADGRL3 complex structure revealed that the LK30 binding site on ADGRL3 overlaps with the binding site for an ADGRL3 ligand - teneurin. In cellular-adhesion assays, LK30 specifically broke the trans-cellular interaction of ADGRL3 with teneurin, but not with another ADGRL3 ligand - FLRT3. Our work provides proof of concept for the modulation of isoform- and ligand-specific aGPCR functions using unique tools, and thus establishes a foundation for the development of fine-tuned aGPCR-targeted therapeutics.

biochemistry↗

bioRxiv2022

Geometric effects in gas vesicle buckling under ultrasound

Acoustic reporter genes based on gas vesicles (GVs) have enabled the use of ultrasound to noninvasively visualize cellular function in vivo. The specific detection of GV signals relative to background acoustic scattering in tissues is facilitated by nonlinear ultrasound imaging techniques taking advantage of the sonomechanical buckling of GVs. However, the effect of geometry on the buckling behavior of GVs under exposure to ultrasound has not been studied. To understand such geometric effects, we developed computational models of GVs of various lengths and diameters and used finite element simulations to predict their threshold buckling pressures and post-buckling deformations. We demonstrated that the GV diameter has an inverse cubic relation to the threshold buckling pressure, whereas length has no substantial effect. To complement these simulations, we experimentally probed the effect of geometry on the mechanical properties of GVs and the corresponding nonlinear ultrasound signals. The results of these experiments corroborate our computational predictions. This study provides fundamental insights into how geometry affects the sonomechanical properties of GVs, which, in turn, can inform further engineering of these nanostructures for high-contrast, nonlinear ultrasound imaging. STATEMENT OF SIGNIFICANCEGas vesicles (GVs) are an emerging class of genetically encodable and engineerable imaging agents for ultrasound whose sonomechanical buckling generates nonlinear contrast to enable sensitive and specific imaging in highly scattering biological systems. Though the effect of protein composition on GV buckling has been studied, the effect of geometry has not previously been addressed. This study reveals that geometry, especially GV diameter, significantly alters the threshold acoustic pressures required to induce GV buckling. Our computational predictions and experimental results provide fundamental understanding of the relationship between GV geometry and buckling properties and underscore the utility of GVs for nonlinear ultrasound imaging. Additionally, our results provide suggestions to further engineer GVs to enable in vivo ultrasound imaging with greater sensitivity and higher contrast.

biophysics↗

bioRxiv2022

Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET

Gas vesicles (GVs) are gas-filled protein nanostructures employed by several species of bacteria and archaea as flotation devices to enable access to optimal light and nutrients. The unique physical properties of GVs have led to their use as genetically-encodable contrast agents for ultrasound and MRI. Currently, however, the structure and assembly mechanism of GVs remain unknown. Here we employ cryo-electron tomography to reveal how the GV shell is formed by a helical filament of highly conserved GvpA subunits. This filament changes polarity at the center of the GV cylinder--a site that may act as an elongation center. High-resolution subtomogram averaging reveals a corrugated pattern of the shell arising from polymerization of GvpA into a {beta}-sheet. The accessory protein GvpC forms a helical cage around the GvpA shell, providing structural reinforcement. Together, our results help explain the remarkable mechanical properties of GVs and their ability to adopt different diameters and shapes.

molecular biology↗

bioRxiv2022

Dynamic structural adaptations enable the endobiotic predation of bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is an endobiotic microbial predator that offers promise as a living antibiotic for its ability to kill Gram-negative bacteria, including human pathogens. Even after six decades of study, fundamental details of its predation cycle remain mysterious. Here, we used cryo-electron tomography to comprehensively image the lifecycle of B. bacteriovorus at nanometer-scale resolution. In addition to providing the first high-resolution images of predation in a native (hydrated, unstained) state, we also discover several surprising features of the process, including novel macromolecular complexes involved in prey attachment/invasion and a flexible portal structure lining a hole in the prey peptidoglycan that tightly seals the prey outer membrane around the predator during entry. Unexpectedly, we find that B. bacteriovorus does not shed its flagellum during invasion, but rather resorbs it into its periplasm for degradation. Finally, following replication and division in the bdelloplast, we observe a transient and extensive ribosomal lattice on the condensed B. bacteriovorus nucleoid. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/496000v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@35da74org.highwire.dtl.DTLVardef@19923f1org.highwire.dtl.DTLVardef@106a616org.highwire.dtl.DTLVardef@181f2fb_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Bimodally oriented cellulose fibers and reticulated homogalacturonan networks - A direct visualization of Allium cepa primary cell walls

One hallmark of plant cells is their pecto-cellulosic cell walls. They protect cells against the environment and high turgor and mediate morphogenesis through the dynamics of their mechanical and chemical properties. The walls are a complex polysaccharidic structure. Although their biochemical composition is well known, how the different components organize in the volume of the cell wall and interact with each other is not well understood and yet is key to the walls mechanical properties. To investigate the ultrastructure of the plant cell wall, we imaged the walls of onion (Allium cepa) bulbs in a near-native state via cryo-Focused Ion Beam milling (cryo-FIB-milling) and cryo-Electron Tomography (cryo-ET). This allowed the high-resolution visualization of cellulose fibers in situ (in muro). We reveal the coexistence of dense fiber fields bathed in a reticulated matrix we termed "meshing," which is more abundant at the inner surface of the cell wall. The fibers adopted a regular bimodal angular distribution at all depths in the cell wall and bundled according to their orientation, creating layers within the cell wall. Concomitantly, employing homogalacturonan (HG)-specific enzymatic digestion, we observed changes in the meshing, suggesting that it is at least in part composed of HG pectins. We propose the following model for the construction of the abaxial epidermal primary cell wall: The cell deposits successive layers of cellulose fibers at -45{degrees} and +45{degrees} relative to the cells long axis and secretes the surrounding HG-rich meshing proximal to the plasma membrane, which then migrates to more distal regions of the cell wall.

plant biology↗

bioRxiv2021

Genomically Mined Acoustic Reporter Genes Enable On-Demand In Vivo Monitoring of Tumor-Homing Bacteria

A major outstanding challenge in the fields of biological research, synthetic biology and cell-based medicine is visualizing the function of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and spatial resolution below 100 m. Recently, the first genetically encoded acoustic reporters were developed based on bacterial gas vesicles to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging gene expression in vivo. Here, we describe second-generation ARGs with greatly improved acoustic properties and expression characteristics, identified through a phylogenetic screen of candidate gene clusters from diverse bacteria and archaea. The resulting constructs offer major qualitative and quantitative improvements, including much stronger ultrasound contrast, the ability to produce nonlinear signals distinguishable from background tissue, and stable long-term expression. We demonstrate the capabilities of these next-generation ARGs by imaging in situ gene expression in mouse models of breast cancer and tumor-homing therapeutic bacteria, noninvasively revealing the unique spatial distributions of tumor growth and colonization by therapeutic cells in living subjects and providing real-time guidance for interventions such as needle biopsies.

bioengineering↗

bioRxiv2021

Measuring gas vesicle dimensions by electron microscopy

Gas vesicles (GVs) are cylindrical or spindle-shaped protein nanostructures filled with air and used for flotation by various cyanobacteria, heterotrophic bacteria, and Archaea. Recently, GVs have gained interest in biotechnology applications due to their ability to serve as imaging agents and actuators for ultrasound, magnetic resonance and several optical techniques. The diameter of GVs is a crucial parameter contributing to their mechanical stability, buoyancy function and evolution in host cells, as well as their properties in imaging applications. Despite its importance, reported diameters for the same types of GV differ depending on the method used for its assessment. Here, we provide an explanation for these discrepancies and utilize electron microscopy (EM) techniques to accurately estimate the diameter of the most commonly studied types of GVs. We show that during air drying on the EM grid, GVs flatten, leading to a ~1.5-fold increase in their apparent diameter. We demonstrate that GVs diameter can be accurately determined by direct measurements from cryo-EM samples or alternatively indirectly derived from widths of flat collapsed and negatively stained GVs. Our findings help explain the inconsistency in previously reported data and provide accurate methods to measure GV dimensions.

biochemistry↗