Biology subjects

Lewis, K. J.

Publications and source records attributed to Lewis, K. J..

11 recordsLinked to original sources

bioRxiv2026

Chromosome-level genome assemblies and annotations of Amaranthus spinosus, Amaranthus acanthochiton, Amaranthus arenicola, and Amaranthus floridanus

Amaranthus L. spans aggressive agricultural weeds, ornamentals, and ancient pseudocereals. Species within the genus vary in morphology, environmental tolerance, and sexual systems, making them well-suited for studying reproductive evolution and plant adaptation. To investigate sex chromosome architecture within the genus, we generated chromosome-level assemblies of a monoecious amaranth (Amaranthus spinosus) and three dioecious species (A. acanthochiton, A. arenicola, and A. floridanus) using PacBio high-fidelity (HiFi) long reads. We paired these data with Dovetail Genomics Omni-C sequencing to achieve haplotype resolution for A. spinosus and A. acanthochiton, and we used reference-guided scaffolding for the remaining two species. The assemblies are highly contiguous, with sizes ranging from 394.24 to 607.10 Mbp, contig N50 from 0.63 to 8.76 Mbp, and scaffold N50 from 22.44 to 37.97 Mbp. Evaluation of the assemblies and annotations revealed 96.3 to 97.6%, and 97.6 to 98.3% BUSCO completeness, respectively. Comparative genomic analysis revealed that the Chromosome 1 inversions and Robertsonian fusion previously reported in A. tuberculatus are conserved in A. acanthochiton and consistent with the architecture of A. arenicola and A. floridanus, suggesting that the evolution of dioecy in this clade predates subsequent speciation. In parallel, multiple homologs of Rf1 on Chromosome 3 of A. spinosus, a monoecious species that exhibits spatial separation of male and female flowers and is closely related to the dioecious A. palmeri, were identified. Together, this study provides foundational resources for advancing evolutionary, ecological, and agronomic research across the genus, including herbicide resistance evolution and weediness traits.

genomics↗

bioRxiv2026

Depth-Dependent Advantages of Three-Photon Microscopy for Imaging Through Intact Murine Cortical Bone and Into the Marrow

Bone adapts to its mechanical and physiological environment through the coordinated actions of osteocytes and marrow-resident cell populations. Because these processes depend on complex interactions within native tissue microenvironments, intravital imaging offers a unique opportunity to reveal cellular behaviors that cannot be fully captured ex vivo. However, the highly scattering nature of mineralized bone limits imaging depth and direct observation of cells within intact tissue. Two-photon (2P) microscopy has enabled important advances in bone biology, while three-photon (3P) microscopy has been proven to extend imaging depth and image quality in skeletal tissues. Here, we directly compared the performance of 2P and 3P microscopy in ex vivo mouse long bones by quantifying laser attenuation, signal-to-noise ratio, signal-to-background ratio, and spatial resolution, as well as assessed the impact of wave-front correction. We found that 2P and 3P microscopy generated comparable image quality through approximately 50 {micro}m of cortical bone. Beyond this depth, however, 3P microscopy provided superior brightness, contrast, and resolution, enabling improved visualization of structures deep within the cortex and at the cortical-marrow interface. To assess compatibility with intravital imaging, we evaluated endogenous markers of cellular stress during 3P imaging. Although prolonged continuous imaging decreased osteocyte spontaneous calcium signaling magnitude and increased autofluorescence, short intermittent imaging bouts produced negligible evidence of cellular damage compared to controls. Leveraging the enhanced penetration depth of 3P microscopy, we further show visualization of immune cell migration within the marrow cavity through intact cortical bone in both the third metatarsal (MT3) and tibia. Together, these findings affirm 3P microscopy as a powerful tool for studying cellular dynamics in living bone. By extending imaging beyond superficial cortical regions and enabling direct visualization of marrow-resident cells through intact bone, 3P microscopy expands opportunities to investigate osteocyte biology, marrow niche function, and skeletal adaptation in vivo.

bioengineering↗

bioRxiv2026

Intravital Cholesterol Depletion Reduces Membrane Dynamics and Increases Mechanosensitivity in Osteocytes In Vivo

Osteocytes detect mechanical forces within bone through signaling processes organized at the plasma membrane. Membrane cholesterol regulates membrane organization, dynamics, and mechanical properties, yet its role in osteocyte mechanotransduction in vivo remains unknown. Here, we developed an intravital multiphoton imaging approach to quantify membrane-associated uptake, retention, and clearance alongside load-induced Ca2+ signaling in osteocytes within the intact metatarsal. Using fluorescent nanoparticles and a membrane-labeling probe, we tracked these processes before and after pharmacological cholesterol depletion. Cholesterol depletion reduced osteocyte membrane uptake and retention and altered clearance in both sexes, while affecting load-induced Ca2+ signaling in a sex-dependent manner. In females, cholesterol depletion increased both the proportion of osteocytes responding to mechanical loading and the magnitude of their responses, whereas neither outcome changed in males. These findings identify plasma membrane cholesterol as a regulator of osteocyte membrane dynamics and mechanical responsiveness in vivo. More broadly, this work establishes an approach for directly examining how membrane composition and turnover regulate mechanotransduction in cells embedded within their native tissue environment.

cell biology↗

bioRxiv2026

Real-Time Axial Motion Compensation for Intravital Two-Photon Imaging of Mechanically Loaded Bone

Motion artifacts present a major challenge for intravital imaging of tissues undergoing physiological movement or mechanical loading. Blurring or artificial changes in image intensity due to shifting on the z-axis reduce data reproducibility and reliability. Existing post-acquisition approaches can partially compensate for motion, but they increase experimental complexity and are not well suited for use in mechanically loaded bone. Therefore, we developed a novel method for real-time correction of axial motion during mechanical loading of bone by synchronizing the movement of the objective to the actuator. Synchronization was achieved by linking the position of the actuator piezo motor to the objective piezo motor with a user refined reduction via potentiometer. Applying axial motion correction effectively removed artificial changes in fluorescent intensity in a static fluorescent marker up to 3000{varepsilon} in bone as measured by similarity and average intensity before and during loading. This improvement was reflected in the improved accuracy in capture of a dynamic fluorescent calcium indicator (GCaMP6f) in osteocytes. Our system provides a user-friendly, robust framework that can be easily adapted to other mechanically loaded tissues, improving data collection and expanding the utility of two-photon imaging across a variety of biological applications.

bioengineering↗

bioRxiv2024

Intestinal stem cells enhance local mucosal immunity through apoptotic body phagocytosis

Modulation of immune tone at mucosal surfaces is critical to maintain homeostasis while facilitating the handling of emerging threats. One dynamic component of immune modulation is the phagocytosis and clearance of apoptotic bodies known as efferocytosis that inhibits inflammation by promoting its resolution. Here, we evaluated the effects of apoptotic body phagocytosis by intestinal epithelial stem and progenitor cells (ISCs). Unexpectedly, instead of immunomodulation through efferocytosis, this process elevated local immune system activity. To achieve this result, ISCs actively engaged apoptotic bodies in a unique fashion, leading to their engulfment and ultimate delivery to lysosomes for processing. We found that ISCs were capable of actively recruiting inert material such as apoptotic bodies by using actin-based intrinsic biomechanical processes. Uptake of apoptotic bodies was facilitated by complement factor C3 produced by apoptotic bodies themselves. ISCs in turn generated signals heightening T cell activity that was driven in part by ISC-generated TNF. Taken together, uptake of apoptotic bodies by ISCs produced a local inflammatory alert to specific immune cells. This altered paradigm for the response to phagocytosed apoptotic bodies fits the needs of active mucosal surfaces and demonstrates that efferocytosis as currently defined is not a universal response of all cell types.

cell biology↗

bioRxiv2024

Ligand-Mediated Endocytosis Is Regulated in a Sexually Dimorphic Way in Osteocytes in vivo

Endocytosis is a critical cellular process involved in many physiological functions. Most research on endocytosis has been performed in vitro, however, understanding this process in vivo is necessary in tissues like bone that have a unique 3D extra-cellular matrix. Here, we present a live-cell study of endocytosis in osteocytes, mechanosensory cells embedded in mouse bone. We visualized real-time fluorescent nanoparticle uptake and trafficking in osteocytes by using intravital imaging combined with multiphoton microscopy within living animals. We applied pharmacologic inhibitors to distinguish between general and receptor-specific endocytosis pathways in vivo. Our findings reveal rapid nanoparticle uptake in osteocytes, with marked differences in the timescale and pattern of uptake depending on nanoparticle surface functionality. We also discovered differences in dynamin-dependent endocytosis in osteocytes between male and female animals. These results offer the first in vivo derived insights into how osteocytes take up materials and provide new evidence for chemically altering receptor-mediated endocytosis in live bone tissue.

bioengineering↗

bioRxiv2024

Novel Approach for Refractive Index Matching in Calcified Tissues

Tissue-clearing techniques have become indispensable in biomedical research, especially for three-dimensional (3D) imaging. These techniques enable the visualization of complex biological structures by rendering tissues transparent while preserving their structural integrity. Calcified tissues, such as bone and cartilage, pose a unique challenge in imaging studies due to their inherent opacity and rigidity. In this work, we introduce a novel modification to the BABB clearing protocol aimed at enhancing the clearing efficacy specifically for calcified tissues. Through this work, we provide an enhanced tissue-clearing method that addresses the specific challenges associated with calcified tissues. This advancement has the potential to facilitate more precise and comprehensive studies in fields such as developmental biology, orthopedics, and regenerative medicine.

cell biology↗

bioRxiv2023

Nicotinic Acetylcholine Receptors on Osteocytes Impact Bone Mechanoadaptation in a Sexually Dimorphic Manner

Recent evidence suggests acetylcholine has a positive influence on bone mechanotransduction. Osteocytes express components for nicotinic acetylcholine receptors (nAChRs), which are known for mediating calcium signaling and may impact mechanosensitivity. Here, we use novel fluorescent imaging approaches to provide the first evidence of direct interaction between osteocytes and cholinergic nerve fibers in cortical bone in vivo. Moreover, we show that osteocytes are functional targets of cholinergic signaling for bone mechanoadaptation. We report sexually dimorphic patterns in bone structure and mechanobiology based on nAChR function. In females, osteocyte mechanosensitivity was decreased at small force magnitudes and tissue level deficits were recovered with anabolic loading. In males, osteocyte mechanosensitivity was increased in some groups and anabolic loading had very little effect on overall tissue architecture. This work establishes a new signaling paradigm wherein osteocytes interface with cholinergic nerves and bone mechanotransduction is regulated by osteocyte cholinergic signaling in a sexually dimorphic way.

physiology↗

bioRxiv2023

Solute Transport in Engineered Living Materials using Bone Inspired Microscale Channel Networks

Engineered living materials (ELMs) are an emerging class of materials that is synthesized and/or populated by living cells. Maintaining living cells within an ELM over prolonged periods remains a major technical challenge that limits the service life of a material. Biological materials regularly maintain robust populations of living cells. Bone maintains living cells for decades by delivering nutrients through a network of nanoscale channels punctuated by microscale pores. Nutrient transfer in bone is enabled by mechanical loading experienced during regular use. Here we identify the characteristics of channel-pore network geometries and external mechanical loading that can be used in engineered living materials to deliver nutrients to resident cell populations. Transport occurs when deformation in the microscale pore network exceeds the volume of the connecting channels. Computational models show that transport is enhanced at greater load magnitudes and lower loading frequencies and are consistent with experimental validation using microfluidic systems. Our findings provide quantitative design principles for channel-pore networks capable of delivering nutrients to materials designed to house living cells.

bioengineering↗

bioRxiv2023

Intravital imaging of osteocyte αvβ3 integrin dynamics with locally injectable fluorescent nanoparticles

Osteocytes are the resident mechanosensory cells in bone. They are responsible for skeletal homeostasis and adaptation to mechanical cues. Integrin proteins play an prominent role in osteocyte mechanotransduction, however the details are not well stratified in vivo. Intravital imaging with multiphoton microscopy presents an opportunity to study molecular level mechanobiological events in vivo, and could be used to study integrin dynamics in osteocytes. However, fluorescent imaging limitations with respect to excessive optical scattering and low signal to noise ratio caused by mineralized bone matrix make such investigations non-trivial. Here we demonstrate that ultra-small and bright fluorescent core-shell silica nanoparticles (<7nm diameter), known as Cornell Prime Dots (CDots), are well-suited for the in vivo bone microenvironment and can improve intravital imaging capabilities. We report validation studies for CDots as a novel, locally injected in vivo osteocyte imaging tool for both non-specific cellular uptake and for targeting integrins. The pharmacokinetics of CDots reveal distinct sex differences in nanoparticle cycling and clearance in osteocytes, which represents a novel topic of study in bone biology. Integrin-targeted CDots were used to study osteocyte integrin dynamics. To the best of our knowledge, we report here the first evidence of osteocyte integrin endocytosis and recycling in vivo. Our results provide novel insights in osteocyte biology and will open up new lines of investigation that were previously unavailable in vivo.

bioengineering↗

bioRxiv2022

Gabapentin Disrupts Binding of Perlecan to the α2δ1 Voltage Sensitive Calcium Channel Subunit and Impairs Skeletal Mechanosensation

Our understanding of how osteocytes, the principal mechanosensors within bone, sense and perceive force remains unclear. Previous work identified "tethering elements" (TEs) spanning the pericellular space of osteocytes and transmitting mechanical information into biochemical signals. While we identified the heparan sulfate proteoglycan perlecan (PLN) as a component of these TEs, PLN must attach to the cell surface to induce biochemical responses. As voltage-sensitive calcium channels (VSCCs) are critical for bone mechanotransduction, we hypothesized that PLN binds the extracellular 2{delta}1 subunit of VSCCs to couple the bone matrix to the osteocyte membrane. Here, we showed co-localization of PLN and 2{delta}1 along osteocyte dendritic processes. Additionally, we quantified the molecular interactions between 2{delta}1 and PLN domains and demonstrated for the first time that 2{delta}1 strongly associates with PLN via its domain III. Furthermore, 2{delta}1 is the binding site for the commonly used pain drug, gabapentin (GBP), which is associated with adverse skeletal effects when used chronically. We found that GBP disrupts PLN::2{delta}1 binding in vitro, and GBP treatment in vivo results in impaired bone mechanosensation. Our work identified a novel mechanosensory complex within osteocytes composed of PLN and 2{delta}1, necessary for bone force transmission and sensitive to the drug GBP. This work provides insights into the mechanisms underlying mechanotransduction and will inform future studies to understand the mechanisms responsible for the negative effects of GBP on bone.

molecular biology↗