Biology subjects

Prahl, L. S.

Publications and source records attributed to Prahl, L. S..

10 recordsLinked to original sources

bioRxiv2026

Synthetic budding morphogenesis by optogenetic receptor tyrosine kinase signaling

The mammalian kidney relies on a branched network of collecting ducts for fluid transport and homeostasis. Replicating this network in vitro would parallelize function in synthetic replacement kidneys, yet current organoids have limited branching capacity. Here, we establish a developmentally-informed strategy to control organoid budding through optogenetic control of a receptor tyrosine kinase, RET. We first show pharmacological manipulation of RET signaling controls the extent of branching in mouse embryonic kidneys and human stem cell-derived kidney organoids. Next, we develop an optogenetic RET receptor (optoRET) that signals in a ligand-independent manner via blue light-mediated clustering. Epithelial cells expressing optoRET reproduce stereotyped RET signaling, scattering, and symmetry breaking in response to blue light. Human kidney organoids undergo budding with controllable orientation in response to spatially patterned optoRET stimulation. Our results establish ligand-free optogenetic control of branching and inspire new synthetic biology strategies for epithelial organoid design. HighlightsGDNF-RET controls branching and tip cell state in mouse and human kidney tissues. OptoRET reproduces endogenous RET signaling and morphogenesis in cell lines. OptoRET enables ligand-free budding in human renal epithelial organoids. Spatially patterned optoRET stimulation controls budding orientation.

bioengineering↗

bioRxiv2024

Engineering kidney developmental trajectory using culture boundary conditions

Kidney explant cultures are traditionally carried out at air-liquid interfaces, which disrupts 3D tissue structure and limits interpretation of developmental data. To overcome this limitation, we developed a 3D culture technique using hydrogel embedding to capture morphogenesis in real time. We show that 3D culture better approximates in vivo-like niche spacing and dynamic tubule tip rearrangement, as well as in vivo-like presentation of branching defects under perturbations to glial cell-derived neurotrophic factor (GDNF)-REarranged during Transfection (RET) tyrosine kinase signaling. We find that the concentration of the embedding matrix influences the number of nephrons per ureteric bud (UB) tip and the spacing between tips. To isolate the effect of specific material properties on explant development, we introduce engineered acrylated hyaluronic acid hydrogels that allow independent tuning of stiffness and adhesion. We find that sufficient stiffness and adhesion are both required to maintain kidney shape. Matrix stiffness has a "Goldilocks effect" on the nephron per UB tip balance centered at [~]2 kPa, while higher matrix adhesion increases nephron per UB tip ratio. Our technique captures large-scale, in vivo-like tissue morphogenesis in 3D, providing a platform suited to contrasting normal and congenital disease contexts. Moreover, understanding the impact of boundary condition mechanics on kidney development benefits fundamental renal research and advances the engineering of next-generation kidney replacement tissues.

developmental biology↗

bioRxiv2024

Measurement of adhesion and traction of cells at high yield (MATCHY) reveals an energetic ratchet driving nephron condensation

Engineering of embryonic strategies for tissue-building has extraordinary promise for regenerative medicine. This has led to a resurgence in interest in the relationship between cell biophysical properties and morphological transitions. However, mapping gene or protein expression data to cell biophysical properties to physical morphogenesis remains challenging with current techniques. Here we present MATCHY (multiplexed adhesion and traction of cells at high yield). MATCHY advances the multiplexing and throughput capabilities of existing traction force and cell-cell adhesion assays using microfabrication and an automated computation scheme with machine learning-driven cell segmentation. Both biophysical assays are coupled with serial downstream immunofluorescence to extract cell type/signaling state information. MATCHY is especially suited to complex primary tissue-, organoid-, or biopsy-derived cell mixtures since it does not rely on a priori knowledge of cell surface markers, cell sorting, or use of lineage-specific reporter animals. We first validate MATCHY on canine kidney epithelial cells engineered for RET tyrosine kinase expression and quantify a relationship between downstream signaling and cell traction. We go on to create a biophysical atlas of primary cells dissociated from the mouse embryonic kidney and use MATCHY to identify distinct biophysical states along the nephron differentiation trajectory. Our data complement expression-level knowledge of adhesion molecule changes that accompany nephron differentiation with quantitative biophysical information. These data reveal an energetic ratchet that explains spatial nephron progenitor cell condensation from the niche as they differentiate, which we validate through agent-based computational simulation. MATCHY offers automated cell biophysical characterization at >104-cell throughput, a highly enabling advance for fundamental studies and new synthetic tissue design strategies for regenerative medicine.

bioengineering↗

bioRxiv2023

Rho/ROCK activity tunes cell compartment segregation and differentiation in nephron-forming niches

Controlling the time and place of nephron formation in vitro would improve nephron density and connectivity in next-generation kidney replacement tissues. Recent developments in kidney organoid technology have paved the way to achieving self-sustaining nephrogenic niches in vitro. The physical and geometric structure of the niche are key control parameters in tissue engineering approaches. However, their relationship to nephron differentiation is unclear. Here we investigate the relationship between niche geometry, cell compartment mixing, and nephron differentiation by targeting the Rho/ROCK pathway, a master regulator of the actin cytoskeleton. We find that the ROCK inhibitor Y-27632 increases mixing between nephron progenitor and stromal compartments in native mouse embryonic kidney niches, and also increases nephrogenesis. Similar increases are also seen in reductionist mouse primary cell and human induced pluripotent stem cell (iPSC)-derived organoids perturbed by Y-27632, dependent on the presence of stromal cells. Our data indicate that niche organization is a determinant of nephron formation rate, bringing renewed focus to the spatial context of cell-cell interactions in kidney tissue engineering efforts.

bioengineering↗

bioRxiv2022

Independent control over cell patterning and adhesion on hydrogel substrates for tissue interface mechanobiology

Replicating organizational principles that establish fine-scale tissue structure is critical to our capacity for building functional replacement tissues. Tissue boundaries such as epithelial-mesenchymal interfaces are engines for morphogenesis in vivo. However, despite a wealth of micropatterning approaches available to control tissue size, shape, and mechanical environment in vitro, fine-scale spatial control of cell composition within tissue constructs remains an engineering challenge. To address this, we augment DNA "velcro" technology for selective patterning of ssDNA-labeled cells with long-term culture on mechanically defined polyacrylamide hydrogels. We co-functionalize photoactive benzophenone-containing polyacrylamide gels (BP-PA gels) with spatially precise ssDNA features that confer temporary cell adhesion and with extracellular matrix (ECM) proteins that confer long-term adhesion. We find that co-functionalization does not compromise ssDNA patterning fidelity or cell capture, nor hydrogel mechanical properties or mechanosensitive fibroblast spreading, enabling mechanobiology studies of precise cell interfaces. We then co-pattern colonies of fibroblasts and epithelial cells to study interface formation and extracellular signal-related kinase (ERK) activity at cellular contacts. Combining DNA velcro and ECM functionalization approaches provides independent control of initial cell placement, adhesion, and mechanical environment, constituting a new tool for studying biological interfaces and for programming multicellular interactions in engineered tissues.

bioengineering↗

bioRxiv2022

Tubule jamming in the developing kidney creates cyclical mechanical stresses instructive to nephron formation

The kidney develops through branching of progressively crowded ureteric bud (UB) tubules at the organ surface. The elongating tubule tips are surrounded by traveling cap mesenchyme niches consisting of nephron progenitors and separated by stromal boundaries. Dynamic interactions between these tissues coordinate a balance between UB tip branching, elongation, and nephron induction that sets nephron numbers for life, impacting the likelihood of adult disease. Such a crowded tissue environment could place geometric limits on the number of niches that can be formed while maintaining mechanical integrity of the tissue. Since space is at a premium, crowding could also force a given niche to prioritize between nephron formation or UB branching differently depending on its spatial context. Here we study the geometric and mechanical consequences of tubule tip crowding at the embryonic kidney surface. Organ curvature reduces and tubule tip domain niches pack more closely over developmental time. These together create a semi-crystalline geometry of tips at the kidney surface and a rigidity transition to more solid-like tissue properties at later developmental stages. To infer mechanical dynamics over the branching timescale, we define a new method to infer tip domain ages relative to their most recent branch events from fixed kidneys. We find that new tip domains overcome mechanical resistance as they branch and displace close-packed neighbors, transiently increasing mechanical stress in the niche. Ongoing efforts to understand geometric and mechanical effects on niche regulation will clarify variation in kidney tissue composition and advance engineering control strategies for synthetic regenerative tissues.

bioengineering↗

bioRxiv2021

The developing kidney actively negotiates geometric packing conflicts to avoid defects

The physiological functions of several organs rely on branched tubular networks, but little is known about conflicts in development between building enough tubules for adequate function and geometric constraints imposed by organ size. We show that the mouse embryonic kidney epithelium negotiates a physical packing conflict between tubule tip duplication and limited area at the organ surface. Imaging, computational, and soft material modeling of tubule families identifies six geometric packing phases, including two defective ones. Experiments in kidney explants show that a retrograde tension on tubule families is necessary and sufficient for them to avoid defects by switching to a vertical orientation that increases packing density. These results reveal developmental contingencies in response to physical limitations, and create a framework for classifying kidney defects. One-Sentence SummaryEpithelial branching in the kidney causes a geometric packing conflict that is resolved through internally generated tensions

developmental biology↗

bioRxiv2019

Kinomorphs: Shape-shifting tissues for developmental engineering

Current methods for building tissues usually start with a non-biological blueprint, or rely on self-organization, which does not extend to organ-scales. This has limited the construction of large tissues that simultaneously encode fine-scale cell organization. Here we bridge scales by mimicking developmental dynamics using \"kinomorphs\", tissue scaffolds that undergo globally programmed shape and density changes to trigger local self-organization of cells in many locations at once. In this first report, we focus on mimicking the extracellular matrix (ECM) compaction and division into leaflets that occurs in kidney collecting duct development. We start by creating single-cell resolution cell patterns in ECM-mimetic hydrogels that are >10x larger than previously described, by leveraging photo-lithographic technology. These patterns are designed to mimic the branch geometry of the embryonic kidney collecting duct tree. We then predict the shape dynamics of kinomorphs driven by cell contractility-based compaction of the ECM using kinematic origami simulations. We show that these dynamics spur centimeter-scale assembly of structurally mature ~50 m-diameter epithelial tubules that are locally self-organized, but globally programmed. Our approach prescribes tubule network geometry at ~5x smaller length-scales than currently possible using 3D printing, and at local cell densities comparable to in vivo tissues. Kinomorphs could be used to scaffold and \"plumb\" arrays of organoids in the future, by guiding the morphogenesis of epithelial networks. Such hybrid globally programmed/locally self-organized tissues address a major gap in our ability to recapitulate organ-scale tissue structure.\n\nSignificance StatementEngineers are attempting to build tissues that mimic human diseases outside of the body. Although stem cells can be coaxed to form small organoids with a diversity of cell types, they do not properly organize over large distances by themselves. We report a strategy to mimic developmental processes using dynamic materials that attempt to guide a cellular \"blueprint\" towards a more complex tissue endpoint. We call these materials kinomorphs, combining the Greek kino (propel, drive) and morfi (form, shape), since they seek to shepherd both the shape and developmental trajectory of cell collectives within them. Kinomorphs could pave the way towards organ-scale synthetic tissues built through a hybrid of engineering and self-organization strategies.

bioengineering↗

bioRxiv2019

Regulation and dynamics of force transmission at individual cell-matrix adhesion bonds

Integrin-based adhesion complexes link the cytoskeleton to the extracellular matrix (ECM) and are central to the construction of multicellular animal tissues. How biological function emerges from the 10s-1000s of proteins present within a single adhesion complex has remained unclear. We used fluorescent molecular tension sensors to visualize force transmission by individual integrins in living cells. These measurements revealed an underlying functional modularity in which integrin class controlled adhesion size and ECM ligand specificity, while the number and type of connections between integrins and F-actin determined the force per individual integrin. In addition, we found that most integrins existed in a state of near-mechanical equilibrium, a result not predicted by existing models of cytoskeletal force transduction. A revised model that includes reversible crosslinks within the F-actin network accounts for this result, and suggests how cellular mechanical homeostasis can arise at the molecular level.

cell biology↗

bioRxiv2018

Glioma cell migration in confined microchannels via a motor-clutch mechanism

Glioma tumor dispersion involves invading cells escaping the tumor bulk and migrating into the healthy brain parenchyma. Here, they encounter linearly aligned track-like tissue structures such as axon bundles and the perivascular space. These environments also contain micrometer-scale pores that impose mechanical confinement on invading cells. To study glioma cell migration in an in vitro system that reproduces some of these features, we used microfluidic devices with 60 m2 cross-sectional area channels that confine cells into one-dimensional (1D) tracks. Individual cell tracking revealed strongly persistent migration at a mean rate of 8.5 {+/-} 0.33 nm s-1. Notably, a 1D computational cell migration simulator predicts migration behaviors of glioma cells without significant adjustment of parameters estimated from previous experiments on two-dimensional (2D) substrates. Pharmacological inhibitors of integrin-mediated adhesions, myosin II activation, or drugs targeting F-actin assembly or microtubule dynamics influence migration consistent with simulations where relevant parameters are changed. These results suggest that cell parameters calibrated to a motor-clutch model on 2D substrates effectively predict 1D confined migration behaviors a priori. Our results outline a method for testing biophysical mechanisms of tumor cell migration in confined spaces and predicting the effects of anti-motility therapy.

biophysics↗