David A. Weitz

The exchange of biological matter between bacterial cells drives adaptation and evolution. However, whether bacteria can exchange functional proteins remains unclear. In this work, we found that antibiotic treatment can induce vesicle-mediated horizontal protein transfer within and between bacterial species. We developed a genetic system in Escherichia coli to track transfer events and performed single-cell transcriptomic profiling on an isogenic population of bacteria. Antibiotics stimulated the differentiation of this isogenic population into distinct cell states: donor cells that activated a membrane stress response to release protein-containing vesicles and recipient cells that suppressed this response to acquire protein from their neighbors. Protein uptake enhanced the antibiotic persistence of recipient cells, revealing that vesicle exchange promotes bacterial survival during antibiotic treatment.

Microbes are increasingly used as living therapeutics, yet their uncontrolled dissemination in the body has remained a clinical roadblock. Physical containment remains largely unattainable owing to eventual bacteria escape. In this work, we present an implantable material that encapsulates and confines bacteria, wherein synthetically engineered microbes produce therapeutic payloads from within. We developed a hydrogel scaffold with dual mechanical features: high stiffness to regulate bacterial proliferation and high toughness to resist material fracture under physiological stress. This design achieved complete bacterial containment for 6 months and withstood multiple forms of mechanical loading that otherwise caused catastrophic material failure. By genetically engineering embedded bacteria, we endowed the material with environmental sensing and on-demand therapeutic release capabilities and demonstrated autonomous treatment in a murine prosthetic joint infection model.

Squeaking is a constant companion in various aspects of our daily lives, whether we slide rubber-soled shoes across hardwood floors1, scrape chalk on a blackboard2, engage the brakes on a bicycle3 or walk with a hip replacement4,5. When two rigid bodies slide over each other, squeaking is widely understood to result from self-excited stick–slip oscillations, triggered by a decrease in the friction coefficient with increasing slip velocity6, 7, 8, 9–10. However, sliding of extended interfaces can involve crack or slip-pulse propagation11, 12, 13, 14, 15, 16, 17, 18, 19, 20–21. This distinction is amplified when a soft body slides on a rigid one, in which large deformations and material mismatch can cause detachment by opening slip pulses22, 23, 24, 25, 26–27. Previous studies focused mainly on slow sliding17,26,28, 29, 30, 31, 32, 33–34, in which pulses are slow and squeaking is absent. Although squeaking at soft–rigid interfaces has been linked to stick–slip oscillations35, 36–37, the mechanisms remain unclear. Here we experimentally investigate soft–rigid interfaces sliding at velocities that produce squeaking. High-speed imaging and acoustic analysis show that opening pulses propagate at approximately the shear wave speed of the soft material, mediating local slip across diverse materials. In flat samples, these pulses are irregular and generate broadband acoustic emissions. Introducing thin surface ridges confines pulse propagation, yielding a consistent repetition frequency matching the first shear mode of the sliding block and squeaking at that frequency. These findings show a structure-driven mechanism that stabilizes rupture in bimaterial friction. Geometric confinement suppresses competing modes, transforming irregular two-dimensional dynamics into coherent one-dimensional pulse trains, offering new insights into frictional rupture from engineered surfaces to geological faults. High-speed imaging reveals that the squeak of soft–rigid frictional interfaces, like sneakers sliding on a basketball court, arises from intersonic opening slip pulses—analogous to earthquake ruptures—that thin ridges on the rubber confine to repeat at a musical frequency.

The physical properties of drug delivery vehicles are important for the development of effective and targeted treatment options for human disease. In this review, we elucidate the role of the fundamental physical properties such as size, charge, elasticity, curvature, fluidity, and asymmetry in optimizing lipid-based drug delivery systems. These properties significantly influence the performance of such drug delivery vehicles in overcoming biological barriers, minimizing clearance, and improving cellular uptake. The optimization of physical properties is important in bridging the translational gap and achieving consistent clinical outcomes. By focusing on the fundamental physical properties, we also provide a comprehensive review that identifies remaining knowledge gaps and guides future development of lipid-based nanocarriers. This Review explores the role of fundamental physical properties, such as size, charge, elasticity, curvature, fluidity, and asymmetry, on optimizing lipid-based drug delivery systems. Knowledge gaps and guidance for the future development of lipid-based nanocarriers are also discussed.

The human antibody repertoire is a promising source for therapeutic-grade antibodies. Yet current methods for strategically mining these B cell repertoires are stymied by throughput and chain pairing considerations. This study presents advancements in fluidics and molecular biology that enable the multi-step encapsulation and capture of B cells from an immunized, humanized mouse in nanoliter sized droplets. Once singularly captured, antigen-specific B-cells can be lysed and individually manipulated via RT-PCR to splice cognate V genes and create a predominantly natively paired library. To explore the importance of these process improvements in library generation, we constructed natively-paired libraries against two therapeutically-relevant human proteins. Through deep sequencing, bioinformatics-driven screening and phage display, we selected functional, target-specific antibodies. Our findings reveal that natively paired libraries contain a higher percentage of target-specific antibodies and demonstrate enhanced potency and improved developability in both in silico and in vitro assessments relative to combinatorial library-derived antibodies. Furthermore, antibodies with native pairing show increased potency as well as improved in silico and in vitro developability compared to their randomly paired counterparts. To this end, we see this droplet microfluidic platform and its capacity to generate and facilitate the high-throughput interrogation of antigen-specific antibody repertoires as an important, orthogonal therapeutic antibody discovery approach.

While PDMS-based microfluidic devices set the rapid prototyping standard, their application is limited by incompatibility with many non-polar solvents. This inability to tolerate organic solvents significantly restricts the types of materials that can be handled and/or synthesized. UV-curable photopolymers, such as NOA 81, present a promising solution to these challenges. NOA 81 enables simple, cost-effective device fabrication, but current limitations on proper fabrication protocols limit its full potential. Here, we present a well-defined, simple, single-step fabrication method for producing NOA 81 microfluidic devices that are compatible with organic solvents. This method allows for the rapid prototyping of devices using similar steps associated with PDMS. We report a rapid heat treatment step that enhances the chemical resistance to a wider range of organic solvents while also increasing the material's elastic modulus by nearly two orders of magnitude. We demonstrate how to control the channel wall wettability for producing water-in-oil-in-water double emulsions which serve as templates for microcapsules and amphiphilic polymer vesicles. This novel method, which we call "hard lithography", reduces the time needed to produce working prototypes to less than one day while expanding the range of solvents that can be used. It simplifies fabrication and enables the production of chemically resistant devices suitable for a wide array of applications.

The identification of specific markers to distinguish resident microglia from infiltrating monocytes has been a long-standing challenge in neuroscience. Recently, proteins such as P2RY12, TMEM119, and FCRLS have been proposed as microglia-specific and are now widely used to define microglial populations in health and disease. The specificity of these markers was predicated on the assumption that circulating monocytes retain their distinct signatures after entering the central nervous system (CNS). Here, we challenge this paradigm. Using a combination of bone marrow chimeras, single-cell RNA sequencing, ATAC-seq, flow cytometry, and immunohistochemistry, we demonstrate that monocytes engrafting into the CNS acquire de novo expression of these established microglia markers. This phenotypic conversion is driven by profound epigenetic reprogramming, characterized by dynamic changes in chromatin accessibility at key gene loci, including P2ry12, Tmem119, and Aif1 (Iba1), and a shift in transcription factor binding motifs toward a microglial profile. We show this process occurs in the retina following injury and, remarkably, under physiological conditions in the brain and spinal cord, where blood-derived monocytes progressively contribute to the resident myeloid pool. Furthermore, engrafted monocytes downregulate canonical monocyte markers (Ly6C, CD45), eventually becoming indistinguishable from embryonic microglia based on conventional phenotyping. Our findings reveal that infiltrating monocytes undergo extensive epigenetic and transcriptional remodeling to adopt a microglia-like fate, challenging the specificity of current markers and necessitating a re-evaluation of the distinct roles of these two cell populations in CNS pathology.

The identification of specific markers to distinguish resident microglia from infiltrating monocytes has been a long-standing challenge in neuroscience. Recently, proteins such as P2RY12, TMEM119, and FCRLS have been proposed as microglia-specific and are now widely used to define microglial populations in health and disease. The specificity of these markers was predicated on the assumption that circulating monocytes retain their distinct signatures after entering the central nervous system (CNS). Here, we challenge this paradigm. Using a combination of bone marrow chimeras, single-cell RNA sequencing, ATAC-seq, flow cytometry, and immunohistochemistry, we demonstrate that monocytes engrafting into the CNS acquire de novo expression of these established microglia markers. This phenotypic conversion is driven by profound epigenetic reprogramming, characterized by dynamic changes in chromatin accessibility at key gene loci, including P2ry12, Tmem119, and Aif1 (Iba1), and a shift in transcription factor binding motifs toward a microglial profile. We show this process occurs in the retina following injury and, remarkably, under physiological conditions in the brain and spinal cord, where blood-derived monocytes progressively contribute to the resident myeloid pool. Furthermore, engrafted monocytes downregulate canonical monocyte markers (Ly6C, CD45), eventually becoming indistinguishable from embryonic microglia based on conventional phenotyping. Our findings reveal that infiltrating monocytes undergo extensive epigenetic and transcriptional remodeling to adopt a microglia-like fate, challenging the specificity of current markers and necessitating a re-evaluation of the distinct roles of these two cell populations in CNS pathology.

Abstract Description The identification of specific markers for microglia has been a long-standing challenge. Recently, markers such as P2ry12, TMEM119, and Fcrls have been proposed as microglia specific. However, the specificity of these markers was based on the assumption that circulating monocytes retain their distinct signatures even after infiltrating the CNS. Our recent findings reveal that infiltrating monocytes can adopt microglia-like characteristics while maintaining a pro-inflammatory profile upon permanent engraftment in the CNS. In this study, we utilize bone marrow chimeras, single-cell RNA sequencing, ATAC-seq, flow cytometry, and immunohistochemistry to demonstrate that engrafted monocytes acquire expression of established microglia markers. These changes are accompanied by alterations in chromatin accessibility and shifts in chromatin binding motifs that are indicative of microglial identity. Moreover, we show that engrafted monocytes dynamically regulate the expression of transcription factors PU.1, CTCF, RUNX, AP-1, CEBP, and IRF2, all of which are crucial for shaping microglial identity. This study is the first to illustrate that engrafted monocytes in the retina undergo both epigenetic and transcriptional changes, enabling them to express microglia-like signatures. These findings highlight the need for future research to account for these changes when assessing the roles of monocytes and microglia in CNS pathology. Funding Sources Boston KPro fund: ME0770 NEI: 5P30EY003790 Topic Categories Neuroimmunology (NEUR)

Abstract Microbes are increasingly utilized as living therapeutic vehicles, yet their uncontrolled dissemination in the body has long remained a roadblock to clinical development. Physical containment, while widely used for mammalian cells, remains largely unattainable due to eventual bacteria escape. Here, we present an implantable material platform that encapsulates and confines bacteria, wherein synthetically engineered microbes produce therapeutic payloads from within. To prevent microbial escape, we developed a hydrogel scaffold with dual mechanical features: high stiffness to regulate bacterial proliferation and high toughness to resist material fracture under physiological stress. This design achieved complete bacterial containment for over six months and withstood multiple forms of mechanical loading that otherwise caused catastrophic material failure. By genetically engineering embedded bacteria, we endowed the material with environmental sensing and on-demand therapeutic release capabilities and demonstrated autonomous treatment in a murine prosthetic joint infection model. This multimodal strategy provides a safe and generalizable framework for deploying microbial medicines in vivo and supports their use as autonomous drug depots across a range of disease settings.

Lipid vesicles consist of aqueous cores surrounded by a bilayer of phospholipids. Hybrid polymer-lipid vesicles incorporate both polymers and lipids, offering promising properties for developing pharmaceuticals, biosensors, and artificial cells. The hybrid vesicles can be symmetric, in which their two leaflets contain identical compositions, or asymmetric, in which the leaflets possess dissimilar compositions and can lead to dramatically modified properties. However, methods to produce both symmetric and asymmetric hybrid vesicles result in heterogenous compositions and sizes, making it challenging to quantify the effect of asymmetry and limiting applications. Here, we use a microfluidic approach to produce hybrid vesicles containing symmetric or asymmetric leaflets with precisely engineered compositions. We find the vesicles with asymmetric leaflets are significantly stiffer and tougher than those with symmetric leaflets; moreover, the lateral diffusivity of lipids is greatly decreased. The structure for improved toughness consists of an inner leaflet that is a stretchable lipid leaflet and an outer leaflet that is a fully continuous polymer leaflet. This technique of precisely engineering asymmetric structures may be applied to hybrid vesicles composed of block copolymers and phospholipids dissolvable in chloroform and hexane, further expanding their applications.

Most surfaces of practical interest are heterogeneous, with natural surfaces often exhibiting microscale variations in wetting characteristics. These variations make it challenging to accurately characterize surface properties and interactions with fluids using conventional bulk contact angle measurements. Existing methods for directly measuring microcontact angles are limited by inadequate spatial resolution or reliance on indirect, force-based approaches. To address these challenges, we present a novel contact angle mapping goniometer that deposits picoliter droplets to visualize and quantify apparent contact angles and contact angle hysteresis with a spatial resolution as low as tens of microns. We validate the device on test surfaces with varying contact angles and apply it to tertiary basalt samples to investigate their wetting properties for geological carbon sequestration. We also examine Bacillus subtilis biofilms, where wettability significantly impacts antibiotic efficacy. This research advances the understanding of surface wettability and its implications across scientific and industrial applications.

Lipase A (BsLipA) for improved thermotolerance. We screen a combinatorial library where 15 residues are saturated and identify multiple variants with 9-12 substitutions and >40 °C improvement in thermotolerance after a single round of mutagenesis. This platform offers a robust, scalable solution for protein engineering.

Abstract With increasing demand for metals but declining ore grades and complex mineralogy, current separation technologies are often inefficient in separating valuable minerals from waste due to low selectivity. Herein, a sustainable biotechnology approach is reported for the selective separation of precious metal particles from waste. Using phage display, peptides (PepMin) are designed to target mineral particles with high specificity and affinity. The results demonstrate that PepMin can selectively separate silver particles from silica, a common waste byproduct, achieving over 98% silver purity with a recovery rate of more than 95%. Furthermore, temperature‐sensitive proteins incorporating PepMin are engineered for recyclable separation, achieving a high separation factor of 23,000 and a high protein recovery of 97%. When tested with real photovoltaic panel leachate, these proteins achieve a 94.6% silver recovery, illustrating practical utility for complex industrial samples. This approach offers a more efficient and economically viable solution, due to the scalability of protein production, reduced purification costs, and protein recyclability. This work demonstrates a new bioinspired technology for sustainable particle separation with high selectivity, highlighting the great potential of biomolecule‐based strategies for mineral particle separation.

Hydrogels often suffer from weak mechanical properties when fully swollen due to reduced polymer chain density, leading to stress concentration and failure under mechanical loads. To address this, we propose an electron-interaction-induced phase separation strategy for synthesizing tough hybrid hydrogels. Introducing Zr 4+ ions during preparation forms nanocrystalline domains via chelation with acid-group monomers, while flexible domains arise from other monomers. This dual-domain structure enhances toughness through efficient energy dissipation and ensures durability. The hybrid hydrogels exhibit a swelling ratio of 15.7 in 10,000 g/L NaCl at 80 °C for 30 days, with a compressive modulus of over 18 kPa and a compressive strength of 0.11 MPa. They maintain integrity after 40 fatigue cycles and show a dissipation energy ratio of ∼44%. Hydrogel particles with the same formula, prepared via inverse emulsion polymerization, demonstrate a compressive modulus of 49.50 MPa and a shear modulus of 13 MPa in the swollen state, retaining structure after navigating capillaries three times smaller than their diameter. This one-step strategy creates hybrid hydrogels with nanocrystalline and flexible domains, providing exceptional toughness and resilience under extreme conditions and offering substantial potential for applications in challenging aquatic environments characterized by high salinity and elevated temperatures.

This work describes a microfluidic high-throughput fabrication method for untethered soft microactuators which, while initially unspecific, develop distinct shapes, surface textures, and actuation modes based on various environmental cues. Analogous to the core concept of cell differentiation, the central idea of this technique is to apply controlled mechanical and chemical stimuli to a deformable hydrogel fiber and transmit the induced geometrical and textural changes to embedded droplets. Using liquid crystal (LC) monomer droplets as a core allows us to orthogonally program the geometric, textural, and molecular architecture of the resulting microactuators upon droplet polymerization. Fine-tuning of the microfluidic parameters yields microdroplets that dry and transform into microparticles with a variety of shapes, including spindle, rod, pancake, dumbbell, pyramid, and worm-like assemblies with a range of aspect ratios. Leveraging mechanical instability via rapid dehydration of hydrogel fibers allows us to generate and impart stable 3D patterns to the core, resulting in microparticles that vary both in global shape and surface texture. After polymerizing these precursor droplets in a magnetic field to encode the mesogenic orientation, LCE microactuators are realized with a rich library of shapes, surface patterns, and molecular structures, each displaying distinct deformations upon heating, validated via finite element analysis.

Cells communicate by transporting vesicles and organelles, which is essential for maintaining cellular homeostasis. However, dysregulated vesicle transfer between cells can contribute to several diseases. In the skin, excessive melanosome transfer from melanocytes to keratinocytes leads to hyperpigmentation and can contribute to the progression of melanoma. Current treatments often rely on eliminating the contents of melanosomes with drugs, which risks significant side effects. Here, we present a drug-free strategy to regulate intercellular transport. We demonstrate our approach by reducing the amount of melanosomes transferred from melanocytes to keratinocytes. To achieve this, we incorporate keratinocyte cell membrane proteins into liposomes formed with microfluidics. Such functionalization enables the liposomes to selectively anchor to the surface of pigment globules, which transport melanosomes between cells. We show that the liposomes passivate the pigment globule surface and inhibit their uptake by keratinocytes, which results in a significant reduction in the level of melanosome transfer. Thus, our findings provide an effective strategy for reducing melanosome transfer and present a generalizable method for modulating cellular communication through extracellular vesicles and organelles.

Microcapsules with an aqueous core and stimuli-responsive shell are attractive as delivery or collection vehicles for repeatedly or reversibly loading and unloading cargo molecules. Applications in complex scenarios often require smart capsules to respond intelligently and dynamically to environmental changes. However, dynamic capsules for release in acidic environmental conditions but blockage under neutral or basic conditions are lacking. In this work, a new design of a dynamic capsule is reported using a cross-linked poly(amine) shell to achieve reversibly pH-responsive hydrogel microcapsules. The poly(amine) hydrogel capsules exhibit dynamic swelling–shrinking behavior together with a corresponding mesh size change of the shell network upon pH switches, with the capability of cyclic capture, trap, and release of cargo molecules with size selectivity. The newly designed poly(amine) capsules enrich the diversity of the smart capsule library and broaden their potential application space.

Nanoparticles (NPs) are promising tools in biomedical applications. Their unique physicochemical properties, such as controllable size, tunable shape, and versatile surface functionality, provide significant advantages in targeted delivery and controlled release. Despite the large progress, NP‐based drug delivery systems still face a major challenge, i.e., NPs often demonstrate less therapeutic improvements than expected. The disparity mainly arises from the incomplete understanding of NP behaviors in the complex biological environments, especially their cellular uptake mechanisms, and thus, the performances of NPs are generally not optimized. A comprehensive understanding of how NP properties influence cellular uptake is essential for the design of high‐performance delivery systems. This review summarizes recent advancements in the investigation of NP cellular uptake pathways and factors, such as NP size, shape, and surface functionality, which affect the cellular uptake processes. The physical and chemical properties of NPs can be modulated to control the cellular uptake pathway and enhance the cellular uptake efficiency, thus ultimately improving the bioavailability, efficacy, and safety. It aims to provide new insights for the design of NPs, ultimately advancing their applications in biomedical therapy.

We describe a shear cell and associated sample preparation methods to be used to measure mPa-scale shear stresses using a confocal microscope. The shear cell can be mounted on an inverted confocal microscope that is used to image the deformation of a calibrated polymer gel with embedded tracers, from which the shear stress is determined. The gels have shear moduli G ∼ 1-10 Pa, which, when combined with the high strain resolution afforded by single-particle locating of the embedded tracers, are sufficiently compliant to measure mPa stresses. While these exceptionally compliant gels are necessary to achieve mPa stress resolution, they are easily deformed by surface tension, leading to several technical challenges with their fabrication and functionalization. To overcome these challenges, a fabrication technique using sacrificial molds is described, alongside processing steps to functionalize the gels without distorting the surface. While the apparatus presented is designed specifically for simple shear deformation of soft solids, the processing and fabrication techniques can be employed to mold and fabricate compliant gels in other geometries.

Abstract Intermediate filaments (IFs) play key roles in cellular mechanics, signaling, and organization, but tools for their rapid, selective disassembly remain limited. Here, we introduce FilaBuster, a photochemical approach for efficient and spatiotemporally controlled IF disassembly in living cells. FilaBuster uses a three-step strategy: (1) targeting HaloTag to IFs, (2) labeling with a covalent photosensitizer ligand, and (3) light-induced generation of localized reactive oxygen species to trigger filament disassembly. This modular strategy applies broadly across IF subtypes—including vimentin, GFAP, desmin, peripherin, and keratin 18—and is compatible with diverse dyes and imaging platforms. Using vimentin IFs as a model system, we establish a baseline implementation in which vimentin-HaloTag labeled with a photosensitizer HaloTag ligand triggers rapid and specific IF disassembly upon light activation. We then refine this approach by (i) expanding targeting strategies to include a vimentin nanobody-HaloTag fusion, (ii) broadening the range of effective photosensitizers, and (iii) optimizing irradiation parameters to enable precise spatial control over filament disassembly. Together, these findings position FilaBuster as a robust platform for acute, selective, and spatiotemporally precise disassembly of IF networks, enabling new investigations into their structural and functional roles in cell physiology and disease.

Biomolecular condensates form via macromolecular phase separation. Here, we report results from our characterization of synthetic condensates formed by phase separation of mixtures comprising two types of RNA molecules and the biocompatible polymer polyethylene glycol. Purine-rich RNAs are scaffolds that drive phase separation via heterotypic interactions. Conversely, pyrimidine-rich RNA molecules are adsorbents defined by weaker heterotypic interactions. They adsorb onto and wet the interfaces of coexisting phases formed by scaffolds. Lattice-based simulations reproduce the phenomenology observed in experiments and these simulations predict that scaffolds and adsorbents have different non-random orientational preferences at interfaces. Dynamics at interfaces were probed using single-molecule tracking of fluorogenic probes bound to RNA molecules. These experiments revealed dynamical anisotropy at interfaces whereby motions of probe molecules parallel to the interface are faster than motions perpendicular to the interface. Taken together, our findings have broad implications for designing synthetic condensates with tunable interfacial properties. Experiments and computations show that the organization and dynamics of molecules within and at interfaces of biomolecular condensates are governed by differential interaction strengths leading to the classification of adsorbents versus scaffolds.

Abstract The delivery of therapeutics to cells is crucial for the treatment and prevention of diseases. To enhance targeting and protect therapeutics from degradation, they are often encapsulated in drug delivery vehicles like lipid nanoparticles, lipid vesicles, and viral vectors. However, there is no universal vehicle for all cargo types including small molecules, nucleic acids, and proteins. Here, a method for engineering lipid vesicles with asymmetric leaflets is presented, and their ability to deliver mRNA and proteins to cells is demonstrated. The results show that leaflet asymmetry enhances vesicle uptake by cells, and increases the transfection efficiency with mRNA up to fivefold. Additionally, it is shown that asymmetric vesicles can deliver a variety of proteins. In particular, the delivery of Cas9 proteins and Cas9/sgRNA complexes for gene‐editing is demonstrated. This work expands the design parameters for drug delivery vehicles, enabling more efficient and universal carriers for drug and protein delivery.

Effective intracellular delivery is essential for successful gene editing of cells. Spatially selective delivery to cells that is simultaneously precise, consistent, and nondestructive remains challenging using conventional state-of-the-art techniques. Here, we introduce a carrier-free method for spatiotemporal delivery of fluorescently labeled cargo into both adherent and suspension cells using carbon-black-embedded polydimethylsiloxane (PDMS) substrates irradiated by nanosecond laser pulses. This low-cost, biocompatible material, coupled with an optical approach, enables scalable, spatially selective, and sequential delivery of multiple cargo molecules, including FITC-Dextran and siRNA, to a broad range of cells. Notably, we achieved siRNA delivery into the cytoplasm of hard-to-transfect K562 cells with 45% efficiency, while maintaining nearly 100% cell viability.

Droplet-based organoid culture offers several advantages over conventional bulk organoid culture, such as improved yield, reproducibility, and throughput. However, organoids grown in droplets typically display only a spherical geometry and lack the intricate structural complexity found in native tissue. By incorporating singularized pancreatic ductal adenocarcinoma cells into collagen droplets, we achieve the growth of branched structures, indicating a more complex interaction with the surrounding hydrogel. A comparison of organoid growth in droplets of different diameters showed that while geometrical confinement improves organoid homogeneity, it also impairs the formation of more complex organoid morphologies. Thus, only in 750 µ m diameter collagen droplets did we achieve the consistent growth of highly branched structures with a morphology closely resembling the structural complexity achieved in traditional bulk organoid culture. Moreover, our analysis of organoid morphology and transcriptomic data suggests an accelerated maturation of organoids cultured in collagen droplets, highlighting a shift in developmental timing compared to traditional systems.

The cytoskeletal protein actin forms a spatially organized biopolymer network that plays a central role in many cellular processes. Actin filaments continuously assemble and disassemble, enabling cells to rapidly reorganize their cytoskeleton. Filament severing accelerates actin turnover, as both polymerization and depolymerization rates depend on the number of free filament ends. Here, we use light to control actin severing in vitro by locally generating reactive oxygen species (ROS) with photosensitive molecules such as fluorophores. We see that ROS sever actin filaments, which increases actin polymerization in our experiments. However, beyond a certain threshold, excessive severing leads to the disassembly of actin networks, allowing us to selectively remove actin structures. Our experimental data is supported by simulations using a kinetic model of actin polymerization, which helps us understand the underlying dynamics. In cells, ROS are known to regulate the actin cytoskeleton, but the molecular mechanisms are poorly understood. Here we show that, in vitro, ROS directly affect actin reorganization.

The cytoskeleton is composed of F-actin, microtubules, and intermediate filaments (IFs). Vimentin is one of the most ubiquitous and well-studied IFs. It is involved in many activities including wound healing, tissue fibrosis, and cancer metastasis, all of which require rapid vimentin IF assembly. In this paper, we report that vimentin forms liquid condensates which appear to enable rapid filament growth. Given the transient nature of these droplets, we focus on properties of vimentin-Y117L, which has a point mutation that leads to formation of condensates but not IFs, enabling us to study these droplets in detail. The droplets dissolve under 1,6-Hexanediol treatment and under decreasing concentration, confirming that they are liquid, and phase separated. These condensates extensively wet actin stress fibers, rendering them resistant to actin-binding drugs and protecting them from depolymerization. We show similar behavior occurs in wild-type vimentin during its assembly into filaments.

Abstract Aggregated fibrillar tangles of the microtubule-associated protein tau are a hallmark of Alzheimer’s disease. It is becoming increasingly clear that a key process that can trigger the formation of such tau tangles is the aberrant ageing of biomolecular condensates of tau formed via liquid-liquid phase separation. This ageing process affects the overall mechanical and structural properties of the condensates, but the molecular-level mechanisms by which aggregation takes place in the condensates have remained elusive. Here, by tracking individual tau molecules inside the condensates using single molecule microscopy, we show that the ageing process is characterized by the coexistence of a growing solid phase of tau within a dense liquid phase. The liquid phase is increasingly confined to the pores of the growing fibril gel network, but maintains its initial viscosity. These findings add a spatial dimension to the ageing of condensates and demonstrate that spatial heterogeneity is a key feature of the liquid-to-solid transition of tau.

Sustained release of bone morphogenetic protein 2 (BMP-2) is used to enhance bone regeneration, but immobilizing BMP-2 in three-dimensional scaffolds could enable spatial regulation of stem cell differentiation and bone formation. Here, we fabricate porous granular hydrogels presenting BMP-2 on the surface to regulate stem cell growth and differentiation. Immobilization of BMP-2 and cell-adhesive ligands is achieved by surface-specific functionalization of microgels, which are jammed to form microporous hydrogels. Varying surface ligand density regulated spreading, proliferation and differentiation of cells. In addition, modulating the distribution of cell-adhesive ligands and BMP-2 allowed spatial control over cell adhesion and osteogenic differentiation.