Surface Topography Induces and Orients Nematic Swarms of Active Filaments: Considerations for Lab-On-A-Chip Devices.

Surface-bound molecular motors can drive the collective motion of cytoskeletal filaments in the form of nematic bands and polar flocks in reconstituted gliding assays. Although these "swarming transitions" are an emergent property of active filament collisions, they can be controlled and guided by tuning the surface chemistry or topography of the substrate. To date, the impact of surface topography on collective motion in active nematics is only partially understood, with most experimental studies focusing on the escape of a single filament from etched channels. Since the late 1990s, significant progress has been made to utilize the nonequilibrium properties of active filaments and create a range of functional nanodevices relevant to biosensing and parallel computation; however, the complexity of these swarming transitions presents a challenge when attempting to increase filament surface concentrations. In this work, we etch shallow, linear trenches into glass substrates to induce the formation of swarming nematic bands and investigate the mechanisms by which surface topography regulates the two-dimensional (2D) collective motion of driven filamentous actin (F-actin). We demonstrate that nematic swarms only appear at intermediate trench spacings and vanish if the trenches are made too narrow, wide, or tortuous. To rationalize these results, we simulate the F-actin as self-propelled, semiflexible chains subject to a soft, spatially modulated potential that encodes the energetic cost of bending a filament along the edge of a trench. In our model, we hypothesize that an individual filament experiences a penalty when its projected end-to-end distance is smaller than the trench spacing ("bending and turning"). However, chains that span the channel width glide above the trenches in a force- and torque-free manner ("crowd-surfing"). Our simulations demonstrate that collections of filaments form nematic bands only at intermediate trench spacings, consistent with our experimental findings.

Molecular basis promoting centriole triplet microtubule assembly.

The triplet microtubule, a core structure of centrioles crucial for the organization of centrosomes, cilia, and flagella, consists of unclosed incomplete microtubules. The mechanisms of its assembly represent a fundamental open question in biology. Here, we discover that the ciliopathy protein HYLS1 and the ß-tubulin isotype TUBB promote centriole triplet microtubule assembly. HYLS1 or a C-terminal tail truncated version of TUBB generates tubulin-based superstructures composed of centriole-like incomplete microtubule chains when overexpressed in human cells. AlphaFold-based structural models and mutagenesis analyses further suggest that the ciliopathy-related residue D211 of HYLS1 physically traps the wobbling C-terminal tail of TUBB, thereby suppressing its inhibitory role in the initiation of the incomplete microtubule assembly. Overall, our findings provide molecular insights into the biogenesis of atypical microtubule architectures conserved for over a billion years.

Protocol for gene knockdown using siRNA in primary cultured neonatal murine microglia.

In this protocol, we describe the small interfering RNA (siRNA)-mediated gene knockdown in primary mouse microglia, providing an approach to investigate functions such as phagocytosis and chemotaxis. The approach includes siRNA design, establishment of mixed glial cultures, microglia isolation, and siRNA transfection. Validation of knockdown efficacy employs quantitative immunoblot analysis. This technique empowers the investigation of specific molecular and cellular functions within the intricate microenvironment of the brain, comprising diverse cell types. For complete details on the use and execution of this protocol, please refer to Iguchi et al. (2023).1.

Lysosomal stress drives the release of pathogenic α-synuclein from macrophage lineage cells via the LRRK2-Rab10 pathway.

α-Synuclein and LRRK2 are associated with both familial and sporadic Parkinson's disease (PD), although the mechanistic link between these two proteins has remained elusive. Treating cells with lysosomotropic drugs causes the recruitment of LRRK2 and its substrate Rab10 onto overloaded lysosomes and induces extracellular release of lysosomal contents. Here we show that lysosomal overload elicits the release of insoluble α-synuclein from macrophages and microglia loaded with α-synuclein fibrils. This release occurred specifically in macrophage lineage cells, was dependent on the LRRK2-Rab10 pathway and involved exosomes. Also, the uptake of α-synuclein fibrils enhanced the LRRK2 phosphorylation of Rab10, which was accompanied by an increased recruitment of LRRK2 and Rab10 onto lysosomal surface. Our data collectively suggest that α-synuclein fibrils taken up in lysosomes activate the LRRK2-Rab10 pathway, which in turn upregulates the extracellular release of α-synuclein aggregates, leading to a vicious cycle that could enhance α-synuclein propagation in PD pathology.

Soft confinement of self-propelled rods: simulation and theory.

We present an analytical framework for evolving the dynamics of active rods under any periodic external potential, including confining channels and arrays of harmonic traps. As a proof of concept, we analyze the structure and dispersion of self-propelled rods under a soft, periodic one-dimensional (1D) confinement potential and under a two-dimensional (2D) periodic radial harmonic trap. While passive rods and polymers nematically order under 1D confinement, their diffusive transport along the director is limited by thermal diffusion. In contrast, self-propelled rods can generate large convective fluxes when combined with nematic ordering, producing a strong dispersion along the director. Combining theory and simulation, we demonstrate that nematic alignment and self-propulsion generates an exponential enhancement in active diffusivity along the director, in contrast to passive rods that experience at most a 2-fold increase.

Colloidal transport phenomena in dynamic, pulsating porous materials.

We study the transport phenomena of colloidal particles embedded within a moving array of obstacles that mimics a dynamic, time-varying porous material. While colloidal transport in an array of stationary obstacles ("passive" porous media) has been well studied, we lack the fundamental understanding of colloidal diffusion in a nonequilibrium porous environment. We combine Taylor dispersion theory, Brownian dynamics simulations, and optical tweezer experiments to study the transport of tracer colloidal particles in an oscillating lattice of obstacles. We discover that the dispersion of tracer particles is a non-monotonic function of oscillation frequency and exhibits a maximum that exceeds the Stokes-Einstein-Sutherland diffusivity in the absence of obstacles. By solving the Smoluchowski equation using a generalized dispersion framework, we demonstrate that the enhanced transport of the tracers depends critically on both the direct interparticle interactions with the obstacles and the fluid-mediated, hydrodynamic interactions generated by the moving obstacles.

TOLLIP acts as a cargo adaptor to promote lysosomal degradation of aberrant ER membrane proteins.

Endoplasmic reticulum (ER) proteostasis is maintained by various catabolic pathways. Lysosomes clear entire ER portions by ER-phagy, while proteasomes selectively clear misfolded or surplus aberrant proteins by ER-associated degradation (ERAD). Recently, lysosomes have also been implicated in the selective clearance of aberrant ER proteins, but the molecular basis remains unclear. Here, we show that the phosphatidylinositol-3-phosphate (PI3P)-binding protein TOLLIP promotes selective lysosomal degradation of aberrant membrane proteins, including an artificial substrate and motoneuron disease-causing mutants of VAPB and Seipin. These cargos are recognized by TOLLIP through its misfolding-sensing intrinsically disordered region (IDR) and ubiquitin-binding CUE domain. In contrast to ER-phagy receptors, which clear both native and aberrant proteins by ER-phagy, TOLLIP selectively clears aberrant cargos by coupling them with the PI3P-dependent lysosomal trafficking without promoting bulk ER turnover. Moreover, TOLLIP depletion augments ER stress after ERAD inhibition, indicating that TOLLIP and ERAD cooperatively safeguard ER proteostasis. Our study identifies TOLLIP as a unique type of cargo-specific adaptor dedicated to the clearance of aberrant ER cargos and provides insights into molecular mechanisms underlying lysosome-mediated quality control of membrane proteins.

Nonequilibrium interactions between multi-scale colloids regulate the suspension microstructure and rheology.

Understanding nonequilibrium interactions of multi-component colloidal suspensions is critical for many dynamical settings such as self-assembly and material processing. A key question is how the nonequilibrium distributions of individual components influence the effective interparticle interactions and flow behavior. In this work, we develop a first-principle framework to study a bidisperse suspension of colloids and depletants using a Smoluchowski equation and corroborated by Brownian dynamics (BD) simulations. Using nonlinear microrheology as a case study, we demonstrate that effective depletion interactions between driven colloids are sensitive to particle timescales out of equilibrium and cannot be predicted by equilibrium-based pair potentials like Asakura-Oosawa. Furthermore, we show that the interplay between Brownian relaxation timescales of different species plays a critical role in governing the viscosity of multi-component suspensions. Our model highlights the limitations of using equilibrium pair potentials to approximate interparticle interactions in nonequilibrium processes such as hydrodynamic flows and presents a useful framework for studying the transport of driven, interacting suspensions.

Active Surface Flows Accelerate the Coarsening of Lipid Membrane Domains.

Phase separation of multicomponent lipid membranes is characterized by the nucleation and coarsening of circular membrane domains that grow slowly in time as ∼t^{1/3}, following classical theories of coalescence and Ostwald ripening. In this Letter, we study the coarsening kinetics of phase-separating lipid membranes subjected to nonequilibrium forces and flows transmitted by motor-driven gliding actin filaments. We experimentally observe that the activity-induced surface flows trigger rapid coarsening of noncircular membrane domains that grow as ∼t^{2/3}, a 2x acceleration in the growth exponent compared to passive coalescence and Ostwald ripening. We analyze these results by developing analytical theories based on the Smoluchowski coagulation model and the phase field model to predict the domain growth in the presence of active flows. Our Letter demonstrates that active matter forces may be used to control the growth and morphology of membrane domains driven out of equilibrium.

Dynamic interfaces for contact-time control of colloidal interactions.

Understanding pairwise interactions between colloidal particles out of equilibrium has a profound impact on dynamical processes such as colloidal self assembly. However, traditional colloidal interactions are effectively quasi-static on colloidal timescales and cannot be modulated out of equilibrium. A mechanism to dynamically tune the interactions during colloidal contacts can provide new avenues for self assembly and material design. In this work, we develop a framework based on polymer-coated colloids and demonstrate that in-plane surface mobility and mechanical relaxation of polymers at colloidal contact interfaces enable an effective, dynamic interaction. Combining analytical theory, simulations, and optical tweezer experiments, we demonstrate precise control of dynamic pair interactions over a range of pico-Newton forces and seconds timescales. Our model helps further the general understanding of out-of-equilibrium colloidal assemblies while providing extensive design freedom via interface modulation and nonequilibrium processing.

Pathological Roles of INPP5D in Alzheimer's Disease.

Current hypothesis of Alzheimer's disease (AD) postulates that amyloid ß (Aß) deposition in the brain causes tau inclusion in neurons and leads to cognitive decline. The discovery of the genetic association between triggering receptor expressed on myeloid cells 2 (TREM2) with increased AD risk points to a causal link between microglia and AD pathogenesis, and revealed a crucial role of TREM2-dependent clustering of microglia around amyloid plaques that prevents Aß toxicity to facilitate tau deposition near the plaques. Here we review the physiological and pathological roles of another AD risk gene expressed in microglia, inositol polyphosphate-5-polyphosphatase D (INPP5D), which encodes a phosphoinositide phosphatase. Evidence suggests that its risk polymorphisms alter the expression level and/or function of INPP5D, while concomitantly affecting tau levels in cerebrospinal fluids. In ß-amyloidosis mice, INPP5D was upregulated upon Aß deposition and negatively regulated the microglial clustering toward amyloid plaques. INPP5D seems to exert its function by acting antagonistically at downstream of the TREM2 signaling pathway, suggesting that it is a novel regulator of the protective barrier by microglia. Further studies to elucidate INPP5D's role in AD may help in developing new therapeutic targets for AD treatment.

Bio-enabled Engineering of Multifunctional "Living" Surfaces.

Through the magic of "active matter"─matter that converts chemical energy into mechanical work to drive emergent properties─biology solves a myriad of seemingly enormous physical challenges. Using active matter surfaces, for example, our lungs clear an astronomically large number of particulate contaminants that accompany each of the 10,000 L of air we respire per day, thus ensuring that the lungs' gas exchange surfaces remain functional. In this Perspective, we describe our efforts to engineer artificial active surfaces that mimic active matter surfaces in biology. Specifically, we seek to assemble the basic active matter components─mechanical motor, driven constituent, and energy source─to design surfaces that support the continuous operation of molecular sensing, recognition, and exchange. The successful realization of this technology would generate multifunctional, "living" surfaces that combine the dynamic programmability of active matter and the molecular specificity of biological surfaces and apply them to applications in biosensors, chemical diagnostics, and other surface transport and catalytic processes. We describe our recent efforts in bio-enabled engineering of living surfaces through the design of molecular probes to understand and integrate native biological membranes into synthetic materials.

Antibody binding reports spatial heterogeneities in cell membrane organization.

The spatial organization of cell membrane glycoproteins and glycolipids is critical for mediating the binding of ligands, receptors, and macromolecules on the plasma membrane. However, we currently do not have the methods to quantify the spatial heterogeneities of macromolecular crowding on live cell surfaces. In this work, we combine experiment and simulation to report crowding heterogeneities on reconstituted membranes and live cell membranes with nanometer spatial resolution. By quantifying the effective binding affinity of IgG monoclonal antibodies to engineered antigen sensors, we discover sharp gradients in crowding within a few nanometers of the crowded membrane surface. Our measurements on human cancer cells support the hypothesis that raft-like membrane domains exclude bulky membrane proteins and glycoproteins. Our facile and high-throughput method to quantify spatial crowding heterogeneities on live cell membranes may facilitate monoclonal antibody design and provide a mechanistic understanding of plasma membrane biophysical organization.

Engineered molecular sensors for quantifying cell surface crowding.

Cells mediate interactions with the extracellular environment through a crowded assembly of transmembrane proteins, glycoproteins and glycolipids on their plasma membrane. The extent to which surface crowding modulates the biophysical interactions of ligands, receptors, and other macromolecules is poorly understood due to the lack of methods to quantify surface crowding on native cell membranes. In this work, we demonstrate that physical crowding on reconstituted membranes and live cell surfaces attenuates the effective binding affinity of macromolecules such as IgG antibodies in a surface crowding-dependent manner. We combine experiment and simulation to design a crowding sensor based on this principle that provides a quantitative readout of cell surface crowding. Our measurements reveal that surface crowding decreases IgG antibody binding by 2 to 20 fold in live cells compared to a bare membrane surface. Our sensors show that sialic acid, a negatively charged monosaccharide, contributes disproportionately to red blood cell surface crowding via electrostatic repulsion, despite occupying only ~1% of the total cell membrane by mass. We also observe significant differences in surface crowding for different cell types and find that expression of single oncogenes can both increase and decrease crowding, suggesting that surface crowding may be an indicator of both cell type and state. Our high-throughput, single-cell measurement of cell surface crowding may be combined with functional assays to enable further biophysical dissection of the cell surfaceome.

INPP5D modulates TREM2 loss-of-function phenotypes in a ß-amyloidosis mouse model.

The genetic associations of TREM2 loss-of-function variants with Alzheimer disease (AD) indicate the protective roles of microglia in AD pathogenesis. Functional deficiencies of TREM2 disrupt microglial clustering around amyloid ß (Aß) plaques, impair their transcriptional response to Aß, and worsen neuritic dystrophy. However, the molecular mechanism underlying these phenotypes remains unclear. In this study, we investigated the pathological role of another AD risk gene, INPP5D, encoding a phosphoinositide PI(3,4,5)P3 phosphatase expressed in microglia. In a Tyrobp-deficient TREM2 loss-of-function mouse model, Inpp5d haplodeficiency restored the association of microglia with Aß plaques, partially restored plaque compaction, and astrogliosis, and reduced phosphorylated tau+ dystrophic neurites. Mechanistic analyses suggest that TREM2/TYROBP and INPP5D exert opposing effects on PI(3,4,5)P3 signaling pathways as well as on phosphoproteins involved in the actin assembly. Our results suggest that INPP5D acts downstream of TREM2/TYROBP to regulate the microglial barrier against Aß toxicity, thereby modulates Aß-dependent pathological conversion of tau.

Enhanced dispersion in an oscillating array of harmonic traps.

Experiment, theory, and simulation are employed to understand the dispersion of colloidal particles in a periodic array of oscillating harmonic traps generated by optical tweezers. In the presence of trap oscillation, a nonmonotonic and anisotropic dispersion is observed. Surprisingly, the stiffest traps produce the largest dispersion at a critical frequency, and the particles diffuse significantly faster in the direction of oscillation than those undergoing passive Stokes-Einstein-Sutherland diffusion. Theoretical predictions for the effective diffusivity of the particles as a function of trap stiffness and oscillation frequency are developed using generalized Taylor dispersion theory and Brownian dynamics simulations. Both theory and simulation demonstrate excellent agreement with the experiments, and reveal a "slingshot" mechanism that predicts a significant enhancement of colloidal diffusion in dynamic external fields.

Boundary design regulates the diffusion of active matter in heterogeneous environments.

Physical boundaries play a key role in governing the overall transport properties of nearby self-propelled particles. In this work, we develop dispersion theories and conduct Brownian dynamics simulations to predict the coupling between surface accumulation and effective diffusivity of active particles in boundary-rich media. We focus on three models that are well-understood for passive systems: particle transport in (i) an array of fixed volume-excluding obstacles; (ii) a pore with spatially heterogeneous width; and (iii) a tortuous path with kinks and corners. While the impact of these entropic barriers on passive particle transport is well established, we find that these classical models of porous media flows break down due to the unique interplay between activity and the microstructure of the internal geometry. We study the activity-induced slowdown of effective diffusivity by formulating a Smoluchowski description of long-time self diffusivity which contains contributions from the density and fluctuation fields of the active particles. Particle-based and finite element simulations corroborate this perspective and reveal important nonequilibrium considerations of active transport.

GPR120 signaling controls amyloid-ß degrading activity of matrix metalloproteinases.

Alzheimer disease (AD) is characterized by the extensive deposition of amyloid-ß peptide (Aß) in the brain. Brain Aß level is regulated by a balance between Aß production and clearance. The clearance rate of Aß is decreased in the brains of sporadic AD patients, indicating that the dysregulation of Aß clearance mechanisms affects the pathological process of AD. Astrocytes are among the most abundant cells in the brain and are implicated in the clearance of brain Aß via their regulation of the blood-brain barrier, glymphatic system, and proteolytic degradation. The cellular morphology and activity of astrocytes are modulated by several molecules, including ω3 polyunsaturated fatty acids, such as docosahexaenoic acid, which is one of the most abundant lipids in the brain, via the G protein-coupled receptor GPR120/FFAR4. In this study, we analyzed the role of GPR120 signaling in the Aß-degrading activity of astrocytes. Treatment with the selective antagonist upregulated the matrix metalloproteinase (MMP) inhibitor-sensitive Aß-degrading activity in primary astrocytes. Moreover, the inhibition of GPR120 signaling increased the levels of Mmp2 and Mmp14 mRNAs, and decreased the expression levels of tissue inhibitor of metalloproteinases 3 (Timp3) and Timp4, suggesting that GPR120 negatively regulates the astrocyte-derived MMP network. Finally, the intracerebral injection of GPR120 specific antagonist substantially decreased the levels of Tris-buffered saline-soluble Aß in male AD model mice, and this effect was canceled by the coinjection of an MMP inhibitor. These data indicate that astrocytic GPR120 signaling negatively regulates the Aß degrading activity of MMPs.SIGNIFICANT STATEMENTThe level of amyloid ß (Aß) in the brain is a crucial determinant of the development of Alzheimer disease. Here we found that astrocytes, which are the most abundant cell type in the central nervous system, harbors degrading activity against amyloid ß, which is regulated by GPR120 signaling. GPR120 is involved in the inflammatory response and obesity in peripheral organs. However, the pathophysiological role of GPR120 in Alzheimer disease remains unknown. We found that selective inhibition of GPR120 signaling in astrocytes increased the Aß-degrading activity of matrix metalloproteases. Our results suggest that GPR120 in astrocytes is a novel therapeutic target for the development of anti-Aß therapeutics.

BORCS6 is involved in the enlargement of lung lamellar bodies in Lrrk2 knockout mice.

Leucine-rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson disease. It has been shown that Lrrk2 knockout (KO) rodents have enlarged lamellar bodies (LBs) in their alveolar epithelial type II cells, although the underlying mechanisms remain unclear. Here we performed proteomic analyses on LBs isolated from Lrrk2 KO mice and found that the LB proteome is substantially different in Lrrk2 KO mice compared with wild-type mice. In Lrrk2 KO LBs, several Rab proteins were increased, and subunit proteins of BLOC-1-related complex (BORC) were decreased. The amount of surfactant protein C was significantly decreased in the bronchoalveolar lavage fluid obtained from Lrrk2 KO mice, suggesting that LB exocytosis is impaired in Lrrk2 KO mice. We also found that the enlargement of LBs is recapitulated in A549 cells upon KO of LRRK2 or by treating cells with LRRK2 inhibitors. Using this model, we show that KO of BORCS6, a BORC subunit gene, but not other BORC genes, causes LB enlargement. Our findings implicate the LRRK2-BORCS6 pathway in the maintenance of LB morphology.

Dietary cis-9, trans-11-conjugated linoleic acid reduces amyloid ß-protein accumulation and upregulates anti-inflammatory cytokines in an Alzheimer's disease mouse model.

Conjugated linoleic acid (CLA) is an isomer of linoleic acid (LA). The predominant dietary CLA is cis-9, trans-11-CLA (c-9, t-11-CLA), which constitutes up to ~ 90% of total CLA and is thought to be responsible for the positive health benefits associated with CLA. However, the effects of c-9, t-11-CLA on Alzheimer's disease (AD) remain to be elucidated. In this study, we investigated the effect of dietary intake of c-9, t-11-CLA on the pathogenesis of an AD mouse model. We found that c-9, t-11-CLA diet-fed AD model mice significantly exhibited (1) a decrease in amyloid-ß protein (Aß) levels in the hippocampus, (2) an increase in the number of microglia, and (3) an increase in the number of astrocytes expressing the anti-inflammatory cytokines, interleukin-10 and 19 (IL-10, IL-19), with no change in the total number of astrocytes. In addition, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatographic analysis revealed that the levels of lysophosphatidylcholine (LPC) containing c-9, t-11-CLA (CLA-LPC) and free c-9, t-11-CLA were significantly increased in the brain of c-9, t-11-CLA diet-fed mice. Thus, dietary c-9, t-11-CLA entered the brain and appeared to exhibit beneficial effects on AD, including a decrease in Aß levels and suppression of inflammation.