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.

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.

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.

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.

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.

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.

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.

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.

Molecular height measurement by cell surface optical profilometry (CSOP).

The physical dimensions of proteins and glycans on cell surfaces can critically affect cell function, for example, by preventing close contact between cells and limiting receptor accessibility. However, high-resolution measurements of molecular heights on native cell membranes have been difficult to obtain. Here we present a simple and rapid method that achieves nanometer height resolution by localizing fluorophores at the tip and base of cell surface molecules and determining their separation by radially averaging across many molecules. We use this method, which we call cell surface optical profilometry (CSOP), to quantify the height of key multidomain proteins on a model cell, as well as to capture average protein and glycan heights on native cell membranes. We show that average height of a protein is significantly smaller than its contour length, due to thermally driven bending and rotation on the membrane, and that height strongly depends on local surface and solution conditions. We find that average height increases with cell surface molecular crowding but decreases with solution crowding by solutes, both of which we confirm with molecular dynamics simulations. We also use experiments and simulations to determine the height of an epitope, based on the location of an antibody, which allows CSOP to profile various proteins and glycans on a native cell surface using antibodies and lectins. This versatile method for profiling cell surfaces has the potential to advance understanding of the molecular landscape of cells and the role of the molecular landscape in cell function.

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.

Photo-oxygenation by a biocompatible catalyst reduces amyloid-ß levels in Alzheimer's disease mice.

Amyloid formation and the deposition of the amyloid-ß peptide are hallmarks of Alzheimer's disease pathogenesis. Immunotherapies using anti-amyloid-ß antibodies have been highlighted as a promising approach for the prevention and treatment of Alzheimer's disease by enhancing microglial clearance of amyloid-ß peptide. However, the efficiency of antibody delivery into the brain is limited, and therefore an alternative strategy to facilitate the clearance of brain amyloid is needed. We previously developed an artificial photo-oxygenation system using a low molecular weight catalytic compound. The photocatalyst specifically attached oxygen atoms to amyloids upon irradiation with light, and successfully reduced the neurotoxicity of aggregated amyloid-ß via inhibition of amyloid formation. However, the therapeutic effect and mode of actions of the photo-oxygenation system in vivo remained unclear. In this study, we demonstrate that photo-oxygenation facilitates the clearance of aggregated amyloid-ß from the brains of living Alzheimer's disease model mice, and enhances the microglial degradation of amyloid-ß peptide. These results suggest that photo-oxygenation may represent a novel anti-amyloid-ß strategy in Alzheimer's disease, which is compatible with immunotherapy.

Active Contact Forces Drive Nonequilibrium Fluctuations in Membrane Vesicles.

We analyze the nonequilibrium shape fluctuations of giant unilamellar vesicles encapsulating motile bacteria. Owing to bacteria-membrane collisions, we experimentally observe a significant increase in the magnitude of membrane fluctuations at low wave numbers, compared to the well-known thermal fluctuation spectrum. We interrogate these results by numerically simulating membrane height fluctuations via a modified Langevin equation, which includes bacteria-membrane contact forces. Taking advantage of the lengthscale and timescale separation of these contact forces and thermal noise, we further corroborate our results with an approximate theoretical solution to the dynamical membrane equations. Our theory and simulations demonstrate excellent agreement with nonequilibrium fluctuations observed in experiments. Moreover, our theory reveals that the fluctuation-dissipation theorem is not broken by the bacteria; rather, membrane fluctuations can be decomposed into thermal and active components.

AP180 N-Terminal Homology (ANTH) and Epsin N-Terminal Homology (ENTH) Domains: Physiological Functions and Involvement in Disease.

The AP180 N-terminal homology (ANTH) and Epsin N-terminal homology (ENTH) domains are crucially involved in membrane budding processes. All the ANTH/ENTH-containing proteins share the phosphoinositide-binding activity and can interact with clathrin or its related proteins via multiple binding motifs. Their function also include promotion of clathrin assembly, induction of membrane curvature, and recruitment of various effector proteins, such as those involved in membrane fission. Furthermore, they play a role in the sorting of specific cargo proteins, thereby enabling the cargos to be accurately transported and function at their appropriate locations. As the structural bases underlying these functions are clarified, contrary to their apparent similarity, the mechanisms by which these proteins recognize lipids and proteins have unexpectedly been found to differ from each other. In addition, studies using knockout mice have suggested that their physiological roles may be more complicated than merely supporting membrane budding processes. In this chapter, we review the current knowledge on the biochemical features of ANTH/ENTH domains, their functions predicted from the phenotypes of animals deficient in these domain-containing proteins, and recent findings on the structural basis enabling specific recognition of their ligands. We also discuss the association of these domains with human diseases. Here we focus on CALM, a protein containing an ANTH domain, which is implicated in the pathogenesis of blood cancers and Alzheimer disease, and discuss how alteration of CALM function is involved in these diseases.

Genetic Risk Factors for Alzheimer Disease: Emerging Roles of Microglia in Disease Pathomechanisms.

The accumulation of aggregated amyloid ß (Aß) peptides in the brain is deeply involved in Alzheimer disease (AD) pathogenesis. Mutations in APP and presenilins play major roles in Aß pathology in rare autosomal-dominant forms of AD, whereas pathomechanisms of sporadic AD, accounting for the majority of cases, remain unknown. In this chapter, we review current knowledge on genetic risk factors of AD, clarified by recent advances in genome analysis technology. Interestingly, TREM2 and many genes associated with disease risk are predominantly expressed in microglia, suggesting that these risk factors are involved in pathogenicity through common mechanisms involving microglia. Therefore, we focus on factors closely associated with microglia and discuss their possible roles in pathomechanisms of AD. Furthermore, we review current views on the pathological roles of microglia and emphasize the importance of microglial changes in response to Aß deposition and mechanisms underlying the phenotypic changes. Importantly, functional outcomes of microglial activation can be both protective and deleterious to neurons. We further describe the involvement of microglia in tau pathology and the activation of other glial cells. Through these topics, we shed light on microglia as a promising target for drug development for AD and other neurological disorders.

Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes.

Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P2 using freeze-fracture electron microscopy. GST-ATG18-4×FLAG was used to label PtdIns(3,5)P2 and its binding to phosphatidylinositol 3-phosphate (PtdIns(3)P) was blocked by an excess of the p40(phox) PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P2 was concentrated in intramembrane particle (IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5)P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P2 metabolism is defective. In HeLa cells, PtdIns(3,5)P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5)P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P2 -rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality.

Partial loss of CALM function reduces Aß42 production and amyloid deposition in vivo.

Aberrant production, clearance and deposition of amyloid-ß protein (Aß) in the human brain have been implicated in the aetiology of Alzheimer disease (AD). γ-Secretase is the enzyme responsible for generating various Aß species, such as Aß40 and toxic Aß42. Recently, genome-wide association studies in late-onset AD patients have identified the endocytosis-related phosphatidylinositol-binding clathrin assembly protein (PICALM) gene as a genetic risk factor for AD. We previously found that the loss of expression of CALM protein encoded by PICALM affects the ratio of production of Aß42, through the regulation of the clathrin-mediated endocytosis of γ-secretase. Here, we show that the binding capacity of the assembly protein 180 N-terminal homology (ANTH) domain of CALM to phosphatidylinositol-4,5-biphosphate, as well as to nicastrin, is critical to the modulation of the internalization of γ-secretase and to the Aß42 production ratio. Moreover, reduction of CALM decreases Aß deposition as well as brain levels of insoluble Aß42 in vivo These results suggest that CALM expression modifies AD risk by regulating Aß pathology.

A theory for the phase behavior of mixtures of active particles.

Systems at equilibrium like molecular or colloidal suspensions have a well-defined thermal energy kBT that quantifies the particles' kinetic energy and gauges how "hot" or "cold" the system is. For systems far from equilibrium, such as active matter, it is unclear whether the concept of a "temperature" exists and whether self-propelled entities are capable of thermally equilibrating like passive Brownian suspensions. Here we develop a simple mechanical theory to study the phase behavior and "temperature" of a mixture of self-propelled particles. A mixture of active swimmers and passive Brownian particles is an ideal system for discovery of the temperature of active matter and the quantities that get shared upon particle collisions. We derive an explicit equation of state for the active/passive mixture to compute a phase diagram and to generalize thermodynamic concepts like the chemical potential and free energy for a mixture of nonequilibrium species. We find that different stability criteria predict in general different phase boundaries, facilitating considerations in simulations and experiments about which ensemble of variables are held fixed and varied.

Microscopic methods to observe the distribution of lipids in the cellular membrane.

Membrane lipids not only provide the structural framework of cellular membranes but also influence protein functions in several different ways. In comparison to proteins, however, relatively little is known about distribution of membrane lipids because of the insufficiency of microscopic methods. The difficulty in studying lipid distribution results from several factors, including their unresponsiveness to chemical fixation, fast translational movement, small molecular size, and high packing density. In this Current Topic, we consider the major microscopic methods and discuss whether and to what degree of precision these methods can reveal membrane lipid distribution in situ. We highlight two fixation methods, chemical and physical, and compare the theoretical limitations to their spatial resolution. Recognizing the strengths and weaknesses of each method should help researchers interpret their microscopic results and increase our understanding of the physiological functions of lipids.

Swim stress, motion, and deformation of active matter: effect of an external field.

We analyze the stress, dispersion, and average swimming speed of self-propelled particles subjected to an external field that affects their orientation and speed. The swimming trajectory is governed by a competition between the orienting influence (i.e., taxis) associated with the external (e.g., magnetic, gravitational, thermal, nutrient concentration) field versus the effects that randomize the particle orientations (e.g., rotary Brownian motion and/or an intrinsic tumbling mechanism like the flagella of bacteria). The swimmers' motion is characterized by a mean drift velocity and an effective translational diffusivity that becomes anisotropic in the presence of the orienting field. Since the diffusivity yields information about the micromechanical stress, the anisotropy generated by the external field creates a normal stress difference in the recently developed "swim stress" tensor [Takatori, Yan, and Brady, Phys. Rev. Lett., 2014]. This property can be exploited in the design of soft, compressible materials in which their size, shape, and motion can be manipulated and tuned by loading the material with active swimmers. Since the swimmers exert different normal stresses in different directions, the material can compress/expand, elongate, and translate depending on the external field strength. Such an active system can be used as nano/micromechanical devices and motors. Analytical solutions are corroborated by Brownian dynamics simulations.