A genome-wide screen links peroxisome regulation with Wnt signaling through RNF146 and TNKS/2.

Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced the import of proteins into peroxisomes. RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerases TNKS and TNKS2, which bind the peroxisomal membrane protein PEX14. We propose that RNF146 and TNKS/2 regulate peroxisome import efficiency by PARsylation of proteins at the peroxisome membrane. Interestingly, we found that the loss of peroxisomes increased TNKS/2 and RNF146-dependent degradation of non-peroxisomal substrates, including the ß-catenin destruction complex component AXIN1, which was sufficient to alter the amplitude of ß-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation but also a novel role in bridging peroxisome function with Wnt/ß-catenin signaling during development.

Engineering water exchange is a safe and effective method for magnetic resonance imaging in diverse cell types.

Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

A genome-wide screen links peroxisome regulation with Wnt signaling through RNF146 and tankyrase.

Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced the import of proteins into peroxisomes. RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerase tankyrase, which binds the peroxisomal membrane protein PEX14. We propose that RNF146 and tankyrase regulate peroxisome import efficiency by PARsylation of proteins at the peroxisome membrane. Interestingly, we found that the loss of peroxisomes increased tankyrase and RNF146-dependent degradation of non-peroxisomal substrates, including the beta-catenin destruction complex component AXIN1, which was sufficient to alter the amplitude of beta-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation, but also a novel role in bridging peroxisome function with Wnt/beta-catenin signaling during development.

Hyperactive Rac stimulates cannibalism of living target cells and enhances CAR-M-mediated cancer cell killing.

The 21kD GTPase Rac is an evolutionarily ancient regulator of cell shape and behavior. Rac2 is predominantly expressed in hematopoietic cells where it is essential for survival and motility. The hyperactivating mutation Rac2E62K also causes human immunodeficiency, although the mechanism remains unexplained. Here, we report that in Drosophila, hyperactivating Rac stimulates ovarian cells to cannibalize neighboring cells, destroying the tissue. We then show that hyperactive Rac2E62K stimulates human HL60-derived macrophage-like cells to engulf and kill living T cell leukemia cells. Primary mouse Rac2+/E62K bone-marrow-derived macrophages also cannibalize primary Rac2+/E62K T cells due to a combination of macrophage hyperactivity and T cell hypersensitivity to engulfment. Additionally, Rac2+/E62K macrophages non-autonomously stimulate wild-type macrophages to engulf T cells. Rac2E62K also enhances engulfment of target cancer cells by chimeric antigen receptor-expressing macrophages (CAR-M) in a CAR-dependent manner. We propose that Rac-mediated cell cannibalism may contribute to Rac2+/E62K human immunodeficiency and enhance CAR-M cancer immunotherapy.

Prior Fc Receptor activation primes macrophages for increased sensitivity to IgG via long term and short term mechanisms.

Macrophages measure the 'eat-me' signal IgG to identify targets for phagocytosis. We wondered if prior encounters with IgG influence macrophage appetite. IgG is recognized by the Fc Receptor. To temporally control Fc Receptor activation, we engineered an Fc Receptor that is activated by light-induced oligomerization of Cry2, triggering phagocytosis. Using this tool, we demonstrate that Fc Receptor activation primes macrophages to be more sensitive to IgG in future encounters. Macrophages that have previously experienced Fc Receptor activation eat more IgG-bound cancer cells. Increased phagocytosis occurs by two discrete mechanisms - a short- and long-term priming. Long term priming requires new protein synthesis and Erk activity. Short term priming does not require new protein synthesis and correlates with an increase in Fc Receptor mobility. Our work demonstrates that IgG primes macrophages for increased phagocytosis, suggesting that therapeutic antibodies may become more effective after initial priming doses.

Engineering water exchange is a safe and effective method for magnetic resonance imaging in diverse cell types.

Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

Leveraging DNA Origami to Study Phagocytosis.

Many plasma membrane receptors and ligands form nanoscale clusters on the plasma membrane surface. However, methods for directly and precisely manipulating nanoscale protein localization are limited, making understanding the effects of this clustering difficult. DNA origami allows precise control over nanoscale protein localization with high fidelity and adaptability. Here, we describe how we have used this technique to study how nanoscale protein clustering affects phagocytosis. We provide protocols for conjugating DNA origami structures to supported lipid bilayer-coated beads to assay phagocytosis and planar glass coverslips for TIRF microscopy. The core aspects of this protocol can be translated to study other immune signaling pathways and should enable the implementation of previously inaccessible investigations.

Evaluation of a Modified Bit Device to Obtain Saliva Samples from Horses.

(1) Background: Accounting for the well-being of equine partners is a responsibility of those engaged in Equine-Assisted Services (EAS). Researchers took heed of this call to action by developing an innovative way to collect data to assess the physiological indicators of stress in equine participants. The collection of saliva is considered to be a minimally invasive method of data collection and is typically performed using a cotton swab; however, in equines, the introduction of a foreign object may induce stress; (2) Methods: Researchers used a modified bit to collect pooled saliva in an effort to further reduce stress during the saliva collection process. Additionally, the collection of pooled saliva, via the bit, increases the opportunity to consider additional analyses, such as oxytocin, which is more reliable in pooled saliva than site-specific saliva captured with a swab; (3) Results: A data analysis demonstrated that ample saliva was captured using the modified bit. Observational data supported that the horses demonstrated fewer physical stress signals to the bit than to the swab. Thus, the modified bit is a feasible and valid method for equine salivary sample collection; (4) Conclusions: The results suggest that the modified bit provides a viable method to collect equine saliva and supports national calls to prioritize animal welfare analysis, specifically for horses used within EAS. Future research should enhance methodological rigor, including in the process and timing, thereby contributing to the bit's validation.

Tight nanoscale clustering of Fcγ receptors using DNA origami promotes phagocytosis.

Macrophages destroy pathogens and diseased cells through Fcγ receptor (FcγR)-driven phagocytosis of antibody-opsonized targets. Phagocytosis requires activation of multiple FcγRs, but the mechanism controlling the threshold for response is unclear. We developed a DNA origami-based engulfment system that allows precise nanoscale control of the number and spacing of ligands. When the number of ligands remains constant, reducing ligand spacing from 17.5 nm to 7 nm potently enhances engulfment, primarily by increasing efficiency of the engulfment-initiation process. Tighter ligand clustering increases receptor phosphorylation, as well as proximal downstream signals. Increasing the number of signaling domains recruited to a single ligand-receptor complex was not sufficient to recapitulate this effect, indicating that clustering of multiple receptors is required. Our results suggest that macrophages use information about local ligand densities to make critical engulfment decisions, which has implications for the mechanism of antibody-mediated phagocytosis and the design of immunotherapies.

The word 'phagocytosis' means cellular eating. It is the process by which cells extend their membranes around foreign particles and engulf them. Macrophages, a type of immune cell found in every tissue of the body, perform phagocytosis to eat pathogens and diseased cells. To avoid eating healthy cells, macrophages focus on targets marked by proteins called antibodies. They look for cells coated with high levels of a type of antibody called immunoglobulin G, or IgG for short, but only eat cells coated with enough IgG, raising the question, can macrophages count? Macrophages recognize IgG antibodies using cell surface receptors called Fc-gamma Receptors. When these receptors bind to IgG, they cluster together. Researchers do not yet know how the number of IgG antibodies per cluster, or the spacing between them, affects phagocytosis. To find this out, researchers need to be able to manipulate the clustering experimentally. One way to do this is using a technique called DNA origami. This technique creates nanoscale patterns of DNA strands on a target surface. If the part of a receptor that interacts with its target is then replaced with a complementary DNA strand to the strands on the target surface, the receptor will bind the surface following the nanoscale pattern. This allows researchers to generate synthetic targets with specific patterns of receptor-target interaction. Kern et al. replaced the part of the macrophage Fc-gamma Receptor that interacts with IgG with a strand of DNA. They then used DNA origami to arrange complementary DNA strands on pegboards and attached these pegboards to silica beads. The different arrangements of DNA on these pegboards mimicked the types of antibody clusters macrophages might encounter on the surfaces of the cells and particles they have to engulf in the body. Kern et al. found that tight clusters of the DNA targets on the pegboards made the macrophages most likely to begin phagocytosis, particularly clusters of eight or more DNA strands spaced less than seven nanometers apart. Macrophages encountering these tight clusters showed an increase in Fc-gamma receptor activation, which is crucial for macrophage attack. Whether or not macrophages can count, they can at least sense the level of clustering of IgG antibodies to determine if a target should be engulfed. Doctors use antibody therapies that rely on Fc-gamma receptor engagement to treat cancer, autoimmune and neurodegenerative diseases. Understanding how clustering affects phagocytosis could aid in the design of new antibody treatments. It could also help improve the design of synthetic receptors to create designer immune cells that can attack specific targets. The next step will be to recreate the results from the synthetic system used by Kern et al. with natural receptors and antibodies.

CD47 Ligation Repositions the Inhibitory Receptor SIRPA to Suppress Integrin Activation and Phagocytosis.

CD47 acts as a "don't eat me" signal that protects cells from phagocytosis by binding and activating its receptor SIPRA on macrophages. CD47 suppresses multiple different pro-engulfment "eat me" signals, including immunoglobulin G (IgG), complement, and calreticulin, on distinct target cells. This complexity has limited understanding of how the "don't eat me" signal is transduced biochemically. Here, we utilized a reconstituted system with a defined set of signals to interrogate the mechanism of SIRPA activation and its downstream targets. CD47 ligation altered SIRPA localization, positioning SIRPA for activation at the phagocytic synapse. At the phagocytic synapse, SIRPA inhibited integrin activation to limit macrophage spreading across the surface of the engulfment target. Chemical reactivation of integrin bypassed CD47-mediated inhibition and rescued engulfment, similar to the effect of a CD47 function-blocking antibody. Thus, the CD47-SIRPA axis suppresses phagocytosis by inhibiting inside-out activation of integrin signaling in the macrophage, with implications to cancer immunotherapy applications.

Chimeric antigen receptors that trigger phagocytosis.

Chimeric antigen receptors (CARs) are synthetic receptors that reprogram T cells to kill cancer. The success of CAR-T cell therapies highlights the promise of programmed immunity and suggests that applying CAR strategies to other immune cell lineages may be beneficial. Here, we engineered a family of Chimeric Antigen Receptors for Phagocytosis (CAR-Ps) that direct macrophages to engulf specific targets, including cancer cells. CAR-Ps consist of an extracellular antibody fragment, which can be modified to direct CAR-P activity towards specific antigens. By screening a panel of engulfment receptor intracellular domains, we found that the cytosolic domains from Megf10 and FcRÉ£ robustly triggered engulfment independently of their native extracellular domain. We show that CAR-Ps drive specific engulfment of antigen-coated synthetic particles and whole human cancer cells. Addition of a tandem PI3K recruitment domain increased cancer cell engulfment. Finally, we show that CAR-P expressing murine macrophages reduce cancer cell number in co-culture by over 40%.

Cell Invasion In Vivo via Rapid Exocytosis of a Transient Lysosome-Derived Membrane Domain.

Invasive cells use small invadopodia to breach basement membrane (BM), a dense matrix that encases tissues. Following the breach, a large protrusion forms to clear a path for tissue entry by poorly understood mechanisms. Using RNAi screening for defects in Caenorhabditis elegans anchor cell (AC) invasion, we found that UNC-6(netrin)/UNC-40(DCC) signaling at the BM breach site directs exocytosis of lysosomes using the exocyst and SNARE SNAP-29 to form a large protrusion that invades vulval tissue. Live-cell imaging revealed that the protrusion is enriched in the matrix metalloprotease ZMP-1 and transiently expands AC volume by more than 20%, displacing surrounding BM and vulval epithelium. Photobleaching and genetic perturbations showed that the BM receptor dystroglycan forms a membrane diffusion barrier at the neck of the protrusion, which enables protrusion growth. Together these studies define a netrin-dependent pathway that builds an invasive protrusion, an isolated lysosome-derived membrane structure specialized to breach tissue barriers.

SPARC Promotes Cell Invasion In Vivo by Decreasing Type IV Collagen Levels in the Basement Membrane.

Overexpression of SPARC, a collagen-binding glycoprotein, is strongly associated with tumor invasion through extracellular matrix in many aggressive cancers. SPARC regulates numerous cellular processes including integrin-mediated cell adhesion, cell signaling pathways, and extracellular matrix assembly; however, the mechanism by which SPARC promotes cell invasion in vivo remains unclear. A main obstacle in understanding SPARC function has been the difficulty of visualizing and experimentally examining the dynamic interactions between invasive cells, extracellular matrix and SPARC in native tissue environments. Using the model of anchor cell invasion through the basement membrane (BM) extracellular matrix in Caenorhabditis elegans, we find that SPARC overexpression is highly pro-invasive and rescues BM transmigration in mutants with defects in diverse aspects of invasion, including cell polarity, invadopodia formation, and matrix metalloproteinase expression. By examining BM assembly, we find that overexpression of SPARC specifically decreases levels of BM type IV collagen, a crucial structural BM component. Reduction of type IV collagen mimicked SPARC overexpression and was sufficient to promote invasion. Tissue-specific overexpression and photobleaching experiments revealed that SPARC acts extracellularly to inhibit collagen incorporation into BM. By reducing endogenous SPARC, we also found that SPARC functions normally to traffic collagen from its site of synthesis to tissues that do not express collagen. We propose that a surplus of SPARC disrupts extracellular collagen trafficking and reduces BM collagen incorporation, thus weakening the BM barrier and dramatically enhancing its ability to be breached by invasive cells.

An active role for basement membrane assembly and modification in tissue sculpting.

Basement membranes are a dense, sheet-like form of extracellular matrix (ECM) that underlie epithelia and endothelia, and surround muscle, fat and Schwann cells. Basement membranes separate tissues and protect them from mechanical stress. Although traditionally thought of as a static support structure, a growing body of evidence suggests that dynamic basement membrane deposition and modification instructs coordinated cellular behaviors and acts mechanically to sculpt tissues. In this Commentary, we highlight recent studies that support the idea that far from being a passive matrix, basement membranes play formative roles in shaping tissues.

B-LINK: a hemicentin, plakin, and integrin-dependent adhesion system that links tissues by connecting adjacent basement membranes.

Basement membrane (BM), a sheet-like form of extracellular matrix, surrounds most tissues. During organogenesis, specific adhesions between adjoining tissues frequently occur; however, their molecular basis is unclear. Using live-cell imaging and electron microscopy, we identify an adhesion system that connects the uterine and gonadal tissues through their juxtaposed BMs at the site of anchor cell (AC) invasion in C. elegans. We find that the extracellular matrix component hemicentin (HIM-4), found between BMs, forms punctate accumulations under the AC and controls BM linkage to promote rapid invasion. Through targeted screening, we identify the integrin-binding cytolinker plakin (VAB-10A) and integrin (INA-1/PAT-3) as key BM-BM linkage regulators: VAB-10A localizes to the AC-BM interface and tethers hemicentin to the AC while integrin promotes hemicentin punctae formation. Together, plakin, integrin, and hemicentin are founding components of a cell-directed adhesion system, which we name a BM-LINKage (B-LINK), that connects adjacent tissues through adjoining BMs.

The netrin receptor DCC focuses invadopodia-driven basement membrane transmigration in vivo.

Though critical to normal development and cancer metastasis, how cells traverse basement membranes is poorly understood. A central impediment has been the challenge of visualizing invasive cell interactions with basement membrane in vivo. By developing live-cell imaging methods to follow anchor cell (AC) invasion in Caenorhabditis elegans, we identify F-actin-based invadopodia that breach basement membrane. When an invadopodium penetrates basement membrane, it rapidly transitions into a stable invasive process that expands the breach and crosses into the vulval tissue. We find that the netrin receptor UNC-40 (DCC) specifically enriches at the site of basement membrane breach and that activation by UNC-6 (netrin) directs focused F-actin formation, generating the invasive protrusion and the cessation of invadopodia. Using optical highlighting of basement membrane components, we further demonstrate that rather than relying solely on proteolytic dissolution, the AC's protrusion physically displaces basement membrane. These studies reveal an UNC-40-mediated morphogenetic transition at the cell-basement membrane interface that directs invading cells across basement membrane barriers.

Cell invasion through basement membrane: The netrin receptor DCC guides the way.

Cell invasion through basement membrane is an essential part of normal development and physiology, and occurs during the pathological progression of human inflammatory diseases and cancer. F-actin-rich membrane protrusions, called invadopodia, have been hypothesized to be the "drill bits" of invasive cells, mediating invasion through the dense, highly cross-linked basement membrane matrix. Though studied in vitro for over 30 y, invadopodia function in vivo has remained elusive. We have recently discovered that invadopodia breach basement membrane during anchor cell invasion in C. elegans, a genetically and visually tractable in vivo invasion event. Further, we found that the netrin receptor DCC localizes to the initial site of basement membrane breach and directs invasion through a single gap in the matrix. In this commentary, we examine how the dynamics and structure of AC-invadopodia compare with in vitro invadopodia and how the netrin receptor guides invasion through a single basement membrane breach. We end with a discussion of our surprising result that the anchor cell pushes the basement membrane aside, instead of completely dissolving it through proteolysis, and provide some ideas for how proteases and physical displacement may work together to ensure efficient and robust invasion.

Zwint-1 is a novel Aurora B substrate required for the assembly of a dynein-binding platform on kinetochores.

Aurora B (AurB) is a mitotic kinase responsible for multiple aspects of mitotic progression, including assembly of the outer kinetochore. Cytoplasmic dynein is an abundant kinetochore protein whose recruitment to kinetochores requires phosphorylation. To assess whether AurB regulates recruitment of dynein to kinetochores, we inhibited AurB using ZM447439 or a kinase-dead AurB construct. Inhibition of AurB reduced accumulation of dynein at kinetochores substantially; however, this reflected a loss of dynein-associated proteins rather than a defect in dynein phosphorylation. We determined that AurB inhibition affected recruitment of the ROD, ZW10, zwilch (RZZ) complex to kinetochores but not zwint-1 or more-proximal kinetochore proteins. AurB phosphorylated zwint-1 but not ZW10 in vitro, and three novel phosphorylation sites were identified by tandem mass spectrometry analysis. Expression of a triple-Ala zwint-1 mutant blocked kinetochore assembly of RZZ-dependent proteins and induced defects in chromosome movement during prometaphase. Expression of a triple-Glu zwint-1 mutant rendered cells resistant to AurB inhibition during prometaphase. However, cells expressing the triple-Glu mutant failed to satisfy the spindle assembly checkpoint (SAC) at metaphase because poleward streaming of dynein/dynactin/RZZ was inhibited. These studies identify zwint-1 as a novel AurB substrate required for kinetochore assembly and for proper SAC silencing at metaphase.

Basement membrane sliding and targeted adhesion remodels tissue boundaries during uterine-vulval attachment in Caenorhabditis elegans.

Large gaps in basement membrane occur at sites of cell invasion and tissue remodelling in development and cancer. Though never followed directly in vivo, basement membrane dissolution or reduced synthesis have been postulated to create these gaps. Using landmark photobleaching and optical highlighting of laminin and type IV collagen, we find that a new mechanism, basement membrane sliding, underlies basement membrane gap enlargement during uterine-vulval attachment in Caenorhabditis elegans. Laser ablation and mutant analysis reveal that the invaginating vulval cells promote basement membrane movement. Further, an RNA interference and expression screen identifies the integrin INA-1/PAT-3 and VAB-19, homologue of the tumour suppressor Kank, as regulators of basement membrane opening. Both concentrate within vulval cells at the basement membrane gap boundary and halt expansion of the shifting basement membrane. Basement membrane sliding followed by targeted adhesion represents a new mechanism for creating precise basement membrane breaches that can be used by cells to break down compartment boundaries.