Cup Formation Research Articles

Spatiotemporal control of actomyosin contractility by MRCKβ signaling drives phagocytosis

Phagocytosis requires actin dynamics, but whether actomyosin contractility plays a role in this morphodynamic process is unclear. Here, we show that in the retinal pigment epithelium (RPE), particle binding to Mer Tyrosine Kinase (MerTK), a widely expressed phagocytic receptor, stimulates phosphorylation of the Cdc42 GEF Dbl3, triggering activation of MRCKβ/myosin-II and its coeffector N-WASP, membrane deformation, and cup formation. Continued MRCKβ/myosin-II activity then drives recruitment of a mechanosensing bridge, enabling cytoskeletal force transmission, cup closure, and particle internalization. In vivo, MRCKβ is essential for RPE phagocytosis and retinal integrity. MerTK-independent activation of MRCKβ signaling by a phosphomimetic Dbl3 mutant rescues phagocytosis in retinitis pigmentosa RPE cells lacking functional MerTK. MRCKβ is also required for efficient particle translocation from the cortex into the cell body in Fc receptor-mediated phagocytosis. Thus, conserved MRCKβ signaling at the cortex controls spatiotemporal regulation of actomyosin contractility to guide distinct phases of phagocytosis in the RPE and represents the principle phagocytic effector pathway downstream of MerTK.

A two characteristic length nonlocal GTN model: Application to cup–cone and slant fracture

Ductile failure prediction is essential to avoid loss of structural integrity or to control crack propagation during forming processes. One of the main difficulties is the prediction of complex crack paths. This study proposes a nonlocal extension of a local Gurson–Tvergaard–Needleman (GTN) ductile damage model at finite strains able to capture cup–cone or slant fracture. The proposed model is based on an implicit gradient formulation which enables to solve the problem of spurious strain and damage localization. The model integrates two different material characteristic lengths, which are used to separately regularize damage by void growth and damage by damage nucleation. After parameter fitting using data for a line pipe steel, conditions to obtain converged solutions are studied, which can be used to select the mesh size in the localization band. With the appropriate mesh design, it was possible to study the effect of the characteristic lengths on the formation of cup–cone fracture and slant fracture. It is observed that, for a given specimen size, larger characteristic lengths favor flat crack advance. Similarly, for given material lengths, size effects can be predicted with small specimens being more prone to flat fracture. This paves the way to a more direct determination of material lengths by using homothetic specimens so as to obtain different crack paths.

Suppression of trabecular meshwork phagocytosis by norepinephrine is associated with nocturnal increase in intraocular pressure in mice

Intraocular pressure (IOP) is an important factor in glaucoma development, which involves aqueous humor (AH) dynamics, with inflow from the ciliary body and outflow through the trabecular meshwork (TM). IOP has a circadian rhythm entrained by sympathetic noradrenaline (NE) or adrenal glucocorticoids (GCs). Herein, we investigated the involvement of GC/NE in AH outflow. Pharmacological prevention of inflow/outflow in mice indicated a diurnal outflow increase, which was related to TM phagocytosis. NE showed a non-self-sustained inhibition in phagocytosis of immortalized human TM cells, but not GC. The pharmacological and reverse genetic approaches identified β1-adrenergic receptor (AR)-mediated exchange proteins directly activated by cyclic adenosine monophosphate (EPAC)-SHIP1 signal activation by ablation of phosphatidylinositol triphosphate, regulating phagocytic cup formation. Furthermore, we revealed the phagocytosis involvement in the β1-AR-EPAC-SHIP1-mediated nocturnal IOP rise in mice. These suggest that TM phagocytosis suppression by NE can regulate IOP rhythm through AH outflow. This discovery may aid glaucoma management.

Loss of zebrafish dzip1 results in inappropriate recruitment of periocular mesenchyme to the optic fissure and ocular coloboma.

Cilia are essential for the development and function of many different tissues. Although cilia machinery is crucial in the eye for photoreceptor development and function, a role for cilia in early eye development and morphogenesis is still somewhat unclear: many zebrafish cilia mutants retain cilia at early stages due to maternal deposition of cilia components. An eye phenotype has been described in the mouse Arl13 mutant, however, zebrafish arl13b is maternally deposited, and an early role for cilia proteins has not been tested in zebrafish eye development. Here we use the zebrafish dzip1 mutant, which exhibits a loss of cilia throughout stages of early eye development, to examine eye development and morphogenesis. We find that in dzip1 mutants, initial formation of the optic cup proceeds normally, however, the optic fissure subsequently fails to close and embryos develop the structural eye malformation ocular coloboma. Further, neural crest cells, which are implicated in optic fissure closure, do not populate the optic fissure correctly, suggesting that their inappropriate localization may be the underlying cause of coloboma. Overall, our results indicate a role for dzip1 in proper neural crest localization in the optic fissure and optic fissure closure.

Mechanical, fracture toughness, and fatigue behavior of spinifex littoreus fiber on echinoidea‐spike toughened epoxy composite

AbstractNovel spinifex littores fiber‐reinforced echinoidea spike particle dispersed epoxy resin hybrid composite has been prepared and characterized. The main objective of this research work was to prepare a new system of epoxy hybrid composite comprises of surface‐treated novel spinifex littores fiber (SLF) and echinoidea spike powder (ESP) for sustainable material development. The fiber was treated with alkali and silane for effective adhesion with matrix. The composites were prepared using hand layup method and post cured at 120°C. The mechanical properties were improved with increasing particle content. The wear result shows a lowest coefficient of friction and sp. wear rate of 0.43 and 0.004 mm3/Nm for SP5 composite designation. Fracture toughness of 32.1 ± 0.69 MPa √m and energy release rate of 1.257 ± 0.071 mJ/m2 was observed for composite designation SP4. A highest fatigue life count of 26,832 was observed for composite contains 1 vol% of silane‐treated ESP and 8 h alkali cum silane‐treated SPF. The fractographs reveals toughness improved matrix phase via shear cup formation. These marine based eco‐friendly reinforcement based epoxy composites could be used in various applications in the cadre of automobile, mini‐aircrafts, structural, sports, and domestic.

Investigation of contact dynamics and cell rheology during early phagocytic binding

Phagocytosis, the process in which phagocytes clear pathogens and apoptotic cells is an important part of our innate immune defense. After cell-target binding and subsequent target detection by receptors in the membrane, the formation of the phagocytic cup around the target is driven by the polymerization of cortical actin. While the main proteins involved in phagosome formation have already been identified, a full model of the uptake process has not been developed yet, in part because mechanical aspects such as the early binding kinetics and the cortical rheology during the process remain largely elusive.

Three-dimensional morphodynamic simulations of macropinocytic cups

SummaryMacropinocytosis refers to the non-specific uptake of extracellular fluid, which plays ubiquitous roles in cell growth, immune surveillance, and virus entry. Despite its widespread occurrence, it remains unclear how its initial cup-shaped plasma membrane extensions form without any external solid support, as opposed to the process of particle uptake during phagocytosis. Here, by developing a computational framework that describes the coupling between the bistable reaction-diffusion processes of active signaling patches and membrane deformation, we demonstrated that the protrusive force localized to the edge of the patches can give rise to a self-enclosing cup structure, without further assumptions of local bending or contraction. Efficient uptake requires a balance among the patch size, magnitude of protrusive force, and cortical tension. Furthermore, our model exhibits cyclic cup formation, coexistence of multiple cups, and cup-splitting, indicating that these complex morphologies self-organize via a common mutually-dependent process of reaction-diffusion and membrane deformation.

Stretching of the retinal pigment epithelium contributes to zebrafish optic cup morphogenesis.

The vertebrate eye primordium consists of a pseudostratified neuroepithelium, the optic vesicle (OV), in which cells acquire neural retina or retinal pigment epithelium (RPE) fates. As these fates arise, the OV assumes a cup shape, influenced by mechanical forces generated within the neural retina. Whether the RPE passively adapts to retinal changes or actively contributes to OV morphogenesis remains unexplored. We generated a zebrafish Tg(E1-bhlhe40:GFP) line to track RPE morphogenesis and interrogate its participation in OV folding. We show that, in virtual absence of proliferation, RPE cells stretch and flatten, thereby matching the retinal curvature and promoting OV folding. Localized interference with the RPE cytoskeleton disrupts tissue stretching and OV folding. Thus, extreme RPE flattening and accelerated differentiation are efficient solutions adopted by fast-developing species to enable timely optic cup formation. This mechanism differs in amniotes, in which proliferation drives RPE expansion with a much-reduced need of cell flattening.

Acute RhoA/Rho Kinase Inhibition Is Sufficient to Restore Phagocytic Capacity to Retinal Pigment Epithelium Lacking the Engulfment Receptor MerTK.

The diurnal phagocytosis of spent photoreceptor outer segment fragments (POS) by retinal pigment epithelial (RPE) cells is essential for visual function. POS internalization by RPE cells requires the assembly of F-actin phagocytic cups beneath surface-tethered POS and Mer tyrosine kinase (MerTK) signaling. The activation of the Rho family GTPase Rac1 is necessary for phagocytic cup formation, and Rac1 is activated normally in MerTK-deficient RPE. We show here that mutant RPE lacking MerTK and wild-type RPE deprived of MerTK ligand both fail to form phagocytic cups regardless of Rac1 activation. However, in wild-type RPE in vivo, a decrease in RhoA activity coincides with the daily phagocytosis burst, while RhoA activity in MerTK-deficient RPE is constant. Elevating RhoA activity blocks phagocytic cup formation and phagocytosis by wild-type RPE. Conversely, inhibiting RhoA effector Rho kinases (ROCKs) rescues both F-actin assembly and POS internalization of primary RPE if MerTK or its ligand are lacking. Most strikingly, acute ROCK inhibition is sufficient to induce the formation and acidification of endogenous POS phagosomes by MerTK-deficient RPE ex vivo. Altogether, RhoA pathway inactivation is a necessary and sufficient downstream effect of MerTK phagocytic signaling such that the acute manipulation of cytosolic ROCK activity suffices to restore phagocytic capacity to MerTK-deficient RPE.

Neutrophil elastase-regulated macrophage sheddome/secretome and phagocytic failure.

Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE), a major inflammatory protease in the CF airway, impairs macrophage phagocytic function. To date, NE-impaired macrophage phagocytic function has been attributed to cleavage of cell surface receptors or opsonins. We applied an unbiased proteomic approach to identify other potential macrophage targets of NE protease activity that may regulate phagocytic function. Using the murine macrophage cell line, RAW 264.7, human blood monocyte-derived macrophages, and primary alveolar macrophages from Cftr-null and wild-type littermate mice, we demonstrated that NE exposure blocked phagocytosis of Escherichia coli bio-particles. We performed liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic analysis of the conditioned media from RAW264.7 treated either with active NE or inactive (boiled) NE as a control. Out of 840 proteins identified in the conditioned media, active NE upregulated 142 proteins and downregulated 211 proteins. NE released not only cell surface proteins into the media but also cytoskeletal, mitochondrial, cytosolic, and nuclear proteins that were detected in the conditioned media. At least 32 proteins were associated with the process of phagocytosis including 11 phagocytic receptors [including lipoprotein receptor-related protein 1 (LRP1)], 7 proteins associated with phagocytic cup formation, and 14 proteins involved in phagocytic maturation (including calpain-2) and phagolysosome formation. NE had a broad effect on the proteome required for regulation of all stages of phagocytosis and phagolysosome formation. Furthermore, the NE sheddome/secretome included proteins from other macrophage cellular domains, suggesting that NE may globally regulate macrophage structure and function.

Antibodies Against Macrophage CD44 Protect Platelets from FcγR-Mediated Phagocytosis: A Potential Mechanism for CD44 Antibody Amelioration of Murine Immune Thrombocytopenia

CD44 is a transmembrane protein that is widely expressed on leukocytes and other cell types. CD44 is the major receptor for hyaluronan, and CD44-hyaluronan binding plays an important role in regulating immune cell migration, inflammation, and wound repair. Interestingly, targeting CD44 with monoclonal antibodies (anti-CD44) has demonstrated therapeutic efficacy in numerous murine autoimmune disease models including passive antibody-mediated immune thrombocytopenia (ITP), where it is effective at a three log-fold-lower dose compared to intravenous immunoglobulin (IVIg). However, the anti-inflammatory mechanisms behind anti-CD44 remain largely unsolved. In passive antibody-mediated ITP, anti-platelet antibody is administered to induce thrombocytopenia by FcγR-dependent and independent mechanisms, and macrophage depletion as well as FcγR blocking antibodies protect platelets from FcγR-dependent removal. The efficacy of anti-CD44 in this model suggest to us that anti-CD44 may be interfering with platelet removal by phagocytes. Here we show that anti-CD44 inhibits macrophage FcγR-mediated phagocytosis and protects platelets from macrophage uptake in vitro. Anti-CD44 inhibited RAW 264.7 macrophage phagocytosis of antibody-opsonized platelets in a dose-dependent manner, up to near complete (>90%) inhibition as compared to isotype controls. Multiple anti-CD44 clones tested were capable of inhibiting macrophage uptake of platelets opsonized with different anti-platelet antibodies. The binding of antibody-opsonized platelets to anti-CD44-treated macrophages was not blocked, suggesting that inhibition of phagocytosis occurs at a post-FcγR binding step. Because CD44 can regulate actin polymerization by recruiting the Rho-family GTPase-activating proteins Tiam1 and Vav2, we asked whether anti-CD44 may be interfering with actin polymerization required for platelet uptake. Live-cell microscopy of RAW 264.7 macrophages transfected with LifeAct (plasmid encoding a fluorescent non-cytotoxic actin binding protein) revealed that anti-CD44 did not affect actin dynamics and filopodia formation in the absence of antibody-opsonized platelets. However, anti-CD44-treated macrophages failed to initiate phagocytic cup formation upon antibody-opsonized-platelet binding, suggesting that inhibition is upstream of actin polymerization initiated by FcγRs. Finally, the ability of anti-CD44 to inhibit platelet uptake also showed complete concordance with a blocking antibody against FcγRIIb/III, implicating that anti-CD44 may be mediating inhibition of the FcγRIII pathway. In conclusion, our data suggests that antibodies against CD44 protect platelets from macrophage phagocytosis in vitro by inhibition of FcγRs, and that inhibition occurs at a step post-binding but before phagocytic cup formation. New therapies for ITP are needed to replace current donor-derived therapeutics and irreversible splenectomy. This research helps to shed light on how anti-CD44 functions to normalize platelet counts in murine passive ITP, and may pave the way for future novel therapeutics to replace IVIg in the treatment of ITP. DisclosuresLazarus:CSL pharmaceuticals: Research Funding; St.Michael's Hosp/The Canadian Blood Services: Other: Patents on monoclonal antibodies as IVIg and anti-D replacements; Rigel pharmaceuticals: Research Funding; Momenta pharmaceuticals: Honoraria.

Experimental Modeling of Pressure in the Hydrostatic Formation of a Cylindrical Cup with Different Materials

Forming complex sheet products using hydrostatic forming technology is currently a focus of the majority of forming processes. However, in order to increase stability during the forming process, it is necessary to identify and analyze the dependency of the forming pressure and the quality of a product on input parameters. For the purpose of modeling the forming pressure, this paper presents empirical research on the product of a cylindrical cup made of various materials, including carbon steel (DC04), copper (CDA260), and stainless steel (SUS 304) with different thicknesses (0.8 mm, 1.0 mm, and 1.2 mm), under a defined range of binder pressures. The regression method is selected to formulate an equation that shows the relationship between the input parameters, including the materials (ultimate strength and yield stress), workpiece thickness, binder pressure and the output parameter, and the formation of fluid pressure. The mathematical equation allows us to determine the extent of the effect of each input on the forming pressure. The experimental results can be used for the easier planning and forecasting of the process and product quality in hydrostatic forming.

Rab10-Positive Tubular Structures Represent a Novel Endocytic Pathway That Diverges From Canonical Macropinocytosis in RAW264 Macrophages.

Using the optogenetic photo-manipulation of photoactivatable (PA)-Rac1, remarkable cell surface ruffling and the formation of a macropinocytic cup (premacropinosome) could be induced in the region of RAW264 macrophages irradiated with blue light due to the activation of PA-Rac1. However, the completion of macropinosome formation did not occur until Rac1 was deactivated by the removal of the light stimulus. Following PA-Rac1 deactivation, some premacropinosomes closed into intracellular macropinosomes, whereas many others transformed into long Rab10-positive tubules without forming typical macropinosomes. These Rab10-positive tubules moved centripetally towards the perinuclear Golgi region along microtubules. Surprisingly, these Rab10-positive tubules did not contain any endosome/lysosome compartment markers, such as Rab5, Rab7, or LAMP1, suggesting that the Rab10-positive tubules were not part of the degradation pathway for lysosomes. These Rab10-positive tubules were distinct from recycling endosomal compartments, which are labeled with Rab4, Rab11, or SNX1. These findings suggested that these Rab10-positive tubules may be a part of non-degradative endocytic pathway that has never been known. The formation of Rab10-positive tubules from premacropinosomes was also observed in control and phorbol myristate acetate (PMA)-stimulated macrophages, although their frequencies were low. Interestingly, the formation of Rab10-positive premacropinosomes and tubules was not inhibited by phosphoinositide 3-kinase (PI3K) inhibitors, while the classical macropinosome formation requires PI3K activity. Thus, this study provides evidence to support the existence of Rab10-positive tubules as a novel endocytic pathway that diverges from canonical macropinocytosis.

An Evolutionarily Conserved Coreceptor Gene Is Essential for CLAVATA Signaling in Marchantia polymorpha

Growth and development of land plants are controlled by CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) family of peptide hormones. In contrast to the genetic diversity of CLE family in flowering plants, the liverwort Marchantia polymorpha possesses a minimal set of CLE, MpCLE1(TDIF homolog), and MpCLE2 (CLV3 homolog). MpCLE1 and MpCLE2 peptides exert distinct function at the apical meristem of M. polymorpha gametophyte via specific receptors, MpTDIF RECEPTOR (MpTDR) and MpCLAVATA1 (MpCLV1), respectively, both belonging to the subclass XI of leucine-rich repeat receptor-like kinases (LRR-RLKs). Biochemical and genetic studies in Arabidopsis have shown that TDR/PXY family and CLV1/BAM family recognize the CLE peptide ligand in a heterodimeric complex with a member of subclass-II coreceptors. Here we show that three LRR-RLK genes of M. polymorpha are classified into subclass II, representing three distinct subgroups evolutionarily conserved in land plants. To address the involvement of subclass-II coreceptors in M. polymorpha CLE signaling, we performed molecular genetic analysis on one of them, MpCLAVATA3 INSENSITIVE RECEPTOR KINASE (MpCIK). Two knockout alleles for MpCIK formed narrow apical meristems marked by promMpYUC2:GUS marker, which were not expanded by MpCLE2 peptide treatment, phenocopying Mpclv1. Loss of sensitivity to MpCLE2 peptide was also observed in gemma cup formation in both Mpclv1 and Mpcik. Biochemical analysis using a Nicotiana benthamiana transient expression system revealed weak association between MpCIK and MpCLV1, as well as MpCIK and MpTDR. While MpCIK may also participate in MpCLE1 signaling, our data show that the conserved CLV3-CLV1-CIK module functions in M. polymorpha, controlling meristem activity for development and organ formation for asexual reproduction.

Loss of Hem1 disrupts macrophage function and impacts migration, phagocytosis, and integrin-mediated adhesion

Hematopoietic-specific protein 1 (Hem1) is an essential subunit of the WAVE regulatory complex (WRC) in immune cells. WRC is crucial for Arp2/3 complex activation and the protrusion of branched actin filament networks. Moreover, Hem1 loss of function in immune cells causes autoimmune diseases in humans. Here, we show that genetic removal of Hem1 in macrophages diminishes frequency and efficacy of phagocytosis as well as phagocytic cup formation in addition to defects in lamellipodial protrusion and migration. Moreover, Hem1-null macrophages displayed strong defects in cell adhesion despite unaltered podosome formation and concomitant extracellular matrix degradation. Specifically, dynamics of both adhesion and de-adhesion as well as concomitant phosphorylation of paxillin and focal adhesion kinase (FAK) were significantly compromised. Accordingly, disruption of WRC function in non-hematopoietic cells coincided with both defects in adhesion turnover and altered FAK and paxillin phosphorylation. Consistently, platelets exhibited reduced adhesion and diminished integrin αIIbβ3 activation upon WRC removal. Interestingly, adhesion phenotypes, but not lamellipodia formation, were partially rescued by small molecule activation of FAK. A full rescue of the phenotype, including lamellipodia formation, required not only the presence of WRCs but also their binding to and activation by Rac. Collectively, our results uncover that WRC impacts on integrin-dependent processes in a FAK-dependent manner, controlling formation and dismantling of adhesions, relevant for properly grabbing onto extracellular surfaces and particles during cell edge expansion, like in migration or phagocytosis.

Effect of different concentrations of corn starch and whey protein on the characteristics of biodegradable cup

An attempt was made to prepare a composite biodegradable cup using corn starch, whey protein, carboxymethyl cellulose and glycerol. For biodegradable cup formation, corn starch (5-7%), WPC-35 (1.5-3%), CMC (0.5-2%) and glycerol (2-4%) range was selected. To decide upon temperature at which the cup forming solution attains final viscosity, samples were tested under rapid visco analyser to conclude that layering was enabled at 50oC. Based on observations three best biodegradable cups combinations were selected Sample A (corn starch 7%, WPC-35 3%, Cellulose 2%, glycerol 2.5%); Sample B (corn starch 7%, WPC-35 2%, Cellulose 2%, glycerol 2.5%); Sample C (corn starch 7%, WPC-35 3%, Cellulose 2%, glycerol 4.0%). Properties like thickness, solubility, water vapour transmission rate, viscosity, moisture, lightness were studied for three samples to select the final cup. Based on observation, Sample C's final composition was selected as corn starch 7%, WPC-35 3%, cellulose 2%, glycerol 4.0%. The value for thickness, solubility, WVTR, moisture and lightness was observed as 0.5±0.025mm, 48.50±0.66%, 1.14±0.086g/m2/hr, 7.74±0.16%, 41.96±1.86 respectively. The lower diameter of the cup was observed as 5.5cm, upper diameter 7.5cm and height of cup was observed as 4.0 cm with the capacity of the final cup as 55.5ml/47.1gm.

\u03b1-Linolenic acid induces clearance of Tau seeds via Actin-remodeling in Microglia

Alzheimer’s disease (AD) is known by characteristic features, extracellular burden of amyloid-β and intracellular neuronal Tau. Microglia, the innate immune cell of the brain has the ability to clear the burden of accumulated proteins via phagocytosis. But the excessive proinflammatory cytokine production, altered cellular signaling and actin remodeling hampers the process of migration and phagocytosis by microglia. Actin remodeling is necessary to initiate the chemotactic migration of microglia towards the target and engulf it. The formation of lamellipodia, filopodia, membrane ruffling and rapid turnover of F-actin is necessary to sense the extracellular target by the cells. Omega-3 fatty acids, are known to impose anti-inflammatory phenotype of microglia by enhancing its ability for migration and phagocytosis. But the role of omega-3 fatty acids in cellular actin remodeling, which is the basis of cellular functions such as migration and phagocytosis, is not well understood. Here, we have focused on the effect of dietary supplement of α-linolenic acid (ALA) on extracellular Tau internalization and assisted actin polymerization for the process. ALA is found to induce membrane ruffling and phagocytic cup formation along with cytoskeletal rearrangement. ALA also enhances the localization of Arp2/3 complex at the leading edge and its colocalization with F-actin to induce the actin polymerization. The excessive actin polymerization might help the cell to protrude forward and perform its migration. The results suggest that dietary supplement of ALA could play a neuroprotective role and slow down the AD pathology.

Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms.

A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events are unclear. Here we used real-time 3D imaging and knockout mouse models to clarify how particles (human red blood cells) are internalized by resident peritoneal F4/80+ cells (macrophages) via CRs and/or FcγRs. We first show that FcγRs mediate highly efficient, rapid (2-3 min) phagocytic cup formation, which is completely abolished by deletion or mutation of the FcR γ-chain or conditional deletion of the signal transducer Syk. FcγR-mediated phagocytic cups robustly arise from any point of cell-particle contact, including filopodia. In the absence of CR3, FcγR-mediated phagocytic cups exhibit delayed closure and become aberrantly elongated. Independent of FcgRs, CR3 mediates sporadic ingestion of complement-opsonized particles by rapid phagocytic cup-like structures, typically emanating from membrane ruffles and largely prevented by deletion of the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ-chain and DAP12 or Syk. Deletion of ITAM adaptors or Syk clearly revealed that there is a slow (10-25 min) sinking mode of phagocytosis via a restricted orifice. In summary, we show that (1) CR3 indeed mediates a slow sinking mode of phagocytosis, which is accentuated by deletion of ITAM adaptors or Syk, (2) CR3 induces phagocytic cup-like structures, driven by ITAM adaptors and Syk, and (3) CR3 is involved in forming and closing FcγR-mediated phagocytic cups.

CR3 ruffles FcγR’s claim over phagocytic cups

Phagocytosis plays diverse roles in biology, but our understanding of the purpose, interplay, and cell signaling mechanisms associated with different modes of phagocytosis is limited, without being able to capture and visualize each step in this rapid process from the beginning to end. A new study by Walbaum et al. uses stunning time-lapse 3D imaging of the engulfment of erythrocytes by macrophages via sinking, ruffling, and cup formation, unequivocally confirming a visionary 44-year-old theory derived from still electron microscopy photos that phagocytosis mediated by complement receptor CR3 occurs via a sinking mechanism and antibody-mediated phagocytosis occurs via phagocytic cup formation. The article also challenges the dogma, showing that phagocytic cup formation is not unique to antibody receptor phagocytosis, rather CR3 plays a complex role in different modes of phagocytosis. For example, inhibition of antibody-mediated phagocytosis leads to a compensatory upregulation of CR3-mediated sinking phagocytosis. These findings animate, in vivid colors, processes previously only captured as stills, exposing interactions between different phagocytic mechanisms and altering our basic understanding of this important process.

Complement receptor 3 mediates both sinking phagocytosis and phagocytic cup formation via distinct mechanisms

A long-standing hypothesis is that complement receptors (CRs), especially CR3, mediate sinking phagocytosis, but evidence is lacking. Alternatively, CRs have been reported to induce membrane ruffles or phagocytic cups, akin to those induced by Fcγ receptors (FcγRs), but the details of these events are unclear. Here we used real-time 3D imaging and KO mouse models to clarify how particles (human red blood cells) are internalized by resident peritoneal F4/80+ cells (macrophages) via CRs and/or FcγRs. We first show that FcγRs mediate highly efficient, rapid (2–3 min) phagocytic cup formation, which is completely abolished by deletion or mutation of the FcR γ chain or conditional deletion of the signal transducer Syk. FcγR-mediated phagocytic cups robustly arise from any point of cell-particle contact, including filopodia. In the absence of CR3, FcγR-mediated phagocytic cups exhibit delayed closure and become aberrantly elongated. Independent of FcγRs, CR3 mediates sporadic ingestion of complement-opsonized particles by rapid phagocytic cup-like structures, typically emanating from membrane ruffles and largely prevented by deletion of the immunoreceptor tyrosine-based activation motif (ITAM) adaptors FcR γ chain and DAP12 or Syk. Deletion of ITAM adaptors or Syk clearly revealed that there is a slow (10–25 min) sinking mode of phagocytosis via a restricted orifice. In summary, we show that (1) CR3 indeed mediates a slow sinking mode of phagocytosis, which is accentuated by deletion of ITAM adaptors or Syk, (2) CR3 induces phagocytic cup-like structures, driven by ITAM adaptors and Syk, and (3) CR3 is involved in forming and closing FcγR-mediated phagocytic cups.