Survival of mycobacteria in macrophages is mediated by coronin 1-dependent activation of calcineurin

Pathogenic mycobacteria survive within macrophages by avoiding lysosomal delivery, instead residing in mycobacterial phagosomes. Upon infection, the leukocyte-specific protein coronin 1 is actively recruited to mycobacterial phagosomes, where it blocks lysosomal delivery by an unknown mechanism. Analysis of macrophages from coronin 1-deficient mice showed that coronin 1 is dispensable for F-actin-dependent processes such as phagocytosis, motility, and membrane ruffling. However, upon mycobacterial infection, coronin 1 was required for activation of the Ca(2+)-dependent phosphatase calcineurin, thereby blocking lysosomal delivery of mycobacteria. In the absence of coronin 1, calcineurin activity did not occur, resulting in lysosomal delivery and killing of mycobacteria. Furthermore, blocking calcineurin activation with cyclosporin A or FK506 led to lysosomal delivery and intracellular mycobacterial killing. These results demonstrate a role for coronin 1 in activating Ca(2+) dependent signaling processes in macrophages and reveal a function for calcineurin in the regulation of phagosome-lysosome fusion upon mycobacterial infection.     

RNA interference in J774 macrophages reveals a role for coronin 1 in mycobacterial trafficking but not in actin-dependent processes

Macrophages are crucial for innate immunity, apoptosis, and tissue remodeling, processes that rely on the capacity of macrophages to internalize and process cargo through phagocytosis. Coronin 1, a member of the WD repeat protein family of coronins specifically expressed in leukocytes, was originally identified as a molecule that is recruited to mycobacterial phagosomes and prevents the delivery of mycobacteria to lysosomes, allowing these to survive within phagosomes. However, a role for coronin 1 in mycobacterial pathogenesis has been disputed in favor for its role in mediating phagocytosis and cell motility. In this study, a role for coronin 1 in actin-mediated cellular processes was addressed using RNA interference in the murine macrophage cell line J774. It is shown that the absence of coronin 1 in J774 macrophages expressing small interfering RNA constructs specific for coronin 1 does not affect phagocytosis, macropinocytosis, cell locomotion, or regulation of NADPH oxidase activity. However, in coronin 1-negative J774 cells, internalized mycobacteria were rapidly transferred to lysosomes and killed. Therefore, these results show that in J774 cells coronin 1 has a specific role in modulating phagosome-lysosome transport upon mycobacterial infection and that it is dispensable for most F-actin-mediated cytoskeletal rearrangements.     

Association of the leukocyte plasma membrane with the actin cytoskeleton through coiled coil-mediated trimeric coronin 1 molecules

Coronin 1 is a member of the coronin protein family specifically expressed in leukocytes and accumulates at sites of rearrangements of the F-actin cytoskeleton. Here, we describe that coronin 1 molecules are coiled coil-mediated homotrimeric complexes, which associate with the plasma membrane and with the cytoskeleton via two distinct domains. Association with the cytoskeleton was mediated by trimerization of a stretch of positively charged residues within a linker region between the N-terminal, WD repeat-containing domain and the C-terminal coiled coil. In contrast, neither the coiled coil nor the positively charged residues within the linker domain were required for plasma membrane binding, suggesting that the N-terminal, WD repeat-containing domain mediates membrane interaction. The capacity of coronin 1 to link the leukocyte cytoskeleton to the plasma membrane may serve to integrate outside-inside signaling with modulation of the cytoskeleton.     

Architectural dynamics and gene replacement of coronin suggest its role in cytokinesis

Coronin is a ubiquitous actin-binding protein representing a member of proteins portraying a WD-repeat sequence, including the beta-subunits of trimeric G-proteins. Coronin has been suggested to participate in multiple, actin-based physiological activities such as cell movement and cell division. Although the slow growth of coronin deletion mutants has been attributed to a defect in the fluid-phase uptake of nutrients, the exact role of coronin in cytoskeletal organization has not been elucidated. In this study, we examined a role of coronin in cytokinesis by analyzing the effect of coronin deletion on the actin cytoskeleton and its dynamic distribution using a green fluorescent protein (GFP)-coronin fusion protein. We show that GFP-coronin works similarly to natural coronin in vivo and in vitro. In live cells, GFP-coronin was found to accumulate into the cleavage furrow during cytokinesis. The fluorescence pattern suggests its association to the contractile ring throughout cytokinesis. Interestingly, a substantial amount of coronin was also bound to F-actin at the prospective posterior cortex of the daughter cells. We also show that the coronin null cells reveal irregularities in organization of actin and myosin II and divide by a process identical to the traction-mediated cytofission reported in myosin II mutants. Overall, this study suggests that coronin is essential for organizing the normal actin cytoskeleton and plays a significant role in cell division.     

Oligomerization,F-actin interaction,and membrane association of the ubiquitous mammalian coronin 3 are mediated by its carboxyl terminus

Coronin 3 is a ubiquitously expressed member of the coronin protein family in mammals. In fibroblasts and HEK 293 cells, it is localized both in the cytosol and in the submembranous cytoskeleton, especially at lamellipodia and membrane ruffles. The carboxyl terminus of all coronins contains a coiled coil suggested to mediate dimerization. We show here that in contrast to other coronin homologues, the recombinant human coronin 3 carboxyl terminus forms oligomers rather than dimers, and that this part is sufficient to bind to and cross-link F-actin in vitro. The carboxyl terminus alone also conferred membrane association in vivo, and removal of the coiled coil abolished membrane localization but not in vitro F-actin binding. Coronin 3 is exclusively extracted as an oligomer from both the cytosol and the membrane fraction. Because oligomerization was not reported for other coronins, it might be a key feature governing coronin 3-specific functions. Cytosolic coronin 3 showed a high degree of phosphorylation, which is likely to regulate the subcellular localization of the protein.     

Coronin is involved in uptake of Mycobacterium bovis BCG in human macrophages but not in phagosome maintenance

By applying density gradient electrophoresis (DGE) to human macrophages infected with Mycobacterium bovis BCG, we were able to separate three different bacterial fractions representing arrested phagosomes, phagolysosomes and mycobacterial clumps. After further purification of the phagosomal population, we found that isolated phagosomes containing live BCG were arrested in maturation as they exhibited only low amounts of the lysosomal glycoprotein LAMP-1 and processing of the lysosomal hydrolase cathepsin D was blocked. In addition, low amounts of MHC class I and class II molecules and the absence of HLA-DM suggest sequestration of mycobacterial phagosomes from antigen-processing pathways. We further investigated the involvement of the actin-binding protein coronin in intracellular survival of mycobacteria and showed that human coronin, as well as F-actin, were associated with early stages of mycobacterial phagocytosis but not with phagosome maintenance. Therefore, we conclude that the unique DGE migration pattern of arrested phagosomes is not as a result of retention of coronin, but that there are other proteins or lipids responsible for the block in maturation in human macrophages.     

Evidence for a pool of coronin in mammalian cells that is sensitive to PI 3-kinase

Coronin, a 57 kDa actin binding protein elutes with an apparent molecular mass of 400-600 kDa from gel filtration columns. This fraction is not unrelated to the reported 200 kDa complex where coronin is associated with phox proteins of the NADPH-oxidase. Phosphatidylinositol 3-kinase (PI 3-kinase) solubilizes coronin from the 400-600 kDa complex, thus constitutive active PI 3-kinase is sufficient to disrupt the complex, whereas wortmannin stabilizes it. Conversely, the phox protein associated pool of coronin is PI 3-kinase independent. During phagocytosis coronin is recruited together with PI 3-kinase to membranes of nascent and early phagosomes co-localizing with the actin cytoskeleton, confirming that coronin contributes to phagocytosis.     

The coronin family of actin-associated proteins.

Coronin was first isolated from Dictyostelium, but similar proteins have been identified in many species and individual cell types. The coronin-like protein in yeast promotes actin polymerization and also interacts with microtubules. Dictyostelium mutants lacking coronin are impaired in cytokinesis and all actin-mediated processes. Analysis of coronin-GFP (green-fluorescent protein) fusions and knockout mutants shows that coronin participates in the remodelling of the cortical actin cytoskeleton that is responsible for phagocytosis and macropinocytosis. Likewise, in mammalian neutrophils, a coronin-like protein is also associated with the phagocytic apparatus. The diversity of function in this family of actin-associated proteins is just beginning to be explored.     

TB or not TB: calcium regulation in mycobacterial survival

Mycobacterium tuberculosis (Mtb)-the bacterium that causes tuberculosis-resides in phagosomes inside macrophages. This bacterium evades destruction by preventing phagosome maturation, which involves the fusion of phagosomes with lysosomes. In this issue of Cell, Jayachandran et al. (2007) suggest that mycobacteria co-opt the actin-binding protein coronin 1 to activate the phosphatase calcineurin, thereby preventing phagosomal maturation.     

Coronin 1C harbours a second actin-binding site that confers co-operative binding to F-actin

Dynamic rearrangement of actin filament networks is critical for cell motility, phagocytosis and endocytosis. Coronins facilitate these processes, in part, by their ability to bind F-actin (filamentous actin). We previously identified a conserved surface-exposed arginine (Arg(30)) in the β-propeller of Coronin 1B required for F-actin binding in vitro and in vivo. However, whether this finding translates to other coronins has not been well defined. Using quantitative actin-binding assays, we show that mutating the equivalent residue abolishes F-actin binding in Coronin 1A, but not Coronin 1C. By mutagenesis and biochemical competition, we have identified a second actin-binding site in the unique region of Coronin 1C. Interestingly, leading-edge localization of Coronin 1C in fibroblasts requires the conserved site in the β-propeller, but not the site in the unique region. Furthermore, in contrast with Coronin 1A and Coronin 1B, Coronin 1C displays highly co-operative binding to actin filaments. In the present study, we highlight a novel mode of coronin regulation, which has implications for how coronins orchestrate cytoskeletal dynamics.     

Dictyostelium mutants lacking the cytoskeletal protein coronin are defective in cytokinesis and cell motility

Coronin is an actin-binding protein in Dictyostelium discoideum that is enriched at the leading edge of the cells and in projections of the cell surface called crowns. The polypeptide sequence of coronin is distinguished by its similarities to the beta-subunits of trimeric G proteins (E. L. de Hostos, B. Bradtke, F. Lottspeich, R. Guggenheim, and G. Gerisch, 1991. EMBO (Eur. Mol. Biol. Organ.) J. 10:4097-4104). To elucidate the in vivo function of coronin, null mutants have been generated by gene replacement. The mutant cells lacking coronin grow and migrate more slowly than wild-type cells. When these cor- cells grow in liquid medium they become multinucleate, indicating a role of coronin in cytokinesis. To explore this role, coronin has been localized in mitotic wild-type cells by immunofluorescence labeling. During separation of the daughter cells, coronin is strongly accumulated at their distal portions including the leading edges. This contrasts with the localization of myosin II in the cleavage furrow and suggests that coronin functions independently of the conventional myosin in facilitating cytokinesis.     

Functional Surfaces on the Actin-binding Protein Coronin Revealed by Systematic Mutagenesis

Coronin is a conserved actin-binding protein that co-functions with ADF/cofilin and Arp2/3 complex to govern cellular actin dynamics. Despite emerging roles for coronin in a range of physiological processes and disease states, a detailed understanding of the molecular interactions of coronin with actin and other binding partners has been lacking. Here, we performed a systematic mutational analysis of surfaces on the yeast coronin β-propeller domain, which binds to F-actin and is conserved in all coronin family members. We generated 21 mutant alleles and analyzed their biochemical effects on actin binding and ADF/cofilin activity. Conserved actin-binding residues mapped to a discrete ridge stretching across one side of the β-propeller. Mutants defective in actin binding showed loss of synergy with ADF/cofilin in severing filaments, diminished localization to actin structures in vivo, and loss of coronin overexpression growth defects. In addition, one allele showed normal actin binding but impaired functional interactions with ADF/cofilin. Another allele showed normal actin binding but failed to cause coronin overexpression defects. Together, these results indicate that actin binding is critical for many of the biochemical and cellular functions of coronin and that the β-propeller domain mediates additional functional interactions with ADF/cofilin and possibly other ligands. Conservation of the actin-binding surfaces across distant species and in all three major classes of coronin isoforms suggests that the nature of the coronin-actin association may be similar in other family members.     

Coronin is a component of the endocytic collar of hyphae of Neurospora crassa and is necessary for normal growth and morphogenesis

Coronin plays a major role in the organization and dynamics of actin in yeast. To investigate the role of coronin in a filamentous fungus (Neurospora crassa), we examined its subcellular localization using fluorescent proteins and the phenotypic consequences of coronin gene (crn-1) deletion in hyphal morphogenesis, Spitzenk?rper behavior and endocytosis. Coronin-GFP was localized in patches, forming a subapical collar near the hyphal apex; significantly, it was absent from the apex. The subapical patches of coronin colocalized with fimbrin, Arp2/3 complex, and actin, altogether comprising the endocytic collar. Deletion of crn-1 resulted in reduced hyphal growth rates, distorted hyphal morphology, uneven wall thickness, and delayed establishment of polarity during germination; it also affected growth directionality and increased branching. The Spitzenk?rper of Δcrn-1 mutant was unstable; it appeared and disappeared intermittently giving rise to periods of hyphoid-like and isotropic growth respectively. Uptake of FM4-64 in Δcrn-1 mutant indicated a partial disruption in endocytosis. These observations underscore coronin as an important component of F-actin remodeling in N. crassa. Although coronin is not essential in this fungus, its deletion influenced negatively the operation of the actin cytoskeleton involved in the orderly deployment of the apical growth apparatus, thus preventing normal hyphal growth and morphogenesis.     

Coronin 1 Regulates Cognition and Behavior through Modulation of cAMP/Protein Kinase A Signaling

Cognitive and behavioral disorders are thought to be a result of neuronal dysfunction, but the underlying molecular defects remain largely unknown. An important signaling pathway involved in the regulation of neuronal function is the cyclic AMP/Protein kinase A pathway. We here show an essential role for coronin 1, which is encoded in a genomic region associated with neurobehavioral dysfunction, in the modulation of cyclic AMP/PKA signaling. We found that coronin 1 is specifically expressed in excitatory but not inhibitory neurons and that coronin 1 deficiency results in loss of excitatory synapses and severe neurobehavioral disabilities, including reduced anxiety, social deficits, increased aggression, and learning defects. Electrophysiological analysis of excitatory synaptic transmission in amygdala revealed that coronin 1 was essential for cyclic–AMP–protein kinase A–dependent presynaptic plasticity. We further show that upon cell surface stimulation, coronin 1 interacted with the G protein subtype Gαs to stimulate the cAMP/PKA pathway. The absence of coronin 1 or expression of coronin 1 mutants unable to interact with Gαs resulted in a marked reduction in cAMP signaling. Strikingly, synaptic plasticity and behavioral defects of coronin 1–deficient mice were restored by in vivo infusion of a membrane-permeable cAMP analogue. Together these results identify coronin 1 as being important for cognition and behavior through its activity in promoting cAMP/PKA-dependent synaptic plasticity and may open novel avenues for the dissection of signal transduction pathways involved in neurobehavioral processes.     

Actin assembly controlled by GDP-Rab27a is essential for endocytosis of the insulin secretory membrane

We have recently reported that GDP-bound Rab27a regulates endocytosis of the insulin secretory membrane via its binding to coronin 3, an actin-binding protein. The aim of this study was to examine the participation of actin assembly in the Rab27a-dependent regulation of endocytosis using a pancreatic beta cell line, MIN6. Coronin 3 promoted F-actin bundling only in the presence of GDP-Rab27a. This effect was independent of coronin-3-binding to the actin-related proteins 2 and 3 (Arp2/3). Uptake of anti-phogrin-lumen antibody into MIN6 was inhibited by anti-coronin-3-C antibody which recognizes the actin-binding site. This inhibition was also observed with coronin-3-R28D, which lacks in actin binding. These results suggest that coronin 3 is a genuine GDP-Rab27a effector, and that controls endocytosis of the secretory membrane via modulating actin assembly in pancreatic β-cells.     

Structural and Functional Diversity of Novel Coronin 1C (CRN2) Isoforms in Muscle

Coronin 1C (synonyms: coronin-3, CRN2), a WD40 repeat-containing protein involved in cellular actin dynamics, is ubiquitously expressed in human tissues. Here, we report on the identification and functional characterization of two novel coronin 1C isoforms, referred to as CRN2i2 and CRN2i3, which also associate with F-actin. Analyses of the coronin 1C gene disclosed a single promoter containing binding sites for myogenic regulatory factors and an alternative first exon 1b present in intron 1, which give rise to the novel isoforms. Chromatin immunoprecipitation studies demonstrate MyoD binding to a region of the CRN2 gene, which contains a highly conserved E-box element in exon 1a. Gel-filtration assays suggest that the largest isoform 3 exists as a monomer, in contrast to isoform 1 and isoform 2 appearing as trimers. CRN2i3, which can be induced by MyoD, is exclusively expressed in well-differentiated myoblasts as well as in mature skeletal muscle tissue. In human skeletal muscle, CRN2i3 is a novel component of postsynaptic neuromuscular junctions and thin filaments of myofibrils. Together, our findings postulate a role for CRN2 isoforms in the structural and functional organization of F-actin in highly ordered protein complexes.     

WISp39 binds phosphorylated Coronin 1B to regulate Arp2/3 localization and Cofilin-dependent motility

We previously identified Waf1 Cip1 stabilizing protein 39 (WISp39) as a binding partner for heat shock protein 90 (Hsp90). We now report that WISp39 has an essential function in the control of directed cell migration, which requires WISp39 interaction with Hsp90. WISp39 knockdown (KD) resulted in the loss of directional motility of mammalian cells and profound changes in cell morphology, including the loss of a single leading edge. WISp39 binds Coronin 1B, known to regulate the Arp2/3 complex and Cofilin at the leading edge. WISp39 preferentially interacts with phosphorylated Coronin 1B, allowing it to complex with Slingshot phosphatase (SSH) to dephosphorylate and activate Cofilin. WISp39 also regulates Arp2/3 complex localization at the leading edge. WISp39 KD-induced morphological changes could be rescued by overexpression of Coronin 1B together with a constitutively active Cofilin mutant. We conclude that WISp39 associates with Hsp90, Coronin 1B, and SSH to regulate Cofilin activation and Arp2/3 complex localization at the leading edge.     

Inflammatory Stimuli Reprogram Macrophage Phagocytosis to Macropinocytosis for the Rapid Elimination of Pathogens

Following an infectious challenge, macrophages have to be activated in order to allow efficient clearance of infectious pathogens, but how macrophage activation is coupled to increased clearance remains largely unknown. We here describe that inflammatory stimuli induced the reprogramming of the macrophage endocytic machinery from receptor-mediated phagocytosis to macropinocytosis, allowing the rapid transfer of internalized cargo to lysosomes in a receptor-independent manner. Reprogramming occurred through protein kinase C-mediated phosphorylation of the macrophage protein coronin 1, thereby activating phosphoinositol (PI)-3-kinase activity necessary for macropinocytic uptake. Expression of a phosphomimetic form of coronin 1 was sufficient to induce PI3-kinase activation and macropinocytosis even in the absence of inflammatory stimuli. Together these results suggest a hitherto unknown mechanism to regulate the internalization and degradation of infectious material during inflammation.