UNC93B1 is essential for TLR11 activation and IL-12-dependent host resistance to Toxoplasma gondii

Toll-like receptor (TLR) activation relies on biochemical recognition of microbial molecules and localization of the TLR within specific cellular compartments. Cell surface TLRs largely recognize bacterial membrane components, and intracellular TLRs are exclusively involved in sensing nucleic acids. Here we show that TLR11, an innate sensor for the Toxoplasma protein profilin, is an intracellular receptor that resides in the endoplasmic reticulum. The 12 membrane-spanning endoplasmic reticulum-resident protein UNC93B1 interacts directly with TLR11 and regulates the activation of dendritic cells in response to Toxoplasma gondii profilin and parasitic infection in vivo. A deficiency in functional UNC93B1 protein abolished TLR11-dependent IL-12 secretion by dendritic cells, attenuated Th1 responses against T. gondii, and dramatically enhanced susceptibility to the parasite. Our results reveal that the association with UNC93B1 and the intracellular localization of TLRs are not unique features of nucleic acid-sensing TLRs but is also essential for TLR11-dependent recognition of T. gondii profilin and for host protection against this parasite.     

A role for complexes of survival of motor neurons (SMN) protein with gemins and profilin in neurite-like cytoplasmic extensions of cultured nerve cells

Spinal muscular atrophy (SMA) is caused by reduced levels of SMN (survival of motor neurons protein) and consequent loss of motor neurons. SMN is involved in snRNP transport and nuclear RNA splicing, but axonal transport of SMN has also been shown to occur in motor neurons. SMN also binds to the small actin-binding protein, profilin. We now show that SMN and profilin II co-localise in the cytoplasm of differentiating rat PC12 cells and in neurite-like extensions, especially at their growth cones. Many components of known SMN complexes were also found in these extensions, including gemin2 (SIP-1), gemin6, gemin7 and unrip (unr-interacting protein). Coilin p80 and Sm core protein immunoreactivity, however, were seen only in the nucleus. SMN is known to associate with beta-actin mRNA and specific hnRNPs in axons and in neurite extensions of cultured nerve cells, and SMN also stimulates neurite outgrowth in cultures. Our results are therefore consistent with SMN complexes, rather than SMN alone, being involved in the transport of actin mRNPs along the axon as in the transport of snRNPs into the nucleus by similar SMN complexes. Antisense knockdown of profilin I and II isoforms inhibited neurite outgrowth of PC12 cells and caused accumulation of SMN and its associated proteins in cytoplasmic aggregates. BIAcore studies demonstrated a high affinity interaction of SMN with profilin IIa, the isoform present in developing neurons. Pathogenic missense mutations in SMN, or deletion of exons 5 and 7, prevented this interaction. The interaction is functional in that SMN can modulate actin polymerisation in vitro by reducing the inhibitory effect of profilin IIa. This suggests that reduced SMN in SMA might cause axonal pathfinding defects by disturbing the normal regulation of microfilament growth by profilins.     

Nuclear localization of profilin during the cell cycle in Tradescantia virginiana stamen hair cells

Summary. The localization of the actin-monomer-binding protein profilin during the cell cycle of living Tradescantia virginiana stamen hair cells has been studied by microinjection of a fluorescently labeled analog of the protein. In contrast to previously published studies performed on chemically fixed animal cells, we do not find a specific colocalization of profilin with actin filament arrays. Our results show that, besides a general cytoplasmic distribution, profilin specifically accumulates in the nucleus in interphase and prophase cells. This nuclear localization was confirmed by means of electron microscopic immunolocalization of endogenous profilin (in Gibasis scheldiana stamen hair cells). During mitosis, as the nuclear envelope and nuclear matrix break down at the onset of prometaphase, the nuclear profilin redistributes equally into the accessible volume (cytosol) of the cell. During metaphase and anaphase no specific localization of profilin can be observed associated with the mitotic apparatus. However, during telophase, as nuclear envelopes and nuclear matrices re-form and the sister chromatids start to decondense, a subset of the microinjected profilin again localizes to the nucleus. No accumulation of profilin could be observed in the phragmoplast, where a distinct array of actin filaments exists. The function of profilin in the nucleus remains unclear.Correspondence and reprints: Department of Biology, 221 Morrill Science Center II, University of Massachusetts, Amherst, MA 01003, U.S.A.Received September 30, 2002; accepted February 12, 2003 Published online September 23, 2003     

Profilin Isoforms Modulate Astrocytic Morphology and the Motility of Astrocytic Processes

The morphology of astrocytic processes determines their close structural association with synapses referred to as the ‘tripartite synapse’. Concerted morphological plasticity processes at tripartite synapses are supposed to shape neuronal communication. Morphological changes in astrocytes as well as the motility of astrocytic processes require remodeling of the actin cytoskeleton. Among the regulators of fast timescale actin-based motility, the actin binding protein profilin 1 has recently been shown to control the activity-dependent outgrowth of astrocytic processes.Here, we demonstrate that cultured murine astrocytes in addition to the ubiquitous profilin 1 also express the neuronal isoform profilin 2a. To analyze the cellular function of both profilins in astrocytes, we took advantage of a shRNA mediated isoform-specific downregulation. Interestingly, consistent with earlier results in neurons, we found redundant as well as isoform-specific functions of both profilins in modulating cellular physiology. The knockdown of either profilin 1 or profilin 2a led to a significant decrease in cell spreading of astrocytes. In contrast, solely the knockdown of profilin 2a resulted in a significantly reduced morphological complexity of astrocytes in both dissociated and slice culture astrocytes. Moreover, both isoforms proved to be crucial for forskolin-induced astrocytic stellation. Furthermore, forskolin treatment resulted in isoform-specific changes in the phosphorylation level of profilin 1 and profilin 2a, leading to a PKA-dependent phosphorylation of profilin 2a. In addition, transwell assays revealed an involvement of both isoforms in the motility of astrocytic processes, while FRAP analysis displayed an isoform-specific role of profilin 1 in the regulation of actin dynamics in peripheral astrocytic processes. Taken together, we suggest profilin isoforms to be important modulators of astrocytic morphology and motility with overlapping as well as isoform-specific functions.     

Preserved morphology and physiology of excitatory synapses in profilin1-deficient mice

Profilins are important regulators of actin dynamics and have been implicated in activity-dependent morphological changes of dendritic spines and synaptic plasticity. Recently, defective presynaptic excitability and neurotransmitter release of glutamatergic synapses were described for profilin2-deficient mice. Both dendritic spine morphology and synaptic plasticity were fully preserved in these mutants, bringing forward the hypothesis that profilin1 is mainly involved in postsynaptic mechanisms, complementary to the presynaptic role of profilin2. To test the hypothesis and to elucidate the synaptic function of profilin1, we here specifically deleted profilin1 in neurons of the adult forebrain by using conditional knockout mice on a CaMKII-cre-expressing background. Analysis of Golgi-stained hippocampal pyramidal cells and electron micrographs from the CA1 stratum radiatum revealed normal synapse density, spine morphology, and synapse ultrastructure in the absence of profilin1. Moreover, electrophysiological recordings showed that basal synaptic transmission, presynaptic physiology, as well as postsynaptic plasticity were unchanged in profilin1 mutants. Hence, loss of profilin1 had no adverse effects on the morphology and function of excitatory synapses. Our data are in agreement with two different scenarios: i) profilins are not relevant for actin regulation in postsynaptic structures, activity-dependent morphological changes of dendritic spines, and synaptic plasticity or ii) profilin1 and profilin2 have overlapping functions particularly in the postsynaptic compartment. Future analysis of double mutant mice will ultimately unravel whether profilins are relevant for dendritic spine morphology and synaptic plasticity.     

Reversible, activity-dependent targeting of profilin to neuronal nuclei

The actin cytoskeleton in pyramidal neurons plays a major role in activity-dependent processes underlying neuronal plasticity. The small actin-binding protein profilin shows NMDA receptor-dependent accumulation in dendritic spines, which is correlated with suppression of actin dynamics and long-term stabilization of synaptic morphology. Here we show that following NMDA receptor activation profilin also accumulates in the nucleus of hippocampal neurons via a process involving rearrangement of the actin cytoskeleton. This simultaneous targeting to dendritic spines and the cell nucleus suggests a novel mechanism of neuronal plasticity in which profilin both tags activated synapses and influences nuclear events.     

PNG1, a yeast gene encoding a highly conserved peptide:N-glycanase

It has been proposed that cytoplasmic peptide:N-glycanase (PNGase) may be involved in the proteasome-dependent quality control machinery used to degrade newly synthesized glycoproteins that do not correctly fold in the ER. However, a lack of information about the structure of the enzyme has limited our ability to obtain insight into its precise biological function. A PNGase-defective mutant (png1-1) was identified by screening a collection of mutagenized strains for the absence of PNGase activity in cell extracts. The PNG1 gene was mapped to the left arm of chromosome XVI by genetic approaches and its open reading frame was identified. PNG1 encodes a soluble protein that, when expressed in Escherichia coli, exhibited PNGase activity. PNG1 may be required for efficient proteasome-mediated degradation of a misfolded glycoprotein. Subcellular localization studies indicate that Png1p is present in the nucleus as well as the cytosol. Sequencing of expressed sequence tag clones revealed that Png1p is highly conserved in a wide variety of eukaryotes including mammals, suggesting that the enzyme has an important function.     

植物细胞中的前纤维蛋白

肌动蛋白组成的微丝骨架是真核细胞中的重要结构,在体内处于高度动态变化之中,受多种肌动蛋白结合蛋白（actin-binding proteins）的调节。前纤维蛋白（profilin）是一种单体肌动蛋白结合蛋白,存在于所有的真核细胞中,在植物细胞中也得到较多的研究。前纤维蛋白除可以结合单体肌动蛋白之外,还可以与磷脂酰肌醇及富含多聚脯氨酸的蛋白质等多种分子结合,在细胞信号转导中行使着重要的功能。本文结合本实验室的研究结果,概述了前纤维蛋白的最新研究进展。     

Profilin binds proline-rich ligands in two distinct amide backbone orientations.

The actin regulatory protein profilin is targeted to specific cellular regions through interactions with highly proline-rich motifs embedded within its binding partners. New X-ray crystallographic results demonstrate that profilin, like SH3 domains, can bind proline-rich ligands in two distinct amide backbone orientations. By further analogy with SH3 domains, these data suggest that non-proline residues in profilin ligands may dictate the polarity and register of binding, and the detailed organization of the assemblies involving profilin. This degeneracy may be a general feature of modules that bind proline-rich ligands, including WW and EVH1 domains, and has implications for the assembly and activity of macromolecular complexes involved in signaling and the regulation of the actin cytoskeleton.     

An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast

The localization of ASH1 mRNA to the distal tip of budding yeast cells is essential for the proper regulation of mating type switching in Saccharomyces cerevisiae. A localization element that is predominantly in the 3'-untranslated region (UTR) can direct this mRNA to the bud. Using this element in the three-hybrid in vivo RNA-binding assay, we identified a protein, Loc1p, that binds in vitro directly to the wild-type ASH1 3'-UTR RNA, but not to a mutant RNA incapable of localizing to the bud nor to several other mRNAs. LOC1 codes for a novel protein that recognizes double-stranded RNA structures and is required for efficient localization of ASH1 mRNA. Accordingly, Ash1p gets symmetrically distributed between daughter and mother cells in a loc1 strain. Surprisingly, Loc1p was found to be strictly nuclear, unlike other known RNA-binding proteins involved in mRNA localization which shuttle between the nucleus and the cytoplasm. We propose that efficient cytoplasmic ASH1 mRNA localization requires a previous interaction with specific nuclear factors.     

High-resolution structural analysis of mammalian profilin 2a complex formation with two physiological ligands: the formin homology 1 domain of mDia1 and the proline-rich domain of VASP

Profilins are small proteins capable of binding actin, poly-l-proline and other proline-rich sequences, and phosphatidylinositol (4,5)-bisphosphate. A number of proline-rich ligands for profilin have been characterised, including proteins of the Ena/VASP and formin families. We have determined the high-resolution crystal structures of mouse profilin 2a in complex with peptides from two functionally important ligands from different families, VASP and mDia1. The structures show that the binding mode of the peptide ligand is strongly affected by the non-proline residues in the sequence, and the peptides from VASP and mDia1 bind to profilin 2a in distinct modes. The high resolution of the crystallographic data allowed us to detect conserved CH-π hydrogen bonds between the peptide and profilin in both complexes. Furthermore, both peptides, which are shown to have micromolar affinity, induced the dimerisation of profilin, potentially leading to functionally different ligand-profilin-actin complexes. The peptides did not significantly affect actin polymerisation kinetics in the presence or in the absence of profilin 2a. Mutant profilins were tested for binding to poly-l-proline and the VASP and mDia1 peptides, and the F139A mutant bound proline-rich ligands with near-native affinity. Peptide blotting using a series of designed peptides with profilins 1 and 2a indicates differences between the two profilins towards proline-rich peptides from mDia1 and VASP. Our data provide structural insights into the mechanisms of mDia1 and VASP regulated actin polymerisation.     

Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility.

To propel itself in infected cells, the pathogen Shigella flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filopodia formation. Using a combination of bacterial motility assays in platelet extracts with Escherichia coli expressing the Shigella IcsA protein and in vitro analysis of reconstituted systems from purified proteins, we show here that the bacterial protein IcsA binds N-WASP and activates it in a Cdc42-like fashion. Dramatic stimulation of actin assembly is linked to the formation of a ternary IcsA-N-WASP-Arp2/3 complex, which nucleates actin polymerization. The Arp2/3 complex is essential in initiation of actin assembly and Shigella movement, as previously observed for Listeria monocytogenes. Activation of N-WASP by IcsA unmasks two domains acting together in insertional actin polymerization. The isolated COOH-terminal domain of N-WASP containing a verprolin-homology region, a cofilin-homology sequence, and an acidic terminal segment (VCA) interacts with G-actin in a unique profilin-like functional fashion. Hence, when N-WASP is activated, its COOH-terminal domain feeds barbed end growth of filaments and lowers the critical concentration at the bacterial surface. On the other hand, the NH(2)-terminal domain of N-WASP interacts with F-actin, mediating the attachment of the actin tail to the bacterium surface. VASP is not involved in Shigella movement, and the function of profilin does not require its binding to proline-rich regions.     

Signal transduction and the actin cytoskeleton: the roles of MARCKS and profilin.

MARCKS and profilin, two actin-binding proteins, are discussed to illustrate the mechanism by which extracellular signals are coupled to changes in the structure of the actin cytoskeleton. MARCKS is a filamentous actin-crosslinking protein that appears to function as an integrator of protein kinase C and calcium (Ca2+)/calmodulin signals in the regulation of actin-membrane interactions. New data suggest that profilin is activated by the coordinated action of receptor tyrosine kinases and phospholipase C-gamma 1 to stimulate the stabilization of actin filaments.     

Nucleolar GTPase NOG-1 Regulates Development,Fat Storage,and Longevity through Insulin/IGF Signaling in C. elegans

NOG1 is a nucleolar GTPase that is critical for 60S ribosome biogenesis. Recently, NOG1 was identified as one of the downstream regulators of target of rapamycin (TOR) in yeast. It is reported that TOR is involved in regulating lifespan and fat storage in Caenorhabditis elegans. Here, we show that the nog1 ortholog (T07A9.9: nog-1) in C. elegans regulates growth, development, lifespan, and fat metabolism. A green fluorescence protein (GFP) promoter assay revealed ubiquitous expression of C. elegans nog-1 from the early embryonic to the adult stage. Furthermore, the GFP-tagged NOG-1 protein is localized to the nucleus, whereas the aberrant NOG-1 protein is concentrated in the nucleolus. Functional studies of NOG-1 in C. elegans further revealed that nog-1 knockdown resulted in smaller broodsize, slower growth, increased life span, and more fat storage. Moreover, nog-1 over-expression resulted in decreased life span. Taken together, our data suggest that nog-1 in C. elegans may be an important player in regulating life span and fat storage via the insulin/IGF pathway.     

Isolation of profilin from embryonic chicken skeletal muscle and evaluation of its interaction with different actin isoforms

An actin-binding protein of 16 kDa was isolated from embryonic chicken skeletal muscle. The protein had the same properties as profilin, exhibited a much higher affinity for cytoskeletal (beta- and gamma-) actins than for sarcomeric (alpha-) actin in the embryonic muscle, and inhibited the polymerization of beta- and gamma-actins more efficiently in a physiological salt solution. These results indicate that the assembly of cytoskeletal and sarcomeric actins is regulated differently by profilin in the developing skeletal muscle, and that the former may not be involved in myofibril assembly.     

Transition of basic protein during spermatogenesis of <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck, 1765)

According to the ultrastructural characteristic observation of the developing male germ cells, spermatogenesis of the crustacean shrimp, Fenneropenaeus chinensis, is classified into spermatogonia, primary spermatocytes, secondary spermatocyte, four stages of spermatids, and mature sperm. The basic protein transition during its spermatogenesis is studied by transmission electron microscopy of ammoniacal silver reaction and immunoelectron microscopical distribution of acetylated histone H4. The results show that basic protein synthesized in cytoplasm of spermatogonia is transferred into the nucleus with deposition on new duplicated DNA. In the spermatocyte stage, some nuclear basic protein combined with RNP is transferred into the cytoplasm and is involved in forming the cytoplasmic vesicle clumps. In the early spermatid, most of the basic protein synthesized in the new spermatid cytoplasm is transferred into the nucleus, and the chromatin condensed gradually, and the rest is shifted into the pre-acrosomal vacuole. In the middle spermatid, the nuclear basic protein linked with DNA is acetylated and transferred into the proacrosomal vacuole and assembled into the acrosomal blastema. At the late spermatid, almost all of the basic protein in the nucleus has been removed into the acrosome. During the stage from late spermatid to mature sperm, some de novo basic proteins synthesized in the cytoplasm belt transfer into the nucleus without a membrane and almost all deposit in the periphery to form a supercoating. The remnant histone H4 accompanied by chromatin fibers is acetylated in the center of the nucleus, leading to relaxed DNA and activated genes making the nucleus non-condensed.     

A Formin Homology protein and a profilin are required for cytokinesis and Arp2/3-independent assembly of cortical microfilaments in C. elegans

BACKGROUND: F-actin is enriched at the cortex of embryonic cells in the nematode Caenorhabditis elegans and is required for multiple processes that include the establishment of an anterior-posterior (A-P) axis and cytokinesis. However, the mechanisms that regulate cortical microfilament (MF) assembly remain poorly understood. RESULTS: We show here that a profilin called PFN-1 accumulates at the cortex independent of the actin cytoskeleton and is required for the assembly or maintenance of cortical MFs and myosin. Reducing PFN-1 levels by RNAi results in cytokinesis and A-P polarity defects. PFN-1 binds to the Formin Homology (FH) protein CYK-1, which also is required for cortical MFs. In contrast to PFN-1 and CYK-1, the Arp2/3 complex appears to be dispensable for the assembly of cortical MFs, for A-P polarity, and for cytokinesis. Instead, the Arp2/3 complex is required for cell migrations that occur during gastrulation and may also be involved in cellular rearrangements required for epidermal enclosure prior to elongation of ovoid embryos into vermiform larvae. CONCLUSIONS: We conclude that the FH protein CYK-1 and the profilin PFN-1 mediate the Arp2/3-independent assembly of MFs and are required for cytokinesis in the early embryo. These data suggest that CYK-1 and PFN-1 may nucleate MFs, as has recently been shown for an FH protein and a profilin in yeast.     

Mechanism of action of Acanthamoeba profilin: demonstration of actin species specificity and regulation by micromolar concentrations of MgCl2

Acanthamoeba profilin strongly inhibits in a concentration-dependent fashion the rate and extent of Acanthamoeba actin polymerization in 50 mM KCl. The lag phase is prolonged indicating reduction in the rate of nucleus formation. The elongation rates at both the barbed and pointed ends of growing filaments are inhibited. At steady state, profilin increases the critical concentration for polymerization but has no effect on the reduced viscosity above the critical concentration. Addition of profilin to polymerized actin causes it to depolymerize until a new steady-state, dependent on profilin concentration, is achieved. These effects of profilin can be explained by the formation of a 1:1 complex with actin with a dissociation constant of 1 to 4 microM. MgCl2 strongly inhibits these effects of profilin, most likely by binding to the high-affinity divalent cation site on the actin. Acanthamoeba profilin has similar but weaker effects on muscle actin, requiring 5 to 10 times more profilin than with amoeba actin.     

Src64 is involved in fusome development and karyosome formation during Drosophila oogenesis

Src family tyrosine kinases respond to a variety of signals by regulating the organization of the actin cytoskeleton. Here, we show that during early oogenesis Src64 mutations lead to uneven accumulation of cortical actin, defects in fusome formation, mislocalization of septins, defective transport of Orb protein into the oocyte, and possible defects in cell division. Similar mutant phenotypes suggest that Src64, the Tec29 tyrosine kinase, and the actin crosslinking protein Kelch act together to regulate actin crosslinking, much as they do later during ring canal growth. Condensation of the oocyte chromatin into a compact karyosome is also defective in Src64, Tec29, and kelch mutants and in mutants for spire and chickadee (profilin), genes that regulate actin polymerization. These data, along with changes in G-actin accumulation in the oocyte nucleus, suggest that Src64 is involved in a nuclear actin function during karyosome condensation. Our results indicate that Src64 regulates actin dynamics at multiple stages of oogenesis.