Distinct Cytoplasmic Domains Research Articles

TRAF6 and TRAF2/3 Binding Motifs in CD40 Differentially Regulate B Cell Function in T-Dependent Antibody Responses and Dendritic Cell Function in Experimental Autoimmune Encephalomyelitis.

Expression of the costimulatory molecule CD40 on both B cells and dendritic cells (DCs) is required for induction of experimental autoimmune encephalomyelitis (EAE), and cell-autonomous CD40 expression on B cells is required for primary T-dependent (TD) Ab responses. We now ask whether the function of CD40 expressed by different cell types in these responses is mediated by the same or different cytoplasmic domains. CD40 has been reported to possess multiple cytoplasmic domains, including distinct TRAF6 and TRAF2/3 binding motifs. To elucidate the invivo function of these motifs in B cells and DCs involved in EAE and TD germinal center responses, we have generated knock-in mice containing distinct CD40 cytoplasmic domain TRAF-binding site mutations and have used these animals, together with bone marrow chimeric mice, to assess the roles that these motifs play in CD40 function. We found that both TRAF2/3 and TRAF6 motifs of CD40 are critically involved in EAE induction and demonstrated that this is mediated by a role of both motifs for priming of pathogenic T cells by DCs. In contrast, the TRAF2/3 binding motif, but not the TRAF6 binding motif, is required for B cell CD40 function in TD high-affinity Ab responses. These data demonstrate that the requirements for expression of specific TRAF-binding CD40 motifs differ for B cells or DCs that function in specific immune responses and thus identify targets for intervention to modulate these responses.

STAC proteins inhibit the voltage dependent inactivation of l-type calcium channels.

The inactivation of voltage-gated calcium channels is governed by two distinct processes: voltage dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The STAC family of adaptor proteins inhibits the CDI of L-type calcium channels (LTCC). In fact, all LTCC (CaV1.2, CaV1.3 and CaV1.4) showed reduced CDI in the presence of any STAC isoform. However, whether STAC proteins also modulate the CDI of the skeletal muscle channel CaV1.1 was initially not investigated, as CaV1.1 requires STAC3 for its functional expression. Interestingly, we could recently demonstrate that STAC3 does not inhibit the CDI, but the VDI of CaV1.1. Therefore, we hypothesized that STAC proteins inhibit also the VDI of LTCC. Indeed, when co-expressed in HEK cells, all STAC isoforms reduced the VDI of CaV1.2 and CaV1.3 currents. As for CDI, also the VDI inhibition is specific for LTCC, as the VDI of CaV2.1 and CaV2.3 is not affected by STAC proteins. As the predominant contribution to VDI is known to arise from the I-II loop interaction with the CaVβ subunit, and that STAC proteins were shown to interact with distinct cytoplasmic domains (II-III loop and C-terminus), we hypothesized that STAC proteins and membrane tethered β subunits utilize different mechanism to inhibit VDI. Indeed, co-expression of STAC3 with CaV1.3/β2a revealed additional deceleration of VDI in HEK cells. Ongoing experiments, involving STAC and channels mutants and chimeras, will reveal the molecular determinants of the VDI inhibition by STAC proteins.

Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM

Ryanodine receptors (RyRs) are main regulators of intracellular Ca2+ release and muscle contraction. The Y522S mutation of RyR1 causes central core disease, a weakening myopathy, and malignant hyperthermia, a sudden and potentially fatal response to anesthetics or heat. Y522 is in the core of the N-terminal subdomain C of RyR1 and the mechanism of how this mutation orchestrates malfunction is unpredictable for this 2-MDa ion channel, which has four identical subunits composed of 15 distinct cytoplasmic domains each. We expressed and purified the RyR1 rabbit homolog, Y523S, from HEK293 cells and reconstituted it in nanodiscs under closed and open states. The high-resolution cryogenic electron microscopic (cryo-EM) three-dimensional (3D) structures show that the phenyl ring of Tyr functions in a manner analogous to a "spacer" within an α-helical bundle. Mutation to the much smaller Ser alters the hydrophobic network within the bundle, triggering rearrangement of its α-helices with repercussions in the orientation of most cytoplasmic domains. Examining the mutation-induced readjustments exposed a series of connected α-helices acting as an ∼100 Å-long lever: One end protrudes toward the dihydropyridine receptor, its molecular activator (akin to an antenna), while the other end reaches the Ca2+ activation site. The Y523S mutation elicits channel preactivation in the absence of any activator and full opening at 1.5 µM free Ca2+, increasing by ∼20-fold the potency of Ca2+ to activate the channel compared with RyR1 wild type (WT). This study identified a preactivated pathological state of RyR1 and a long-range lever that may work as a molecular switch to open the channel.

Role of KASH domain lengths in the regulation of LINC complexes.

The linker of the nucleoskeleton and cytoskeleton (LINC) complex is formed by the conserved interactions between Sad-1 and UNC-84 (SUN) and Klarsicht, ANC-1, SYNE homology (KASH) domain proteins, providing a physical coupling between the nucleoskeleton and cytoskeleton that mediates the transfer of physical forces across the nuclear envelope. The LINC complex can perform distinct cellular functions by pairing various KASH domain proteins with the same SUN domain protein. For example, in Caenorhabditis elegans, SUN protein UNC-84 binds to two KASH proteins UNC-83 and ANC-1 to mediate nuclear migration and anchorage, respectively. In addition to distinct cytoplasmic domains, the luminal KASH domain also varies among KASH domain proteins of distinct functions. In this study, we combined in vivo C. elegans genetics and in silico molecular dynamics simulations to understand the relation between the length and amino acid composition of the luminal KASH domain, and the function of the SUN–KASH complex. We show that longer KASH domains can withstand and transfer higher forces and interact with the membrane through a conserved membrane proximal EEDY domain that is unique to longer KASH domains. In agreement with our models, our in vivo results show that swapping the KASH domains of ANC-1 and UNC-83, or shortening the KASH domain of ANC-1, both result in a nuclear anchorage defect in C. elegans.

High-affinity heterotetramer formation between the large myelin-associated glycoprotein and the dynein light chain DYNLL1.

The close association of myelinated axons and their myelin sheaths involves numerous intercellular molecular interactions. For example, myelin-associated glycoprotein (MAG) mediates myelin-to-axon adhesion and signalling via molecules on the axonal surface. However, knowledge about intracellular binding partners of myelin proteins, including MAG, has remained limited. The two splice isoforms of MAG, S- and L-MAG, display distinct cytoplasmic domains and spatiotemporal expression profiles. We used yeast two-hybrid screening to identify interaction partners of L-MAG and found the dynein light chain DYNLL1 (also termed dynein light chain 8). DYNLL1 homodimers are known to facilitate dimerization of target proteins. L-MAG and DYNLL1 associate with high affinity, as confirmed with recombinant proteins invitro. Structural analyses of the purified complex indicate that the DYNLL1-binding segment is localized close to the L-MAG C terminus, next to the Fyn kinase Tyr phosphorylation site. The crystal structure of the complex between DYNLL1 and its binding segment on L-MAG shows 2:2 binding in a parallel arrangement, indicating a heterotetrameric complex. The homology between L-MAG and previously characterized DYNLL1-ligands is limited, and some details of binding site interactions are unique for L-MAG. The structure of the complex between the entire L-MAG cytoplasmic domain and DYNLL1, as well as that of the extracellular domain of MAG, were modelled based on small-angle X-ray scattering data, allowing structural insights into L-MAG interactions on both membrane surfaces. Our data imply that DYNLL1 dimerizes L-MAG, but not S-MAG, through the formation of a specific 2:2 heterotetramer. This arrangement is likely to affect, in an isoform-specific manner, the functions of MAG in adhesion and myelin-to-axon signalling. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/. Read the Editorial Highlight for this article on page712.

GPR56/ADGRG1 regulates development and maintenance of peripheral myelin.

Myelin is a multilamellar sheath generated by specialized glia called Schwann cells (SCs) in the peripheral nervous system (PNS), which serves to protect and insulate axons for rapid neuronal signaling. In zebrafish and rodent models, we identify GPR56/ADGRG1 as a conserved regulator of PNS development and health. We demonstrate that, during SC development, GPR56-dependent RhoA signaling promotes timely radial sorting of axons. In the mature PNS, GPR56 is localized to distinct SC cytoplasmic domains, is required to establish proper myelin thickness, and facilitates organization of the myelin sheath. Furthermore, we define plectin-a scaffolding protein previously linked to SC domain organization, myelin maintenance, and a series of disorders termed "plectinopathies"-as a novel interacting partner of GPR56. Finally, we show that Gpr56 mutants develop progressive neuropathy-like symptoms, suggesting an underlying mechanism for peripheral defects in some human patients with GPR56 mutations. In sum, we define Gpr56 as a new regulator in the development and maintenance of peripheral myelin.

Neto-mediated intracellular interactions shape postsynaptic composition at the Drosophila neuromuscular junction.

The molecular mechanisms controlling the subunit composition of glutamate receptors are crucial for the formation of neural circuits and for the long-term plasticity underlying learning and memory. Here we use the Drosophila neuromuscular junction (NMJ) to examine how specific receptor subtypes are recruited and stabilized at synaptic locations. In flies, clustering of ionotropic glutamate receptors (iGluRs) requires Neto (Neuropillin and Tolloid-like), a highly conserved auxiliary subunit that is essential for NMJ assembly and development. Drosophila neto encodes two isoforms, Neto-α and Neto-β, with common extracellular parts and distinct cytoplasmic domains. Mutations that specifically eliminate Neto-β or its intracellular domain were generated. When Neto-β is missing or is truncated, the larval NMJs show profound changes in the subtype composition of iGluRs due to reduced synaptic accumulation of the GluRIIA subunit. Furthermore, neto-β mutant NMJs fail to accumulate p21-activated kinase (PAK), a critical postsynaptic component implicated in the synaptic stabilization of GluRIIA. Muscle expression of either Neto-α or Neto-β rescued the synaptic transmission at neto null NMJs, indicating that Neto conserved domains mediate iGluRs clustering. However, only Neto-β restored PAK synaptic accumulation at neto null NMJs. Thus, Neto engages in intracellular interactions that regulate the iGluR subtype composition by preferentially recruiting and/or stabilizing selective receptor subtypes.

Distinct Cytoplasmic Domains in Plexin-A4 Mediate Diverse Responses to Semaphorin 3A in Developing Mammalian Neurons

Guidance receptor signaling is crucial for neural circuit formation and elicits diverse cellular events in specific neurons. We found that signaling from the guidance cue semaphorin 3A diverged through distinct cytoplasmic domains in its receptor Plexin-A4 to promote disparate cellular behavior in different neuronal cell types. Plexin-A4 has three main cytoplasmic domains--C1, Hinge/RBD, and C2--and interacts with family members of the Rho guanine nucleotide exchange factor FARP proteins. We show that growth cone collapse occurred in Plexin-A4-deficient dorsal root ganglion sensory neurons reconstituted with Plexin-A4 containing either the Hinge/RBD or C2 domain, whereas both of the Hinge/RBD and C1 domains were required for dendritic arborization in cortical neurons. Although knockdown studies indicated that both the collapse and arborization responses involved FARP2, mutations in the cytoplasmic region of Plexin-A4 that reduced its interaction with FARP2 strongly inhibited semaphorin 3A-induced dendritic branching but not growth cone collapse, suggesting that different degrees of interaction are required for the two responses or that developing axons have an indirect path to FARP2 activation. Thus, our study provided insights into the multifunctionality of guidance receptors, in particular showing that the semaphorin 3A signal diverges through specific functions of the modular domains of Plexin-A4.

Integrin α6A splice variant regulates proliferation and the Wnt/β-catenin pathway in human colorectal cancer cells

The integrin α6 subunit pre-messenger RNA undergoes alternative splicing to generate two different splice variants, named α6A and α6B, having distinct cytoplasmic domains. In the human colonic gland, these splice variants display different patterns of expression suggesting specific functions for each variant. We have previously found an up-regulation of the α6β4 integrin in colon adenocarcinomas as well as an increase in the α6A/α6B ratio, but little is known about the involvement of α6Aβ4 versus α6Bβ4 in this context. The aim of this study was to elucidate the function of the α6Aβ4 integrin in human colorectal cancer (CRC) cells. Expression studies on a panel of primary CRCs confirmed that the up-regulation of the α6 subunit in CRC is a direct consequence of the increase of the α6A variant. To investigate the functional significance of an α6A up-regulation in CRC, we specifically knocked down its expression in well-established CRC cell lines using a small-hairpin RNA approach. Results showed a growth rate reduction in all α6A knockdown CRC cell lines studied. The α6A silencing was also found to be associated with a significant repression of a number of Wnt/β-catenin pathway end points. Moreover, it was accompanied by a reduction in the capacity of these cells to develop tumours in xenografts. Taken together, these results demonstrate that the α6A variant is a pro-proliferative form of the α6 integrin subunit in CRC cells and appears to mediate its effects through the Wnt/β-catenin pathway.

Recent progress in the single-cell C4 photosynthesis in terrestrial plants

Currently, single-cell C4 photosynthesis has been reported in four terrestrial plant species, Bienertia cycloptera, B. sinuspersici, B. kavirense and Suaeda aralocaspica, of family Chenopodiaceae. These species possess novel mechanisms of C4 photosynthesis through spatial partitioning of organelles and key enzymes in distinct cytoplasmic domains within single chlorenchyma cells. Anatomical and biochemical studies have shown that the three Bienertia species and S. aralocaspica utilize biochemical and organellar compartmentation to achieve the equivalent spatial separation of Kranz anatomy but within a single photosynthetic cell. These discoveries have challenged the paradigm for C4 photosynthesis in terrestrial plants which had suggested for more than 40 years that the Kranz feature was indispensably required for its C4 function. In this review, we focus on the recent progress in understanding the cellular and molecular mechanisms that control the spatial relationship of organelles in these unique single-cell C4 systems. The demonstrated interaction of dimorphic chloroplasts with microtubules and actin filaments has shed light on the importance of these cytoskeleton components in the intracellular partitioning of organelles. Future perspectives on the potential function of the cytoskeleton in targeting gene products to specific subcellular compartments are discussed.

A multidomain hub anchors the chromosome segregation and chemotactic machinery to the bacterial pole

The cell poles constitute key subcellular domains that are often critical for motility, chemotaxis, and chromosome segregation in rod-shaped bacteria. However, in nearly all rods, the processes that underlie the formation, recognition, and perpetuation of the polar domains are largely unknown. Here, in Vibrio cholerae, we identified HubP (hub of the pole), a polar transmembrane protein conserved in all vibrios, that anchors three ParA-like ATPases to the cell poles and, through them, controls polar localization of the chromosome origin, the chemotactic machinery, and the flagellum. In the absence of HubP, oriCI is not targeted to the cell poles, chemotaxis is impaired, and a small but increased fraction of cells produces multiple, rather than single, flagella. Distinct cytoplasmic domains within HubP are required for polar targeting of the three ATPases, while a periplasmic portion of HubP is required for its localization. HubP partially relocalizes from the poles to the mid-cell prior to cell division, thereby enabling perpetuation of the polar domain in future daughter cells. Thus, a single polar hub is instrumental for establishing polar identity and organization.

Common Themes in RNA Subcellular Transport, Stress Granule Formation and Abnormal Protein Aggregation

Control of protein synthesis and quality are critical steps to support eukaryotic cells maintenance and survival. Two very distinctive mechanisms emerge as key checkpoints of protein synthesis regulation. The first one is the delivery of mRNA molecules, packed into ribonucleoprotein (mRNP) granules, to specific subcellular regions in order to restrict protein synthesis to distinct cytoplasmic domains. In the presence of cellular stress or injury, translation is aborted by sequestering mRNA molecules into a sub-type of RNP particles called stress granules (SGs). The second mechanism deals with the folding state and further processing of synthesized proteins. Misbehavior of a particular protein, affecting its processing, functioning, and/or conformation can cause the formation of protein inclusions called aggresomes. Interestingly, self-aggregation of abnormal proteins is one of the leading causes of neurodegenerative disorders. Recently, intracellular transport directed by microtubule-motors, has emerged as an important step in the assembly and dynamic of SGs and aggresomes. This mechanism allows for a precise temporal and spatial trafficking of RNA and protein complexes. Furthermore, it facilitates the regulation of the RNA silencing domains and targets abnormal protein aggregates for degradation. In this review we will explore the specific and common features of mRNA transport and of SG and aggresome formation, and will provide details on the role of the microtubule network and motors in their movement and dynamics. Keywords: Abnormal protein aggregates, aggresome, cell stress, cytoskeleton, dynein, kinesin, RNP, stress granules, RNA Subcellular Transport, Drosophila S2 cells, bicoid mRNA, prion-like proteins, neurodegenerative disorders, Double knockdown (KD) experiments

The interaction of PKC, S100A4, and the cytoplasmic domain of FcγRIIIa (CD16A) α-chain regulates receptor mediated functions (116.1)

Abstract FcγRIIIa(CD16A) pairs with FcR-common γ-chain for surface expression and function. Since many functionally distinct immune receptors utilize γ-chains as signaling subunit, we hypothesized that elements for specificity in receptor signaling may be determined by the distinct FcγR cytoplasmic domains (CY). In silico analysis identified a PKC phosphorylation consensus motif within CD16A-CY; and in vitro kinase assays demonstrated that it is specifically phosphorylated by PKC. Non-phosphorylated CD16A is more effective at activation of the Gab2/PI3K pathway, leading to enhanced degranulation, while phosphorylated CD16A mediates a more robust calcium flux, tyrosine phosphorylation of Syk and pro-inflammatory cytokine production. S100A4, a specific protein binding partner for CD16A-CY, inhibits phosphorylation of CD16A-CY by PKC in vitro. Reduction of S100A4 levels in vivo enhanced receptor phosphorylation upon cross-linking. Taken together, we show that among IgG and IgA receptors, PKC-mediated phosphorylation of CD16A is specific and modulates distinct signaling pathways engaged by the receptor. Calcium activated binding of S100A4 to CD16A, promoted by the initial calcium flux and attenuating the phosphorylation of CY, may serve as a negative feedback on cytokine production pathways during sustained receptor engagement and favor a shift to degranulation, consistent with the importance of degranulation following conjugate formation between CD16A+ effector cells and target cells.

A Structural Determinant for the Control of PIP2 Sensitivity in G Protein-gated Inward Rectifier K+ Channels

Inward rectifier K+ (Kir) channels are activated by phosphatidylinositol-(4,5)-bisphosphate (PIP2), but G protein-gated Kir (KG) channels further require either G protein βγ subunits (Gβγ) or intracellular Na+ for their activation. To reveal the mechanism(s) underlying this regulation, we compared the crystal structures of the cytoplasmic domain of KG channel subunit Kir3.2 obtained in the presence and the absence of Na+. The Na+-free Kir3.2, but not the Na+-plus Kir3.2, possessed an ionic bond connecting the N terminus and the CD loop of the C terminus. Functional analyses revealed that the ionic bond between His-69 on the N terminus and Asp-228 on the CD loop, which are known to be critically involved in Gβγ- and Na+-dependent activation, lowered PIP2 sensitivity. The conservation of these residues within the KG channel family indicates that the ionic bond is a character that maintains the channels in a closed state by controlling the PIP2 sensitivity.

Functional Interactions between Distinct Sodium Channel Cytoplasmic Domains through the Action of Calmodulin

Sodium channels are fundamental signaling molecules in excitable cells, and are molecular targets for local anesthetic agents and intracellular free Ca(2+) ([Ca(2+)](i)). Two regions of Na(V)1.5 have been identified previously as [Ca(2+)](i)-sensitive modulators of channel inactivation. These include a C-terminal IQ motif that binds calmodulin (CaM) in different modes depending on Ca(2+) levels, and an immediately adjacent C-terminal EF-hand domain that directly binds Ca(2+). Here we show that a mutation of the IQ domain (A1924T; Brugada Syndrome) that reduces CaM binding stabilizes Na(V)1.5 inactivation, similarly and more extensively than even reducing [Ca(2+)](i). Because the DIII-DIV linker is an essential structure in Na(V)1.5 inactivation, we evaluated this domain for a potential CaM binding interaction. We identified a novel CaM binding site within the linker, validated its interaction with CaM by NMR spectroscopy, and revealed its micromolar affinity by isothermal titration calorimetry. Mutation of three consecutive hydrophobic residues (Phe(1520)-Ile(1521)-Phe(1522)) to alanines in this CaM-binding domain recapitulated the electrophysiology phenotype observed with mutation of the C-terminal IQ domain: Na(V)1.5 inactivation was stabilized; moreover, mutations of either CaM-binding domain abolish the well described stabilization of inactivation by lidocaine. The direct physical interaction of CaM with the C-terminal IQ domain and the DIII-DIV linker, combined with the similarity in phenotypes when CaM-binding sites in either domain are mutated, suggests these cytoplasmic structures could be functionally coupled through the action of CaM. These findings have bearing upon Na(+) channel function in genetically altered channels and under pathophysiologic conditions where [Ca(2+)](i) impacts cardiac conduction.

α- and γ-Protocadherins Negatively Regulate PYK2

Genetic studies demonstrate that gamma-protocadherins (PCDH-gamma) are required for the survival and synaptic connectivity in neuronal subpopulations of the central nervous system. However, the intracellular signaling mechanisms for PCDH-gamma are poorly understood. Here, we show that PCDH-gamma binds two tyrosine kinases, PYK2 and focal adhesion kinase (FAK), and interaction with PCDH-gamma inhibits kinase activity. Consistent with this, PYK2 activity is abnormally up-regulated in the Pcdh-gamma-deficient neurons. Overexpression of PYK2 induces apoptosis in the chicken spinal cord. Thus, negative regulation of PYK2 activity by PCDH could contribute to the survival of subsets of neurons. Surprisingly, we found that PCDH-alpha interacts similarly with PYK2 and FAK despite containing a distinct cytoplasmic domain. In neural tissue, PCDH-gamma, together with PCDH-alpha, forms functional complexes with PYK2 and/or FAK. Therefore, the identification of common intracellular effectors for PCDH-gamma and PCDH-alpha suggests that dozens of protocadherins generated by Pcdh-alpha and Pcdh-gamma gene clusters can converge different extracellular signals into common intracellular pathways.

An alternatively spliced isoform of PECAM-1 is expressed at high levels in human and murine tissues, and suggests a novel role for the C-terminus of PECAM-1 in cytoprotective signaling

The Ig-ITIM family member PECAM-1 is expressed in vascular and endothelial cells, and its functions include suppression of mitochondria-dependent apoptosis. Previous studies have identified distinct PECAM-1 cytoplasmic domain splice variants at the mRNA, but not protein, level. Several relatively abundant mRNA isoforms lack exon 15 (Delta15) and would theoretically encode a protein with a truncated cytoplasmic domain and a unique C-terminal sequence. Using a novel rabbit polyclonal antibody that specifically recognizes Delta15 PECAM-1, we found that the Delta15 PECAM-1 isoform was expressed in human tissues, including brain, testes and ovary. This isoform was also expressed on the cell surface of human platelets, human umbilical vein endothelial cells (HUVECs) and the Jurkat T-cell leukemia, human erythroleukemia (HEL) and U937 histiocytic lymphoma cell lines. Furthermore, murine platelets and lung lysates demonstrated abundant amounts of exon-15-deficient PECAM-1. Functional studies revealed that Delta15 PECAM-1 retains both its homophilic binding capacity and its ability to signal by means of its immunoreceptor tyrosine-based inhibitory motif (ITIM) domains. Delta15 PECAM-1 was unable, however, to protect against apoptosis induced by overexpression of Bax or treatment with the chemotherapy agent etoposide. These studies suggest a novel role for the PECAM-1 C-terminus in cytoprotective signaling and highlight a need for further characterization of expression of PECAM-1 isoforms in normal and malignant tissues.

Structural studies on Vibrio cholerae ToxR periplasmic and cytoplasmic domains

The transcription activator ToxR controls the expression of cholera toxin, pilus colonization factor and outer membrane protein in Vibrio cholerae. It binds to the 5′-TTTTGAT-3′ tandemly repeated DNA sequence in the cholera toxin promoter region. ToxR is a membrane protein having distinct periplasmic and cytoplasmic domains. The two domains have been cloned, over-expressed and purified for structural studies. The cytoplasmic domain is more compact than the periplasmic domain. The periplasmic domain exists as dimer due to the presence of an interchain disulfide linkage, while the cytoplasmic domain is monomeric in solution implying the importance of the disulfide bond to homodimerize the native ToxR. By replacing one of the cysteines C293 with alanine, using site-directed mutagenesis, a C293A mutant was created at the periplasmic domain to elucidate the role of cysteine in dimerization of ToxR.

Caenorhabditis elegans decapping proteins: localization and functional analysis of Dcp1, Dcp2, and DcpS during embryogenesis.

Though posttranscriptional regulation is important for early embryogenesis, little is understood regarding control of mRNA decay during development. Previous work defined two major pathways by which normal transcripts are degraded in eukaryotes. However it is not known which pathways are key in mRNA decay during early patterning or whether developmental transcripts are turned over via specific pathways. Here we show that Caenorhabditis elegans Dcp2 is localized to distinct foci during embryogenesis, reminiscent of P-bodies, the sites of mRNA degradation in yeast and mammals. However the decapping enzyme of the 3' to 5' transcript decay system (DcpS) localizes throughout the cytoplasm, suggesting this degradation pathway is not highly organized. In addition we find that Dcp2 is localized to P-granules, showing that Dcp2 is stored and/or active in these structures. However RNAi of these decapping enzymes has no obvious effect on embryogenesis. In contrast we find that nuclear cap binding proteins (CBP-20 and 80), eIF4G, and PAB-1 are absolutely required for development. Together our data provides further evidence that pathways of general mRNA metabolism can be remarkably organized during development, with two different decapping enzymes localized in distinct cytoplasmic domains.

Identification of novel Prominin-1/CD133 splice variants with alternative C-termini and their expression in epididymis and testis.

Prominin-1/CD133 is a five-membrane-span glycoprotein that is thought to act as an organizer of plasma-membrane protrusions. Here, we report the molecular and cell-biological characterization of four novel prominin-1 splice variants isolated from a mouse testis cDNA library and referred to as prominin-1.s3 to prominin-1.s6. Compared with kidney-derived prominin-1.s1, the s3, s4 and s5 variants contain a distinct cytoplasmic C-terminal domain. The s4 and s5 variants bear, in addition, two and one inframe deletion(s), respectively, in the extracellular domains. The s6 variant displays a truncated C-terminal domain caused by a premature termination resulting from intron retention. Upon their ectopic expression in Chinese hamster ovary cells, the s3 and s6 variants were found to be concentrated in plasma-membrane protrusions, whereas the s4 and s5 variants did not reach the cell surface. Biochemical analyses suggest that most of the prominin-1 in the adult male reproductive system is expressed as the s6 variant. Immunohistological and electron microscopic analyses show that prominin-1 is: (1) confined to the apical surface of the epithelium all along the epididymal duct, with the exception of the initial segment; (2) concentrated in stereocilia of the epididymal duct epithelium; and (3) found on the tail of developing spermatozoa in seminiferous tubules. Our data suggest that prominin-1 is involved in the formation and/or stabilization of epididymal stereocilia and the tail of spermatozoa, and hence might play a dual role in the biogenesis of spermatozoa.