Mitochondria are essential and dynamic organelles undergoing constant fission and fusion. The primary players in mitochondrial morphology (MFN1/2, OPA1, DRP1) have been identified, but their mechanism(s) of regulation are still being elucidated. ARL2 is a regulatory GTPase that has previously been shown to play a role in the regulation of mitochondrial morphology. Here we demonstrate that ELMOD2, an ARL2 GTPase-activating protein (GAP), is necessary for ARL2 to promote mitochondrial elongation. We show that loss of ELMOD2 causes mitochondrial fragmentation and a lower rate of mitochondrial fusion, while ELMOD2 overexpression promotes mitochondrial tubulation and increases the rate of fusion in a mitofusin-dependent manner. We also show that a mutant of ELMOD2 lacking GAP activity is capable of promoting fusion, suggesting that ELMOD2 does not need GAP activity to influence mitochondrial morphology. Finally, we show that ELMOD2, ARL2, Mitofusins 1 and 2, Miros 1 and 2, and mitochondrial phospholipase D (mitoPLD) all localize to discrete, regularly spaced puncta along mitochondria. These results suggest that ELMOD2 is functioning as an effector downstream of ARL2 and upstream of the mitofusins to promote mitochondrial fusion. Our data provide insights into the pathway by which mitochondrial fusion is regulated in the cell.
The regulatory GTPase Arl13b localizes to primary cilia, where it regulates Sonic hedgehog (Shh) signaling. Missense mutations in ARL13B can cause the ciliopathy Joubert syndrome (JS), and the mouse null allele is embryonic lethal. We used mouse embryonic fibroblasts as a system to determine the effects of Arl13b mutations on Shh signaling. We tested seven different mutants-Three JS-causing variants, two point mutants predicted to alter guanine nucleotide handling, one that disrupts cilia localization, and one that prevents palmitoylation and thus membrane binding-in assays of transcriptional and nontranscriptional Shh signaling. We found that mutations disrupting Arl13b's palmitoylation site, cilia localization signal, or GTPase handling altered the Shh response in distinct assays of transcriptional or nontranscriptional signaling. In contrast, JS-causing mutations in Arl13b did not affect Shh signaling in these same assays, suggesting that these mutations result in more subtle defects, likely affecting only a subset of signaling outputs. Finally, we show that restricting Arl13b from cilia interferes with its ability to regulate Shh-stimulated chemotaxis, despite previous evidence that cilia themselves are not required for this nontranscriptional Shh response. This points to a more complex relationship between the ciliary and nonciliary roles of this regulatory GTPase than previously envisioned.
Two urea transporters, UT-A1 and UT-A3, are expressed in the kidney terminal inner medullary collecting duct (IMCD) and are important for the production of concentrated urine. UT-A1, as the largest isoform of all UT-A urea transporters, has gained much attention and been extensively studied; however, the role and the regulation of UT-A3 are less explored. In this study, we investigated UT-A3 regulation by glycosylation modification. A site-directed mutagenesis verified a single glycosylation site in UT-A3 at Asn279. Loss of the glycosylation reduced forskolin-stimulated UT-A3 cell membrane expression and urea transport activity. UT-A3 has two glycosylation forms, 45 and 65 kDa. Using sugar-specific binding lectins, the UT-A3 glycosylation profile was examined. The 45-kDa form was pulled down by lectin concanavalin A (Con A) and Galant husnivalis lectin (GNL), indicating an immature glycan with a high amount of mannose (Man), whereas the 65-kDa form is a mature glycan composed of acetylglucosamine (GlcNAc) and poly-N-acetyllactosame (poly-LacNAc) that was pulled down by wheat germ agglutinin (WGA) and tomato lectin, respectively. Interestingly, the mature form of UT-A3 glycan contains significant amounts of sialic acid. We explored the enzymes responsible for directing UT-A3 sialylation. Sialyltransferase ST6GalI, but not ST3GalIV, catabolizes UT-A3 α2,6-sialylation. Activation of protein kinase C (PKC) by PDB treatment promoted UT-A3 glycan sialylation and membrane surface expression. The PKC inhibitor chelerythrine blocks ST6GalI-induced UT-A3 sialylation. Increased sialylation by ST6GalI increased UT-A3 protein stability and urea transport activity. Collectively, our study reveals a novel mechanism of UT-A3 regulation by ST6GalI-mediated sialylation modification that may play an important role in kidney urea reabsorption and the urinary concentrating mechanism.
At the nexus of specialized cellular responses are localized enrichments of protein activity. The localization of messenger RNA (mRNA) coupled with translational control often plays a crucial role in the generation of protein concentrations at defined subcellular domains. Although mRNA localization is classically associated with large specialized cells, such as neurons and embryos, RNA localization is a highly conserved paradigm of post-transcriptional regulation observed in diverse cellular contexts. Functions of localized mRNAs extend far beyond the well-studied examples of neuronal polarization and developmental patterning. Since the initial discovery of the intracellular localization of cytoskeletal mRNAs within migrating cells, hundreds of mRNAs are now known to be enriched at specific organelles where they contribute to cell function. In this short review, we discuss basic principles regulating RNA localization and consider the contribution of localized mRNA to several essential cellular behaviors. We consider RNA localization as a mechanism with widespread implications for cellular function.
by
Prachee Avasthi;
Masayuki Onishi;
Joel Karpiak;
Ryosuke Yamamoto;
Luke Mackinder;
Martin C. Jonikas;
Winfield Sale;
Brian Shoichet;
John R. Pringle;
Wallace F. Marshall
Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin Dresulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length.
by
Stéphanie Morchoisne-Bolhy;
Marie-Claude Geoffroy;
Imène B. Bouhlel;
Annabelle Alves;
Nicolas Auduge;
Xavier Baudin;
Kevin Van Bortle;
Maureen Powers;
Valérie Doye
Nup98 is a glycine-leucine-phenylalanine-glycine (GLFG) repeat-containing nucleoporin that, in addition to nuclear transport, contributes to multiple aspects of gene regulation. Previous studies revealed its dynamic localization within intranuclear structures known as GLFG bodies. Here we show that the mammalian Nup107-160 complex (Y-complex), a major scaffold module of the nuclear pore, together with its partner Elys, colocalizes with Nup98 in GLFG bodies. The frequency and size of GLFG bodies vary among HeLa sublines, and we find that an increased level of Nup98 is associated with the presence of bodies. Recruitment of the Y-complex and Elys into GLFG bodies requires the C-terminal domain of Nup98. During cell division, Y-Nup-containing GLFG bodies are disassembled in mitotic prophase, significantly ahead of nuclear pore disassembly. FRAP studies revealed that, unlike at nuclear pores, the Y-complex shuttles into and out of GLFG bodies. Finally, we show that within the nucleoplasm, a fraction of Nup107, a key component of the Y-complex, displays reduced mobility, suggesting interaction with other nuclear components. Together our data uncover a previously neglected intranuclear pool of the Y-complex that may underscore a yet-uncharacterized function of these nucleoporins inside the nucleus, even in cells that contain no detectable GLFG bodies.
The localization of β-actin mRNA to the leading lamellae of chicken fibroblasts and neurite growth cones of developing neurons requires a 54-nt localization signal (the zipcode) within the 3′ untranslated region. In this study we have identified and isolated five proteins binding to the zipcode. One of these we previously identified as zipcode binding protein (ZBP)1, a 4-KH domain protein. A second is now investigated in detail: a 92-kD protein, ZBP2, that is especially abundant in extracts from embryonic brain. We show that ZBP2 is a homologue of the human hnRNP protein, KSRP, that appears to mediate premRNA splicing. However, ZBP2 has a 47-amino acid (aa) sequence not present in KSRP. Various portions of ZBP2 fused to GFP indicate that the protein most likely shuttles between the nucleus and the cytoplasm, and that the 47-aa insert promotes the nuclear localization. Expression of a truncated ZBP2 inhibits the localization of β-actin mRNA in both fibroblast and neurons. These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of β-actin mRNA.
The role of Rab coupling protein (RCP) has not been previously investigated in squamous cell carcinoma of the head and neck (SCCHN). The aim of this study was to explore RCP protein expression and its clinicopathological significance in SCCHN. RCP protein expression in 95 SCCHN samples, 18 vocal nodule epithelia and 16 leukoplakia epithelia samples was analyzed by immunohistochemistry and correlated with clinicopathological parameters and patient outcome. Our data indicated that vocal nodule epithelia, leukoplakia epithelia and SCCHN showed a gradual increase in the expression of RCP protein. RCP overexpression was significantly associated with T classification, clinical staging, lymph node metastasis and recurrence. Survival analysis revealed that a high RCP expression was significantly correlated with shorter overall survival and disease-free survival. In conclusion, RCP protein may contribute to the malignant progression of SCCHN, and serves as a novel prognostic marker in patients with SCCHN.
The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α-And γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.
by
Christoph Brockmann;
Sharon Soucek;
Sonja I. Kuhlmann;
Katherine Mills-Lujan;
Seth Kelly;
Ji-Chun Yang;
Nahid Iglesias;
Francoise Stutz;
Anita Corbett;
David Neuhaus;
Murray Stewart
Polyadenylation regulation and efficient nuclear export of mature mRNPs both require the polyadenosine-RNA-binding protein, Nab2, which contains seven CCCH Zn fingers. We describe here the solution structure of fingers 5-7, which are necessary and sufficient for high-affinity polyadenosine-RNA binding, and identify key residues involved. These Zn fingers form a single structural unit. Structural coherence is lost in the RNA-binding compromised Nab2-C437S mutant, which also suppresses the rat8-2 allele of RNA helicase Dbp5. Structure-guided Nab2 variants indicate that dbp5(rat8-2) suppression is more closely linked to hyperadenylation and suppression of mutant alleles of the nuclear RNA export adaptor, Yra1, than to affinity for polyadenosine-RNA. These results indicate that, in addition to modulating polyA tail length, Nab2 has an unanticipated function associated with generating export-competent mRNPs, and that changes within fingers 5-7 lead to suboptimal assembly of mRNP export complexes that are more easily disassembled by Dbp5 upon reaching the cytoplasm.