scholarly journals A novel RAB11-containing adaptor complex anchoring myosin-5 to secretory vesicles

2021 ◽  
Author(s):  
Mario Pinar ◽  
Ana Alonso ◽  
Vivian de los Ríos ◽  
Ignacio Bravo-Plaza ◽  
Álvaro Gandara ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Analytical Ultracentrifugation ◽  
Coiled Coil ◽  
Shotgun Proteomics ◽  
Secretory Vesicles ◽  
Dependent Manner ◽  
Apical Dome ◽  
Basal Septum ◽  
Dependent Transport

AbstractHyphal fungi grow rapidly by apical extension, providing a notorious example of polarized growth. The continuous supply of secretory vesicles necessary to meet the demands of the extending tip and the long intracellular distances existing between the tip and the basal septum, often localized > 100 µm away from the former, impose the need of efficient networks of intracellular traffic involving exquisite cooperation between microtubule- and actin-mediated transport. In Aspergillus nidulans kinesin-1 conveys secretory vesicles to the hyphal tip, where they are transferred to myosin-5, which focuses them at the growing apex, thereby determining cell shape. This relay mechanism and the central role played by myosin-5 in hyphal morphogenesis suggested that the mechanisms anchoring secretory vesicles to this motor should involve specific adaptor(s) ensuring the robustness of actomyosin-dependent transport.Secretory vesicles are charged with RAB11, a regulatory GTPase that determines the Golgi to post-Golgi identity transition. By using a combination of shotgun proteomics, GST-RAB pull-down assays, in vitro reconstitution experiments, targeted reverse genetics and multidimensional fluorescence microscopy with endogenously tagged proteins we show that RAB11, the master regulator of fungal exocytosis, mediates myosin-5 engagement both by contacting the motor and by recruiting UDS1, a homologue of an as yet uncharacterized Schizosaccharomyces protein ‘upregulated during mitosis’, which we demonstrate to be a novel RAB11 effector. Analytical ultracentrifugation determined that UDS1 is an elongated dimer and negative-stain electron microscopy showed that, in agreement, UDS1 is rod-shaped. UDS1 does not contact myosin-5 directly, but rather recruits the coiled-coil HMSV, which bridges RAB11/UDS1 to myosin-5. An HMSV-scaffolded complex containing UDS1 and myosin-5 is present in cells, and a RAB11-UDS1-HMSV complex can be reconstituted in vitro in a RAB nucleotide state-dependent manner. In the absence of UDS1/HMSV the steady state levels of myosin-5 at the apical vesicle supply center diminish markedly, such that microtubule-dependent transport spreading vesicles across the apical dome predominates over apex-focused actin-mediated transport. As a consequence, RAB11 and chitin-synthase B (a cargo of the RAB11 pathway) are not focused at the apex, being distributed instead across the apical dome. Therefore, the RAB11 effector UDS1/HMSV cooperates with the GTPase to adapt secretory vesicles to myosin-5, which is required for the apical targeting of RAB11 cargoes and thus for the normal morphology of the hyphae.

scholarly journals Aurora A activates D-TACC–Msps complexes exclusively at centrosomes to stabilize centrosomal microtubules

2005 ◽  
Vol 170 (7) ◽  
pp. 1039-1046 ◽  
Author(s):  
Teresa P. Barros ◽  
Kazuhisa Kinoshita ◽  
Anthony A. Hyman ◽  
Jordan W. Raff
Keyword(s):  
Drosophila Melanogaster ◽  
Coiled Coil ◽  
Mutant Form ◽  
Dependent Manner ◽  
Aurora A ◽  
Animal Cells

Centrosomes are the dominant sites of microtubule (MT) assembly during mitosis in animal cells, but it is unclear how this is achieved. Transforming acidic coiled coil (TACC) proteins stabilize MTs during mitosis by recruiting Minispindles (Msps)/XMAP215 proteins to centrosomes. TACC proteins can be phosphorylated in vitro by Aurora A kinases, but the significance of this remains unclear. We show that Drosophila melanogaster TACC (D-TACC) is phosphorylated on Ser863 exclusively at centrosomes during mitosis in an Aurora A–dependent manner. In embryos expressing only a mutant form of D-TACC that cannot be phosphorylated on Ser863 (GFP-S863L), spindle MTs are partially destabilized, whereas astral MTs are dramatically destabilized. GFP-S863L is concentrated at centrosomes and recruits Msps there but cannot associate with the minus ends of MTs. We propose that the centrosomal phosphorylation of D-TACC on Ser863 allows D-TACC–Msps complexes to stabilize the minus ends of centrosome-associated MTs. This may explain why centrosomes are such dominant sites of MT assembly during mitosis.

scholarly journals Yeast Rgd3 is a phospho-regulated F-BAR-containing RhoGAP involved in the regulation of Rho3 distribution and cell morphology

2020 ◽  
Author(s):  
Robert M. Gingras ◽  
Kyaw Myo Lwin ◽  
Abigail M. Miller ◽  
Anthony Bretscher
Keyword(s):  
Cell Polarity ◽  
Binding Motif ◽  
Restrictive Temperature ◽  
Secretory Vesicles ◽  
Polarized Growth ◽  
Related Protein ◽  
Myosin V ◽  
Over Expression ◽  
Dependent Transport

AbstractPolarized growth requires the integration of polarity pathways with the delivery of exocytic vesicles for cell expansion and counterbalancing endocytic uptake. In budding yeast, the myosin-V Myo2 is aided by the kinesin-related protein Smy1 in carrying out the essential Sec4-dependent transport of secretory vesicles to sites of polarized growth. Over-expression suppressors of a conditional myo2 smy1 mutant identified a novel F-BAR-containing RhoGAP, Rgd3, that has activity primarily on Rho3, but also Cdc42. Internally tagged Rho3 is restricted to the plasma membrane in a gradient corresponding to cell polarity that is altered upon Rgd3 over-expression. Rgd3 itself is localized to dynamic polarized vesicles that, while distinct from constitutive secretory vesicles, are dependent on actin and Myo2 function. In vitro Rgd3 associates with liposomes in a PIP2-enhanced manner. Further, the Rgd3 C-terminal region contains several phosphorylatable residues within a reported SH3-binding motif. An unphosphorylated mimetic construct is active and highly polarized, while the phospho-mimetic form is not. Rgd3 is capable of activating Myo2, dependent on its phospho-state and Rgd3 overexpression rescues aberrant Rho3 localization and cell morphologies seen at the restrictive temperature in the myo2 smy1 mutant. We propose a model where Rgd3 functions to modulate and maintain Rho3 polarity during growth.

scholarly journals In Vitro Inhibitory Mechanism Effect of TRAIP on the Function of TRAF2 Revealed by Characterization of Interaction Domains

2018 ◽  
Vol 19 (8) ◽  
pp. 2457 ◽  
Author(s):  
Eijaz Bhat ◽  
Chang Kim ◽  
Sunghwan Kim ◽  
Hyun Park
Keyword(s):  
Coiled Coil ◽  
Adaptor Protein ◽  
Direct Interaction ◽  
Negative Regulator ◽  
Dependent Manner ◽  
Inhibitory Mechanism ◽  
Concentration Dependent Manner ◽  
Critical Function

TRAF-interacting protein (TRAIP), a negative regulator of TNF-induced-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, inhibits adaptor protein TRAF2 by direct interaction and is critical in apoptosis, cell proliferation, antiviral response, and embryonic development. Although the critical function of TRAIP in NF-κB signaling is well-known, the molecular inhibitory mechanism of TRAIP remains unclear. We found that the TRAIP coiled-coil domain altered its stoichiometry between dimer and trimer in a concentration-dependent manner. Additionally, the TRAIP RING domain induced even higher-ordered assembly, which was necessary for interacting with the TRAF-N domain of TRAF2 but not TRAF1. Characterization of the TRAF-N domains of TRAF1 and TRAF2, the tentative TRAIP-binding region of TRAFs, suggested the molecular basis of the inhibitory effect of TRAIP on TRAF2 in NF-κB signaling.

scholarly journals Mutations Q93H and E97K in TPM2 Disrupt Ca-Dependent Regulation of Actin Filaments

2021 ◽  
Vol 22 (8) ◽  
pp. 4036
Author(s):  
Małgorzata Śliwinska ◽  
Katarzyna Robaszkiewicz ◽  
Piotr Wasąg ◽  
Joanna Moraczewska
Keyword(s):  
Skeletal Muscle ◽  
Actin Filaments ◽  
Thin Filament ◽  
Molecular Mechanisms ◽  
Coiled Coil ◽  
Dependent Manner ◽  
Gene Encoding ◽  
Troponin Complex ◽  
Increased Sensitivity

Tropomyosin is a two-chain coiled coil protein, which together with the troponin complex controls interactions of actin with myosin in a Ca2+-dependent manner. In fast skeletal muscle, the contractile actin filaments are regulated by tropomyosin isoforms Tpm1.1 and Tpm2.2, which form homo- and heterodimers. Mutations in the TPM2 gene encoding isoform Tpm2.2 are linked to distal arthrogryposis and congenital myopathy—skeletal muscle diseases characterized by hyper- and hypocontractile phenotypes, respectively. In this work, in vitro functional assays were used to elucidate the molecular mechanisms of mutations Q93H and E97K in TPM2. Both mutations tended to decrease actin affinity of homo-and heterodimers in the absence and presence of troponin and Ca2+, although the effect of Q93H was stronger. Changes in susceptibility of tropomyosin to trypsin digestion suggested that the mutations diversified dynamics of tropomyosin homo- and heterodimers on the filament. The presence of Q93H in homo- and heterodimers strongly decreased activation of the actomyosin ATPase and reduced sensitivity of the thin filament to [Ca2+]. In contrast, the presence of E97K caused hyperactivation of the ATPase and increased sensitivity to [Ca2+]. In conclusion, the hypo- and hypercontractile phenotypes associated with mutations Q93H and E97K in Tpm2.2 are caused by defects in Ca2+-dependent regulation of actin–myosin interactions.

scholarly journals Utilizing fowlpox virus recombinants to generate defective RNAs of the coronavirus infectious bronchitis virus

2000 ◽  
Vol 81 (12) ◽  
pp. 2855-2865 ◽  
Author(s):  
Sharon Evans ◽  
David Cavanagh ◽  
Paul Britton
Keyword(s):  
Infectious Bronchitis Virus ◽  
Mammalian Cells ◽  
Reverse Genetics ◽  
Leader Sequence ◽  
Dependent Manner ◽  
T7 Promoter ◽  
Fowlpox Virus ◽  
Infectious Bronchitis ◽  
Defective Rnas

Coronavirus defective RNAs (D-RNAs) have been used as RNA vectors for the expression of heterologous genes and as vehicles for reverse genetics by modifying coronavirus genomes by targetted recombination. D-RNAs based on the avian coronavirus infectious bronchitis virus (IBV) D-RNA CD-61 have been rescued (replicated and packaged into virions) in a helper virus-dependent manner following electroporation of in vitro-generated T7 transcripts into IBV-infected cells. In order to increase the efficiency of rescue of IBV D-RNAs, cDNAs based on CD-61, under the control of a T7 promoter, were integrated into the fowlpox virus (FPV) genome. The 3′-UTR of the D-RNAs was flanked by a hepatitis delta antigenomic ribozyme and T7 terminator sequence to generate suitable 3′ ends for rescue by helper IBV. Cells were co-infected simultaneously with IBV, the recombinant FPV (rFPV) containing the D-RNA sequence and a second rFPV expressing T7 RNA polymerase for the initial expression of the D-RNA transcript, subsequently rescued by helper IBV. Rescue of rFPV-derived CD-61 occurred earlier and with higher efficiency than demonstrated previously for electroporation of in vitro T7-generated RNA transcripts in avian cells. Rescue of CD-61 was also demonstrated for the first time in mammalian cells. The rescue of rFPV-derived CD-61 by M41 helper IBV resulted in leader switching, in which the Beaudette-type leader sequence on CD-61 was replaced with the M41 leader sequence, confirming that helper IBV virus replicated the rFPV-derived D-RNA. An rFPV-derived D-RNA containing the luciferase gene under the control of an IBV transcription-associated sequence was also rescued and expressed luciferase on serial passage.

scholarly journals The stoichiometry of the nucleoporin 62 subcomplex of the nuclear pore in solution

2014 ◽  
Vol 25 (9) ◽  
pp. 1484-1492 ◽  
Author(s):  
Alexander Ulrich ◽  
James R. Partridge ◽  
Thomas U. Schwartz
Keyword(s):  
Analytical Ultracentrifugation ◽  
Protein Complexes ◽  
Gel Filtration ◽  
Coiled Coil ◽  
In Vitro Study ◽  
Nuclear Pore ◽  
Binding Partner ◽  
Repeat Proteins ◽  
Complex Forms

The nuclear pore complex (NPC) regulates transport between the nucleus and cytoplasm. Soluble cargo-protein complexes navigate through the pore by binding to phenylalanine-glycine (FG)-repeat proteins attached to the channel walls. The Nup62 complex contains the FG-repeat proteins Nup62, Nup54, and Nup58 and is located in the center of the NPC. The three proteins bind each other via conserved coiled-coil segments. To determine the stoichiometry of the Nup62 complex, we undertook an in vitro study using gel filtration and analytical ultracentrifugation. Our results reveal a 1:1:1 stoichiometry of the Nup62 complex, where Nup54 is central with direct binding to Nup62 and Nup58. At high protein concentration, the complex forms larger assemblies while maintaining the Nup62:Nup54:Nup58 ratio. For the homologous Nsp1 complex from Saccharomyces cerevisiae, we determine the same stoichiometry, indicating evolutionary conservation. Furthermore, we observe that eliminating one binding partner can result in the formation of complexes with noncanonical stoichiometry, presumably because unpaired coiled-coil elements tend to find a promiscuous binding partner. We suggest that these noncanonical stoichiometries observed in vitro are unlikely to be physiologically relevant.

scholarly journals Kinetochore–microtubule attachment throughout mitosis potentiated by the elongated stalk of the kinetochore kinesin CENP-E

2014 ◽  
Vol 25 (15) ◽  
pp. 2272-2281 ◽  
Author(s):  
Benjamin Vitre ◽  
Nikita Gudimchuk ◽  
Ranier Borda ◽  
Yumi Kim ◽  
John E. Heuser ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Biological Role ◽  
Wild Type ◽  
Microtubule Attachment ◽  
Chromosome Congression ◽  
Chromosome Alignment ◽  
Type Motor ◽  
Dependent Transport

Centromere protein E (CENP-E) is a highly elongated kinesin that transports pole-proximal chromosomes during congression in prometaphase. During metaphase, it facilitates kinetochore–microtubule end-on attachment required to achieve and maintain chromosome alignment. In vitro CENP-E can walk processively along microtubule tracks and follow both growing and shrinking microtubule plus ends. Neither the CENP-E–dependent transport along microtubules nor its tip-tracking activity requires the unusually long coiled-coil stalk of CENP-E. The biological role for the CENP-E stalk has now been identified through creation of “Bonsai” CENP-E with significantly shortened stalk but wild-type motor and tail domains. We demonstrate that Bonsai CENP-E fails to bind microtubules in vitro unless a cargo is contemporaneously bound via its C-terminal tail. In contrast, both full-length and truncated CENP-E that has no stalk and tail exhibit robust motility with and without cargo binding, highlighting the importance of CENP-E stalk for its activity. Correspondingly, kinetochore attachment to microtubule ends is shown to be disrupted in cells whose CENP-E has a shortened stalk, thereby producing chromosome misalignment in metaphase and lagging chromosomes during anaphase. Together these findings establish an unexpected role of CENP-E elongated stalk in ensuring stability of kinetochore–microtubule attachments during chromosome congression and segregation.

scholarly journals Interactome and F-Actin Interaction Analysis of Dictyostelium discoideum Coronin A

2020 ◽  
Vol 21 (4) ◽  
pp. 1469 ◽  
Author(s):  
Tohnyui Ndinyanka Fabrice ◽  
Thomas Fiedler ◽  
Vera Studer ◽  
Adrien Vinet ◽  
Francesco Brogna ◽  
...  
Keyword(s):  
Interaction Analysis ◽  
Coiled Coil ◽  
Cellular Interaction ◽  
Rabbit Muscle ◽  
Dependent Manner ◽  
Independent Manner ◽  
Multicellular Development ◽  
Cellular Processes ◽  
Myosin Subunits

Coronin proteins are evolutionary conserved WD repeat containing proteins that have been proposed to carry out different functions. In Dictyostelium, the short coronin isoform, coronin A, has been implicated in cytoskeletal reorganization, chemotaxis, phagocytosis and the initiation of multicellular development. Generally thought of as modulators of F-actin, coronin A and its mammalian homologs have also been shown to mediate cellular processes in an F-actin-independent manner. Therefore, it remains unclear whether or not coronin A carries out its functions through its capacity to interact with F-actin. Moreover, the interacting partners of coronin A are not known. Here, we analyzed the interactome of coronin A as well as its interaction with F-actin within cells and in vitro. Interactome analysis showed the association with a diverse set of interaction partners, including fimbrin, talin and myosin subunits, with only a transient interaction with the minor actin10 isoform, but not the major form of actin, actin8, which was consistent with the absence of a coronin A-actin interaction as analyzed by co-sedimentation from cells and lysates. In vitro, however, purified coronin A co-precipitated with rabbit muscle F-actin in a coiled-coil-dependent manner. Our results suggest that an in vitro interaction of coronin A and rabbit muscle actin may not reflect the cellular interaction state of coronin A with actin, and that coronin A interacts with diverse proteins in a time-dependent manner.

Chlorobutanol, a Preservative of Desmopressin, Inhibits Human Platelet Aggregation and Release In Vitro

1990 ◽  
Vol 64 (03) ◽  
pp. 473-477 ◽  
Author(s):  
Shih-Luen Chen ◽  
Wu-Chang Yang ◽  
Tung-Po Huang ◽  
Shiang Wann ◽  
Che-ming Teng
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Atp Release ◽  
Free Calcium ◽  
Dependent Manner ◽  
Antiplatelet Effect ◽  
Human Platelet Aggregation ◽  
Acid Pathway ◽  
Arachidonic Acid Pathway

SummaryTherapeutic preparations of desmopressin for parenteral use contain the preservative chlorobutanol (5 mg/ml). We show here that chlorobutanol is a potent inhibitor of platelet aggregation and release. It exhibited a significant inhibitory activity toward several aggregation inducers in a concentration- and time-dependent manner. Thromboxane B2 formation, ATP release, and elevation of cytosolic free calcium caused by collagen, ADP, epinephrine, arachidonic acid and thrombin respectively were markedly inhibited by chlorobutanol. Chlorobutanol had no effect on elastase- treated platelets and its antiplatelet effect could be reversed. It is concluded that the antiplatelet effect of chlorobutanol is mainly due to its inhibition on the arachidonic acid pathway but it is unlikely to have a nonspecitic toxic effect. This antiplatelet effect of chlorobutanol suggests that desmopressin, when administered for improving hemostasis, should not contain chlorobutanol as a preservative.

Oxygenation of 18-hydroxycorticosterone as the final reaction for aldosterone biosynthesis

1984 ◽  
Vol 107 (3) ◽  
pp. 395-400 ◽  
Author(s):  
Itaru Kojima ◽  
Etsuro Ogata ◽  
Hiroshi Inano ◽  
Bun-ichi Tamaoki
Keyword(s):  
Carbon Monoxide ◽  
Hydroxysteroid Dehydrogenase ◽  
Dependent Manner ◽  
In Vitro Production ◽  
Mitochondrial Preparation ◽  
Final Reaction ◽  
Vitro Production ◽  
Dose Dependent ◽  
Cytochrome P 450

Abstract. Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000. SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H ]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

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