unc-53 controls longitudinal migration in C. elegans

Development ◽  
2002 ◽  
Vol 129 (14) ◽  
pp. 3367-3379 ◽  
Author(s):  
Eve Stringham ◽  
Nathalie Pujol ◽  
Joel Vandekerckhove ◽  
Thierry Bogaert
Keyword(s):  
Binding Sites ◽  
Adaptor Protein ◽  
Direct Interaction ◽  
Actin Binding ◽  
C Elegans ◽  
Guidance Cues ◽  
Preferred Direction ◽  
Excretory Canals ◽  
Novel Protein

Cell migration and outgrowth are thought to be based on analogous mechanisms that require repeated cycles of process extension, reading and integration of multiple directional signals, followed by stabilisation in a preferred direction, and renewed extension. We have characterised a C. elegans gene, unc-53, that appears to act cell autonomously in the migration and outgrowth of muscles, axons and excretory canals. Abrogation of unc-53 function disrupts anteroposterior outgrowth in those cells that normally express the gene. Conversely, overexpression of unc-53 in bodywall muscles leads to exaggerated outgrowth. UNC-53 is a novel protein conserved in vertebrates that contains putative SH3- and actin-binding sites. unc-53 interacts genetically with sem-5 and we demonstrated a direct interaction in vitro between UNC-53 and the SH2-SH3 adaptor protein SEM-5/GRB2. Thus, unc-53 is involved in longitudinal navigation and might act by linking extracellular guidance cues to the intracellular cytoskeleton.

scholarly journals Acentrosomal spindle assembly and stability in C. elegans oocytes requires a kinesin-12 non-motor microtubule interaction domain

2021 ◽  
Author(s):  
Ian Daniel Wolff ◽  
Jeremy Alden Hollis ◽  
Sarah Marie Wignall
Keyword(s):  
Adaptor Protein ◽  
Direct Interaction ◽  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Interaction Domain ◽  
Microtubule Binding ◽  
C Elegans ◽  
Insight Into

During the meiotic divisions in oocytes, microtubules are sorted and organized by motor proteins to generate a bipolar spindle in the absence of centrosomes. In most organisms, kinesin-5 family members crosslink and slide microtubules to generate outward force that promotes acentrosomal spindle bipolarity. However, the mechanistic basis for how other kinesin families act on acentrosomal spindles has not been explored. We investigated this question in C. elegans oocytes, where kinesin-5 is not required to generate outward force. Instead, the kinesin-12 family motor KLP-18 performs this function. KLP-18 acts with adaptor protein MESP-1 (meiotic spindle 1) to sort microtubule minus ends to the periphery of a microtubule array, where they coalesce into spindle poles. If either of these proteins is depleted, outward sorting of microtubules is lost and minus ends converge to form a monoaster. Here we use a combination of in vitro biochemical assays and in vivo mutant analysis to provide insight into the mechanism by which these proteins collaborate to promote acentrosomal spindle assembly. We identify a microtubule binding site on the C-terminal stalk of KLP-18 and demonstrate that a direct interaction between the KLP-18 stalk and MESP-1 activates non-motor microtubule binding. We also provide evidence that this C-terminal domain is required for KLP-18 activity during spindle assembly and show that KLP-18 is continuously required to maintain spindle bipolarity. This study thus provides new insight into the construction and maintenance of the oocyte acentrosomal spindle as well as into kinesin-12 mechanism and regulation.

scholarly journals Nebulin Interacts with CapZ and Regulates Thin Filament Architecture within the Z-Disc

2008 ◽  
Vol 19 (5) ◽  
pp. 1837-1847 ◽  
Author(s):  
Christopher T. Pappas ◽  
Nandini Bhattacharya ◽  
John A. Cooper ◽  
Carol C. Gregorio
Keyword(s):  
Actin Filaments ◽  
Thin Filament ◽  
Actin Polymerization ◽  
Striated Muscle ◽  
Solid Phase ◽  
Molecular Model ◽  
Direct Interaction ◽  
Tryptophan Fluorescence ◽  
Actin Binding

The barbed ends of actin filaments in striated muscle are anchored within the Z-disc and capped by CapZ; this protein blocks actin polymerization and depolymerization in vitro. The mature lengths of the thin filaments are likely specified by the giant “molecular ruler” nebulin, which spans the length of the thin filament. Here, we report that CapZ specifically interacts with the C terminus of nebulin (modules 160–164) in blot overlay, solid-phase binding, tryptophan fluorescence, and SPOTs membrane assays. Binding of nebulin modules 160–164 to CapZ does not affect the ability of CapZ to cap actin filaments in vitro, consistent with our observation that neither of the two C-terminal actin binding regions of CapZ is necessary for its interaction with nebulin. Knockdown of nebulin in chick skeletal myotubes using small interfering RNA results in a reduction of assembled CapZ, and, strikingly, a loss of the uniform alignment of the barbed ends of the actin filaments. These data suggest that nebulin restricts the position of thin filament barbed ends to the Z-disc via a direct interaction with CapZ. We propose a novel molecular model of Z-disc architecture in which nebulin interacts with CapZ from a thin filament of an adjacent sarcomere, thus providing a structural link between sarcomeres.

scholarly journals The Human FSGS-Causing ANLN R431C Mutation Induces Dysregulated PI3K/AKT/mTOR/Rac1 Signaling in Podocytes

2018 ◽  
Vol 29 (8) ◽  
pp. 2110-2122 ◽  
Author(s):  
Gentzon Hall ◽  
Brandon M. Lane ◽  
Kamal Khan ◽  
Igor Pediaditakis ◽  
Jianqiu Xiao ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Adaptor Protein ◽  
Pi3k Pathway ◽  
Actin Binding ◽  
Loss Of Function ◽  
Signaling Axis ◽  
Phosphoinositide 3 Kinase

BackgroundWe previously reported that mutations in the anillin (ANLN) gene cause familial forms of FSGS. ANLN is an F-actin binding protein that modulates podocyte cell motility and interacts with the phosphoinositide 3-kinase (PI3K) pathway through the slit diaphragm adaptor protein CD2-associated protein (CD2AP). However, it is unclear how the ANLN mutations cause the FSGS phenotype. We hypothesized that the R431C mutation exerts its pathogenic effects by uncoupling ANLN from CD2AP.MethodsWe conducted in vivo complementation assays in zebrafish to determine the effect of the previously identified missense ANLN variants, ANLNR431C and ANLNG618C during development. We also performed in vitro functional assays using human podocyte cell lines stably expressing wild-type ANLN (ANLNWT) or ANLNR431C.ResultsExperiments in anln-deficient zebrafish embryos showed a loss-of-function effect for each ANLN variant. In human podocyte lines, expression of ANLNR431C increased cell migration, proliferation, and apoptosis. Biochemical characterization of ANLNR431C-expressing podocytes revealed hyperactivation of the PI3K/AKT/mTOR/p70S6K/Rac1 signaling axis and activation of mTOR-driven endoplasmic reticulum stress in ANLNR431C-expressing podocytes. Inhibition of mTOR, GSK-3β, Rac1, or calcineurin ameliorated the effects of ANLNR431C. Additionally, inhibition of the calcineurin/NFAT pathway reduced the expression of endogenous ANLN and mTOR.ConclusionsThe ANLNR431C mutation causes multiple derangements in podocyte function through hyperactivation of PI3K/AKT/mTOR/p70S6K/Rac1 signaling. Our findings suggest that the benefits of calcineurin inhibition in FSGS may be due, in part, to the suppression of ANLN and mTOR. Moreover, these studies illustrate that rational therapeutic targets for familial FSGS can be identified through biochemical characterization of dysregulated podocyte phenotypes.

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 Repression of the interleukin-6 promoter by estrogen receptor is mediated by NF-kappa B and C/EBP beta.

1995 ◽  
Vol 15 (9) ◽  
pp. 4971-4979 ◽  
Author(s):  
B Stein ◽  
M X Yang
Keyword(s):  
Estrogen Receptor ◽  
Bone Resorption ◽  
Interleukin 6 ◽  
Binding Sites ◽  
Hormone Replacement ◽  
Direct Interaction ◽  
Nf Kappa B ◽  
Two Factors

Bone metabolism is regulated by a balance between bone resorption caused by osteoclasts and bone formation caused by osteoblasts. This balance is disturbed in postmenopausal women as a result of lower serum estrogen levels. Estrogen, which is used in hormone replacement therapy to prevent postmenopausal osteoporosis, downregulates expression of the interleukin 6 (IL-6) gene in osteoblasts and bone marrow stromal cells. IL-6 is directly involved in bone resorption by activating immature osteoclasts. We show here that NF-kappa B and C/EBP beta are important regulators of IL-6 gene expression in human osteoblasts. Importantly, the IL-6 promoter is inhibited by estrogen in the absence of a functional estrogen receptor (ER) binding site. This inhibition is mediated by the transcription factors NF-kappa B and C/EBP beta. Evidence is presented for a direct interaction between these two factors and ER. We characterized the protein sequence requirements for this association in vitro and in vivo. The physical and functional interaction depends in part on the DNA binding domain and region D of ER and on the Rel homology domain of NF-kappa B and the bZIP region of C/EBP beta. The cross-coupling between ER, NF-kappa B, and C/EBP beta also results in reduced activity of promoters with ER binding sites. We further show that the mechanism of IL-6 gene repression by estrogen is clearly different from that of activation of promoters with ER binding sites. Therefore, drugs that separate the transactivation and transrepression functions of ER will be very helpful for treatment of osteoporosis without causing undesirable side effects.

scholarly journals Loss of cargo binding in the human myosin VI deafness mutant (R1166X) leads to increased actin filament binding

2016 ◽  
Vol 473 (19) ◽  
pp. 3307-3319 ◽  
Author(s):  
Susan D. Arden ◽  
David A. Tumbarello ◽  
Tariq Butt ◽  
John Kendrick-Jones ◽  
Folma Buss
Keyword(s):  
Amino Acids ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Actin Binding ◽  
Binding Motif ◽  
Myosin Vi ◽  
Binding Ability ◽  
Activated State ◽  
Cargo Binding

Mutations in myosin VI have been associated with autosomal-recessive (DFNB37) and autosomal-dominant (DFNA22) deafness in humans. Here, we characterise an myosin VI nonsense mutation (R1166X) that was identified in a family with hereditary hearing loss in Pakistan. This mutation leads to the deletion of the C-terminal 120 amino acids of the myosin VI cargo-binding domain, which includes the WWY-binding motif for the adaptor proteins LMTK2, Tom1 as well as Dab2. Interestingly, compromising myosin VI vesicle-binding ability by expressing myosin VI with the R1166X mutation or with single point mutations in the adaptor-binding sites leads to increased F-actin binding of this myosin in vitro and in vivo. As our results highlight the importance of cargo attachment for regulating actin binding to the motor domain, we perform a detailed characterisation of adaptor protein binding and identify single amino acids within myosin VI required for binding to cargo adaptors. We not only show that the adaptor proteins can directly interact with the cargo-binding tail of myosin VI, but our in vitro studies also suggest that multiple adaptor proteins can bind simultaneously to non-overlapping sites in the myosin VI tail. In conclusion, our characterisation of the human myosin VI deafness mutant (R1166X) suggests that defects in cargo binding may leave myosin VI in a primed/activated state with an increased actin-binding ability.

A Pyk2–Vav1 complex is recruited to β3-adhesion sites to initiate Rho activation

2009 ◽  
Vol 420 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Chunlei Gao ◽  
Scott D. Blystone
Keyword(s):  
Cell Adhesion ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Fluorescent Protein ◽  
Rho Gtpase ◽  
Adaptor Protein ◽  
Direct Interaction ◽  
Nucleotide Exchange ◽  
Tyrosine Kinase 2

Integrin αvβ3-mediated adhesion of haemopoietic cells to vitronectin results in β3 tyrosine phosphorylation and Rho activation which is necessary for adhesion. Previously, we have shown that the RhoGEF (Rho guanine-nucleotide-exchange factor) Vav1 could associate indirectly with αvβ3 during leucocyte adhesion to vitronectin. In the present study, we have identified the non-receptor tyrosine kinase Pyk2 (proline-rich tyrosine kinase 2) as the adaptor protein that links Vav1 with αvβ3. The association of Pyk2 and Vav1 with β3 relies on the presence of Tyr747 in β3, the primary site of β3 phosphorylation. However, association of Pyk2 with Vav1 is independent of β3 tyrosine phosphorylation. Formation of a Pyk2–Vav1 complex occurs upon cell adhesion and Pro717 of Pyk2 plays a key role in Pyk2 interaction with Vav1. Utilizing purified recombinant proteins, we confirmed the direct interaction between Pyk2 and Vav1 In vitro. Cells transfected with GFP (green fluorescent protein)–Pyk2-P717A demonstrated severely suppressed cytoskeletal reorganization, impaired Vav1 recruitment, decreased Rho GTPase activation and loss of cell adhesion. Using siRNA (small interfering RNA) to specifically reduce Pyk2 levels in cells resulted in disrupted association between Vav1 and β3 and impaired cell adhesion. These results indicate that Pyk2 is a critical signalling molecule downstream of β3 integrin tyrosine phosphorylation and mediates Vav1 recruitment to accomplish actin reorganization necessary for adhesion.

scholarly journals Identification of the actin-binding domain of Ins(1,4,5)P3 3-kinase isoform B (IP3K-B)

2004 ◽  
Vol 382 (1) ◽  
pp. 353-362 ◽  
Author(s):  
Maria A. BREHM ◽  
Isabell SCHREIBER ◽  
Uwe BERTSCH ◽  
Albrecht WEGNER ◽  
Georg W. MAYR
Keyword(s):  
Endoplasmic Reticulum ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Direct Interaction ◽  
Actin Binding ◽  
Binding Domains ◽  
Isolated Segment ◽  
Green Fluorescent ◽  
Isoform B

Dewaste et al. [Dewaste, Moreau, De Smedt, Bex, De Smedt, Wuytaack, Missiaen and Erneux (2003) Biochem. J. 374, 41–49] showed that over-expressed EGFP (enhanced green fluorescent protein) fused to Ins(1,4,5)P3 3-kinase B (IP3K-B) co-localizes with the cytoskeleton, as well as with the endoplasmic reticulum and the plasma membrane. The domains responsible for these subcellular localizations are not yet identified. For the endogenous enzyme, we confirmed both actin and endoplasmic reticulum localization by employing a high affinity antibody against IP3K-B. F-actin targeting is exclusively dependent on the non-catalytic N-terminal region of IP3K-B. By expressing fragments of this N-terminal domain as EGFP-fusion proteins and inspecting transfected cells by confocal microscopy, we characterized a distinct 63-amino-acid domain comprising amino acids 108–170 of the enzyme which is responsible for F-actin targeting. A truncation of this fragment from both sides revealed that the full size of this segment is essential for this function. Deletion of this segment in a full-length over-expressed IP3K-B–EGFP-fusion protein completely abolished F-actin interaction. Direct interaction of this actin-binding segment with only F-actin, but not with G-actin, was observed in vitro using a bacterially expressed, affinity-purified GST (glutathione S-transferase)–Rattus norvegicus IP3K (aa 108–170) fusion protein. Helix-breaking mutations within this isolated segment abolished the F-actin binding properties both in vitro and when over-expressed in cells, indicating that an intact secondary structure is essential for actin targeting. The segment shows sequence similarities to the actin-binding region in IP3K-A, but no similarity to other actin-binding domains.

scholarly journals A Genetic Dissection of Aip1p's Interactions Leads to a Model for Aip1p-Cofilin Cooperative Activities

2006 ◽  
Vol 17 (4) ◽  
pp. 1971-1984 ◽  
Author(s):  
Michael G. Clark ◽  
Joseph Teply ◽  
Brian K. Haarer ◽  
Susan C. Viggiano ◽  
David Sept ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Actin Filaments ◽  
Random Mutagenesis ◽  
Site Directed Mutagenesis ◽  
Actin Binding ◽  
Interacting Protein ◽  
Actin Binding Site

Actin interacting protein 1 (Aip1p) and cofilin cooperate to disassemble actin filaments in vitro and are thought to promote rapid turnover of actin networks in vivo. The precise method by which Aip1p participates in these activities has not been defined, although severing and barbed-end capping of actin filaments have been proposed. To better describe the mechanisms and biological consequences of Aip1p activities, we undertook an extensive mutagenesis of AIP1 aimed at disrupting and mapping Aip1p interactions. Site-directed mutagenesis suggested that Aip1p has two actin binding sites, the primary actin binding site lies on the edge of its N-terminal β-propeller and a secondary actin binding site lies in a comparable location on its C-terminal β-propeller. Random mutagenesis followed by screening for separation of function mutants led to the identification of several mutants specifically defective for interacting with cofilin but still able to interact with actin. These mutants suggested that cofilin binds across the cleft between the two propeller domains, leaving the actin binding sites exposed and flanking the cofilin binding site. Biochemical, genetic, and cell biological analyses confirmed that the actin binding- and cofilin binding-specific mutants are functionally defective, whereas the genetic analyses further suggested a role for Aip1p in an early, internalization step of endocytosis. A complementary, unbiased molecular modeling approach was used to derive putative structures for the Aip1p-cofilin complex, the most stable of which is completely consistent with the mutagenesis data. We theorize that Aip1p-severing activity may involve simultaneous binding to two actin subunits with cofilin wedged between the two actin binding sites of the N- and C-terminal propeller domains.

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