scholarly journals Giant ankyrin-B suppresses stochastic collateral axon branching through direct interaction with microtubules

2020 ◽  
Vol 219 (8) ◽  
Author(s):  
Keyu Chen ◽  
Rui Yang ◽  
Yubing Li ◽  
Jin Chuan Zhou ◽  
Mingjie Zhang
Keyword(s):  
Tandem Repeats ◽  
Direct Interaction ◽  
Binding Activity ◽  
Axon Collateral ◽  
Axon Branching ◽  
Microtubule Binding ◽  
Ankyrin G ◽  
Binding Avidity ◽  
Ankyrin B

Giant ankyrin-B (gAnkB) is a 440-kD neurospecific ankyrin-B isoform and a high-confidence target for autism mutations. gAnkB suppresses axon branching through coordination of cortical microtubules, and autism-related mutation of gAnkB results in ectopic neuronal connectivity. We identified a bipartite motif from gAnkB, which bundles and avidly binds to microtubules in vitro. This motif is composed of a module of 15 tandem repeats followed by a short, conserved fragment also found in giant ankyrin-G (BG-box). Combination of these two parts synergistically increases microtubule-binding avidity. Transfection of astrocytes (which lack gAnkB) with WT gAnkB resulted in prominent bundling of microtubules, which did not occur with mutant gAnkB with impaired microtubule-binding activity. Similarly, rescue of gAnkB-deficient neurons with WT gAnkB suppressed axonal branching and invasion of EB3-tagged microtubules into filopodia, which did not occur with the same mutant gAnkB. Together, these findings demonstrate that gAnkB suppresses axon collateral branching and prevents microtubule invasion of nascent axon branches through direct interaction with microtubules.

scholarly journals A novel Netrin-1–sensitive mechanism promotes local SNARE-mediated exocytosis during axon branching

2014 ◽  
Vol 205 (2) ◽  
pp. 217-232 ◽  
Author(s):  
Cortney C. Winkle ◽  
Leslie M. McClain ◽  
Juli G. Valtschanoff ◽  
Charles S. Park ◽  
Christopher Maglione ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cortical Neurons ◽  
Direct Interaction ◽  
Vesicle Fusion ◽  
Dependent Manner ◽  
Axon Branching ◽  
Spatial Regulation ◽  
Novel Model

Developmental axon branching dramatically increases synaptic capacity and neuronal surface area. Netrin-1 promotes branching and synaptogenesis, but the mechanism by which Netrin-1 stimulates plasma membrane expansion is unknown. We demonstrate that SNARE-mediated exocytosis is a prerequisite for axon branching and identify the E3 ubiquitin ligase TRIM9 as a critical catalytic link between Netrin-1 and exocytic SNARE machinery in murine cortical neurons. TRIM9 ligase activity promotes SNARE-mediated vesicle fusion and axon branching in a Netrin-dependent manner. We identified a direct interaction between TRIM9 and the Netrin-1 receptor DCC as well as a Netrin-1–sensitive interaction between TRIM9 and the SNARE component SNAP25. The interaction with SNAP25 negatively regulates SNARE-mediated exocytosis and axon branching in the absence of Netrin-1. Deletion of TRIM9 elevated exocytosis in vitro and increased axon branching in vitro and in vivo. Our data provide a novel model for the spatial regulation of axon branching by Netrin-1, in which localized plasma membrane expansion occurs via TRIM9-dependent regulation of SNARE-mediated vesicle fusion.

scholarly journals Targeting and Stability of Na/Ca Exchanger 1 in Cardiomyocytes Requires Direct Interaction with the Membrane Adaptor Ankyrin-B

2006 ◽  
Vol 282 (7) ◽  
pp. 4875-4883 ◽  
Author(s):  
Shane R. Cunha ◽  
Naina Bhasin ◽  
Peter J. Mohler
Keyword(s):  
Molecular Mechanisms ◽  
Cardiac Tissue ◽  
Direct Interaction ◽  
Binding Activity ◽  
Membrane Domains ◽  
Excitable Cells ◽  
Expression Activity ◽  
Cellular Pathways ◽  
Ankyrin B ◽  
Calcium Extrusion

Na/Ca exchanger activity is important for calcium extrusion from the cardiomyocyte cytosol during repolarization. Animal models exhibiting altered Na/Ca exchanger expression display abnormal cardiac phenotypes. In humans, elevated Na/Ca exchanger expression/activity is linked with pathophysiological conditions including arrhythmia and heart failure. Whereas the molecular mechanisms underlying Na/Ca exchanger biophysical properties are widely studied and generally well characterized, the cellular pathways and molecular partners underlying the specialized membrane localization of Na/Ca exchanger in cardiac tissue are essentially unknown. In this report, we present the first direct evidence for a protein pathway required for Na/Ca exchanger localization and stability in primary cardiomyocytes. We define the minimal structural requirements on ankyrin-B for direct Na/Ca exchanger interactions. Moreover, using ankyrin-B mutants that lack Na/Ca exchanger binding activity, and primary cardiomyocytes with reduced ankyrin-B expression, we demonstrate that direct interaction with the membrane adaptor ankyrin-B is required for the localization and post-translational stability of Na/Ca exchanger 1 in neonatal mouse cardiomyocytes. These results raise exciting new questions regarding potentially dynamic roles for ankyrin proteins in the biogenesis and maintenance of specialized membrane domains in excitable cells.

scholarly journals A membrane-bound form of glutamate dehydrogenase possesses an ATP-dependent high-affinity microtubule-binding activity

1993 ◽  
Vol 295 (2) ◽  
pp. 447-455 ◽  
Author(s):  
F Rajas ◽  
B Rousset
Keyword(s):  
Membrane Protein ◽  
Glutamate Dehydrogenase ◽  
Rat Liver ◽  
Binding Activity ◽  
Binding Studies ◽  
Microtubule Binding ◽  
Phase Partitioning ◽  
Membrane Bound ◽  
Commercial Sources

We previously identified a 50 kDa membrane protein which bound to in vitro assembled microtubules [Mithieux and Rousset (1989) J. Biol. Chem. 264, 4664-4668]. This protein exhibited the expected properties for mediating the ATP-dependent association of vesicles with microtubules [Mithieux, Audebet and Rousset (1988) Biochim. Biophys. Acta 969, 121-130]. The 50 kDa membrane protein (MP50), initially extracted in very low amount from isolated pig thyroid lysosomes/endosomes, has now been purified from membrane preparations of crude vesicle fractions from pig liver and brain. MP50 was isolated from detergent-solubilized membrane protein by affinity chromatography on immobilized ATP; 3-5 mg of MP50 was obtained from 100 g of liver tissue. Phase partitioning in Triton X-114 indicated that MP50 is a peripheral membrane protein. Radioiodinated liver MP50 bound to microtubules assembled in vitro. The binding was inhibited by ATP (Ki = 0.76 mM) and displaced by unlabelled liver or brain MP50. Equilibrium binding studies yielded KD values of 1.8 x 10(-7) M. By N-terminal amino acid sequence analysis, MP50 was identified as glutamate dehydrogenase (GDH), by comparison of V8 protease peptide maps of MP50 with purified liver GDH. Liver MP50 exhibited a low GDH activity; 4-5 units/mg compared with 18 and 34 units/mg for purified bovine and rat liver GDH respectively. Bovine and rat liver GDH yielded six spots from pI 5.7 to 7.2 when analysed by two-dimensional electrophoresis; in contrast, MP50 gave one main spot (corresponding to spot 2 of liver GDH) with a pI of approx. 6.5. Soluble liver GDH from commercial sources exhibited a very low or no microtubule-binding activity. In conclusion, we have found a membrane-bound form of GDH capable of specific and nucleotide-sensitive interaction with microtubules. Our data suggest that GDH isoproteins, the number of which has been undervalued up to now, could have cellular functions other than that of an enzyme.

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 Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Blake A Creighton ◽  
Simone Afriyie ◽  
Deepa Ajit ◽  
Cristine R Casingal ◽  
Kayleigh M Voos ◽  
...  
Keyword(s):  
Brain Connectivity ◽  
Autism Spectrum ◽  
Axon Collateral ◽  
Collateral Branch ◽  
Cellular Assays ◽  
Behavioral Abnormalities ◽  
Neuropilin 1 ◽  
Branch Formation ◽  
Ankyrin B

Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.

Abstract 1073: Cardiac Voltage-gated Nav channel Na v 1.5 Requires An AnkyrinG-dependent Pathway For Targeting in Cardiomyocytes

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
John S Lowe ◽  
Oleg Palygin ◽  
Patrick Wright ◽  
Erwin Shibata ◽  
Peter Mohler
Keyword(s):  
Ion Channels ◽  
Site Directed Mutagenesis ◽  
Loss Of Function ◽  
Excitable Cells ◽  
Membrane Expression ◽  
Voltage Gated ◽  
Ankyrin G ◽  
Ankyrin B ◽  
Dependent Pathway

Membrane localization of ion channels is very important for normal function in excitable cells. In heart, voltage-gated Na + channels are necessary for the rapid upstroke of the cardiomyocyte action potential, and variants in SCN5A (encodes Na v 1.5) are associated with fatal arrhythmias. We have identified ankyrin family proteins as critical components for normal ion channel and transporter targeting in cardiomyocytes. Humans with ANK2 (encodes ankyrin-B) loss of function variants display abnormal cardiac phenotypes and risk for sudden cardiac death. Mice that lack ankyrin-B expression display a similar phenotype. Our most recent results demonstrate that a second ankyrin gene product, ankyrin-G (encoded by ANK 3) is critical for targeting Na v 1.5 to specific cardiomyocyte membrane domains. We assessed the hypothesis that Na v 1.5 membrane expression and localization is controlled by an ankyrin-G-dependent pathway and disruption of ankyrin-G/Na v 1.5 interactions lead to human cardiac disease in this study. We used a combination of techniques including biochemistry, confocal microscopy, lentiviral expression, and electrophysiology to evaluate the functional relationship between ankyrin-G and Na v 1.5. We defined the structural elements on ankyrin-G and Na v 1.5 for their interaction using site-directed mutagenesis and in vitro binding assays. Lentiviral expression of shRNA targeted to rat 190 kD ankyrin-G effectively reduced the expression of ankyrin-G with a concomitant reduction of Na v 1.5 in immunofluorescence and immunoblot assays. Further-more, primary cardiomyocytes with reduced ankyrin-G expression have a significant reduction in Na + current density with no evident biophysical effects on Ca 2+ current or inactivation-gating of Na v 1.5 These results confirm the importance of ankyrin polypeptides for normal cardiac function and shed new light on the importance of intracellular trafficking pathways for the delivery and stability of critical ion channels and transporters in excitable cells.

scholarly journals Dynein, Dynactin, and Kinesin II's Interaction with Microtubules Is Regulated during Bidirectional Organelle Transport

2000 ◽  
Vol 151 (1) ◽  
pp. 155-166 ◽  
Author(s):  
Eric L. Reese ◽  
Leah T. Haimo
Keyword(s):  
Pigment Granule ◽  
Cytoplasmic Dynein ◽  
Binding Activity ◽  
The State ◽  
Organelle Transport ◽  
Microtubule Binding ◽  
Pigment Granules ◽  
Microtubule Motors

The microtubule motors, cytoplasmic dynein and kinesin II, drive pigmented organelles in opposite directions in Xenopus melanophores, but the mechanism by which these or other motors are regulated to control the direction of organelle transport has not been previously elucidated. We find that cytoplasmic dynein, dynactin, and kinesin II remain on pigment granules during aggregation and dispersion in melanophores, indicating that control of direction is not mediated by a cyclic association of motors with these organelles. However, the ability of dynein, dynactin, and kinesin II to bind to microtubules varies as a function of the state of aggregation or dispersion of the pigment in the cells from which these molecules are isolated. Dynein and dynactin bind to microtubules when obtained from cells with aggregated pigment, whereas kinesin II binds to microtubules when obtained from cells with dispersed pigment. Moreover, the microtubule binding activity of these motors/dynactin can be reversed in vitro by the kinases and phosphatase that regulate the direction of pigment granule transport in vivo. These findings suggest that phosphorylation controls the direction of pigment granule transport by altering the ability of dynein, dynactin, and kinesin II to interact with microtubules.

scholarly journals Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor Sema 3A

2021 ◽  
Author(s):  
Damaris N Lorenzo ◽  
Blake A Creighton ◽  
Deepa Ajit ◽  
Simone Afriyie ◽  
Julia C Bay
Keyword(s):  
Brain Connectivity ◽  
Autism Spectrum ◽  
Axonal Guidance ◽  
Axon Collateral ◽  
Collateral Branch ◽  
Behavioral Abnormalities ◽  
Neuropilin 1 ◽  
Branch Formation ◽  
Ankyrin B

Variants in the high confident autism spectrum disorder gene ANK2 target both ubiquitously expressed 220-kDa ankyrin- B and neurospecific 440-kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3A (Sema 3A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal guidance and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.

scholarly journals Ebola Virus VP30 Is an RNA Binding Protein

2007 ◽  
Vol 81 (17) ◽  
pp. 8967-8976 ◽  
Author(s):  
Sinu P. John ◽  
Tan Wang ◽  
Scott Steffen ◽  
Sonia Longhi ◽  
Connie S. Schmaljohn ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Ebola Virus ◽  
Rna Binding ◽  
Transcriptional Start Site ◽  
Direct Interaction ◽  
Binding Activity ◽  
Stem Loop ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions

ABSTRACT The Ebola virus (EBOV) genome encodes for several proteins that are necessary and sufficient for replication and transcription of the viral RNAs in vitro; NP, VP30, VP35, and L. VP30 acts in trans with an RNA secondary structure upstream of the first transcriptional start site to modulate transcription. Using a bioinformatics approach, we identified a region within the N terminus of VP30 with sequence features that typify intrinsically disordered regions and a putative RNA binding site. To experimentally assess the ability of VP30 to directly interact with the viral RNA, we purified recombinant EBOV VP30 to >90% homogeneity and assessed RNA binding by UV cross-linking and filter-binding assays. VP30 is a strongly acidophilic protein; RNA binding became stronger as pH was decreased. Zn2+, but not Mg2+, enhanced activity. Enhancement of transcription by VP30 requires a RNA stem-loop located within nucleotides 54 to 80 of the leader region. VP30 showed low binding affinity to the predicted stem-loop alone or to double-stranded RNA but showed a good binding affinity for the stem-loop when placed in the context of upstream and downstream sequences. To map the region responsible for interacting with RNA, we constructed, purified, and assayed a series of N-terminal deletion mutations of VP30 for RNA binding. The key amino acids supporting RNA binding activity map to residues 26 to 40, a region rich in arginine. Thus, we show for the first time the direct interaction of EBOV VP30 with RNA and the importance of the N-terminal region for binding RNA.

scholarly journals The Helicobacter pylori HspR-Modulator CbpA Is a Multifunctional Heat-Shock Protein

2020 ◽  
Vol 8 (2) ◽  
pp. 251
Author(s):  
Simona Pepe ◽  
Vincenzo Scarlato ◽  
Davide Roncarati
Keyword(s):  
Helicobacter Pylori ◽  
Heat Shock ◽  
Functional Characterization ◽  
Direct Interaction ◽  
Binding Activity ◽  
Regulatory Role ◽  
Heat Shock Gene ◽  
Dna Binding Activity ◽  
H Pylori

The medically important human pathogen Helicobacter pylori relies on a collection of highly conserved heat-shock and chaperone proteins to preserve the integrity of cellular polypeptides and to control their homeostasis in response to external stress and changing environmental conditions. Among this set of chaperones, the CbpA protein has been shown to play a regulatory role in heat-shock gene regulation by directly interacting with the master stress-responsive repressor HspR. Apart from this regulatory role, little is known so far about CbpA functional activities. Using biochemistry and molecular biology approaches, we have started the in vitro functional characterization of H. pylori CbpA. Specifically, we show that CbpA is a multifunctional protein, being able to bind DNA and to stimulate the ATPase activity of the major chaperone DnaK. In addition, we report a preliminary observation suggesting that CbpA DNA-binding activity can be affected by the direct interaction with the heat-shock master repressor HspR, supporting the hypothesis of a reciprocal crosstalk between these two proteins. Thus, our work defines novel functions for H. pylori CbpA and stimulates further studies aimed at the comprehension of the complex regulatory interplay among chaperones and heat-shock transcriptional regulators.

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