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Article ; Online: β-III-spectrin N-terminus is required for high-affinity actin binding and SCA5 neurotoxicity.

Denha, Sarah A / Atang, Alexandra E / Hays, Thomas S / Avery, Adam W

2022 Volume 12, Issue 1, Page(s) 1726

Abstract: Recent structural studies of β-III-spectrin and related cytoskeletal proteins revealed N-terminal sequences that directly bind actin. These sequences are variable in structure, and immediately precede a conserved actin-binding domain composed of tandem ... ...

Abstract	Recent structural studies of β-III-spectrin and related cytoskeletal proteins revealed N-terminal sequences that directly bind actin. These sequences are variable in structure, and immediately precede a conserved actin-binding domain composed of tandem calponin homology domains (CH1 and CH2). Here we investigated in Drosophila the significance of the β-spectrin N-terminus, and explored its functional interaction with a CH2-localized L253P mutation that underlies the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5). We report that pan-neuronal expression of an N-terminally truncated β-spectrin fails to rescue lethality resulting from a β-spectrin loss-of-function allele, indicating that the N-terminus is essential to β-spectrin function in vivo. Significantly, N-terminal truncation rescues neurotoxicity and defects in dendritic arborization caused by L253P. In vitro studies show that N-terminal truncation eliminates L253P-induced high-affinity actin binding, providing a mechanistic basis for rescue. These data suggest that N-terminal sequences may be useful therapeutic targets for small molecule modulation of the aberrant actin binding associated with SCA5 β-spectrin and spectrin-related disease proteins.
MeSH term(s)	Actins/metabolism ; Animals ; Animals, Genetically Modified ; Binding Sites ; Drosophila Proteins/genetics ; Drosophila Proteins/metabolism ; Drosophila melanogaster/genetics ; Drosophila melanogaster/metabolism ; Female ; Male ; Mutation ; Neuronal Plasticity ; Neurons/metabolism ; Neurons/pathology ; Protein Binding ; Protein Interaction Domains and Motifs ; Spectrin/genetics ; Spectrin/metabolism ; Spinocerebellar Ataxias/genetics ; Spinocerebellar Ataxias/metabolism ; Spinocerebellar Ataxias/pathology
Chemical Substances	Actins ; Drosophila Proteins ; Spectrin (12634-43-4)
Language	English
Publishing date	2022-02-02
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-022-05762-2
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Article ; Online: Conduction-Dominated Cryomesh for Organism Vitrification.

Guo, Zongqi / Zuchowicz, Nikolas / Bouwmeester, Jessica / Joshi, Amey S / Neisch, Amanda L / Smith, Kieran / Daly, Jonathan / Etheridge, Michael L / Finger, Erik B / Kodandaramaiah, Suhasa B / Hays, Thomas S / Hagedorn, Mary / Bischof, John C

Advanced science (Weinheim, Baden-Wurttemberg, Germany)

2023 Volume 11, Issue 3, Page(s) e2303317

Abstract: Vitrification-based cryopreservation is a promising approach to achieving long-term storage of biological systems for maintaining biodiversity, healthcare, and sustainable food production. Using the "cryomesh" system achieves rapid cooling and rewarming ... ...

Abstract	Vitrification-based cryopreservation is a promising approach to achieving long-term storage of biological systems for maintaining biodiversity, healthcare, and sustainable food production. Using the "cryomesh" system achieves rapid cooling and rewarming of biomaterials, but further improvement in cooling rates is needed to increase biosystem viability and the ability to cryopreserve new biosystems. Improved cooling rates and viability are possible by enabling conductive cooling through cryomesh. Conduction-dominated cryomesh improves cooling rates from twofold to tenfold (i.e., 0.24 to 1.2 × 10
MeSH term(s)	Animals ; Vitrification ; Zebrafish ; Cryopreservation ; Cold Temperature ; Nitrogen
Chemical Substances	Nitrogen (N762921K75)
Language	English
Publishing date	2023-11-28
Publishing country	Germany
Document type	Journal Article
ZDB-ID	2808093-2
ISSN	2198-3844 ; 2198-3844
ISSN (online)	2198-3844
ISSN	2198-3844
DOI	10.1002/advs.202303317
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Article ; Online: β-III-spectrin spinocerebellar ataxia type 5 mutation reveals a dominant cytoskeletal mechanism that underlies dendritic arborization.

Avery, Adam W / Thomas, David D / Hays, Thomas S

Proceedings of the National Academy of Sciences of the United States of America

2017 Volume 114, Issue 44, Page(s) E9376–E9385

Abstract: A spinocerebellar ataxia type 5 (SCA5) L253P mutation in the actin-binding domain (ABD) of β-III-spectrin causes high-affinity actin binding and decreased thermal stability in vitro. Here we show in mammalian cells, at physiological temperature, that the ...

Abstract	A spinocerebellar ataxia type 5 (SCA5) L253P mutation in the actin-binding domain (ABD) of β-III-spectrin causes high-affinity actin binding and decreased thermal stability in vitro. Here we show in mammalian cells, at physiological temperature, that the mutant ABD retains high-affinity actin binding. Significantly, we provide evidence that the mutation alters the mobility and recruitment of β-III-spectrin in mammalian cells, pointing to a potential disease mechanism. To explore this mechanism, we developed a
MeSH term(s)	Animals ; Ankyrins/genetics ; Cells, Cultured ; Cytoskeleton/genetics ; Dendrites/genetics ; Drosophila/genetics ; Drosophila/physiology ; HEK293 Cells ; Humans ; Mutation/genetics ; Neuronal Plasticity/genetics ; Neurons/physiology ; Protein Binding/genetics ; Purkinje Cells/physiology ; Spectrin/genetics ; Spinocerebellar Ataxias/genetics
Chemical Substances	Ankyrins ; Spectrin (12634-43-4)
Language	English
Publishing date	2017-10-16
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1707108114
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Article ; Online: A STRIPAK complex mediates axonal transport of autophagosomes and dense core vesicles through PP2A regulation.

Neisch, Amanda L / Neufeld, Thomas P / Hays, Thomas S

The Journal of cell biology

2017 Volume 216, Issue 2, Page(s) 441–461

Abstract: Autophagy plays an essential role in the cellular homeostasis of neurons, facilitating the clearance of cellular debris. This clearance process is orchestrated through the assembly, transport, and fusion of autophagosomes with lysosomes for degradation. ... ...

Abstract	Autophagy plays an essential role in the cellular homeostasis of neurons, facilitating the clearance of cellular debris. This clearance process is orchestrated through the assembly, transport, and fusion of autophagosomes with lysosomes for degradation. The motor protein dynein drives autophagosome motility from distal sites of assembly to sites of lysosomal fusion. In this study, we identify the scaffold protein CKA (connector of kinase to AP-1) as essential for autophagosome transport in neurons. Together with other core components of the striatin-interacting phosphatase and kinase (STRIPAK) complex, we show that CKA associates with dynein and directly binds Atg8a, an autophagosomal protein. CKA is a regulatory subunit of PP2A, a component of the STRIPAK complex. We propose that the STRIPAK complex modulates dynein activity. Consistent with this hypothesis, we provide evidence that CKA facilitates axonal transport of dense core vesicles and autophagosomes in a PP2A-dependent fashion. In addition, CKA-deficient flies exhibit PP2A-dependent motor coordination defects. CKA function within the STRIPAK complex is crucial to prevent transport defects that may contribute to neurodegeneration.
MeSH term(s)	Adaptor Proteins, Signal Transducing/genetics ; Adaptor Proteins, Signal Transducing/metabolism ; Animals ; Animals, Genetically Modified ; Autophagosomes/enzymology ; Axonal Transport ; Axons/enzymology ; Cell Line ; Drosophila Proteins/genetics ; Drosophila Proteins/metabolism ; Drosophila melanogaster/enzymology ; Drosophila melanogaster/genetics ; Dyneins/genetics ; Dyneins/metabolism ; Genotype ; Microscopy, Fluorescence ; Multiprotein Complexes/genetics ; Multiprotein Complexes/metabolism ; Mutation ; Nerve Tissue Proteins/genetics ; Nerve Tissue Proteins/metabolism ; Phenotype ; Presynaptic Terminals/metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Phosphatase 2/genetics ; Protein Phosphatase 2/metabolism ; RNA Interference ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/metabolism ; Secretory Vesicles/enzymology ; Signal Transduction ; Transfection
Chemical Substances	Adaptor Proteins, Signal Transducing ; Atg8a protein, Drosophila ; Cka protein, Drosophila ; Drosophila Proteins ; Mob4 protein, Drosophila ; Multiprotein Complexes ; Nerve Tissue Proteins ; Recombinant Fusion Proteins ; mts protein, Drosophila ; Protein Phosphatase 2 (EC 3.1.3.16) ; Dyneins (EC 3.6.4.2)
Language	English
Publishing date	2017-01-18
Publishing country	United States
Document type	Journal Article ; Video-Audio Media
ZDB-ID	218154-x
ISSN	1540-8140 ; 0021-9525
ISSN (online)	1540-8140
ISSN	0021-9525
DOI	10.1083/jcb.201606082
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Article ; Online: Early-phase drug discovery of β-III-spectrin actin-binding modulators for treatment of spinocerebellar ataxia type 5.

Guhathakurta, Piyali / Rebbeck, Robyn T / Denha, Sarah A / Keller, Amanda R / Carter, Anna L / Atang, Alexandra E / Svensson, Bengt / Thomas, David D / Hays, Thomas S / Avery, Adam W

The Journal of biological chemistry

2023 Volume 299, Issue 3, Page(s) 102956

Abstract: β-III-Spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 L253P mutation in the cytoskeletal protein β-III- ... ...

Abstract	β-III-Spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje cells and is required for dendritic arborization and signaling. A spinocerebellar ataxia type 5 L253P mutation in the cytoskeletal protein β-III-spectrin causes high-affinity actin binding. Previously we reported a cell-based fluorescence assay for identification of small-molecule actin-binding modulators of the L253P mutant β-III-spectrin. Here we describe a complementary, in vitro, fluorescence resonance energy transfer (FRET) assay that uses purified L253P β-III-spectrin actin-binding domain (ABD) and F-actin. To validate the assay for high-throughput compatibility, we first confirmed that our 50% FRET signal was responsive to swinholide A, an actin-severing compound, and that this yielded excellent assay quality with a Z' value > 0.77. Second, we screened a 2684-compound library of US Food and Drug Administration-approved drugs. Importantly, the screening identified numerous compounds that decreased FRET between fluorescently labeled L253P ABD and F-actin. The activity and target of multiple Hit compounds were confirmed in orthologous cosedimentation actin-binding assays. Through future medicinal chemistry, the Hit compounds can potentially be developed into a spinocerebellar ataxia type 5-specific therapeutic. Furthermore, our validated FRET-based in vitro high-throughput screening platform is poised for screening large compound libraries for β-III-spectrin ABD modulators.
MeSH term(s)	Humans ; Actins/genetics ; Actins/metabolism ; Drug Discovery ; Neurons/metabolism ; Spectrin/metabolism ; Spinocerebellar Ataxias/drug therapy ; Spinocerebellar Ataxias/genetics ; Spinocerebellar Ataxias/metabolism
Chemical Substances	Actins ; Spectrin (12634-43-4) ; SPTBN2 protein, human
Language	English
Publishing date	2023-01-31
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1016/j.jbc.2023.102956
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Article: Kinesin-5 mediated chromosome congression in insect spindles.

Tubman, Emily / He, Yungui / Hays, Thomas S / Odde, David J

Cellular and molecular bioengineering

2017 Volume 11, Issue 1, Page(s) 25–36

Abstract: Introduction: The microtubule motor protein kinesin-5 is well known to establish the bipolar spindle by outward sliding of antiparallel interpolar microtubules. In yeast, kinesin-5 also facilitates chromosome alignment "congression" at the spindle ... ...

Abstract	Introduction: The microtubule motor protein kinesin-5 is well known to establish the bipolar spindle by outward sliding of antiparallel interpolar microtubules. In yeast, kinesin-5 also facilitates chromosome alignment "congression" at the spindle equator by preferentially depolymerizing long kinetochore microtubules (kMTs). The motor protein kinesin-8 has also been linked to chromosome congression. Therefore, we sought to determine whether kinesin-5 or kinesin-8 facilitates chromosome congression in insect spindles. Methods: RNAi of the kinesin-5 Klp61F and kinesin-8 Klp67A were performed separately in Results: RNAi of Klp61F with a weak Klp61F knockdown resulted in longer kMTs and less congressed kinetochores compared to control over a range of conditions, consistent with kinesin-5 length-dependent depolymerase activity. RNAi of the kinesin-8 Klp67A revealed that kMTs relative to the spindle lengths were not longer compared to control, but rather that the spindles were longer, indicating that Klp67A acts preferentially as a length-dependent depolymerase on interpolar microtubules without significantly affecting kMT length and chromosome congression. Conclusions: This study demonstrates that in addition to establishing the bipolar spindle, kinesin-5 regulates kMT length to facilitate chromosome congression in insect spindles. It expands on previous yeast studies, and it expands the role of kinesin-5 to include kMT assembly regulation in eukaryotic mitosis.
Language	English
Publishing date	2017-08-21
Publishing country	United States
Document type	Journal Article
ZDB-ID	2416037-4
ISSN	1865-5033 ; 1865-5025
ISSN (online)	1865-5033
ISSN	1865-5025
DOI	10.1007/s12195-017-0500-0
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Article ; Online: Novel drug discovery platform for spinocerebellar ataxia, using fluorescence technology targeting β-III-spectrin.

Rebbeck, Robyn T / Andrick, Anna K / Denha, Sarah A / Svensson, Bengt / Guhathakurta, Piyali / Thomas, David D / Hays, Thomas S / Avery, Adam W

The Journal of biological chemistry

2020 Volume 296, Page(s) 100215

Abstract: Numerous diseases are linked to mutations in the actin-binding domains (ABDs) of conserved cytoskeletal proteins, including β-III-spectrin, α-actinin, filamin, and dystrophin. A β-III-spectrin ABD mutation (L253P) linked to spinocerebellar ataxia type 5 ( ...

Abstract	Numerous diseases are linked to mutations in the actin-binding domains (ABDs) of conserved cytoskeletal proteins, including β-III-spectrin, α-actinin, filamin, and dystrophin. A β-III-spectrin ABD mutation (L253P) linked to spinocerebellar ataxia type 5 (SCA5) causes a dramatic increase in actin binding. Reducing actin binding of L253P is thus a potential therapeutic approach for SCA5 pathogenesis. Here, we validate a high-throughput screening (HTS) assay to discover potential disrupters of the interaction between the mutant β-III-spectrin ABD and actin in live cells. This assay monitors FRET between fluorescent proteins fused to the mutant ABD and the actin-binding peptide Lifeact, in HEK293-6E cells. Using a specific and high-affinity actin-binding tool compound, swinholide A, we demonstrate HTS compatibility with an excellent Z'-factor of 0.67 ± 0.03. Screening a library of 1280 pharmacologically active compounds in 1536-well plates to determine assay robustness, we demonstrate high reproducibility across plates and across days. We identified nine Hits that reduced FRET between Lifeact and ABD. Four of those Hits were found to reduce Lifeact cosedimentation with actin, thus establishing the potential of our assay for detection of actin-binding modulators. Concurrent to our primary FRET assay, we also developed a high-throughput compatible counter screen to remove undesirable FRET Hits. Using the FRET Hits, we show that our counter screen is sensitive to undesirable compounds that cause cell toxicity or ABD aggregation. Overall, our FRET-based HTS platform sets the stage to screen large compound libraries for modulators of β-III-spectrin, or disease-linked spectrin-related proteins, for therapeutic development.
MeSH term(s)	Actins/chemistry ; Actins/genetics ; Actins/metabolism ; Binding Sites/drug effects ; Fluorescence Resonance Energy Transfer ; Gene Expression ; Green Fluorescent Proteins/genetics ; Green Fluorescent Proteins/metabolism ; HEK293 Cells ; High-Throughput Screening Assays ; Humans ; Kinetics ; Luminescent Proteins/genetics ; Luminescent Proteins/metabolism ; Marine Toxins/pharmacology ; Models, Biological ; Models, Molecular ; Mutation ; Neuroprotective Agents/pharmacology ; Protein Binding/drug effects ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Recombinant Fusion Proteins/chemistry ; Recombinant Fusion Proteins/genetics ; Recombinant Fusion Proteins/metabolism ; Reproducibility of Results ; Spectrin/chemistry ; Spectrin/genetics ; Spectrin/metabolism ; Spinocerebellar Ataxias/genetics ; Spinocerebellar Ataxias/metabolism ; Spinocerebellar Ataxias/pathology ; Red Fluorescent Protein
Chemical Substances	Actins ; Luminescent Proteins ; Marine Toxins ; Neuroprotective Agents ; Recombinant Fusion Proteins ; SPTBN2 protein, human ; Spectrin (12634-43-4) ; Green Fluorescent Proteins (147336-22-9) ; swinholide A (95927-67-6)
Language	English
Publishing date	2020-12-24
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.RA120.015417
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Article ; Online: A human β-III-spectrin spinocerebellar ataxia type 5 mutation causes high-affinity F-actin binding.

Avery, Adam W / Crain, Jonathan / Thomas, David D / Hays, Thomas S

Scientific reports

2016 Volume 6, Page(s) 21375

Abstract: Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P ...

Abstract	Spinocerebellar ataxia type 5 (SCA5) is a human neurodegenerative disease that stems from mutations in the SPTBN2 gene encoding the protein β-III-spectrin. Here we investigated the molecular consequence of a SCA5 missense mutation that results in a L253P substitution in the actin-binding domain (ABD) of β-III-spectrin. We report that the L253P substitution in the isolated β-III-spectrin ABD causes strikingly high F-actin binding affinity (Kd = 75.5 nM) compared to the weak F-actin binding affinity of the wild-type ABD (Kd = 75.8 μM). The mutation also causes decreased thermal stability (Tm = 44.6 °C vs 59.5 °C). Structural analyses indicate that leucine 253 is in a loop at the interface of the tandem calponin homology (CH) domains comprising the ABD. Leucine 253 is predicted to form hydrophobic contacts that bridge the CH domains. The decreased stability of the mutant indicates that these bridging interactions are probably disrupted, suggesting that the high F-actin binding affinity of the mutant is due to opening of the CH domain interface. These results support a fundamental role for leucine 253 in regulating opening of the CH domain interface and binding of the ABD to F-actin. This study indicates that high-affinity actin binding of L253P β-III-spectrin is a likely driver of neurodegeneration.
MeSH term(s)	Actins/metabolism ; Amino Acid Sequence ; Humans ; Hydrophobic and Hydrophilic Interactions ; Models, Molecular ; Mutation ; Protein Binding ; Protein Conformation ; Protein Interaction Domains and Motifs/genetics ; Protein Stability ; Spectrin/chemistry ; Spectrin/genetics ; Spectrin/metabolism ; Spinocerebellar Ataxias/genetics ; Spinocerebellar Ataxias/metabolism ; Thermodynamics
Chemical Substances	Actins ; SPTBN2 protein, human ; Spectrin (12634-43-4)
Language	English
Publishing date	2016-02-17
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/srep21375
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Article: Methods to identify and analyze gene products involved in neuronal intracellular transport using Drosophila.

Neisch, Amanda L / Avery, Adam W / Machamer, James B / Li, Min-gang / Hays, Thomas S

Methods in cell biology

2016 Volume 131, Page(s) 277–309

Abstract: Proper neuronal function critically depends on efficient intracellular transport and disruption of transport leads to neurodegeneration. Molecular pathways that support or regulate neuronal transport are not fully understood. A greater understanding of ... ...

Abstract	Proper neuronal function critically depends on efficient intracellular transport and disruption of transport leads to neurodegeneration. Molecular pathways that support or regulate neuronal transport are not fully understood. A greater understanding of these pathways will help reveal the pathological mechanisms underlying disease. Drosophila melanogaster is the premier model system for performing large-scale genetic functional screens. Here we describe methods to carry out primary and secondary genetic screens in Drosophila aimed at identifying novel gene products and pathways that impact neuronal intracellular transport. These screens are performed using whole animal or live cell imaging of intact neural tissue to ensure integrity of neurons and their cellular environment. The primary screen is used to identify gross defects in neuronal function indicative of a disruption in microtubule-based transport. The secondary screens, conducted in both motoneurons and dendritic arborization neurons, will confirm the function of candidate gene products in intracellular transport. Together, the methodologies described here will support labs interested in identifying and characterizing gene products that alter intracellular transport in Drosophila.
MeSH term(s)	Animals ; Axonal Transport/genetics ; Axonal Transport/physiology ; Axons/metabolism ; Dendrites/physiology ; Drosophila Proteins/genetics ; Drosophila Proteins/metabolism ; Drosophila melanogaster/genetics ; Drosophila melanogaster/metabolism ; Dynactin Complex ; Dyneins/genetics ; Dyneins/metabolism ; Kinesin/metabolism ; Larva/metabolism ; Microtubule-Associated Proteins/metabolism ; Microtubules/genetics ; Microtubules/metabolism ; Neurodegenerative Diseases/pathology ; Neuronal Plasticity/genetics ; Neuronal Plasticity/physiology ; RNA Interference ; RNA, Small Interfering/genetics
Chemical Substances	Drosophila Proteins ; Dynactin Complex ; Microtubule-Associated Proteins ; RNA, Small Interfering ; Dyneins (EC 3.6.4.2) ; Kinesin (EC 3.6.4.4)
Language	English
Publishing date	2016
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	0091-679X
ISSN	0091-679X
DOI	10.1016/bs.mcb.2015.06.015
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Article ; Online: Structural basis for high-affinity actin binding revealed by a β-III-spectrin SCA5 missense mutation.

Avery, Adam W / Fealey, Michael E / Wang, Fengbin / Orlova, Albina / Thompson, Andrew R / Thomas, David D / Hays, Thomas S / Egelman, Edward H

Nature communications

2017 Volume 8, Issue 1, Page(s) 1350

Abstract: Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the cytoskeletal protein β-III-spectrin. Previously, a SCA5 mutation resulting in a leucine-to-proline substitution (L253P) in the actin-binding domain (ABD) was ... ...

Abstract	Spinocerebellar ataxia type 5 (SCA5) is a neurodegenerative disease caused by mutations in the cytoskeletal protein β-III-spectrin. Previously, a SCA5 mutation resulting in a leucine-to-proline substitution (L253P) in the actin-binding domain (ABD) was shown to cause a 1000-fold increase in actin-binding affinity. However, the structural basis for this increase is unknown. Here, we report a 6.9 Å cryo-EM structure of F-actin complexed with the L253P ABD. This structure, along with co-sedimentation and pulsed-EPR measurements, demonstrates that high-affinity binding caused by the CH2-localized mutation is due to opening of the two CH domains. This enables CH1 to bind actin aided by an unstructured N-terminal region that becomes α-helical upon binding. This helix is required for association with actin as truncation eliminates binding. Collectively, these results shed light on the mechanism by which β-III-spectrin, and likely similar actin-binding proteins, interact with actin, and how this mechanism can be perturbed to cause disease.
MeSH term(s)	Actins/metabolism ; Binding Sites ; Cryoelectron Microscopy ; Electron Spin Resonance Spectroscopy ; Humans ; Models, Molecular ; Mutation, Missense ; Protein Conformation ; Protein Domains ; Spectrin/chemistry ; Spectrin/genetics ; Spectrin/metabolism
Chemical Substances	Actins ; SPTBN2 protein, human ; Spectrin (12634-43-4)
Language	English
Publishing date	2017-11-07
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-017-01367-w
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