scholarly journals DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein–dynactin–cargo adaptor complexes

2017 ◽  
Vol 114 (9) ◽  
pp. E1597-E1606 ◽  
Author(s):  
Ha Thi Hoang ◽  
Max A. Schlager ◽  
Andrew P. Carter ◽  
Simon L. Bullock
Keyword(s):  
Single Molecule ◽  
Heavy Chain ◽  
Muscular Atrophy ◽  
Recombinant Expression ◽  
Neurological Diseases ◽  
Expression System ◽  
Cytoplasmic Dynein ◽  
In Vitro Motility

Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4-MDa motor complex that traffics organelles, vesicles, and macromolecules toward microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterize 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein’s core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor Bicaudal-D2 (BICD2). Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2 and are therefore expected to result in linkage of cargos to dynein–dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo–motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

scholarly journals DYNC1H1 mutations associated with neurological diseases compromise processivity of dynein-dynactin-cargo adaptor complexes

2016 ◽  
Author(s):  
Ha Thi Hoang ◽  
Max A. Schlager ◽  
Andrew P. Carter ◽  
Simon L Bullock
Keyword(s):  
Single Molecule ◽  
Heavy Chain ◽  
Muscular Atrophy ◽  
Recombinant Expression ◽  
Neurological Diseases ◽  
Expression System ◽  
Cytoplasmic Dynein ◽  
Recombinant Expression System

Mutations in the human DYNC1H1 gene are associated with neurological diseases. DYNC1H1 encodes the heavy chain of cytoplasmic dynein-1, a 1.4 MDa motor complex that traffics organelles, vesicles and macromolecules towards microtubule minus ends. The effects of the DYNC1H1 mutations on dynein motility, and consequently their links to neuropathology, are not understood. Here, we address this issue using a recombinant expression system for human dynein coupled to single-molecule resolution in vitro motility assays. We functionally characterise 14 DYNC1H1 mutations identified in humans diagnosed with malformations in cortical development (MCD) or spinal muscular atrophy with lower extremity predominance (SMALED), as well as three mutations that cause motor and sensory defects in mice. Two of the human mutations, R1962C and H3822P, strongly interfere with dynein’s core mechanochemical properties. The remaining mutations selectively compromise the processive mode of dynein movement that is activated by binding to the accessory complex dynactin and the cargo adaptor BICD2. Mutations with the strongest effects on dynein motility in vitro are associated with MCD. The vast majority of mutations do not affect binding of dynein to dynactin and BICD2, and are therefore expected to result in linkage of cargoes to dynein-dynactin complexes that have defective long-range motility. This observation offers an explanation for the dominant effects of DYNC1H1 mutations in vivo. Collectively, our results suggest that compromised processivity of cargo-motor assemblies contributes to human neurological disease and provide insight into the influence of different regions of the heavy chain on dynein motility.

scholarly journals C9orf72-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility

2019 ◽  
Author(s):  
Laura Fumagalli ◽  
Florence L. Young ◽  
Steven Boeynaems ◽  
Mathias De Decker ◽  
Arpan R. Mehta ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Motor Neurons ◽  
Single Molecule ◽  
Induced Pluripotent Stem Cell ◽  
Cytoplasmic Dynein ◽  
Inhibitory Interaction ◽  
Hexanucleotide Repeat ◽  
Repeat Expansions

ABSTRACTHexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we use human induced pluripotent stem cell-derived motor neurons to show that C9orf72 repeat expansions impair microtubule-based transport of mitochondria, a process critical for maintenance of neuronal function. Cargo transport defects are recapitulated by treating healthy neurons with the arginine-rich dipeptide repeat proteins (DPRs) that are produced by the hexanucleotide repeat expansions. Single-molecule imaging shows that these DPRs perturb motility of purified kinesin-1 and cytoplasmic dynein-1 motors along microtubules in vitro. Additional in vitro and in vivo data indicate that the DPRs impair transport by interacting with both microtubules and the motor complexes. We also show that kinesin-1 is enriched in DPR inclusions in patient brains and that increasing the level of this motor strongly suppresses the toxic effects of arginine-rich DPR expression in a Drosophila model. Collectively, our study implicates an inhibitory interaction of arginine-rich DPRs with the axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to novel potential therapeutic strategies.

scholarly journals Dynein 1 supports spermatid transport and spermiation during spermatogenesis in the rat testis

2018 ◽  
Vol 315 (5) ◽  
pp. E924-E948 ◽  
Author(s):  
Qing Wen ◽  
Elizabeth I. Tang ◽  
Wing-yee Lui ◽  
Will M. Lee ◽  
Chris K. C. Wong ◽  
...  
Keyword(s):  
Germ Cell ◽  
Heavy Chain ◽  
Sertoli Cells ◽  
Cytoplasmic Dynein ◽  
Seminiferous Epithelium ◽  
Organelle Transport ◽  
Cellular Events

In the mammalian testis, spermatogenesis is dependent on the microtubule (MT)-specific motor proteins, such as dynein 1, that serve as the engine to support germ cell and organelle transport across the seminiferous epithelium at different stages of the epithelial cycle. Yet the underlying molecular mechanism(s) that support this series of cellular events remain unknown. Herein, we used RNAi to knockdown cytoplasmic dynein 1 heavy chain (Dync1h1) and an inhibitor ciliobrevin D to inactivate dynein in Sertoli cells in vitro and the testis in vivo, thereby probing the role of dynein 1 in spermatogenesis. Both treatments were shown to extensively induce disruption of MT organization across Sertoli cells in vitro and the testis in vivo. These changes also perturbed the transport of spermatids and other organelles (such as phagosomes) across the epithelium. These changes thus led to disruption of spermatogenesis. Interestingly, the knockdown of dynein 1 or its inactivation by ciliobrevin D also perturbed gross disruption of F-actin across the Sertoli cells in vitro and the seminiferous epithelium in vivo, illustrating there are cross talks between the two cytoskeletons in the testis. In summary, these findings confirm the role of cytoplasmic dynein 1 to support the transport of spermatids and organelles across the seminiferous epithelium during the epithelial cycle of spermatogenesis.

scholarly journals An N-terminal truncation of the ncd motor protein supports diffusional movement of microtubules in motility assays

1993 ◽  
Vol 104 (3) ◽  
pp. 899-906
Author(s):  
R. Chandra ◽  
S.A. Endow ◽  
E.D. Salmon
Keyword(s):  
Heavy Chain ◽  
Atp Hydrolysis ◽  
Motor Protein ◽  
Longitudinal Axis ◽  
Opposite Polarity ◽  
Truncated Protein ◽  
In Vitro Motility ◽  
Kinesin Heavy Chain

The nonclaret disjunctional (ncd) protein is a kinesin-related microtubule motor protein that is encoded at the claret locus in Drosophila and is required for proper chromosome distribution in meiosis and early mitosis. The protein contains a region with 41% amino acid sequence identity to kinesin heavy chain, but translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends. The overall structure of ncd also differs from kinesin heavy chain, in that the proposed motor domain is present at the C terminus of the molecule instead of the N terminus, as in kinesin heavy chain. In studies to define the molecular determinants of ncd function, we constructed and expressed a protein with a deletion of the N-terminal 208 amino acids of the non-motor region. Analysis of the truncated protein shows that the protein exhibits microtubule-stimulated Mg(2+)-ATPase activity and binds microtubules in pelleting assays. In contrast to near full-length ncd, the truncated protein does not support directional movement of microtubules in in vitro motility assays. Instead, microtubules show nucleotide-sensitive binding to the truncated protein on glass surfaces and bound microtubules exhibit one-dimensional diffusional movement that is constrained to their longitudinal axis. The diffusional movement reveals a weak binding state of the ncd motor that may represent a mechanochemical intermediate in its ATP hydrolysis cycle. If diffusional movement is a characteristic intrinsic to the claret motor, it is likely to be important in the in vivo function of the protein.

scholarly journals Lis1 has two opposing modes of regulating cytoplasmic dynein

2017 ◽  
Author(s):  
Morgan E. DeSantis ◽  
Michael A. Cianfrocco ◽  
Zaw Min Htet ◽  
Phuoc Tien Tran ◽  
Samara L. Reck-Peterson ◽  
...  
Keyword(s):  
Single Molecule ◽  
Intracellular Transport ◽  
Cytoplasmic Dynein ◽  
The Novel ◽  
Microtubule Binding ◽  
In Vivo Experiments ◽  
Cryo Electron Microscopy ◽  
Functional Versatility

SummaryRegulation is central to the functional versatility of cytoplasmic dynein, a motor involved in intracellular transport, cell division, and neurodevelopment. Previous work established that Lis1, a conserved and ubiquitous regulator of dynein, binds to its motor domain and induces a tight microtubule-binding state in dynein. The work we present here—a combination of biochemistry, single-molecule assays, cryo-electron microscopy and in vivo experiments—led to the surprising discovery that Lis1 has two opposing modes of regulating dynein, being capable of inducing both low and high affinity for the microtubule. We show that these opposing modes depend on the stoichiometry of Lis1 binding to dynein and that this stoichiometry is regulated by the nucleotide state of dynein’s AAA3 domain. We present data on the in vitro and in vivo consequences of abolishing the novel Lis1-induced weak microtubule-binding state in dynein and propose a new model for the regulation of dynein by Lis1.

scholarly journals A microscopy-based screen employing multiplex genome sequencing identifies cargo-specific requirements for dynein velocity

2014 ◽  
Vol 25 (5) ◽  
pp. 669-678 ◽  
Author(s):  
Kaeling Tan ◽  
Anthony J. Roberts ◽  
Mark Chonofsky ◽  
Martin J. Egan ◽  
Samara L. Reck-Peterson
Keyword(s):  
Genome Sequencing ◽  
Single Molecule ◽  
Intracellular Transport ◽  
Screening Method ◽  
Cytoplasmic Dynein ◽  
Catalytic Core ◽  
Single Molecule Studies ◽  
Novel Alleles

The timely delivery of membranous organelles and macromolecules to specific locations within the majority of eukaryotic cells depends on microtubule-based transport. Here we describe a screening method to identify mutations that have a critical effect on intracellular transport and its regulation using mutagenesis, multicolor-fluorescence microscopy, and multiplex genome sequencing. This screen exploits the filamentous fungus Aspergillus nidulans, which has many of the advantages of yeast molecular genetics but uses long-range microtubule-based transport in a manner more similar to metazoan cells. Using this method, we identified seven mutants that represent novel alleles of components of the intracellular transport machinery: specifically, kinesin-1, cytoplasmic dynein, and the dynein regulators Lis1 and dynactin. The two dynein mutations identified in our screen map to dynein's AAA+ catalytic core. Single-molecule studies reveal that both mutations reduce dynein's velocity in vitro. In vivo these mutants severely impair the distribution and velocity of endosomes, a known dynein cargo. In contrast, another dynein cargo, the nucleus, is positioned normally in these mutants. These results reveal that different dynein functions have distinct stringencies for motor performance.

Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

2020 ◽  
Author(s):  
James Frederich ◽  
Ananya Sengupta ◽  
Josue Liriano ◽  
Ewa A. Bienkiewicz ◽  
Brian G. Miller
Keyword(s):  
Protein Interactions ◽  
Neurological Diseases ◽  
Adapter Proteins ◽  
Protein Protein Interactions ◽  
Specific Expression ◽  
Individual Client ◽  
Isoform Specificity ◽  
Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions <i>in vivo</i>. Herein, we report the isoform-specificity profile of FC <i>in vitro</i>using recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals The effect of dipeptidyl peptidase IV on disease-associated microglia phenotypic transformation in epilepsy

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Zhicheng Zheng ◽  
Peiyu Liang ◽  
Baohua Hou ◽  
Xin Lu ◽  
Qianwen Ma ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Neurological Diseases ◽  
Dipeptidyl Peptidase ◽  
Dipeptidyl Peptidase Iv ◽  
Sequencing Data ◽  
Migration Ability ◽  
Dpp4 Inhibitor ◽  
Phenotypic Transformation

Abstract Background Accumulating evidence suggests that disease-associated microglia (DAM), a recently discovered subset of microglia, plays a protective role in neurological diseases. Targeting DAM phenotypic transformation may provide new therapeutic options. However, the relationship between DAM and epilepsy remains unknown. Methods Analysis of public RNA-sequencing data revealed predisposing factors (such as dipeptidyl peptidase IV; DPP4) for epilepsy related to DAM conversion. Anti-epileptic effect was assessed by electroencephalogram recordings and immunohistochemistry in a kainic acid (KA)-induced mouse model of epilepsy. The phenotype, morphology and function of microglia were assessed by qPCR, western blotting and microscopic imaging. Results Our results demonstrated that DPP4 participated in DAM conversion and epilepsy. The treatment of sitagliptin (a DPP4 inhibitor) attenuated KA-induced epilepsy and promoted the expression of DAM markers (Itgax and Axl) in both mouse epilepsy model in vivo and microglial inflammatory model in vitro. With sitagliptin treatment, microglial cells did not display an inflammatory activation state (enlarged cell bodies). Furthermore, these microglia exhibited complicated intersections, longer processes and wider coverage of parenchyma. In addition, sitagliptin reduced the activation of NF-κB signaling pathway and inhibited the expression of iNOS, IL-1β, IL-6 and the proinflammatory DAM subset gene CD44. Conclusion The present results highlight that the DPP4 inhibitor sitagliptin can attenuate epilepsy and promote DAM phenotypic transformation. These DAM exhibit unique morphological features, greater migration ability and better surveillance capability. The possible underlying mechanism is that sitagliptin can reduce the activation of NF-κB signaling pathway and suppress the inflammatory response mediated by microglia. Thus, we propose DPP4 may act as an attractive direction for DAM research and a potential therapeutic target for epilepsy.

Sign in / Sign up

Export Citation Format

Share Document