Dataset Information

Conformational Flexibility of p150Glued(1-191) Subunit of Dynactin Assembled with Microtubules.

ABSTRACT: Microtubule (MT)-associated proteins perform diverse functions in cells. These functions are dependent on their interactions with MTs. Dynactin, a cofactor of dynein motor, assists the binding of dynein to various organelles and is crucial to the long-distance processivity of dynein-based complexes. The largest subunit of dynactin, the p150Glued, contains an N-terminus segment that is responsible for the MT-binding interactions and long-range processivity of dynactin. We employed solution and magic angle spinning NMR spectroscopy to characterize the structure and dynamics of the p150Glued N-terminal region, both free and in complex with polymerized MTs. This 191-residue region encompasses the cytoskeleton-associated protein glycine-rich domain, the basic domain, and serine/proline-rich (SP-rich) domain. We demonstrate that the basic and SP-rich domains are intrinsically disordered in solution and significantly enhance the binding affinity to MTs as these regions contain the second MT-binding site on the p150Glued subunit. The majority of the basic and SP-rich domains are predicted to be random coil, whereas the segments S111-I116, A124-R132, and K144-T146 in the basic domain contain short ?-helical or ?-sheet structures. These three segments possibly encompass the MT-binding site. Surprisingly, the protein retains a high degree of flexibility upon binding to MTs except for the regions that are directly involved in the binding interactions with MTs. This conformational flexibility may be essential for the biological functions of the p150Glued subunit.

SUBMITTER: Guo C

PROVIDER: S-EPMC6731383 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Conformational Flexibility of p150<sup>Glued</sup>(1-191) Subunit of Dynactin Assembled with Microtubules.

Guo Changmiao C Williams John C JC Polenova Tatyana T

Biophysical journal 20190730 5

Microtubule (MT)-associated proteins perform diverse functions in cells. These functions are dependent on their interactions with MTs. Dynactin, a cofactor of dynein motor, assists the binding of dynein to various organelles and is crucial to the long-distance processivity of dynein-based complexes. The largest subunit of dynactin, the p150<sup>Glued</sup>, contains an N-terminus segment that is responsible for the MT-binding interactions and long-range processivity of dynactin. We employed solu ...[more]

PMID: 31445682

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Project description:BACKGROUND:Dynactin p150Glued, the largest subunit of the dynactin macromolecular complex, binds to both microtubules and tubulin dimers through the N-terminal cytoskeleton-associated protein and glycine-rich (CAP-Gly) and basic domains, and serves as an anti-catastrophe factor in stabilizing microtubules in neurons. P150Glued also initiates dynein-mediated axonal retrograde transport. Multiple missense mutations at the CAP-Gly domain of p150Glued are associated with motor neuron diseases and other neurodegenerative disorders, further supporting the importance of microtubule domains (MTBDs) in p150Glued functions. However, most functional studies were performed in vitro. Whether p150Glued is required for neuronal function and survival in vivo is unknown. METHODS:Using Cre-loxP genetic manipulation, we first generated a line of p150Glued knock-in mice by inserting two LoxP sites flanking the MTBD-coding exons 2 to 4 of p150Glued-encoding Dctn1 gene (Dctn1LoxP/), and then crossbred the resulting Dctn1LoxP/ mice with Thy1-Cre mice to generate the bigenic p150Glued (Dctn1LoxP/LoxP; Thy1-Cre) conditional knockout (cKO) mice for the downstream motor behavioral and neuropathological studies. RESULTS:P150Glued expression was completely abolished in Cre-expressing postnatal neurons, including corticospinal motor neurons (CSMNs) and spinal motor neurons (SMNs), while the MTBD-truncated forms remained. P150Glued ablation did not affect the formation of dynein/dynactin complex in neurons. The p150Glued cKO mice did not show any obvious developmental phenotypes, but exhibited impairments in motor coordination and rearing after 12 months of age. Around 20% loss of SMNs was found in the lumbar spinal cord of 18-month-old cKO mice, in company with increased gliosis, neuromuscular junction (NMJ) disintegration and muscle atrophy. By contrast, no obvious degeneration of CSMNs, striatal neurons, midbrain dopaminergic neurons, cerebellar granule cells or Purkinje cells was observed. Abnormal accumulation of acetylated ?-tubulin, and autophagosome/lysosome proteins was found in the SMNs of aged cKO mice. Additionally, the total and cell surface levels of glutamate receptors were also substantially elevated in the p150Glued-depleted spinal neurons, in correlation with increased vulnerability to excitotoxicity. CONCLUSION:Overall, our findings demonstrate that p150Glued is particularly required to maintain the function and survival of SMNs during aging. P150Glued may exert its protective function through regulating the transportation of autophagosomes, lysosomes, and postsynaptic glutamate receptors in neurons.

Project description:Amyotrophic lateral sclerosis (ALS) is the most common degenerative motor neuron disorder. Although most cases of ALS are sporadic, 5-10% of cases are familial, with mutations associated with over 40 genes. There is variation of ALS symptoms within families carrying the same mutation; the disease may develop in one sibling and not in another despite the presence of the mutation in both. Although the cause of this phenotypic variation is unknown, it is likely related to genetic modifiers of disease expression. The identification of ALS causing genes has led to the development of transgenic mouse models of motor neuron disease. Similar to families with familial ALS, there are background-dependent differences in disease phenotype in transgenic mouse models of ALS suggesting that, as in human ALS, differences in phenotype may be ascribed to genetic modifiers. These genetic modifiers may not cause ALS rather their expression either exacerbates or ameliorates the effect of the mutant ALS causing genes. We have reported that in both the G93A-hSOD1 and G59S-hDCTN1 mouse models, SJL mice demonstrated a more severe phenotype than C57BL6 mice. From reciprocal intercrosses between G93A-hSOD1 transgenic mice on SJL and C57BL6 strains, we identified a major quantitative trait locus (QTL) on mouse chromosome 17 that results in a significant shift in lifespan. In this study we generated reciprocal intercrosses between transgenic G59S-hDCTN1 mice on SJL and C57BL6 strains and identified survival QTLs on mouse chromosomes 17 and 18. The chromosome 17 survival QTL on G93A-hSOD1 and G59S-hDCTN1 mice partly overlap, suggesting that the genetic modifiers located in this region may be shared by these two ALS models despite the fact that motor neuron degeneration is caused by mutations in different proteins. The overlapping region contains eighty-seven genes with non-synonymous variations predicted to be deleterious and/or damaging. Two genes in this segment, NOTCH3 and Safb/SAFB1, have been associated with motor neuron disease. The identification of genetic modifiers of motor neuron disease, especially those modifiers that are shared by SOD1 and dynactin-1 transgenic mice, may result in the identification of novel targets for therapies that can alter the course of this devastating illness.

Dataset Information

Conformational Flexibility of p150Glued(1-191) Subunit of Dynactin Assembled with Microtubules.

Publications

Conformational Flexibility of p150<sup>Glued</sup>(1-191) Subunit of Dynactin Assembled with Microtubules.

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