Variable Field Analytical Ultracentrifugation: II. Gravitational Sweep Sedimentation Velocity

Ma, Jia; Zhao, Huaying; Sandmaier, Julia; Liddle, J. Alexander; Schuck, Peter

doi:10.1016/j.bpj.2015.11.027

Cited by 31 publications

(24 citation statements)

References 36 publications

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“…The samples assembled in the absence of ATP was centrifuged at 15,000 rpm (∼16,000 × g). Samples containing ATP were analyzed by the gravtional sweep method (45), and centrifuged initially for 1 h at 20,000 rpm (∼29,000 × g), immediately followed by centrifugation at 50,000 rpm (∼182,000 × g). Interference (refractive index) and 295-nm absorbance scans were started immediately, and were collected until no further sedimentation boundary movement was observed.…”

Section: Methodsmentioning

confidence: 99%

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Liu

Billington

Shu

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Addition of 1 mM ATP substantially reduces the light scattering of solutions of polymerized unphosphorylated nonmuscle myosin IIs (NM2s), and this is reversed by phosphorylation of the regulatory light chain (RLC). It has been proposed that these changes result from substantial depolymerization of unphosphorylated NM2 filaments to monomers upon addition of ATP, and filament repolymerization upon RLC-phosphorylation. We now show that the differences in myosin monomer concentration of RLC-unphosphorylated and -phosphorylated recombinant mammalian NM2A, NM2B, and NM2C polymerized in the presence of ATP are much too small to explain their substantial differences in light scattering. Rather, we find that the decrease in light scattering upon addition of ATP to polymerized unphosphorylated NM2s correlates with the formation of dimers, tetramers, and hexamers, in addition to monomers, an increase in length, and decrease in width of the bare zones of RLC-unphosphorylated filaments. Both effects of ATP addition are reversed by phosphorylation of the RLC. Our data also suggest that, contrary to previous models, assembly of RLCphosphorylated NM2s at physiological ionic strength proceeds from folded monomers to folded antiparallel dimers, tetramers, and hexamers that unfold and polymerize into antiparallel filaments. This model could explain the dynamic relocalization of NM2 filaments in vivo by dephosphorylation of RLC-phosphorylated filaments, disassembly of the dephosphorylated filaments to folded monomers, dimers, and small oligomers, followed by diffusion of these species, and reassembly of filaments at the new location following rephosphorylation of the RLC.C lass II myosins, the first of 37 known myosin classes (1, 2) to be discovered and originally thought to be specific for muscle, are now known to occur widely in nonmuscle cells in numerous species. Human nonmuscle cells express three different myosin 2 paralogs: nonmuscle myosins (NM) NM2A, NM2B, and NM2C. Like all class II myosins (3, 4), NM2 monomers are hexapeptides of two identical heavy chains (HC), two essential light chains (ELC), and two regulatory light chains (RLC). The HCs of NM2A, NM2B, and NM2C are encoded by different genes, have 60-80% sequence similarity, and isoforms of each are derived from alternative splicing (4).Each HC has an N-terminal globular motor domain with an ATPase site and an actin-binding site, followed by a lever arm that binds one ELC and one RLC, and a long tail that homodimerizes to form a coiled-coil α-helix. The HC ends with a short C-terminal nonhelical tailpiece (5). Like most class II myosins, NM2 monomers assemble into bipolar filaments by antiparallel association of their elongated coiled-coil helical tails. The filaments of NM2s are appreciably smaller than filaments of muscle myosins, with lengths of about 300 nm, consisting of ∼30 monomers for filaments of NM2A and NM2B and ∼14 monomers for filaments of NM2C (5).As described originally for smooth muscle myosin based on light-scattering data (6-8), filaments of unphosp...

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Section: Methodsmentioning

confidence: 99%

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Liu

Billington

Shu

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…The boundary data (Figure 6A, C) show quite a bit of noise, which can be contrasted to that shown in Figures 2 and 3, as is anticipated from Mie scattering effects. 27 These effects are similar to the haziness of samples seen by eye for very large particles. Nevertheless, the data are of sufficient quality to extract a set of peaks in a model-free continuous sedimentation distribution, ls- g *( s ), resulting in random residuals derived from the fitting of the data.…”

Section: Resultsmentioning

confidence: 53%

“…26 However, implementation of this approach for data analysis has only recently been developed in Sedfit, subsequent to the work described here. 27 A similar approach that has been implemented in Sedanal some time ago is the MSM approach, which collects data serially over a discrete set of rotor speeds, 24,25 and is the method that is used in this study. The analysis algorithm, WDA, plots the data in the form of s * g ( s *) as a function of s *.…”

mentioning

confidence: 99%

Sedimentation Velocity Analysis with Fluorescence Detection of Mutant Huntingtin Exon 1 Aggregation in Drosophila melanogaster and Caenorhabditis elegans

et al. 2017

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At least nine neurodegenerative diseases that are caused by the aggregation induced by long tracts of glutamine sequences have been identified. One such polyglutamine-containing protein is huntingtin, which is the primary factor responsible for Huntington’s disease. Sedimentation velocity with fluorescence detection is applied to perform a comparative study of the aggregation of the huntingtin exon 1 protein fragment upon transgenic expression in Drosophila melanogaster and Caenorhabditis elegans. This approach allows the detection of aggregation in complex mixtures under physiologically relevant conditions. Complementary methods used to support this biophysical approach included fluorescence microscopy and semidenaturing detergent agarose gel electrophoresis, as a point of comparison with earlier studies. New analysis tools developed for the analytical ultracentrifuge have made it possible to readily identify a wide range of aggregating species, including the monomer, a set of intermediate aggregates, and insoluble inclusion bodies. Differences in aggregation in the two animal model systems are noted, possibly because of differences in levels of expression of glutamine-rich sequences. An increased level of aggregation is shown to correlate with increased toxicity for both animal models. Co-expression of the human Hsp70 in D. melanogaster showed some mitigation of aggregation and toxicity, correlating best with inclusion body formation. The comparative study emphasizes the value of the analytical ultracentrifuge equipped with fluorescence detection as a useful and rigorous tool for in situ aggregation analysis to assess commonalities in aggregation across animal model systems.

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“…One of the major virtues of the integral approach is that all experimental data can be incorporated, and as an explicit model for the sedimentation process, ls- g *( s ) can be compared transparently with the experimental data in the original data space to examine the quality of fit. The approach is very general, and without any further complications refinements on χ 1, nd are possible to account for solvent compressibility, 51 time-varying centrifugal fields, 52 , the finite scanning speed of the scanning systems, 3 optical signal magnification gradients, 53 or spatiotemporal modulation of signal increments in fluorescence detection. 54 …”

Section: Theorymentioning

confidence: 99%

Sedimentation coefficient distributions of large particles

Schuck

2016

Analyst

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The spatial and temporal evolution of concentration boundaries in sedimentation velocity analytical ultracentrifugation reports on the size distribution of particles with high hydrodynamic resolution. For large particles such as large protein complexes, fibrils, viral particles, or nanoparticles, sedimentation conditions usually allow migration from diffusion to be neglected relative to sedimentation. In this case, the shape of the sedimentation boundaries of polydisperse mixtures relates directly to the underlying size-distributions. Integral and derivative methods for calculating sedimentation coefficient distributions g*(s) of large particles from experimental boundary profiles have been developed previously, and are recapitulated here in a common theoretical framework. This leads to a previously unrecognized relationship between g*(s) and the time-derivative of concentration profiles. Of closed analytical form, it is analogous to the well-known Bridgman relationship for the radial derivative. It provides a quantitative description of the effect of substituting the time-derivative by scan differences with finite time intervals, which appears as a skewed box average of the true distribution. This helps to theoretically clarify the differences between results from time-derivative method and the approach of directly fitting the integral definition of g*(s) to the entirety of experimental boundary data.

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Variable Field Analytical Ultracentrifugation: II. Gravitational Sweep Sedimentation Velocity

Cited by 31 publications

References 36 publications

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Effect of ATP and regulatory light-chain phosphorylation on the polymerization of mammalian nonmuscle myosin II

Sedimentation Velocity Analysis with Fluorescence Detection of Mutant Huntingtin Exon 1 Aggregation in Drosophila melanogaster and Caenorhabditis elegans

Sedimentation coefficient distributions of large particles

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