The effects of detergents on the polymerization properties of actin

Ujfalusi-Pozsonyi, Kinga; Hild, Gábor; Gróf, Pál; Gutay-Tóth, Zsuzsanna; Bacsó, Zsolt; Nyitrai, Miklós

doi:10.1002/cyto.a.20855

Cited by 8 publications

(6 citation statements)

References 48 publications

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“…These results suggest that the enzymatic cocktail used could be compromising the cell membrane as well as the magnetosome membrane. In addition, MamK, the actin-like protein that arranges magnetosomes in a chain, could also be affected by the EZ treatment as it has been seen that actin polymerization can be affected by the presence of detergents ( Ujfalusi-Pozsonyi et al, 2010 ). The EZ kit used contained a mild non-ionic detergent that may contribute to MamK degradation, causing more magnetosome chain cleavage.…”

Section: Resultsmentioning

confidence: 99%

Evaluation of cell disruption technologies on magnetosome chain length and aggregation behaviour from Magnetospirillum gryphiswaldense MSR-1

Masó-Martínez

Fryer

Aubert³

et al. 2023

Front. Bioeng. Biotechnol.

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Magnetosomes are biologically-derived magnetic nanoparticles (MNPs) naturally produced by magnetotactic bacteria (MTB). Due to their distinctive characteristics, such as narrow size distribution and high biocompatibility, magnetosomes represent an attractive alternative to existing commercially-available chemically-synthesized MNPs. However, to extract magnetosomes from the bacteria, a cell disruption step is required. In this study, a systematic comparison between three disruption techniques (enzymatic treatment, probe sonication and high-pressure homogenization) was carried out to study their effect on the chain length, integrity and aggregation state of magnetosomes isolated from Magnetospirillum gryphiswaldense MSR-1 cells. Experimental results revealed that all three methodologies show high cell disruption yields (>89%). Transmission electron microscopy (TEM), dynamic light scattering (DLS) and, for the first time, nano-flow cytometry (nFCM) were employed to characterize magnetosome preparations after purification. TEM and DLS showed that high-pressure homogenization resulted in optimal conservation of chain integrity, whereas enzymatic treatment caused higher chain cleavage. The data obtained suggest that nFCM is best suited to characterize single membrane-wrapped magnetosomes, which can be particularly useful for applications that require the use of individual magnetosomes. Magnetosomes were also successfully labelled (>90%) with the fluorescent CellMask™ Deep Red membrane stain and analysed by nFCM, demonstrating the promising capacity of this technique as a rapid analytical tool for magnetosome quality assurance. The results of this work contribute to the future development of a robust magnetosome production platform.

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Section: Resultsmentioning

confidence: 99%

Evaluation of cell disruption technologies on magnetosome chain length and aggregation behaviour from Magnetospirillum gryphiswaldense MSR-1

Masó-Martínez

Fryer

Aubert³

et al. 2023

Front. Bioeng. Biotechnol.

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“…To maintain intact protein complexes in each microwell during PAGE fractionation, the F-actin stabilization buffer slows the natural depolymerization kinetics. The non-ionic detergent Triton X-100 at ~1% v/v lyses the cell and minimally alters in vitro polymerization rates of actin 25 , 34 , 35 . The addition of 2 mM MgCl 2 stabilizes F-actin complexes 25 , as Mg 2+ binds G-actin to lower depolymerization rates 33 .…”

Section: Resultsmentioning

confidence: 99%

Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

et al. 2021

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Multimeric cytoskeletal protein complexes orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed, differential detergent fractionation to simultaneously detect protein complexes in hundreds of individual cells. Fractionation occurs by 60 s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Proteins are measured with a ~5-hour immunoassay. Co-detection of cytoskeletal protein complexes in U2OS cells treated with filamentous actin (F-actin) destabilizing Latrunculin A detects a unique subpopulation (~2%) exhibiting downregulated F-actin, but upregulated microtubules. Thus, some cells may upregulate other cytoskeletal complexes to counteract the stress of Latrunculin A treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin. We find heat shock may dysregulate filamentous and globular actin correlation. In this work, our assay overcomes selectivity limitations to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.

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“…To maintain intact F-actin complexes in each microwell during G-actin EP fractionation, the F-actin stabilization buffer slows the natural depolymerization kinetics. The non-ionic detergent Triton X-100 19,23 at ~1% v/v lyses the cell and minimally alters in vitro polymerization rates of actin 24 . Addition of 2 mM MgCl2 stabilizes F-actin complexes 19 , as Mg 2+ binds each G-actin to lower depolymerization rates 22 .…”

Section: Sifter Design Principles and Characterizationmentioning

confidence: 99%

Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

Vlassakis

Hansen

Higuchi‐Sanabria

et al. 2020

Preprint

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Multimeric cytoskeletal protein complexes, including filamentous F-actin, orchestrate normal cellular function. However, protein-complex distributions in stressed, heterogeneous cell populations remain unknown. Cell staining and proximity-based methods have limited selectivity and/or sensitivity for robust endogenous multimeric protein-complex quantification from single cells. We introduce micro-arrayed differential detergent fractionation to simultaneously detect protein complexes in 100s of individual cells. Fractionation occurs by 60s size-exclusion electrophoresis with protein complex-stabilizing buffer that minimizes depolymerization. Validating with actin-destabilizing Latrunculin A (LatA), we quantify 2.7-fold lower median F-actin complex-levels in LatA-treated single cells. Further clustering analysis of U2OS cells treated with LatA detects a subpopulation (~11%) exhibiting downregulated F-actin, but upregulated microtubule and intermediate filament protein complexes. Thus, some cells upregulate other cytoskeletal complexes to counteract the stress of LatA treatment. We also sought to understand the effect of non-chemical stress on cellular heterogeneity of F-actin, and find heat shock dysregulates F and monomeric G-actin correlation. The assay overcomes selectivity limitations of existing methods to biochemically quantify single-cell protein complexes perturbed with diverse stimuli.

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The effects of detergents on the polymerization properties of actin

Cited by 8 publications

References 48 publications

Evaluation of cell disruption technologies on magnetosome chain length and aggregation behaviour from Magnetospirillum gryphiswaldense MSR-1

Evaluation of cell disruption technologies on magnetosome chain length and aggregation behaviour from Magnetospirillum gryphiswaldense MSR-1

Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

Measuring expression heterogeneity of single-cell cytoskeletal protein complexes

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