Semi-retentive cytoskeletal fractionation (SERCYF): A novel method for the biochemical analysis of the organization of microtubule and actin cytoskeleton networks

Biochemical and Biophysical Research Communications
Yuta SatoMasayuki Takahashi

Abstract

A variety of biochemical fractionation methods are available for the quantification of cytoskeletal components. However, each method is designed to target only one cytoskeletal network, either the microtubule (MT) or actin cytoskeleton, and non-targeted cytoskeletal networks are ignored. Considering the importance of MT-actin crosstalk, the organization of both the targeted and non-targeted cytoskeletal networks must be retained intact during fractionation for the accurate analysis of cytoskeletal organization. In this study, we reveal that existing fractionation methods, represented by the MT-sedimentation-method for MTs and the Triton X-100 solubility assay-method for actin cytoskeletons, disrupt the organizations of the non-targeted cytoskeletons. We demonstrate a novel fractionation method for the accurate analysis of the cytoskeletal organizations using a taxol-containing PEM-based permeabilization buffer, which we name "semi-retentive cytoskeletal fractionation (SERCYF)-method". The organizations of both MTs and actin cytoskeletons were retained intact even after permeabilization with this buffer. By using the SERCYF-method, we analyzed the effects of nocodazole on the cytoskeletal organizations biochemically and showed p...Continue Reading

Citations

Oct 11, 2019·Journal of Biochemistry·Yuta SatoMasayuki Takahashi
Mar 21, 2020·Journal of Biochemistry·Yuta SatoMasayuki Takahashi
Oct 15, 2021·The Journal of Cell Biology·Fabian BockRoy Zent

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