Single-molecule tracking for studying protein dynamics and target-search mechanism in live cells of S. cerevisiae

Nitesh Kumar Podh; Ayan Das; Partha Dey; Sheetal Paliwal; Gunjan Mehta

doi:10.1016/j.xpro.2022.101900

Single-molecule tracking for studying protein dynamics and target-search mechanism in live cells of S. cerevisiae

STAR Protoc. 2022 Dec 16;3(4):101900. doi: 10.1016/j.xpro.2022.101900. Epub 2022 Dec 5.

Authors

Nitesh Kumar Podh¹, Ayan Das¹, Partha Dey¹, Sheetal Paliwal¹, Gunjan Mehta²

Affiliations

¹ Laboratory of Chromosome Dynamics and Gene Regulation, Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, Sangareddy, Telangana 502285, India.
² Laboratory of Chromosome Dynamics and Gene Regulation, Department of Biotechnology, Indian Institute of Technology Hyderabad, Kandi, Sangareddy, Telangana 502285, India. Electronic address: gunjanmehta@bt.iith.ac.in.

Abstract

Single-molecule tracking (SMT) is a powerful approach to quantify the biophysical parameters of protein dynamics in live cells. Here, we describe a protocol for SMT in live cells of the budding yeast Saccharomyces cerevisiae. We detail how to genetically engineer yeast strains for SMT, how to set up image acquisition parameters, and how different software programs can be used to quantify a variety of biophysical parameters such as diffusion coefficient, residence time, bound fraction, jump angles, and target-search parameters. For complete details on the use and execution of this protocol, please refer to Mehta et al. ¹ and Ball et al..².

Keywords: Cell Biology; Microscopy; Model Organisms; Single-molecule Assays.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Biophysics
Saccharomyces cerevisiae* / genetics
Saccharomycetales*
Single Molecule Imaging
Software