Motor Function of the Two-Component EEA1-Rab5 Revealed by dcFCCS

Joan Antoni Soler; Anupam Singh; Marino Zerial; Shashi Thutupalli

doi:10.1007/978-1-0716-4280-1_4

Motor Function of the Two-Component EEA1-Rab5 Revealed by dcFCCS

Methods Mol Biol. 2025:2881:87-115. doi: 10.1007/978-1-0716-4280-1_4.

Authors

Joan Antoni Soler¹, Anupam Singh^{1

2}, Marino Zerial¹, Shashi Thutupalli^{3

4}

Affiliations

¹ Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
² Simons Centre for the Study of Living Machines, National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India.
³ Simons Centre for the Study of Living Machines, National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore, India. [email protected].
⁴ International Centre for Theoretical Sciences, Tata Institute of Fundamental Research, Bangalore, India. [email protected].

PMID: 39704939
DOI: 10.1007/978-1-0716-4280-1_4

Abstract

Fluorescence correlation spectroscopy (FCS) enables the measurement of fluctuations at fast timescales (typically few nanoseconds) and with high spatial resolution (tens of nanometers). This single-molecule measurement has been used to characterize single-molecule transport and flexibility of polymers and biomolecules such as DNA and RNA. Here, we apply this technique as dual-color fluorescence cross-correlation spectroscopy (dcFCCS) to identify the motor function of the tethering protein EEA1 and the small GTPase Rab5 by probing the flexibility changes through end-monomer fluctuations.

Keywords: Dual labeling; Dual-color fluorescence cross-correlation spectroscopy; Small GTPase; Tethering proteins.

MeSH terms

Humans
Single Molecule Imaging / methods
Spectrometry, Fluorescence* / methods
Vesicular Transport Proteins / genetics
Vesicular Transport Proteins / metabolism
rab5 GTP-Binding Proteins* / genetics
rab5 GTP-Binding Proteins* / metabolism

Substances

rab5 GTP-Binding Proteins
Vesicular Transport Proteins