Myosin-actin interaction in Dictyostelium cells revealed by GFP-based strain sensor and validated linear spectral unmixing

Myosin is an actin-based motor protein that is involved in a wide range of cellular motile processes. Although in vitro properties of the myosin-actin interaction have been extensively studied, the interaction in vivo remains poorly understood. Recently, we developed a GFP-based strain sensor termed...

Full description

Saved in:
Bibliographic Details
Published in:Cytometry. Part A 2010-08, Vol.77A (8), p.743-750
Main Authors: Iwai, Sosuke, Uyeda, Taro Q.P
Format: Article
Language:English
Subjects:
Actin
Actins - metabolism
Animals
autofluorescence
Biosensing Techniques - methods
Cortex
Cytometry
Dictyostelium
Dictyostelium - cytology
Dictyostelium - metabolism
Filaments
Fluorescence
Fusion protein
Green Fluorescent Proteins - metabolism
imaging
Intracellular Space - metabolism
least squares
linear unmixing
Myosin
Myosins - chemistry
Myosins - metabolism
Protein Binding
Recombinant Fusion Proteins - metabolism
Reproducibility of Results
serial correlation
Spectrometry, Fluorescence
Tails
Wavelength
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Myosin is an actin-based motor protein that is involved in a wide range of cellular motile processes. Although in vitro properties of the myosin-actin interaction have been extensively studied, the interaction in vivo remains poorly understood. Recently, we developed a GFP-based strain sensor termed PriSSM (PRIM-based strain sensor module), by using the proximity imaging (PRIM) technique, which detects spectral changes of two GFP molecules that are in direct contact. Using PriSSM-myosin II fusion proteins, the interaction between myosin II and F-actin can be detected in Dictyostelium cells. In the spectroscopic measurements of PriSSM, to decompose the measured spectra of the cells expressing the sensor proteins into the contributions from the sensor and the background autofluorescence, we applied the linear spectral unmixing approach, which was based on the assumption that the errors at each wavelength were independent. Cellular autofluorescence, however, often includes systematic errors, so that the unmixing procedures might lead to biased estimates. Here, to validate our spectral unmixing procedures, we estimate the possible maximum errors in the fluorescence ratio values that are obtained by unmixing spectra including such systematic errors. This estimation provided a general criterion to validate the results obtained by linear unmixing of spectra including serially correlated error terms. Using the proposed criterion and PriSSM-myosin II fusion proteins, we examined the interaction between myosin II and F-actin in Dictyostelium cells under several different conditions. The spectroscopic results, together with the microscopic observations of the cells expressing the proteins, suggest that the formation of myosin filaments through the tail region has only a slight effect on binding to F-actin but has significant effects on the cortical localization of myosin II. © 2010 International Society for Advancement of Cytometry
ISSN:1552-4922
1552-4930
1552-4930
DOI:10.1002/cyto.a.20900