Quantifying superimposed protein flow dynamics in live cells using spatial filtering and spatiotemporal image correlation spectroscopy.

Autor: Migueles-Ramírez RA; Department of Quantitative Life Sciences, McGill University, Montreal, Quebec, Canada.; Department of Chemistry, McGill University, Montreal, Quebec, Canada.; Department of Physics, McGill University, Montreal, Quebec, Canada.; Department of Biology, McGill University, Montreal, Quebec, Canada., Cambi A; Department of Medical BioSciences, Radboud university medical center, Nijmegen, Netherlands., Hayer A; Department of Biology, McGill University, Montreal, Quebec, Canada., Wiseman PW; Department of Chemistry, McGill University, Montreal, Quebec, Canada.; Department of Physics, McGill University, Montreal, Quebec, Canada., van den Dries K; Department of Medical BioSciences, Radboud university medical center, Nijmegen, Netherlands.
Jazyk: angličtina
Zdroj: Journal of microscopy [J Microsc] 2024 Jul 04. Date of Electronic Publication: 2024 Jul 04.
DOI: 10.1111/jmi.13342
Abstrakt: Flow or collective movement is a frequently observed phenomenon for many cellular components including the cytoskeletal proteins actin and myosin. To study protein flow in living cells, we and others have previously used spatiotemporal image correlation spectroscopy (STICS) analysis on fluorescence microscopy image time series. Yet, in cells, multiple protein flows often occur simultaneously on different scales resulting in superimposed fluorescence intensity fluctuations that are challenging to separate using STICS. Here, we exploited the characteristic that distinct protein flows often occur at different spatial scales present in the image series to disentangle superimposed protein flow dynamics. We employed a newly developed and an established spatial filtering algorithm to alternatively accentuate or attenuate local image intensity heterogeneity across different spatial scales. Subsequently, we analysed the spatially filtered time series with STICS, allowing the quantification of two distinct superimposed flows within the image time series. As a proof of principle of our analysis approach, we used simulated fluorescence intensity fluctuations as well as time series of nonmuscle myosin II in endothelial cells and actin-based podosomes in dendritic cells and revealed simultaneously occurring contiguous and noncontiguous flow dynamics in each of these systems. Altogether, this work extends the application of STICS for the quantification of multiple protein flow dynamics in complex biological systems including the actomyosin cytoskeleton.
(© 2024 The Author(s). Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)
Databáze: MEDLINE
Externí odkaz: