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Capturing Metabolism-Dependent Solvent Dynamics in the Lumen of a Trafficking Lysosome
journal contribution
posted on 2019-01-16, 00:00 authored by Filippo Begarani, Francesca D’Autilia, Giovanni Signore, Ambra Del Grosso, Marco Cecchini, Enrico Gratton, Fabio Beltram, Francesco CardarelliThe eukaryotic cell
compartmentalizes into spatially confined,
membrane-enclosed, intracellular structures (e.g., organelles, endosomes, and vesicles). Here, peculiar
physicochemical properties of the local environment occur and participate
in the regulation of ongoing molecular processes. In spite of the huge amount of available environmental probes, experiments
on subcellular structures are severely challenged by their three-dimensional
(3D) movement. This bottleneck is tackled here by focusing an excitation
light beam in a periodic orbit around the structure of interest. The
recorded signal is used as feedback to localize the structure position
at high temporal resolution: microseconds along the orbit, milliseconds
between orbits. The lysosome is selected as the intracellular target,
together with 6-acetyl-2-dimethylaminonaphthalene (ACDAN) as probe
of the physicochemical properties of the intralysosomal environment.
Generalized polarization (GP) analysis of ACDAN emission is used to
get a quantitative view on
intralysosomal solvent dipolar relaxation. Thus, raster image correlation
spectroscopy (RICS) analysis reveals that the ACDAN GP signal is fluctuating
in the micro-to-millisecond time range during natural organelle 3D
trafficking. We show that ACDAN GP fluctuations are characteristic
of lysosomes in living cells, are selectively abolished by lysosomal
basification, and depend on metabolic energy in the form of ATP. We
argue that intralysosomal ACDAN GP fluctuates according to the ongoing
organelle metabolism. Indeed, we report alterations in amplitude and
timing of GP fluctuations in a cellular model of lysosomal storage
disorder (LSD). The strategy proposed provides insight into the elusive
local environment of a trafficking lysosome and supports similar molecular
investigations at the subcellular level.
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lysosomeprobeintralysosomal ACDAN GPexcitation light beamraster image correlation spectroscopyRICSATPACDAN GP signalLSDeukaryotic cell compartmentalizesmicro-to-millisecond time rangesubcellularorganelle 3 D traffickingintracellularlysosomal storage disorderACDAN GP fluctuationsphysicochemical propertiesCapturing Metabolism-Dependent Solvent Dynamicsorbitanalysis6- acetyl -2-dimethylaminonaphthalene
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