In vivo macromolecular crowding is differentially modulated by aquaporin 0 in zebrafish lens: Insights from a nanoenvironment sensor and spectral imaging

Abstract

Macromolecular crowding is crucial for cellular homeostasis. In vivo studies of macromolecular crowding and water dynamics are needed to understand their roles in cellular physiology and fate determination. Macromolecular crowding in the lens is essential for normal optics, and an understanding of its regulation will help prevent cataract and presbyopia. Here, we combine the use of the nanoenvironmental sensor [6-acetyl-2-dimethylaminonaphthalene (ACDAN)] to visualize lens macromolecular crowding with in vivo studies of aquaporin 0 zebrafish mutants that disrupt its regulation. Spectral phasor analysis of ACDAN fluorescence reveals water dipolar relaxation and demonstrates that mutations in two zebrafish aquaporin 0s, Aqp0a and Aqp0b, alter water state and macromolecular crowding in living lenses. Our results provide in vivo evidence that Aqp0a promotes fluid influx in the deeper lens cortex, whereas Aqp0b facilitates fluid efflux. This evidence reveals previously unidentified spatial regulation of macromolecular crowding and spatially distinct roles for Aqp0 in the lens.

Biomolecular condensates modulate membrane lipid packing and hydration

“…Conversely, in an environment where the dynamics of the solvent molecules is precluded, and water cannot reorient around the dye at the excited state, the spectrum falls towards the blue region. One of the probes we employ is ACDAN, which is a water-soluble dye that has been previously used to characterize the aqueous environment in yeast 28, 32 , HeLa cells 33 , and most recently for the study of in-vivo macromolecular crowding of zebra fish lenses 27 . Here, we exploit the sensitivity of ACDAN by using hyperspectral imaging combined with the phasor plot analysis 27 , to probe the nanoenvironement in biomolecular condensates formed by glycinin.…”

“…One of the probes we employ is ACDAN, which is a water-soluble dye that has been previously used to characterize the aqueous environment in yeast 28, 32 , HeLa cells 33 , and most recently for the study of in-vivo macromolecular crowding of zebra fish lenses 27 . Here, we exploit the sensitivity of ACDAN by using hyperspectral imaging combined with the phasor plot analysis 27 , to probe the nanoenvironement in biomolecular condensates formed by glycinin. Glycinin is one of the most abundant storage proteins in the soybean.…”

“…While in water, ACDAN has an emission spectrum centered at 520 nm, when increasing the protein-water ratio the spectrum is blue shifted reflecting the associated reduction of dipolar relaxation. ACDAN spectroscopic properties provide an accurate quantitative readout of the water activity (confinement) and, in consequence, protein-water ratio and crowding 27 . An easy way of visualizing the spectral shifts consists of extracting the histogram for the pixel distribution along a trajectory defined by the data, i.e.…”

Adaptive optical two-photon fluorescence microscopy probes cellular organization of ocular lenses in vivo

“…Ocular lens is of considerable interest to biology due to both its importance to vision and its continuous growth over the lifespan of the organism 40 . Prior research has attempted in vivo microscopic examination of lens development in zebrafish, a simpler vertebrate model [41][42][43][44] .…”

Stress‐dependent macromolecular crowding in the mitochondrial matrix

“…In this sense, macromolecular crowding will reduce the diffusion coefficient of larger tracer molecules to a greater extent than predicted by the Stokes–Einstein equation (Mika et al , 2010 ; Delarue et al , 2018 ; Junker et al , 2019 ). Experimental and theoretical studies suggest that modulation of macromolecular crowding can act as a (patho)physiological control mechanism in prokaryotes and eukaryotes (Scalettar et al , 1991 ; Akabayov et al , 2013 ; Poggi & Slade, 2015 ; Cravens et al , 2015 ; Joyner et al , 2015 ; Hansen et al , 2016 ; Delarue et al , 2018 ; Schavemaker et al , 2018 ; Fuentes‐Lemus et al , 2021 ; Hochmair et al , 2022 ; Vorontsova et al , 2022 ). The mitochondrial matrix compartment is classically recognized as highly protein‐rich.…”