The endoplasmic reticulum (ER) contacts mitochondria through specialized subdomains called mitochondria-associated membranes (MAMs) at mitochondria-ER contact sites (MERCs). These contacts are vital for lipid and calcium transfer, maintaining organelle homeostasis. A handful of probes exist to study MERC formation and dynamics. GFP complementation probes induce irreversible artificial tethering due to low Kd values. FRET-based probes, while reversible, pose challenges for in vitro and in vivo imaging, limiting simultaneous fluorophore wavelengths. To overcome these limitations, we developed STACCATO, a probe array enabling MERC imaging across diverse interorganellar distances. STACCATO employs a split version of the 14 kDa Fluorescence-Activating and Absorption-Shifting Tag (FAST) protein, evolved from the photoactive yellow protein (PYP). It fluoresces when the ER- and mitochondria-targeted components of SplitFAST complement each other and is bound to the cell-permeant, non-fluorescent fluorogenic chromophores derived from 4-hydroxybenzylidene rhodanine (HBR). STACCATO ensures a high signal-to-noise ratio, does not induce artificial tethering and allows imaging of MERCs at different distances and across various regions of the fluorescence spectrum. STACCATO is a versatile probe for comprehensive MERC analysis.
Imaging various interorganellar distances with STACCATO, a reversible, multispectral Split-FAST Mitochondria-ER Contacts probe
Satoko Shinjo;Luca Scorrano
2024
Abstract
The endoplasmic reticulum (ER) contacts mitochondria through specialized subdomains called mitochondria-associated membranes (MAMs) at mitochondria-ER contact sites (MERCs). These contacts are vital for lipid and calcium transfer, maintaining organelle homeostasis. A handful of probes exist to study MERC formation and dynamics. GFP complementation probes induce irreversible artificial tethering due to low Kd values. FRET-based probes, while reversible, pose challenges for in vitro and in vivo imaging, limiting simultaneous fluorophore wavelengths. To overcome these limitations, we developed STACCATO, a probe array enabling MERC imaging across diverse interorganellar distances. STACCATO employs a split version of the 14 kDa Fluorescence-Activating and Absorption-Shifting Tag (FAST) protein, evolved from the photoactive yellow protein (PYP). It fluoresces when the ER- and mitochondria-targeted components of SplitFAST complement each other and is bound to the cell-permeant, non-fluorescent fluorogenic chromophores derived from 4-hydroxybenzylidene rhodanine (HBR). STACCATO ensures a high signal-to-noise ratio, does not induce artificial tethering and allows imaging of MERCs at different distances and across various regions of the fluorescence spectrum. STACCATO is a versatile probe for comprehensive MERC analysis.File | Dimensione | Formato | |
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