Upon illumination, photosensitizer molecules produce reactive oxygen
species (ROS) that can be utilized for functional manipulation of living cells,
including protein inactivation, targeted damage introduction, and cellular
ablation. Photosensitizers used to date have been either exogenous, resulting in
delivery and removal challenges, or genetically encoded proteins that form or
bind a native photosensitizing molecule, resulting in a constitutively active
photosensitizer inside the cell. By binding a heavy-atom substituted fluorogenic
dye with a genetically encoded Fluorogen Activating Protein (FAP), we
demonstrate an ‘on-demand’ activated photosensitizer that
produces singlet oxygen and fluorescence only when FAP-bound and activated with
near infrared light. This Targeted and Activated Photosensitizer (TAPs) approach
enables protein inactivation and targeted cell killing in cultured cells and
rapid targeted lineage ablation in living larval and adult zebrafish. The
near-infrared excitation and emission of this FAP-TAPs photosensitizer module
provides a new spectral range for photosensitizer proteins, useful for imaging,
manipulation and cellular ablation deep within living organisms.
Zebrafish regenerate cardiac tissue through proliferation of pre-existing cardiomyocytes and neovascularization. Secreted growth factors such as FGFs, IGF, PDGFs and Neuregulin play essential roles in stimulating cardiomyocyte proliferation. These factors activate the Ras/MAPK pathway, which is tightly controlled by the feedback attenuator Dual specificity phosphatase 6 (Dusp6), an ERK phosphatase. Here, we show that suppressing Dusp6 function enhances cardiac regeneration. Inactivation of Dusp6 by small molecules or by gene inactivation increased cardiomyocyte proliferation, coronary angiogenesis, and reduced fibrosis after ventricular resection. Inhibition of Erbb or PDGF receptor signaling suppressed cardiac regeneration in wild-type zebrafish, but had a milder effect on regeneration in mutants. Moreover, in rat primary cardiomyocytes, NRG1-stimulated proliferation can be enhanced upon chemical inhibition of Dusp6 with BCI. Our results suggest that Dusp6 attenuates Ras/MAPK signaling during regeneration and that suppressing Dusp6 can enhance cardiac repair.
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