Light-mediated remote control of signaling pathways

Abstract

Summary Cell signaling networks display an extraordinary range of temporal and spatial plasticity. Our programmatic approach focuses on the construction of intracellular probes, including sensors, inhibitors, and functionally unique proteins that can be temporally and spatially controlled by the investigator even after they have entered the cell. We have designed and evaluated protein kinase sensors that furnish a fluorescent readout upon phosphorylation. In addition, since the sensors are inert (i.e. cannot be phosphorylated) until activated by light, they can be carried through the various stages of any given cell-based behavior without being consumed. Using this strategy, we have shown that PKCβ is essential for nuclear envelope breakdown and thus the transition from prophase to metaphase in actively dividing cells. Photoactivatable proteins furnish the means to initiate cellular signaling pathways with a high degree of spatial and temporal control. We have used this approach to demonstrate that cofilin serves as a component of the steering apparatus of the cell. Finally, inhibitors are commonly used to assess the participation of specific enzymes in signaling pathways that control cellular behavior. We have constructed a photo-deactivatable inhibitor, an inhibitory species that can be switched off with light. In the absence of light, the target enzyme is inactive due to the presence of the potent inhibitory molecule. Upon photolysis, the inhibitory molecule is destroyed and enzymatic activity is released.

Efficient Far-Red/Near-IR Absorbing BODIPY Photocages by Blocking Unproductive Conical Intersections

“…For example, considerable efforts have been made to red-shift the absorbances of popular photocages beyond the most cell-damaging deep-UV wavelengths and toward the visible, , while retaining reasonable photorelease efficiencies. Alternatively, multiphoton absorption methods allow UV-absorbing photocages to be excited with two or more visible light photons. , Other methods to achieve photorelease with visible light include photorelease initiated via photoinduced electron transfer, − via metal–ligand photocleavage, ,, by exploiting internal photoredox reactions of quinones , or by using photosensitizers that generate reactive singlet oxygen that can initiate reaction cascades that result in release of a covalently linked substrate. , …”

“…Alternatively, multiphoton absorption methods allow UV-absorbing photocages to be excited with two or more visible light photons. 33,34 Other methods to achieve photorelease with visible light include photorelease initiated via photoinduced electron transfer, 35−37 via metal−ligand photocleavage, 6,38,39 by exploiting internal photoredox reactions of quinones 40,41 or by using photosensitizers that generate reactive singlet oxygen that can initiate reaction cascades that result in release of a covalently linked substrate. 42,43 Still, an organic photocage that retains the "plug and play" simplicity that makes o-nitrobenzyl photocages so popular but that directly undergoes efficient photorelease using single photons of red/near-IR light rather than UV light would be highly desirable.…”

Metabolism of peptide reporters in cell lysates and single cells

“…For these reasons, intact cells are also used as an assay format in which peptides act as inhibitors or substrates for enzymes. [18][19][20] The low molecular weight of peptides offers the advantage of efficient loading into cells relative to intact proteins. 21 The loading of substrate peptides into cells followed by lysis and separation of substrate and product by electrophoresis has been performed to track a variety of enzymatic reactions including those of kinases, acyl transferases, and proteases.…”

Chemical modification of proteins with photocleavable groups

“…Cellular processes such as protein localization and protein-protein interactions have also been studied using this approach. (Caldwell et al, 2018;Priestman & Lawrence, 2010) In our lab, we have used light to control the release of therapeutic proteins from injectable dermal depots.(P. K. Jain, D. Karunakaran, & S. H. Friedman, 2013;K.…”