Gerald Radziwill

Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand. OptoMitoImport is based on a two-step process: first, light-induced cleavage by a TEV protease cuts off a plasma membrane-anchored fusion construct in close proximity to a mitochondrial targeting sequence; second, the mitochondrial targeting sequence preceding the protein of interest recruits to the outer mitochondrial membrane and imports the protein fused to it into mitochondria. Upon reaching the mitochondrial matrix, the matrix processing peptidase cuts off the mitochondrial targeting sequence and releases the protein of interest. OptoMitoImport is available as a two-plasmid system as well as a P2A peptide or IRES sequence-based bicistronic system. Fluorescence studies demonstrate the release of the plasma membrane-anchored protein of interest through light-induced TEV protease cleavage and its localization to mitochondria. Cell fractionation experiments confirm the presence of the peptidase-cleaved protein of interest in the mitochondrial fraction. The processed product is protected from proteinase K treatment. Depletion of the membrane potential across the inner mitochondria membrane prevents the mitochondrial protein import, indicating an import of the protein of interest by the presequence pathway. These data demonstrate the functionality of OptoMitoImport as a generic system with which to control the post-translational mitochondrial import of proteins via the presequence pathway.

The progression through the cell cycle phases is driven by cyclin-dependent kinases and cyclins as their regulatory subunits. As nuclear protein, the cell cycle inhibitor p21/CDKN1A arrests the cell cycle at the growth phase G1 by inhibiting the activity of cyclin-dependent kinases. The G1 phase correlates with increased cell size and cellular productivity. Here, we applied an optogenetic approach to control the subcellular localization of p21 and its nuclear functions. To generate light-controllable p21, appropriate fusions with the blue light switch cryptochrome 2/CIBN and the AsLOV-based light-inducible nuclear localization signal, LINuS, were used. Both systems, p21-CRY2/CIB1 and p21-LINuS, increased the amounts of cells arrested in the G1 phase correlating with the increased cell-specific productivity of the reporter-protein-secreted alkaline phosphatase. Varying the intervals of blue LED light exposure and the light dose enable the fine-tuning of the systems. Light-controllable p21 implemented in producer cell lines could be applied to steer the uncoupling of cell proliferation and cell cycle arrest at the G1 phase optimizing the production of biotherapeutic proteins.

Several tissues contain cells with multiple motile cilia that generate a fluid or particle flow to support development and organ functions; defective motility causes human disease. Developmental cues orient motile cilia, but how cilia are locked into their final position to maintain a directional flow is not understood. Here we find that the actin cytoskeleton is highly dynamic during early development of multiciliated cells (MCCs). While apical actin bundles become increasingly more static, subapical actin filaments are nucleated from the distal tip of ciliary rootlets. Anchorage of these subapical actin filaments requires the presence of microridge-like structures formed during MCC development, and the activity of Nonmuscle Myosin II. Optogenetic manipulation of Ezrin, a core component of the microridge actin-anchoring complex, or inhibition of Myosin Light Chain Kinase interfere with rootlet anchorage and orientation. These observations identify microridge-like structures as an essential component of basal body rootlet anchoring in MCCs.

Biological signals are sensed by their respective receptors and are transduced and processed by a sophisticated intracellular signaling network leading to a signal-specific cellular response. Thereby, the response to the signal depends on the strength, the frequency, and the duration of the stimulus as well as on the subcellular signal progression. Optogenetic tools are based on genetically encoded light-sensing proteins facilitating the precise spatiotemporal control of signal transduction pathways and cell fate decisions in the absence of natural ligands. In this review, we provide an overview of optogenetic approaches connecting light-regulated protein-protein interaction or caging/uncaging events with steering the function of signaling proteins. We briefly discuss the most common optogenetic switches and their mode of action. The main part deals with the engineering and application of optogenetic tools for the control of transmembrane receptors including receptor tyrosine kinases, the T cell receptor and integrins, and their effector proteins. We also address the hallmarks of optogenetics, the spatial and temporal control of signaling events.

Die molekulare Optogenetik hat zum Ziel, molekulare Prozesse in Zellen durch Lichtsignale zu steuern, indem genetisch kodierte Photorezeptoren an zelluläre Proteine fusioniert werden. Dadurch kann die zelluläre Signalleitung mit einer bisher nicht möglich gewesenen zeitlichen und örtlichen Präzision gesteuert werden. Hierzu wurden in den letzten Jahren zahlreiche Methoden entwickelt und neue Einblicke gewonnen. Beispielsweise konnte durch lichtabhängige Aktivierung des T-Zell-Rezeptors das kinetic proofreading Modell bestätigt werden. Außerdem ermöglichen lichtinduzierbare Cre-Rekombinasen eine noch präzisere Kontrolle über Genmanipulationen. Dieser Übersichtsartikel soll als Inspiration dafür dienen, wie die molekulare Optogenetik in der präklinischen Forschung Anwendung finden kann.

The precise control of signaling proteins is a prerequisite to decipher the complexity of the signaling network and to reveal and to study pathways involved in regulating cellular metabolism and gene expression. Optogenetic approaches play an emerging role as they enable the spatiotemporal control of signaling processes. Herein, a multichromatic system is developed by combining the blue light cryptochrome 2 system and the red/far‐red light phytochrome B system. The use of three wavelengths allows the orthogonal control of the RAF/ERK and the AKT signaling pathway. Continuous exposure of cells to blue light leads to activation of AKT while simultaneous pulses of red and far‐red light enable the modulation of ERK signaling in cells with constantly active AKT signaling. The optimized, orthogonal multichromatic system presented here is a valuable tool to better understand the fine grained and intricate processes involved in cell fate decisions.

Subcellular localization of signal molecules is a hallmark in organizing the signal network. OpEn-Tag is a modular optogenetic endomembrane targeting toolbox that allows alteration of the localization and therefore the activity of signaling processes with the spatio-temporal resolution of optogenetics. OpEn-Tag is a two-component system employing 1) a variety of targeting peptides, fused to and thereby dictating the localization of mCherry-labeled cryptochrome 2 binding protein CIBN towards distinct endomembranes and 2) the cytosolic, fluorescence-labeled blue light photoreceptor cryptochrome 2 as customizable building block that can be fused to proteins of interest. The combination of OpEn-Tag with growth factor stimulation or the use of two membrane anchor sequences allows investigation of multilayered signal transduction processes as demonstrated here for the protein kinase AKT.

Spatiotemporal control is a common mechanism that modulates activity and function of signal transducers in the signaling network. We identified acetylation of CNK1 (connector enhancer of kinase suppressor of Ras-1) as a late step in the activation of CNK1 signaling, accompanied with prolonged stimulation of extracellular signal–regulated kinase (ERK). We identified the acetyltransferase CREB (cyclic adenosine 3′,5′-monophosphate response element–binding protein)–binding protein and the deacetylase SIRT2 (sirtuin type 2) as novel binding partners of CNK1, modulating the acetylation state of CNK1. Acetylation of CNK1 at position Lys⁴¹⁴ located in the pleckstrin homology domain drives membrane localization of CNK1 in growth factor–stimulated cells. Inhibition of ERK signaling abolishes CNK1 acetylation. Cosmic database search identified CNK1 mutants at position Arg⁴²⁶ near the acetylation site in several human tumor types. These mutants show constitutive acetylation and membrane localization. CNK1 mutants substituting Arg⁴²⁶, the acetylation mimetic mutant CNK1-K414Q, and membrane-anchored CNK1 mutants all interact with the protein kinase CRAF and stimulate ERK-dependent cell proliferation and cell migration. In RAS-transformed cells, CNK1 is acetylated and membrane-bound and drives cell proliferation. Thus, growth factor–stimulated ERK signaling induces CNK1 acetylation, and acetylated CNK1 promotes ERK signaling, demonstrating a novel function of CNK1 as positive feedback regulator of the RAF/MEK (mitogen-activated protein kinase kinase)/ERK pathway. In addition, acetylation of CNK1 is an important step in oncogenic signaling, promoting cell proliferation and migration.