Genetically encoded optogenetic tools are increasingly popular and useful for perturbing signaling pathways with high spatial and temporal resolution in living cells. Here, we show basic procedures employed to implement optogenetics of Rho GTPases in a macrophage cell line. Methods described here are generally applicable to other genetically encoded optogenetic tools utilizing the blue-green spectrum of light for activation, designed for specific proteins and enzymatic targets important for immune cell functions.
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http://dx.doi.org/10.1007/978-1-0716-0247-8_24 | DOI Listing |
EMBO J
July 2024
Mechanobiology Institute, National University of Singapore, Singapore, Singapore.
Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly.
View Article and Find Full Text PDFDuring development, epithelia function as malleable substrates that undergo extensive remodeling to shape developing embryos. Optogenetic control of Rho signaling provides an avenue to investigate the mechanisms of epithelial morphogenesis, but transgenic optogenetic tools can be limited by variability in tool expression levels and deleterious effects of transgenic overexpression on development. Here, we use CRISPR/Cas9 to tag RhoGEF2 and Cysts/Dp114RhoGEF with components of the iLID/SspB optogenetic heterodimer, permitting light-dependent control over endogenous protein activities.
View Article and Find Full Text PDFACS Synth Biol
January 2024
Department of Chemistry, Saint Louis University, St. Louis, Missouri 63103, United States.
Cells experience time-varying and spatially heterogeneous chemokine signals , activating cell surface proteins including G protein-coupled receptors (GPCRs). The Gαq pathway activation by GPCRs is a major signaling axis with broad physiological and pathological significance. Compared with other Gα members, GαqGTP activates many crucial effectors, including PLCβ (Phospholipase Cβ) and Rho GEFs (Rho guanine nucleotide exchange factors).
View Article and Find Full Text PDFPflugers Arch
December 2023
Department of Systemic Cell Biology, Fakultät für Chemie und Chemische Biologie, Max Planck Institute of Molecular Physiology, and Dortmund University of Technology, Room CP-02-157, Otto-Hahn-Str. 4a, 44227, Dortmund, Germany.
Cell contraction plays an important role in many physiological and pathophysiological processes. This includes functions in skeletal, heart, and smooth muscle cells, which lead to highly coordinated contractions of multicellular assemblies, and functions in non-muscle cells, which are often highly localized in subcellular regions and transient in time. While the regulatory processes that control cell contraction in muscle cells are well understood, much less is known about cell contraction in non-muscle cells.
View Article and Find Full Text PDFMol Brain
October 2023
Center for Cognition and Sociality, Institute for Basic Science (IBS), Daejeon, 34126, Republic of Korea.
Calcium ions (Ca) play pivotal roles in regulating diverse brain functions, including cognition, emotion, locomotion, and learning and memory. These functions are intricately regulated by a variety of Ca-dependent cellular processes, encompassing synaptic plasticity, neuro/gliotransmitter release, and gene expression. In our previous work, we developed 'monster OptoSTIM1' (monSTIM1), an improved OptoSTIM1 that selectively activates Ca-release-activated Ca (CRAC) channels in the plasma membrane through blue light, allowing precise control over intracellular Ca signaling and specific brain functions.
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