Curated Optogenetic Publication Database

Search precisely and efficiently by using the advantage of the hand-assigned publication tags that allow you to search for papers involving a specific trait, e.g. a particular optogenetic switch or a host organism.

Showing 1 - 6 of 6 results
1.

Coupling between protrusion dynamics and polarized trafficking steers persistent cell migration.

blue iLID hTERT RPE-1 Control of cytoskeleton / cell motility / cell shape
bioRxiv, 22 Mar 2021 DOI: 10.1101/2021.03.20.436273 Link to full text
Abstract: Migrating cells present a variety of paths, from non-persistent random walks to highly directional trajectories. While random movement can be easily explained by an intrinsic basal activity of the cell, persistent movement requires the cell to be stably polarized. It remains unclear how this is achieved from the regulation of underlying subcellular processes. In the context of mesenchymal migration, the ability of cells to migrate persistently over several hours require a mechanism stabilizing their protruding activity at their front. Here, we address this mechanism using human RPE1 cell line as our model. We measure, manipulate, and quantitatively perturb cell protrusive activity of the cortex as well as intracellular organization of the endomembrane trafficking system using dynamic micropatterning, pharmacological and trafficking assays, optogenetics and live-cell imaging with tracking. First, we demonstrate that the Nucleus-Golgi axis aligns with the direction of migration and its alignment with the protrusive activity leads to efficient cell movement. Then, using low doses of Nocodazole to disrupt internal cell organization, we show that long-lived polarity breaks down and migration becomes random. Next, we indicate that a flow of vesicles is directed towards the protrusive activity with a delay of 20 min. Eventually, by applying a sustained optogenetic activation, we prove that a localized Cdc42 gradient is able to orient the Nucleus-Golgi axis over a couple of hours. Taken together, our results suggest that the internal polarity axis, provided by the polarized trafficking of vesicles, is stabilizing the protrusive activity of the cell, while the protrusive activity biases this polarity axis. Using a novel minimal physical model, we show that this feedback is sufficient by itself to recapitulate the quantitative properties of cell migration in the timescale of hours. Our work highlights the importance of the coupling between high-level cellular functions in stabilizing the direction of migration over long timescales.
2.

Optogenetic control of small GTPases reveals RhoA mediates intracellular calcium signaling.

blue CRY2/CIB1 iLID HEK293T HeLa hTERT RPE-1 MDCK Signaling cascade control
J Biol Chem, 13 Jan 2021 DOI: 10.1016/j.jbc.2021.100290 Link to full text
Abstract: Rho/Ras family small GTPases are known to regulate numerous cellular processes, including cytoskeletal reorganization, cell proliferation, and cell differentiation. These processes are also controlled by Ca2+, and consequently, crosstalk between these signals is considered likely. However, systematic quantitative evaluation has not yet been reported. To fill this gap, we constructed optogenetic tools to control the activity of small GTPases (RhoA, Rac1, Cdc42, Ras, Rap, and Ral) using an improved light-inducible dimer system (iLID). We characterized these optogenetic tools with genetically encoded red fluorescence intensity-based small GTPase biosensors and confirmed these optogenetic tools' specificities. Using these optogenetic tools, we investigated calcium mobilization immediately after small GTPase activation. Unexpectedly, we found that a transient intracellular calcium elevation was specifically induced by RhoA activation in RPE1 and HeLa cells. RhoA activation also induced transient intracellular calcium elevation in MDCK and HEK293T cells, suggesting that generally RhoA induces calcium signaling. Interestingly, the molecular mechanisms linking RhoA activation to calcium increases were shown to be different among the different cell types: In RPE1 and HeLa cells, RhoA activated phospholipase C epsilon (PLCε) at the plasma membrane, which in turn induced Ca2+ release from the endoplasmic reticulum (ER). The RhoA-PLCε axis induced calcium-dependent NFAT nuclear translocation, suggesting it does activate intracellular calcium signaling. Conversely, in MDCK and HEK293T cells, RhoA-ROCK-myosin II axis induced the calcium transients. These data suggest universal coordination of RhoA and calcium signaling in cellular processes, such as cellular contraction and gene expression.
3.

Combination of photoactivation with lattice light-sheet imaging reveals untemplated, lamellar ruffle generation by PA-Rac1.

blue AsLOV2 hTERT RPE-1 Control of cytoskeleton / cell motility / cell shape
bioRxiv, 6 Sep 2020 DOI: 10.1101/2020.09.01.276824 Link to full text
Abstract: Membrane protrusions that occur on the dorsal surface of a cell are an excellent experimental system to study actin machinery at work in a living cell. Small GTPase Rac1 controls the membrane protrusions that form and encapsulate extracellular volumes to perform pinocytic or phagocytic functions. Here, capitalizing on rapid volumetric imaging capabilities of lattice light-sheet microscopy (LLSM), we describe optogenetic approaches using photoactivable Rac1 (PA-Rac1) for controlled ruffle generation. We demonstrate that PA-Rac1 activation needs to be continuous, suggesting a threshold local concentration for sustained actin polymerization leading to ruffling. We show that Rac1 activation leads to actin assembly at the dorsal surface of the cell membrane that result in sheet-like protrusion formation without any requirement of a template. Further, this approach can be used to study the complex morpho-dynamics of the protrusions or to investigate specific proteins that may be enriched in the ruffles. Deactivating PA-Rac1 leads to complex contractile processes resulting in formation of macropinosomes. Using multicolour imaging in combination with these approaches, we find that Myo1e specifically is enriched in the ruffles.
4.

The mitotic protein NuMA plays a spindle-independent role in nuclear formation and mechanics.

blue LOVTRAP hTERT RPE-1
bioRxiv, 3 May 2020 DOI: 10.1101/2020.05.02.070680 Link to full text
Abstract: Eukaryotic cells typically form a single, round nucleus after mitosis, and failures to do so can compromise genomic integrity. How mammalian cells form such a nucleus remains incompletely understood. NuMA is a spindle protein whose disruption results in nuclear fragmentation. What role NuMA plays in nuclear integrity, or whether its perceived role stems from its spindle function, is unclear. Here, we use live imaging to demonstrate that NuMA plays a spindle-independent role in forming a single, round nucleus. NuMA keeps the decondensing chromosome mass compact at mitotic exit, and promotes a mechanically robust nucleus. NuMA’s C-terminus binds DNA in vitro and chromosomes in interphase, while its coiled-coil acts as a regulatory and structural hub: it prevents NuMA from binding chromosomes at mitosis, regulates its nuclear mobility and is essential for nuclear formation. Thus, NuMA plays a long-range structural role in building and maintaining an intact nucleus, as it does for the spindle, playing a protective role over the cell cycle.
5.

Optogenetic stimulation of phosphoinositides reveals a critical role of primary cilia in eye pressure regulation.

blue CRY2/CIB1 GM01676 hTERT RPE-1 human retinal pigment epithelium cells mouse in vivo Control of cytoskeleton / cell motility / cell shape
Sci Adv, 29 Apr 2020 DOI: 10.1126/sciadv.aay8699 Link to full text
Abstract: Glaucoma is a group of progressive optic neuropathies that cause irreversible vision loss. Although elevated intraocular pressure (IOP) is associated with the development and progression of glaucoma, the mechanisms for its regulation are not well understood. Here, we have designed CIBN/CRY2-based optogenetic constructs to study phosphoinositide regulation within distinct subcellular compartments. We show that stimulation of CRY2-OCRL, an inositol 5-phosphatase, increases aqueous humor outflow and lowers IOP in vivo, which is caused by a calcium-dependent actin rearrangement of the trabecular meshwork cells. Phosphoinositide stimulation also rescues defective aqueous outflow and IOP in a Lowe syndrome mouse model but not in IFT88fl/fl mice that lack functional cilia. Thus, our study is the first to use optogenetics to regulate eye pressure and demonstrate that tight regulation of phosphoinositides is critical for aqueous humor homeostasis in both normal and diseased eyes.
6.

ESCRT-mediated phagophore sealing during mitophagy.

blue LOVTRAP hTERT RPE-1 Organelle manipulation
Autophagy, 1 Aug 2019 DOI: 10.1080/15548627.2019.1639301 Link to full text
Abstract: Inactivation of the endosomal sorting complex required for transport (ESCRT) machinery has been reported to cause autophagic defects, but the exact functions of ESCRT proteins in macroautophagy/autophagy remain incompletely understood. Using live-cell fluorescence microscopy we found that the filament-forming ESCRT-III subunit CHMP4B was recruited transiently to nascent autophagosomes during starvation-induced autophagy and mitophagy, with residence times of about 1 and 2 min, respectively. Correlative light microscopy and electron tomography revealed CHMP4B recruitment at a late step in mitophagosome formation. The autophagosomal dwell time of CHMP4B was strongly increased by depletion of the regulatory ESCRT-III subunit CHMP2A. Using a novel optogenetic closure assay we observed that depletion of CHMP2A inhibited phagophore sealing during mitophagy. Consistent with this, depletion of CHMP2A and other ESCRT-III subunits inhibited both PRKN/PARKIN-dependent and -independent mitophagy. We conclude that the ESCRT machinery mediates phagophore closure, and that this is essential for mitophagic flux. Abbreviations: BSA: bovine serum albumin; CHMP: chromatin-modifying protein; CLEM: correlative light and electron microscopy; EGFP: enhanced green fluorescent protein; ESCRT: endosomal sorting complex required for transport; HEPES: 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; HRP: horseradish peroxidase; ILV: intralumenal vesicle; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; LOV2: light oxygen voltage 2; MLS: mitochondrial localization sequence; MT-CO2: mitochondrially encoded cytochrome c oxidase II; O+A: oligomycin and antimycin A; PBS: phosphate-buffered saline; PIPES: piperazine-N,N-bis(2-ethanesulfonic acid); PRKN/PARKIN: parkin RBR E3 ubiquitin protein ligase; RAB: RAS-related in brain; SD: standard deviation; SEM: standard error of the mean; TOMM20: TOMM20: translocase of outer mitochondrial membrane 20; VCL: vinculin; VPS4: vacuolar protein sorting protein 4; Zdk1: Zdark 1; TUBG: Tubulin gamma chain.
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