Browsing by Subject "BIOSENSORS"

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  • Leopold, Anna; Pletnev, Sergei; Verkhusha, Vladislav V. (2020)
    Optically controlled receptor tyrosine kinases (opto-RTKs) allow regulation of RTK signaling using light. Until recently, the majority of opto-RTKs were activated with blue-green light. Fusing a photosensory core module of Deinococcus radiodurans bacterial phytochrome (DrBphP-PCM) to the kinase domains of neurotrophin receptors resulted in opto-RTKs controlled with light above 650 nm. To expand this engineering approach to RTKs of other families, here we combined the DrBpP-PCM with the cytoplasmic domains of EGFR and FGFR1. The resultant Dr-EGFR and Dr-FGFR1 opto-RTKs are rapidly activated with near-infrared and inactivated with far-red light. The opto-RTKs efficiently trigger ERK1/2, PI3K/Akt, and PLC gamma signaling. Absence of spectral crosstalk between the opto-RTKs and green fluorescent protein-based biosensors enables simultaneous Dr-FGFR1 activation and detection of calcium transients. Action mechanism of the DrBphP-PCM-based opto-RTKs is considered using the available RTK structures. DrBphP-PCM represents a versatile scaffold for engineering of opto-RTKs that are reversibly regulated with far-red and near-infrared light. (C) 2020 Elsevier Ltd. All rights reserved.
  • Shcherbakova, Daria M.; Cammer, Natasha Cox; Huisman, Tsipora M.; Verkhusha, Vladislav V.; Hodgson, Louis (2018)
    Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Forster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.
  • Muurinen, Johanna; Pasupulate, Avinash; Lappalainen, Juha; Virta, Marko (2019)
    Whole-cell bioreporters are living organisms and thus using them for detecting environmental contaminants would reflect biological effects of these pollutants. However, bioreporters are not widely used in field studies. Many of the bioreporter field protocols are suitable for liquid samples or include pipetting steps, which is a demanding task outside the laboratory. We present a bioreporter protocol without pipetting or sample type requirements. The protocol utilizes polyester swabs, commonly used in cleanroom technology. As an example contaminant, we used tetracycline and generated test samples with known concentrations up to the maximum tetracycline residue limit of milk set by the European Union (EU) regulation. The matrices of the test samples were Milli-Q water, milk and soil. The swabs were first dipped in the bioreporter cell cultures and then to test samples and luminescence was measured after incubation. The standard deviation of measurements from ten replicate swabs was in the same range as commonly in pipetting protocols (4-19%). The test samples with lowest tetracycline concentration (5 ng mL(-1)) were distinguished from the control samples (0 ng mL(-1) tetracycline). Our results show that swabs can be used together with luminescent whole cell bioreporters, making it possible to conduct the measurements in field conditions.
  • Shcherbakova, Daria M.; Stepanenko, Olesya V.; Turoverov, Konstantin K.; Verkhusha, Vladislav V. (2018)
    Since mammalian tissue is relatively transparent to near-infrared (NIR) light, NIR fluorescentproteins(FPs) engineeredfrombacterialphytochromeshave become widely used probes for non-invasive in vivo imaging. Recently, these genetically encoded NIR probes have been substantially improved, enabling imaging experiments that were not possible previously. Here, we discuss the use of monomeric NIR FPs and NIR biosensors for multiplexed imaging with common visible GFP-based probes and blue light-activatable optogenetic tools. These NIR probes are suitable for visualization of functional activities from molecular to organismal levels. In combination with advanced imaging techniques, such as two-photon microscopy with adaptive optics, photoacoustic tomography and its recent modification reversibly switchable photoacoustic computed tomography, NIR probes allow subcellular resolution at millimeter depths.
  • Redchuk, Taras A.; Karasev, Maksim M.; Omelina, Evgeniya S.; Verkhusha, Vladislav V. (2018)
    Near-infrared (NIR) light-inducible binding of bacterial phytochrome BphP1 to its engineered partner, QPAS1, is used for optical protein regulation in mammalian cells. However, there are no data on the application of the BphP1-QPAS1 pair in cells derived from various mammalian tissues. Here, we tested the functionality of two BphP1-QPAS1-based optogenetic toolsan NIR- and blue-light-sensing system for control of protein localization (iRIS) and an NIR light-sensing system for transcription activation (TA)in several cell types, including cortical neurons. We found that the performance of these optogenetic tools often relied on physiological properties of a specific cell type, such as nuclear transport, which could limit the applicability of the blue-light-sensitive component of iRIS. In contrast, the NIR-light-sensing component of iRIS performed well in all tested cell types. The TA system showed the best performance in cervical cancer (HeLa), bone cancer (U-2 OS), and human embryonic kidney (HEK-293) cells. The small size of the QPAS1 component allowed the design of adeno-associated virus (AAV) particles, which were applied to deliver the TA system to neurons.
  • Leopold, Anna V.; Chernov, Konstantin G.; Shemetov, Anton A.; Verkhusha, Vladislav V. (2019)
    Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.
  • Redchuk, Taras A; Karasev, Maksim M; Verkhusha, Polina V; Donnelly, Sara K; Hülsemann, Maren; Virtanen, Jori; Moore, Henna M; Vartiainen, Maria K; Hodgson, Louis; Verkhusha, Vladislav V (2020)
    Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets. Optogenetic approaches to control protein-protein interactions usually require overexpression of the target proteins. Here the authors integrate intrabodies into near-infrared- and blue-light activatable optogenetic tools to control endogenous proteins in mammalian cells.
  • Liu, Wei; Shcherbakova, Daria M.; Kurupassery, Neel; Li, Yang; Zhou, Qifa; Verkhusha, Vladislav V.; Yao, Junjie (2018)
    A conventional photoacoustic microscopy (PAM) system typically has to make tradeoffs between its spatial resolution and penetration depth, by choosing a fixed configuration of optical excitation and acoustic detection. The single-scale imaging capability of PAM may limit its applications in biomedical studies. Here, we report a quad-mode photoacoustic microscopy (QM-PAM) system with four complementary spatial resolutions and maximum penetration depths. For this we first developed a ring-shaped focused ultrasound transducer that has two independent elements with respective central frequencies at 20 MHz and 40 MHz, providing complementary acoustically-determined spatial resolutions and penetration depths. To accommodate the dual-element ultrasound transducer, we implemented two optical excitation modes to provide tightly-and weakly-focused light illumination. The dual-element acoustic detection combined with the two optical focusing modes can thus provide four imaging scales in a single imaging device, with consistent contrast mechanisms and co-registered field of views. We have demonstrated the multiscale morphological, functional, and molecular imaging capability of QM-PAM in the mouse head, leg and ear in vivo. We expect the high scale flexibility of QM-PAM will enable broad applications in preclinical studies.
  • Li, Lei; Shemetov, Anton A.; Baloban, Mikhail; Hu, Peng; Zhu, Liren; Shcherbakova, Daria M.; Zhang, Ruiying; Shi, Junhui; Yao, Junjie; Wang, Lihong V.; Verkhusha, Vladislav V. (2018)
    Photoacoustic (PA) computed tomography (PACT) benefits from genetically encoded probes with photochromic behavior, which dramatically increase detection sensitivity and specificity through photoswitching and differential imaging. Starting with a DrBphP bacterial phytochrome, we have engineered a near-infrared photochromic probe, DrBphP-PCM, which is superior to the full-length RpBphP1 phytochrome previously used in differential PACT. DrBphP-PCM has a smaller size, better folding, and higher photoswitching contrast. We have imaged both DrBphP-PCM and RpBphP1 simultaneously on the basis of their unique signal decay characteristics, using a reversibly switchable single-impulse panoramic PACT (RS-SIP-PACT) with a single wavelength excitation. The simple structural organization of DrBphPPCM allows engineering a bimolecular PA complementation reporter, a split version of DrBphP-PCM, termed DrSplit. DrSplit enables PA detection of protein-protein interactions in deep-seated mouse tumors and livers, achieving 125-mu m spatial resolution and 530-cell sensitivity in vivo. The combination of RS-SIP-PACT with DrBphP-PCM and DrSplit holds great potential for noninvasive multi-contrast deep-tissue functional imaging.