Cells generate a highly diverse microtubule network to carry out different activities. This network is comprised of distinct tubulin isotypes, tubulins with different post-translational ...modifications, and many microtubule-based structures. Defects in this complex system cause numerous human disorders. However, how different microtubule subtypes in this network regulate cellular architectures and activities remains largely unexplored. Emerging tools such as photosensitive pharmaceuticals, chemogenetics, and optogenetics enable the spatiotemporal manipulation of structures, dynamics, post-translational modifications, and cross-linking with actin filaments in target microtubule subtypes. This review summarizes the design rationale and applications of these new approaches and aims to provide a roadmap for researchers navigating the intricacies of microtubule dynamics and their post-translational modifications in cellular contexts, thereby opening new avenues for therapeutic interventions.
Light-induced dimerizing systems, e.g. iLID, are an increasingly utilized optogenetics tool to perturb cellular signaling. The major benefit of this technique is that it allows external ...spatiotemporal control over protein localization with sub-cellular specificity. However, when it comes to local recruitment of signaling components to the plasmamembrane, this precision in localization is easily lost due to rapid diffusion of the membrane anchor. In this study, we explore different approaches of countering the diffusion of peripheral membrane anchors, to the point where we detect immobilized fractions with iFRAP on a timescale of several minutes. One method involves simultaneous binding of the membrane anchor to a secondary structure, the microtubules. The other strategy utilizes clustering of the anchor into large immobile structures, which can also be interlinked by employing tandem recruitable domains. For both approaches, the anchors are peripheral membrane constructs, which also makes them suitable for in vitro use. Upon combining these slower diffusing anchors with recruitable guanine exchange factors (GEFs), we show that we can elicit much more localized morphological responses from Rac1 and Cdc42 as compared to a regular CAAX-box based membrane anchor in living cells. Thanks to these new slow diffusing anchors, more precisely defined membrane recruitment experiments are now possible.
Regulated secretion is critical for diverse biological processes ranging from immune and endocrine signaling to synaptic transmission. Botulinum and tetanus neurotoxins, which specifically proteolyze ...vesicle fusion proteins involved in regulated secretion, have been widely used as experimental tools to block these processes. Genetic expression of these toxins in the nervous system has been a powerful approach for disrupting neurotransmitter release within defined circuitry, but their current utility in the brain and elsewhere remains limited by lack of spatial and temporal control. Here we engineered botulinum neurotoxin B so that it can be activated with blue light. We demonstrate the utility of this approach for inducibly disrupting excitatory neurotransmission, providing a first-in-class optogenetic tool for persistent, light-triggered synaptic inhibition. In addition to blocking neurotransmitter release, this approach will have broad utility for conditionally disrupting regulated secretion of diverse bioactive molecules, including neuropeptides, neuromodulators, hormones, and immune molecules.
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•Few tools allow inhibition of neural activity for long time periods with light•We engineered botulinum neurotoxin B so that it can be activated with blue light•Photoactivated botulinum toxin efficiently cleaves the SNARE protein VAMP2•We demonstrate utility in diverse systems, from mammalian brain slices to worms
Few tools in neuroscience allow long-term synapse inhibition with light. We engineered botulinum neurotoxin B so that it can be switched on with blue light. Light activation results in robust proteolysis of VAMP2, a SNARE protein universally required for neurotransmitter release.
Compartmentalized protein recruitment is a fundamental feature of signal transduction. Accordingly, the cell cortex is a primary site of signaling supported by the recruitment of protein regulators ...to the plasma membrane. Recent emergence of optogenetic strategies designed to control localized protein recruitment has offered valuable toolsets for investigating spatiotemporal dynamics of associated signaling mechanisms. However, determining proper recruitment parameters is important for optimizing synthetic control. In this chapter, we describe a stepwise process for building linear differential equation models that characterize the kinetics and spatial distribution of optogenetic protein recruitment to the plasma membrane. Specifically, we outline how to construct (1) ordinary differential equations that capture the kinetics, efficiency, and magnitude of recruitment and (2) partial differential equations that model spatial recruitment dynamics and diffusion. Additionally, we explore how these models can be used to evaluate the overall system performance and determine how component parameters can be tuned to optimize synthetic recruitment.
Cells experience time-varying and spatially heterogeneous chemokine signals in vivo, 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). PLCβ regulates many key processes, such as hematopoiesis, synaptogenesis, and cell cycle, and is therefore implicated in terminal-debilitating diseases, including cancer, epilepsy, Huntington’s Disease, and Alzheimer’s Disease. However, due to a lack of genetic and pharmacological tools, examining how the dynamic regulation of PLCβ signaling controls cellular physiology has been difficult. Since activated PLCβ induces several abrupt cellular changes, including cell morphology, examining how the other pathways downstream of Gq-GPCRs contribute to the overall signaling has also been difficult. Here we show the engineering, validation, and application of a highly selective and efficient optogenetic inhibitor (Opto-dHTH) to completely disrupt GαqGTP-PLCβ interactions reversibly in user-defined cellular-subcellular regions on optical command. Using this newly gained PLCβ signaling control, our data indicate that the molecular competition between RhoGEFs and PLCβ for GαqGTP determines the potency of Gq-GPCR-governed directional cell migration.
Light-inducible dimers are powerful tools for cellular optogenetics, as they can be used to control the localization and activity of proteins with high spatial and temporal resolution. Despite the ...generality of the approach, application of light-inducible dimers is not always straightforward, as it is frequently necessary to test alternative dimer systems and fusion strategies before the desired biological activity is achieved. This process is further hindered by an incomplete understanding of the biophysical/biochemical mechanisms by which available dimers behave and how this correlates to in vivo function. To better inform the engineering process, we examined the biophysical and biochemical properties of three blue-light-inducible dimer variants (cryptochrome2 (CRY2)/CIB1, iLID/SspB, and LOVpep/ePDZb) and correlated these characteristics to in vivo colocalization and functional assays. We find that the switches vary dramatically in their dark and lit state binding affinities and that these affinities correlate with activity changes in a variety of in vivo assays, including transcription control, intracellular localization studies, and control of GTPase signaling. Additionally, for CRY2, we observe that light-induced changes in homo-oligomerization can have significant effects on activity that are sensitive to alternative fusion strategies.
Rho GTPase family members are known regulators of directed migration and therefore play key roles in processes including development, the immune response and cancer metastasis. However, their ...individual contributions to these processes are complex. Here, we modify the activity of the two Rho GTPase family members Rac and Cdc42 by optogenetically recruiting specific guanine nucleotide exchange factor (GEF) DH or PH domains to defined regions of the cell membrane. We find that the localized activation of both GTPases produces lamellipodia in cells plated on a fibronectin substrate. By using a novel optotaxis assay, we show that biased activation can drive directional migration. Interestingly, in the absence of exogenous fibronectin, Rac activation is insufficient to produce stable lamellipodia or directional migration whereas Cdc42 activation is sufficient for these processes. We find that a remarkably small amount of fibronectin (<10 puncta per protrusion) is necessary to support stable GTPase-driven lamellipodia formation. Cdc42 bypasses the need for exogenous fibronectin by stimulating cellular fibronectin deposition under the newly formed lamellipodia.This article has an associated First Person interview with the first author of the paper.
The objective of this study was to evaluate the safety and effectiveness of the TearCare
System in adult patients with clinically significant dry eye disease (DED).
This was a prospective, ...single-center, randomized, parallel-group, clinical trial. Subjects with DED were randomized to either a single TearCare treatment conducted at the clinic or 4 weeks of daily warm compress (WC) therapy. The TearCare procedure consisted of 12 minutes of thermal eyelid treatment immediately followed by manual expression of the meibomian glands. WC therapy consisted of once daily application of the compresses to the eyelids for 5 minutes. Subjects were followed until 6 months post-treatment. The primary effectiveness end point was defined as change from baseline to 4 weeks for tear breakup time (TBUT). Secondary effectiveness end points included meibomian gland assessment, corneal and conjunctival staining scores, and assessment of dry eye symptoms using validated questionnaires. Safety was evaluated by collecting device-related adverse events, intraocular pressure, and best spectacle-corrected Snellen Visual acuity.
Twenty-four subjects were enrolled and all subjects completed 6 months follow-up. At the 1-month follow-up, TearCare subjects demonstrated an improvement from baseline in mean (±SD) TBUT of 11.7±2.6 seconds compared with an average worsening of -0.3±1.1 seconds for subjects in the WC group (
<0.0001). Significantly greater improvements in the change from baseline in meibomian gland scores, as well as corneal and conjunctival staining scores, were observed in the TearCare group. Subjects in the TearCare group also showed significantly greater improvement in dry eye symptoms as measured by the 3 questionnaires. Both treatments were well-tolerated.
The findings of this pilot study suggest that the TearCare System is an effective treatment option for patients with DED, with the effects on the signs and symptoms of DED persisting for at least 6 months.
Several light-inducible hetero-dimerization tools have been developed to spatiotemporally control subcellular localization and activity of target proteins or their downstream signaling. In contrast ...to other genetic technologies, such as CRISPR-mediated genome editing, these optogenetic tools can locally control protein localization on the second timescale. In addition, these tools can be used to understand the sufficiency of target proteins' function and manipulate downstream events. In this chapter, I will present methods for locally activating cytoplasmic dynein at the mitotic cell cortex in human cells, with a focus on how to generate knock-in cell lines and set up a microscope system.
The organelle interface emerges as a dynamic platform for a variety of biological responses. However, their study has been limited by the lack of tools to manipulate their occurrence in live cells ...spatiotemporally. Here, we report the development of a genetically encoded light-inducible tethering (LIT) system allowing the induction of contacts between endoplasmic reticulum (ER) and mitochondria, taking advantage of a pair of light-dependent heterodimerization called an iLID system. We demonstrate that the iLID-based LIT approach enables control of ER–mitochondria tethering with high spatiotemporal precision in various cell types including primary neurons, which will facilitate the functional study of ER–mitochondrial contacts.
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