Search

Chemically induced dimerization of FKBP and FRB using rapamycin and rapamycin analogs has been utilized in a variety of biological applications. Formation of the FKBP-rapamycin-FRB ternary complex is typically used to activate a biological process and this interaction has proven to be essentially irreversible. In many cases, it would be beneficial to also have temporal control over deactivating a biological process once it has been initiated. Thus, we developed the first reactive oxygen species-generating rapamycin analog toward this goal. The BODIPY-rapamycin analog BORap is capable of dimerizing FKBP and FRB to form a ternary complex, and upon irradiation with 530 nm light, generates singlet oxygen to oxidize and inactivate proteins of interest fused to FKBP/FRB., Utilization of a ROS-generating chromophore for the development of reversible control of rapamycin-induced protein dimerization via targeted oxidation.

Cell surface receptors are important proteins that mediate communication between the cells and their outside environment, and also play essential roles in the control of a wide variety of biological processes, such as cell cycle, proliferation, communication, migration and apoptosis. Receptor oligomerization is an essential signal transduction mechanism that cell surface receptors use to transmit extracellular signals into the internal cytosol cellular machinery. Therefore, regulating receptor oligomerization provides an opportunity to customize cellular signaling and to direct cellular behavior in a user-defined manner. Some techniques have been developed for receptor oligomerization regulation, such as chemically induced dimerization (CID) and optogenetics, which involve traditional genetic engineering. However, the process of genetic manipulation is time-consuming, unpredictable and inefficient. Thus, development of nongenetic strategies for precisely regulating receptor oligomerization remains a desirable goal. Recently, along with the utilization of DNA, protein, small molecules and stimuli-responsive materials-based nongenetic engineering strategies, various receptor oligomerization and multiple cellular behaviors could be regulated, including migration, proliferation, apoptosis, differentiation and immune responses, etc. In this review, we aim to systematically introduce advances in the development of nongenetic engineering strategies for regulating receptor oligomerization, and provide insights into the existing challenges and future perspectives of this field.

Light‐inducible approaches provide a means to control biological systems with spatial and temporal resolution that is unmatched by traditional genetic perturbations. Recent developments of optogenetic and chemo‐optogenetic systems for induced proximity in cells facilitate rapid and reversible manipulation of highly dynamic cellular processes and have become valuable tools in diverse biological applications. New expansions of the toolbox facilitate control of signal transduction, genome editing, “painting” patterns of active molecules onto cellular membranes, and light‐induced cell cycle control. A combination of light‐ and chemically induced dimerization approaches have also seen interesting progress. Herein, an overview of optogenetic systems and emerging chemo‐optogenetic systems is provided, and recent applications in tackling complex biological problems are discussed., Proteins under the spotlight: Various optogenetic systems that involve photosensitive proteins are summarized and emerging chemo‐optogenetic systems with caged or photocleavable chemical dimerizers are reviewed (see figure). The advantages and disadvantages of these systems are discussed. Recent biological applications of these strategies are presented.

Chemically induced dimerization (CID) systems, in which two proteins dimerize only in the presence of a small molecule ligand, offer versatile tools for small molecule sensing and actuation. However, only a handful of CID systems exist and creating one with the desired sensitivity and specificity for any given ligand is an unsolved problem. Here, we developed a combinatorial binders-enabled selection of CID (COMBINES-CID) method broadly applicable to different ligands. We demonstrated a proof-of-principle by generating nanobody-based heterodimerization systems induced by cannabidiol with high ligand selectivity. We applied the CID system to a sensitive sandwich enzyme-linked immunosorbent assay-like assay of cannabidiol in body fluids with a detection limit of ∼0.25 ng/mL. COMBINES-CID provides an efficient, cost-effective solution for expanding the biosensor toolkit for small molecule detection.

As part of the ‘Central Dogma’ of molecular biology, the function of proteins and nucleic acids within a cell is determined by their primary sequence. Recent work, however, has shown that within living cells the role of many proteins and RNA molecules can be influenced by the physical state in which the molecule is found. Within living cells, both protein and RNA molecules are observed to condense into non-membrane-bound yet distinct structures such as liquid droplets, hydrogels and insoluble aggregates. These unique intracellular organizations, collectively termed biomolecular condensates, have been found to be vital in both normal and pathological conditions. Here, we review the latest studies that have developed molecular tools attempting to recreate artificial biomolecular condensates in living cells. We will describe their design principles, implementation and unique characteristics, along with limitations. We will also introduce how these tools can be used to probe and perturb normal and pathological cell functions, which will then be complemented with discussions of remaining areas for technological advance under this exciting theme.

Various RNA‐targeting approaches have been engineered to modify specific sites on endogenous transcripts, breaking new ground for a variety of basic research tools and promising clinical applications in the future. Here, we combine site‐directed adenosine‐to‐inosine RNA editing with chemically induced dimerization. Specifically, we achieve tight and dose‐dependent control of the editing reaction with gibberellic acid, and obtain editing yields up to 20 % and 44 % in the endogenous STAT1 and GAPDH transcript in cell culture. Furthermore, the disease‐relevant MECP2 R106Q mutation was repaired with editing yields up to 42 %. The introduced principle will enable new applications where temporal or spatiotemporal control of an RNA‐targeting mechanism is desired., RNA editing: Site‐directed adenosine‐to‐inosine RNA editing was engineered to be under control of the plant hormone gibberellic acid, applying the mechanism of chemically induced dimerization. Tight control and editing yields up to 44 % where achieved on endogenous targets in human cell culture.

Compartmentation of proteins into biomolecular condensates or membraneless organelles formed by phase separation is an emerging principle for the regulation of cellular processes. Creating synthetic condensates that accommodate specific intracellular proteins on demand would have various applications in chemical biology, cell engineering, and synthetic biology. Here, we report the construction of synthetic protein condensates capable of recruiting and/or releasing proteins of interest in living mammalian cells in response to a small molecule or light. By a modular combination of a tandem fusion of two oligomeric proteins, which forms phase-separated synthetic protein condensates in cells, with a chemically induced dimerization tool, we first created a chemogenetic protein condensate system that can rapidly recruit target proteins from the cytoplasm to the condensates by addition of a small-molecule dimerizer. We next coupled the protein-recruiting condensate system with an engineered proximity-dependent protease, which gave a second protein condensate system wherein target proteins previously expressed inside the condensates are released into the cytoplasm by small-molecule-triggered protease recruitment. Furthermore, an optogenetic condensate system that allows reversible release and sequestration of protein activity in a repeatable manner using light was constructed successfully. These condensate systems were applicable to control protein activity and cellular processes such as membrane ruffling and ERK signaling in a time scale of minutes. This proof-of-principle work provides a new platform for chemogenetic and optogenetic control of protein activity in mammalian cells and represents a step toward tailor-made engineering of synthetic protein condensate-based soft materials with various functionalities for biological and biomedical applications.

Chemogenetic methods that enable the rapid translocation of specific signaling proteins in living cells using small molecules are powerful tools for manipulating and interrogating intracellular signaling networks. However, existing techniques rely on chemically induced dimerization of two protein components and have certain limitations, such as a lack of reversibility, bioorthogonality, and usability. Here, by expanding our self-localizing ligand-induced protein translocation (SLIPT) approach, we have developed a versatile chemogenetic system for plasma membrane (PM)-targeted protein translocation. In this system, a novel engineered Escherichia coli dihydrofolate reductase in which a hexalysine (K6) sequence is inserted in a loop region (iK6DHFR) is used as a universal protein tag for PM-targeted SLIPT. Proteins of interest that are fused to the iK6DHFR tag can be specifically recruited from the cytoplasm to the PM within minutes by addition of a myristoyl-d-Cys-tethered trimethoprim ligand (mDcTMP). We demonstrated the broad applicability and robustness of this engineered protein–synthetic ligand pair as a tool for the conditional activation of various types of signaling molecules, including protein and lipid kinases, small GTPases, heterotrimeric G proteins, and second messengers. In combination with a competitor ligand and a culture-medium flow chamber, we further demonstrated the application of the system for chemically manipulating protein localization in a reversible and repeatable manner to generate synthetic signal oscillations in living cells. The present bioorthogonal iK6DHFR/mDcTMP-based SLIPT system affords rapid, reversible, and repeatable control of the PM recruitment of target proteins, offering a versatile and easy-to-use chemogenetic platform for chemical and synthetic biology applications.

A vast range of biomedical applications relies on the specificity of interactions between an antigen and its cognate receptor or antibody. This specificity can be highest when said antigen is a non-natural (synthetic) molecule introduced into a biological setting as a bio-orthogonal ligand. This review aims to present the development of this methodology from the early discovery of haptens a century ago to the recent clinical trials. We discuss such methodologies as antibody recruitment, artificial internalizing receptors and chemically induced dimerization, present the use of chimeric receptors and/or bispecific antibodies to achieve drug targeting and transcytosis, and illustrate how these platforms most impressively found use in the engineering of therapeutic cells such as the chimeric antigen receptor cells. This review aims to be of interest to a broad scientific audience and to spur the development of synthetic artificial ligands for biomedical applications.

The photoactivatable chemically induced dimerization (photo-CID) technique for tag-fused proteins is one of the most promising methods for regulating subcellular protein translocations and protein-protein interactions. However, light-induced covalent protein dimerization in living cells has yet to be established, despite its various advantages. Herein, we developed a photoactivatable covalent protein-labeling technology by applying a caged ligand to the BL-tag system, a covalent protein labeling system that uses mutant β-lactamase. We further developed CBHD, a caged protein dimerizer, using caged BL-tag and HaloTag ligands, and achieved light-induced protein translocation from the cytoplasm to subcellular regions. In addition, this covalent photo-CID system enabled quick protein translocation to a laser-illuminated microregion. These results indicate that the covalent photo-CID system will expand the scope of CID applications in the optical manipulation of cellular functions.

Biomolecular condensates concentrate macromolecules into discrete cellular foci without an encapsulating membrane. Condensates are often presumed to increase enzymatic reaction rates through increased concentrations of enzymes and substrates (mass action), although this idea has not been widely tested and other mechanisms of modulation are possible. Here we describe a synthetic system where the SUMOylation enzyme cascade is recruited into engineered condensates generated by liquid–liquid phase separation of multidomain scaffolding proteins. SUMOylation rates can be increased up to 36-fold in these droplets compared to the surrounding bulk, depending on substrate KM. This dependency produces substantial specificity among different substrates. Analyses of reactions above and below the phase-separation threshold lead to a quantitative model in which reactions in condensates are accelerated by mass action and changes in substrate KM, probaby due to scaffold-induced molecular organization. Thus, condensates can modulate reaction rates both by concentrating molecules and physically organizing them. A chemically induced dimerization strategy was used to recruit SUMOylation enzymes into condensates, enabling quantification of the effect of phase separation on the activity of a SUMOylation enzyme cascade reaction.

Telomerase-free cancer cells employ a recombination-based alternative lengthening of telomeres (ALT) pathway that depends on ALT-associated promyelocytic leukemia nuclear bodies (APBs), whose function is unclear. We find that APBs behave as liquid condensates in response to telomere DNA damage, suggesting two potential functions: condensation to enrich DNA repair factors and coalescence to cluster telomeres. To test these models, we developed a chemically induced dimerization approach to induce de novo APB condensation in live cells without DNA damage. We show that telomere-binding protein sumoylation nucleates APB condensation via interactions between small ubiquitin-like modifier (SUMO) and SUMO interaction motif (SIM), and that APB coalescence drives telomere clustering. The induced APBs lack DNA repair factors, indicating that APB functions in promoting telomere clustering can be uncoupled from enriching DNA repair factors. Indeed, telomere clustering relies only on liquid properties of the condensate, as an alternative condensation chemistry also induces clustering independent of sumoylation. Our findings introduce a chemical dimerization approach to manipulate phase separation and demonstrate how the material properties and chemical composition of APBs independently contribute to ALT, suggesting a general framework for how chromatin condensates promote cellular functions.

A genetically encoded caffeine‐operated synthetic module (COSMO) is introduced herein as a robust chemically induced dimerization (CID) system. COSMO enables chemogenetic manipulation of biological processes by caffeine and its metabolites, as well as caffeinated beverages, including coffee, tea, soda, and energy drinks. This CID tool, evolved from an anti‐caffeine nanobody via cell‐based high‐throughput screening, permits caffeine‐inducible gating of calcium channels, tumor killing via necroptosis, growth factors‐independent activation of tyrosine receptor kinase signaling, and enhancement of nanobody‐mediated antigen recognition for the severe acute respiratory distress coronavirus 2 (SARS‐CoV‐2) spike protein. Further rationalized engineering of COSMO leads to 34–217‐fold enhancement in caffeine sensitivity (EC50 = 16.9 nanomolar), which makes it among the most potent CID systems like the FK506 binding protein (FKBP)–FKBP rapamycin binding domain (FRB)–rapamycin complex. Furthermore, bivalent COSMO (biCOMSO) connected with a long linker favors intramolecular dimerization and acts as a versatile precision switch when inserted in host proteins to achieve tailored function. Given the modularity and high transferability of COMSO and biCOSMO, these chemical biology tools are anticipated to greatly accelerate the development of therapeutic cells and biologics that can be switched on and off by caffeinated beverages commonly consumed in the daily life., A robust chemically induced dimerization (CID) system, caffeine‐operated synthetic module (COSMO), is developed for chemogenetic control of various protein activities in live cells. Bivalent COSMOs with various linkers are engineered to enhance the caffeine sensitivity toward the low nanomolar range, and to enable intramolecular dimerization for precise control of protein functions with caffeine and its metabolites, as well as caffeinated beverages.

Trypanosomal and leishmanial infections claim tens of thousands of lives each year. The metabolism of these unicellular eukaryotic parasites differs from the human host and their enzymes thus constitute promising drug targets. Tryparedoxin (Tpx) from Trypanosoma brucei is the essential oxidoreductase in the parasite's hydroperoxide-clearance cascade. In vitro and in vivo functional assays show that a small, selective inhibitor efficiently inhibits Tpx. With X-ray crystallography, SAXS, analytical SEC, SEC-MALS, MD simulations, ITC, and NMR spectroscopy, we show how covalent binding of this monofunctional inhibitor leads to Tpx dimerization. Intra- and intermolecular inhibitor-inhibitor, protein-protein, and inhibitor-protein interactions stabilize the dimer. The behavior of this efficient antitrypanosomal molecule thus constitutes an exquisite example of chemically induced dimerization with a small, monovalent ligand that can be exploited for future drug design.

Many dynamic biological processes are regulated by protein-protein interactions and protein localization. Experimental techniques to probe such processes with temporal and spatial precision include photoactivatable proteins and chemically-induced dimerization (CID) of proteins. CID has been used to study several cellular events, especially cell signaling networks, which are often reversible. However, chemical dimerizers that can be both rapidly activated and deactivated with high spatiotemporal resolution are currently limited. Herein, we present a novel chemical inducer of protein dimerization that can be rapidly turned on and off using single pulses of light at two orthogonal wavelengths. We demonstrate the utility of this molecule by controlling peroxisome transport and mitotic checkpoint signaling in living cells. Our system highlights and enhances the spatiotemporal control offered by CID. This tool addresses biological questions on sub-cellular levels by controlling protein-protein interactions.

Small-molecule regulation is a powerful switching tool to manipulate cell signal transduction for a desired function; however, most available methods usually require genetic engineering to endow cells with responsiveness to user-defined small molecules. Herein, we demonstrate a nongenetic approach for small-molecule-controlled receptor activation and consequent cell behavior manipulation that is based on DNA-mediated chemically induced dimerization (D-CID). D-CID uses a programmable chemical-responsive DNA nanodevice to trigger DNA strand displacement and induce the activation of c-Met, a tyrosine kinase receptor cognate for hepatocyte growth factor, through dimerization. Through the use of various functional nucleic acids, including aptamers and DNAzymes, as recognition modules, the versatility of D-CID in inducing c-Met signaling upon addition of various small-molecular or ionic cues, including ATP, histidine, and Zn2+ , is demonstrated. Moreover, owing its multi-input properties, D-CID can be used to manipulate the behaviors of multiple cell populations simultaneously in a selective and programmable fashion.

The spatiotemporal dynamics of proteins and organelles play an important role in controlling diverse cellular processes. Optogenetic tools using photosensitive proteins and chemically induced dimerization (CID), which allow control of protein dimerization, have been used to elucidate the dynamics of biological systems and to dissect the complicated biological regulatory networks. However, the inherent limitations of current optogenetic and CID systems remain a significant challenge for the fine‐tuning of cellular activity at precise times and locations. Herein, we present a novel chemo‐optogenetic approach, photoswitchable chemically induced dimerization (psCID), for controlling cellular function by using blue light in a rapid and reversible manner. Moreover, psCID is tunable; that is, the dimerization and dedimerization degrees can be fine‐tuned by applying different doses of illumination. Using this approach, we control the localization of proteins and positioning of organelles in live cells with high spatial (μm) and temporal (ms) precision.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) “anchor binders” that first bind to a ligand of interest and then 2) “dimerization binders” that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 10(9) complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Compositions of proteins and lipids within cilia and on the ciliary membrane are maintained to be distinct from those of the cytoplasm and plasma membrane, respectively, by the presence of the ciliary gate. INPP5E is a phosphoinositide 5-phosphatase that is localized on the ciliary membrane by anchorage via its C-terminal prenyl moiety. In addition, the ciliary membrane localization of INPP5E is determined by the small GTPase ARL13B. However, it remained unclear as to how ARL13B participates in the localization of INPP5E. We here show that wild-type INPP5E, INPP5E(WT), in ARL13B-knockout cells and an INPP5E mutant defective in ARL13B binding, INPP5E(ΔCTS), in control cells were unable to show steady-state localization on the ciliary membrane. However, not only INPP5E(WT) but also INPP5E(ΔCTS) was able to rescue the abnormal localization of ciliary proteins in INPP5E-knockout cells. Analysis using the chemically induced dimerization system demonstrated that INPP5E(WT) in ARL13B-knockout cells and INPP5E(ΔCTS) in control cells were able to enter cilia, but neither was retained on the ciliary membrane due to the lack of the INPP5E–ARL13B interaction. Thus, our data demonstrate that binding of INPP5E to ARL13B is essential for its steady-state localization on the ciliary membrane but is dispensable for its entry into cilia., Summary: We here demonstrate that the interaction of INPP5E with ARL13B is crucial for its retention on the ciliary membrane but dispensable for its entry into cilia.

Sintezna biologija stremi k pripravi novih molekularnih metod za preučevanje in uravnavanje procesov v celicah sesalcev. Diplomska naloga predstavlja nov, z majhnimi molekulami nadzorovan dimerizacijski sistem na osnovi ločenih enoverižnih fragmentov protiteles. Za dokaz koncepta smo si izbrali enoverižni variabilni fragment protitelesa proti estradiolu v fuziji s kresničkino luciferazo, ki je delovala kot poročevalski sistem. Z dodatkom estradiola v gojišče smo v celicah sesalcev uspešno sprožili dimerizacijo ločeno izražene lahke in težke verige protitelesa. Z MTT testom smo dokazali, da sistem deluje tudi pri koncentraciji estradiola, ki za celice ni toksična. S takšnim sistemom bi lahko na preprost način nadzorovali celične procese, kot so transkripcija, lokalizacija proteinov ali celična signalizacija. V delu smo predstavili le eno izmed protiteles in ligandov, sistem pa bi z uporabo drugih protiteles in ligandov lahko postal univerzalen. Synthetic biology strives to engineer new molecular methods for investigation and regulation of mammalian cell processes. This thesis presents a new dimerization system, controlled by small molecules, based on separated single chain Fab variable fragments. To prove the concept, we chose the anti-β-estradiol single chain Fab variable fragment, fused to a firefly’s luciferase, which served as the reporter system. By adding estradiol in the media of mammalian cells, we successfully initiated the dimerization of the individually expressed light- and heavy- antibody chains. The MTT assay proved that the system functions even when the estradiol concentration is not toxic to cells. With this system, we could easily control cell processes such as transcription, protein localisation and cellular signallization. While we examined only one of the antibodies and ligands, the application of others can make this system universal.

Chemically induced dimerization (CID) has been applied to study numerous biological processes and has important pharmacological applications. However, the complex multistep interactions under various physical constraints involved in CID impose a great challenge for the quantification of the interactions. Furthermore, the mechanical stability of the ternary complexes has not been characterized; hence, their potential application in mechanotransduction studies remains unclear. Here, we report a single-molecule detector that can accurately quantify almost all key interactions involved in CID and the mechanical stability of the ternary complex, in a label-free manner. Its application is demonstrated using rapamycin-induced heterodimerization of FRB and FKBP as an example. We revealed the sufficient mechanical stability of the FKBP/rapamycin/FRB ternary complex and demonstrated its utility in the precise switching of talin-mediated force transmission in integrin-based cell adhesions.

The concerted action of multiple genes in a time-dependent manner controls complex cellular phenotypes, yet the temporal regulation of gene expressions is restricted on a single-gene level, which limits our ability to control higher-order gene networks and understand the consequences of multiplex genetic perturbations. Here we developed a system for temporal regulation of multiple genes. This system combines the simplicity of CRISPR/Cas9 activators for orthogonal targeting of multiple genes and the orthogonality of chemically induced dimerizing (CID) proteins for temporal control of CRISPR/Cas9 activator function. In human cells, these transcription activators exerted simultaneous activation of multiple genes and orthogonal regulation of different genes in a ligand-dependent manner with minimal background. We envision that our system will enable the perturbation of higher-order gene networks with high temporal resolution and accelerate our understanding of gene-gene interactions in a complex biological setting.

Cryo–electron tomography and structural labeling show that the intermediate and light chains of the outer dynein arm (ODA) form a distinct complex, designated ODA-Beak, which can transmit mechanosignals from the nexin–dynein regulatory complex to the heavy chains of ODA., The outer dynein arm (ODA) is a molecular complex that drives the beating motion of cilia/flagella. Chlamydomonas ODA is composed of three heavy chains (HCs), two ICs, and 11 light chains (LCs). Although the three-dimensional (3D) structure of the whole ODA complex has been investigated, the 3D configurations of the ICs and LCs are largely unknown. Here we identified the 3D positions of the two ICs and three LCs using cryo–electron tomography and structural labeling. We found that these ICs and LCs were all localized at the root of the outer-inner dynein (OID) linker, designated the ODA-Beak complex. Of interest, the coiled-coil domain of IC2 extended from the ODA-Beak to the outer surface of ODA. Furthermore, we investigated the molecular mechanisms of how the OID linker transmits signals to the ODA-Beak, by manipulating the interaction within the OID linker using a chemically induced dimerization system. We showed that the cross-linking of the OID linker strongly suppresses flagellar motility in vivo. These results suggest that the ICs and LCs of the ODA form the ODA-Beak, which may be involved in mechanosignaling from the OID linker to the HCs.

The Hippo pathway limits cell number and differentiation by blocking the transcriptional activity of YAP. The fidelity of signal transduction relies on the coordinated activity of a core kinase cassette that includes the kinases Mst1/2 and Lats1/2 and the accessory proteins Mob1 and hSalvador. Mst1/2 contains a kinase domain, linker region, and a C-terminal coiled-coil domain termed SARAH and is responsible for the phosphorylation of downstream components. Activation of Mst1/2 requires phosphorylation of the activation loop, a process believed to be a consequence of trans-autophosphorylation that is promoted by SARAH-domain mediated homodimerization. We aimed to understand what mechanistically triggers Mst1/2 autophosphorylation. Using purified proteins, we show that the kinase domain of Mst2 is sufficient for autophosphorylation but full-length Mst2 undergoes autophosphorylation faster than variants lacking the SARAH domain. This increased autophosphorylation could arise from either a specific contribution of a SARAH-domain mediated homodimer (allostery) or the increased proximity of kinase domains following homodimerization (effective local concentration). We investigated both possibilities using a series of biochemical assays and revealed that autophosphorylation of Mst2 variants lacking a SARAH domain could be stimulated by either chemically induced dimerization or increased localization following recruitment to the surface of a lipid-vesicle. Our results suggest that SARAH-domain mediated homodimerization is not the only event that promotes autophosphorylation of Mst1/2 and, instead, supports a more inclusive model that relies on colocalization. Any cellular event that increases the proximity of Mst1/2 could activate the kinase such as SARAH-domain mediated homodimerization, higher-order complex formation with hSalvador, or membrane recruitment. Citation Format: Thao Tran, Jennifer M. Kavran. Increasing proximity triggers Mst2 autophosphorylation [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr A27.

Cellular protrusions are typically considered as distinct structures associated with specific regulators. However, we found that these regulators coordinately localize as propagating cortical waves, suggesting a common underlying mechanism. These molecular events fell into two excitable networks, the signal transduction network STEN and the cytoskeletal network CEN with different wave substructures. Computational studies using a coupled‐network model reproduced these features and showed that the morphology and kinetics of the waves depended on strengths of feedback loops. Chemically induced dimerization at multiple nodes produced distinct, coordinated alterations in patterns of other network components. Taken together, these studies indicate: STEN positive feedback is mediated by mutual inhibition between Ras/Rap and PIP2, while negative feedback depends on delayed PKB activation; PKBs link STEN to CEN; CEN includes positive feedback between Rac and F‐actin, and exerts fast positive and slow negative feedbacks to STEN. The alterations produced protrusions resembling filopodia, ruffles, pseudopodia, or lamellipodia, suggesting that these structures arise from a common regulatory mechanism and that the overall state of the STEN‐CEN system determines cellular morphology.

Small-molecule perturbation of biological systems is able to tackle biological problems that are not accessible by classical genetic interference methods. Chemically induced dimerization (CID) has been used as a valuable tool to study various biological processes. Recent years have seen tremendous progress in the development of orthogonal and reversible CID systems. These new systems allow control over protein function with unprecedented precision and spatiotemporal resolution. While the primary application of CID has been on dissecting signal transductions, new emerging approaches have extended the scope of this technique to elucidating membrane and protein trafficking.