Curated Optogenetic Publication Database

Abstract: The regulation of PKC epsilon (PKCε) and its downstream effects is still not fully understood, making it challenging to develop targeted therapies or interventions. A more precise tool that enables spatiotemporal control of PKCε activity is thus required. Here, we describe a photo-activatable optogenetic PKCε probe (Opto-PKCε) consisting of an engineered PKCε catalytic domain and a blue-light inducible dimerization domain. Molecular dynamics and AlphaFold simulations enable rationalization of the dark-light activity of the optogenetic probe. We first characterize the binding partners of Opto-PKCε, which are similar to those of PKCε. Subsequent validation of the Opto-PKCε tool is performed with phosphoproteome analysis, which reveals that only PKCε substrates are phosphorylated upon light activation. Opto-PKCε could be engineered for recruitment to specific subcellular locations. Activation of Opto-PKCε in isolated hepatocytes reveals its sustained activation at the plasma membrane is required for its phosphorylation of the insulin receptor at Thr1160. In addition, Opto-PKCε recruitment to the mitochondria results in its lowering of the spare respiratory capacity through phosphorylation of complex I NDUFS4. These results demonstrate that Opto-PKCε may have broad applications for the studies of PKCε signaling with high specificity and spatiotemporal resolution.

Abstract: Komagataella phaffii, also known as Pichia pastoris, is a powerful host for recombinant protein production, in part due to its exceptionally strong and tightly controlled PAOX1 promoter. Most K. phaffii bioprocesses for recombinant protein production rely on PAOX1 to achieve dynamic control in two-phase processes. Cells are first grown under conditions that repress PAOX1 (growth phase), followed by methanol-induced recombinant protein expression (production phase). In this study, we propose a methanol-free approach for dynamic metabolic control in K. phaffii using optogenetics, which can help enhance input tunability and flexibility in process optimization and control. The light-responsive transcription factor EL222 from Erythrobacter litoralis is used to regulate protein production from the PC120 promoter in K. phaffii with blue light. We used two system designs to explore the advantages and disadvantages of coupling or decoupling EL222 integration with that of the gene of interest. We investigate the relationship between EL222 gene copy number and light dosage to improve production efficiency for intracellular and secreted proteins. Experiments in lab-scale bioreactors demonstrate the feasibility of the outlined optogenetic systems as potential alternatives to conventional methanol-inducible bioprocesses using K. phaffii.

Abstract: Cells under high confinement form highly polarized hydrostatic pressure-driven, stable leader blebs that enable efficient migration in low adhesion, environments. Here we investigated the basis of the polarized bleb morphology of metastatic melanoma cells migrating in non-adhesive confinement. Using high-resolution time-lapse imaging and specific molecular perturbations, we found that EGF signaling via PI3K stabilizes and maintains a polarized leader bleb. Protein activity biosensors revealed a unique EGFR/PI3K activity gradient decreasing from rear-to-front, promoting PIP3 and Rac1-GTP accumulation at the bleb rear, with its antagonists PIP2 and RhoA-GTP concentrated at the bleb tip, opposite to the front-to-rear organization of these signaling modules in integrin-mediated mesenchymal migration. Optogenetic experiments showed that disrupting this gradient caused bleb retraction, underscoring the role of this signaling gradient in bleb stability. Mathematical modeling and experiments identified a mechanism where, as the bleb initiates, CD44 and ERM proteins restrict EGFR mobility in a membrane-apposed cortical actin meshwork in the bleb rear, establishing a rear-to-front EGFR-PI3K-Rac activity gradient. Thus, our study reveals the biophysical and molecular underpinnings of cell polarity in bleb-based migration of metastatic cells in non-adhesive confinement, and underscores how alternative spatial arrangements of migration signaling modules can mediate different migration modes according to the local microenvironment.

Abstract: Corynebacterium glutamicum is a key industrial workhorse for producing amino acids and high-value chemicals. Balancing metabolic flow between cell growth and product synthesis is crucial for enhancing production efficiency. Developing dynamic, broadly applicable, and minimally toxic gene regulation tools for C. glutamicum remains challenging, as optogenetic tools ideal for dynamic regulatory strategies have not yet been developed. This study introduces an advanced light-controlled gene expression system using light-controlled RNA-binding proteins (RBP), a first for Corynebacterium glutamicum. We established a gene expression regulation system, 'LightOnC.glu', utilizing the light-controlled RBP to construct light-controlled transcription factors in C. glutamicum. Simultaneously, we developed a high-performance light-controlled gene interference system using CRISPR/Cpf1 tools. The metabolic flow in the synthesis network was designed to enable the production of chitin oligosaccharides (CHOSs) and chondroitin sulphate oligosaccharides A (CSA) for the first time in C. glutamicum. Additionally, a light-controlled bioreactor was constructed, achieving a CHOSs production concentration of 6.2 g/L, the highest titer recorded for CHOSs biosynthesis to date. Herein, we have established a programmable light-responsive genetic circuit in C. glutamicum, advancing the theory of dynamic regulation based on light signaling. This breakthrough has potential applications in optimizing metabolic modules in other chassis cells and synthesizing other compounds.

Abstract: In the field of tissue engineering, achieving precise spatiotemporal control over engineered cells is critical for sculpting functional 2D cell cultures into intricate morphological shapes. In this study, we engineer light-responsive mammalian cells and target them with dynamic light patterns to realize 2D cell culture patterning control. To achieve this, we developed μPatternScope (μPS), a modular framework for software-controlled projection of high-resolution light patterns onto microscope samples. μPS comprises hardware and software suite governing pattern projection and microscope maneuvers. Together with a 2D culture of the engineered cells, we utilize μPS for controlled spatiotemporal induction of apoptosis to generate desired 2D shapes. Furthermore, we introduce interactive closed-loop patterning, enabling a dynamic feedback mechanism between the measured cell culture patterns and the light illumination profiles to achieve the desired target patterning trends. Our work offers innovative tools for advanced tissue engineering applications through seamless fusion of optogenetics, optical engineering, and cybernetics.

Abstract: Microtubule acetylation is implicated in regulating cell motility, yet its physiological role in directional migration and the underlying molecular mechanisms have remained unclear. This knowledge gap has persisted primarily due to a lack of tools capable of rapidly manipulating microtubule acetylation in actively migrating cells. To overcome this limitation and elucidate the causal relationship between microtubule acetylation and cell migration, we developed a novel optogenetic actuator, optoTAT, which enables precise and rapid induction of microtubule acetylation within minutes in live cells. Using optoTAT, we observed striking and rapid responses at both molecular and cellular level. First, microtubule acetylation triggers release of the RhoA activator GEF-H1 from sequestration on microtubules. This release subsequently enhances actomyosin contractility and drives focal adhesion maturation. These subcellular processes collectively promote sustained directional cell migration. Our findings position GEF-H1 as a critical molecular responder to microtubule acetylation in the regulation of directed cell migration, revealing a dynamic crosstalk between the actin and microtubule cytoskeletal networks.

Abstract: Recent advances in tissue engineering have been remarkable, yet the precise control of cellular behavior in 2D and 3D cultures remains challenging. One approach to address this limitation is to genomically engineer optogenetic control of cellular processes into tissues using gene switches that can operate with only a few genomic copies. Here, we implement blue and red light-responsive gene switches to engineer genomically stable two- and three-dimensional mammalian tissue models. Notably, we achieve precise control of cell death and morphogen-directed patterning in 2D and 3D tissues by optogenetically regulating cell necroptosis and synthetic WNT3A signaling at high spatiotemporal resolution. This is accomplished using custom-built patterned LED systems, including digital mirrors and photomasks, as well as laser techniques. These advancements demonstrate the capability of precise spatiotemporal modulation in tissue engineering and open up new avenues for developing programmable 3D tissue and organ models, with significant implications for biomedical research and therapeutic applications.

Abstract: Morphogen gradients convey essential spatial information during tissue patterning. While both concentration and timing of morphogen exposure are crucial, how cells interpret these graded inputs remains challenging to address. We employed an optogenetic system to acutely and reversibly modulate the nuclear concentration of the morphogen Dorsal (DL), homologue of NF-κB, which orchestrates dorso-ventral patterning in the Drosophila embryo. By controlling DL nuclear concentration while simultaneously recording target gene outputs in real time, we identified a critical window for DL action that is required to instruct patterning, and characterized the resulting effect on spatio-temporal transcription of target genes in terms of timing, coordination, and bursting. We found that a transient decrease in nuclear DL levels at nuclear cycle 13 leads to reduced expression of the mesoderm-associated gene snail (sna) and partial derepression of the neurogenic ectoderm-associated target short gastrulation (sog) in ventral regions. Surprisingly, the mispatterning elicited by this transient change in DL is detectable at the level of single cell transcriptional bursting kinetics, specifically affecting long inter-burst durations. Our approach of using temporally-resolved and reversible modulation of a morphogen in vivo, combined with mathematical modeling, establishes a framework for understanding the stimulus-response relationships that govern embryonic patterning.

Abstract: Light-Oxygen-Voltage (LOV) domains are the protein-based light switches used in nature to trigger and regulate various processes. They allow light signals to be converted into metabolic signaling cascades. Various LOV-domain proteins have been characterized in the last few decades and have been used to develop light-sensitive tools in cell biology research. LOV-based applications exploit the light-driven regulation of effector elements to activate signaling pathways, activate genes, or locate proteins within cells. A relatively new application of an engineered small LOV-domain protein called miniSOG (mini singlet oxygen generator) is based on the light-induced formation of reactive oxygen species (ROS). The first miniSOG was engineered from a LOV domain from Arabidopsis thaliana. This engineered 14 kDa light-responsive flavin-containing protein can be exploited as protein tag for the light-triggered localized production of ROS. Such tunable ROS production by miniSOG or similarly redesigned LOV-domains can be of use in studies focused on subcellular phenomena but may also allow new light-fueled catalytic processes. This review provides an overview of the discovery of LOV domains and their development into tools for cell biology. It also highlights recent advancements in engineering LOV domains for various biotechnological applications and cell biology studies.

Abstract: In metabolic engineering, increasing chemical production usually involves manipulating the expression levels of key enzymes. However, limited synthetic tools exist for modulating enzyme activity beyond the transcription level. Inspired by natural post-translational mechanisms, we present targeted enzyme degradation mediated by optically controlled nanobodies. We applied this method to a branched biosynthetic pathway, deoxyviolacein, and observed enhanced product specificity and yield. We then extend the biosynthesis pathway to violacein and show how simultaneous degradation of two target enzymes can further shift production profiles. Through the redirection of metabolic flux, we demonstrate how targeted enzyme degradation can be used to minimize unwanted intermediates and boost the formation of desired products.

Abstract: Overexpression of a single enzyme in a multigene heterologous pathway may be out of balance with the other enzymes in the pathway, leading to accumulated toxic intermediates, imbalanced carbon flux, reduced productivity of the pathway, or an inhibited growth phenotype. Therefore, optimal, balanced, and synchronized expression levels of enzymes in a particular metabolic pathway is critical to maximize production of desired compounds while maintaining cell fitness in a growing culture. Furthermore, the optimal intracellular concentration of an enzyme is determined by the expression strength, specific timing/duration, and degradation rate of the enzyme. Here, we modulated the intracellular concentration of a key enzyme, namely HMG-CoA synthase (HMGS), in the heterologous mevalonate pathway by tuning its expression level and period of transcription to enhance limonene production in Escherichia coli. Facilitated by the tuned blue-light inducible BLADE/pBad system, we observed that limonene production was highest (160 mg/L) with an intermediate transcription level of HMGS from moderate light illumination (41 au, 150 s ON/150 s OFF) throughout the growth. Owing to the easy penetration and removal of blue-light illumination from the growing culture which is hard to obtain using conventional chemical-based induction, we further explored different induction patterns of HMGS under strong light illumination (2047 au, 300 s ON) for different durations along the growth phases. We identified a specific timing of HMGS expression in the log phase (3-9 h) that led to optimal limonene production (200 mg/L). This is further supported by a mathematical model that predicts several periods of blue-light illumination (3-9 h, 0-9 h, 3-12 h, 0-12 h) to achieve an optimal expression level of HMGS that maximizes limonene production and maintains cell fitness. Compared to moderate and prolonged transcription (41 au, 150 s ON/150 s OFF, 0-73 h), strong but time-limited transcription (2047 au, 300 s ON, 3-9 h) of HMGS could maintain its optimal intracellular concentration and further increased limonene production up to 92% (250 mg/L) in the longer incubation (up to 73 h) without impacting cell fitness. This work has provided new insight into the "right amount" and "just-in-time" expression of a critical metabolite enzyme in the upper module of the mevalonate pathway using optogenetics. This study would complement previous findings in modulating HMGS expression and potentially be applicable to heterologous production of other terpenoids in E. coli.

Abstract: Alternative splicing is a fundamental process that contributes to the functional diversity and complexity of proteins. The regulation of each alternative splicing event involves the coordinated action of multiple RNA-binding proteins, creating a diverse array of alternatively spliced products. Dysregulation of alternative splicing is associated with various diseases, including neurodegeneration. Here we demonstrate that CELF2, a splicing regulator and a GWAS-identified risk factor for Alzheimer’s disease, binds to mRNAs associated with neurodegenerative diseases, with a specific interaction observed in the intron adjacent to exon 10 on Tau mRNA. Loss of CELF2 in the mouse brain results in a decreased inclusion of Tau exon 10, leading to a reduced 4R:3R ratio. Further exploration shows that the hinge domain of CELF2 possesses an intrinsically disordered region (IDR), which mediates CELF2 condensation and function. The functionality of IDR in regulating CELF2 function is underscored by its substitutability with IDRs from FUS and TAF15. Using TurboID we identified proteins that interact with CELF2 through its IDR. We revealed that CELF2 co-condensate with NOVA2 and SFPQ, which coordinate with CELF2 to regulate the alternative splicing of Tau exon 10. A negatively charged residue within the IDR (D388), which is conserved among CELF proteins, is critical for CELF2 condensate formation, interactions with NOVA2 and SFPQ, and function in regulating tau exon 10 splicing. Our data allow us to propose that CELF2 regulates Tau alternative splicing by forming condensates through its IDR with other splicing factors, and that the composition of the proteins within the condensates determines the outcomes of alternative splicing events.

Abstract: Spatial organization of microbes in biofilms enables crucial community function such as division of labor. However, quantitative understanding of such emergent community properties remains limited due to a scarcity of tools for patterning heterogeneous biofilms. Here we develop a synthetic optogenetic toolkit 'Multipattern Biofilm Lithography' for rational engineering and orthogonal patterning of multi-strain biofilms, inspired by successive adhesion and phenotypic differentiation in natural biofilms. We apply this toolkit to profile the growth dynamics of heterogeneous biofilm communities, and observe the emergence of spatially modulated commensal relationships due to shared antibiotic protection against the beta-lactam ampicillin. Supported by biophysical modeling, these results yield in-vivo measurements of key parameters, e.g., molecular beta-lactamase production per cell and length scale of antibiotic zone of protection. Our toolbox and associated findings provide quantitative insights into the spatial organization and distributed antibiotic protection within biofilms, with direct implications for future biofilm research and engineering.

Abstract: Studies utilizing electron microscopy and live fluorescence microscopy have significantly enhanced our understanding of the molecular mechanisms that regulate junctional dynamics during homeostasis, development and disease. To fully grasp the enormous complexity of cell-cell adhesions, it is crucial to study the nanoscale architectures of tight junctions, adherens junctions and desmosomes. It is important to integrate these junctional architectures with the membrane morphology and cellular topography in which the junctions are embedded. In this Review, we explore new insights from studies using super-resolution and volume electron microscopy into the nanoscale organization of these junctional complexes as well as the roles of the junction-associated cytoskeleton, neighboring organelles and the plasma membrane. Furthermore, we provide an overview of junction- and cytoskeletal-related biosensors and optogenetic probes that have contributed to these advances and discuss how these microscopy tools enhance our understanding of junctional dynamics across cellular environments.

Abstract: Transcriptional control is fundamental to cellular function. However, despite knowing that transcription factors can repress or activate specific genes, how these functions are implemented at the molecular level has remained elusive, particularly in the endogenous context of developing animals. Here, we combine optogenetics, single-cell live-imaging, and mathematical modeling to study how a zinc-finger repressor, Knirps, induces switch-like transitions into long-lived quiescent states. Using optogenetics, we demonstrate that repression is rapidly reversible (~1 min) and memoryless. Furthermore, we show that the repressor acts by decreasing the frequency of transcriptional bursts in a manner consistent with an equilibrium binding model. Our results provide a quantitative framework for dissecting the in vivo biochemistry of eukaryotic transcriptional regulation.

Abstract: Biomolecular condensates appear throughout cell physiology and pathology, but the specific role of condensation or its dynamics is often difficult to determine. Optogenetics offers an expanding toolset to address these challenges, providing tools to directly control condensation of arbitrary proteins with precision over their formation, dissolution, and patterning in space and time. In this review, we describe the current state of the field for optogenetic control of condensation. We survey the proteins and their derivatives that form the foundation of this toolset, and we discuss the factors that distinguish them to enable appropriate selection for a given application. We also describe recent examples of the ways in which optogenetic condensation has been used in both basic and applied studies. Finally, we discuss important design considerations when engineering new proteins for optogenetic condensation, and we preview future innovations that will further empower this toolset in the coming years.

Abstract: Chimeric antigen receptor T cell therapies have achieved great success in eradicating some liquid tumors, whereas the preclinical results in treating solid tumors have proven less decisive. One of the principal challenges in solid tumor treatment is the physical barrier composed of a dense extracellular matrix, which prevents immune cells from penetrating the tissue to attack intratumoral cancer cells. Here, we improve immune cell infiltration into solid tumors by manipulating septin-7 functions in cells. Using protein allosteric design, we reprogram the three-dimensional structure of septin-7 and insert a blue light-responsive light-oxygen-voltage-sensing domain 2 (LOV2), creating a light-controllable septin-7-LOV2 hybrid protein. Blue light inhibits septin-7 function in live cells, inducing extended cell protrusions and cell polarization, enhancing cell transmigration efficiency through confining spaces. We genetically edited human natural killer cell line (NK92) and mouse primary CD8+ T-cells expressing the engineered protein, and we demonstrated improved penetration and cytotoxicity against various tumor spheroid models. Our proposed strategy to enhance immune cell infiltration is compatible with other methodologies and therefore, could be used in combination to further improve cell-based immunotherapies against solid tumors.

Abstract: Endogenous condensates with transient constituents are notoriously difficult to study with common biological assays like mass spectrometry and other proteomics profiling. Here, we report a method for light-induced targeting of endogenous condensates (LiTEC) in living cells. LiTEC combines the identification of molecular zip codes that target the endogenous condensates with optogenetics to enable controlled and reversible partitioning of an arbitrary cargo, such as enzymes commonly used in proteomics, into the condensate in a blue light-dependent manner. We demonstrate a proof of concept by combining LiTEC with proximity-based biotinylation (BioID) and uncover putative components of transcriptional condensates in mouse embryonic stem cells. Our approach opens the road to genome-wide functional studies of endogenous condensates.

Abstract: Motility is a hallmark of life’s dynamic processes, enabling cells to actively chase prey, repair wounds, and shape organs. Recreating these intricate behaviors using well-defined molecules remains a major challenge at the intersection of biology, physics, and molecular engineering. Although the polymerization force of the actin cytoskeleton is characterized as a primary driver of cell motility, recapitulating this process in protocellular systems has proven elusive. The difficulty lies in the daunting task of distilling key components from motile cells and integrating them into model membranes in a physiologically relevant manner. To address this, we developed a method to optically control actin polymerization with high spatiotemporal precision within cell-mimetic lipid vesicles known as giant unilamellar vesicles (GUVs). Within these active protocells, the reorganization of actin networks triggered outward membrane extensions as well as the unidirectional movement of GUVs at speeds of up to 0.43 µm/min, comparable to typical adherent mammalian cells. Notably, our findings reveal a synergistic interplay between branched and linear actin forms in promoting membrane protrusions, highlighting the cooperative nature of these cytoskeletal elements. This approach offers a powerful platform for unraveling the intricacies of cell migration, designing synthetic cells with active morphodynamics, and advancing bioengineering applications, such as self-propelled delivery systems and autonomous tissue-like materials.

Abstract: Intracellular trafficking, an extremely complex network, dynamically orchestrates nearly all cellular activities. A versatile method that enables the manipulation of target transport pathways with high spatiotemporal accuracy in vitro and in vivo is required to study how this network coordinates its functions. Here, a new method called RIVET (Rapid Immobilization of target Vesicles on Engaged Tracks) is presented. Utilizing inducible dimerization between target vesicles and selective cytoskeletons, RIVET can spatiotemporally halt numerous intracellular trafficking pathways within seconds in a reversible manner. Its highly specific perturbations allow for the real-time dissection of the dynamic relationships among different trafficking pathways. Moreover, RIVET is capable of inhibiting receptor-mediated endocytosis. This versatile system can be applied from the cellular level to whole organisms. RIVET opens up new avenues for studying intracellular trafficking under various physiological and pathological conditions and offers potential strategies for treating trafficking-related disorders.

Abstract: Synucleinopathies, including Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, are triggered by α-synuclein aggregation, triggering progressive neurodegeneration. However, the intracellular α-synuclein aggregation mechanism remains unclear. Herein, we demonstrate that RNA G-quadruplex assembly forms scaffolds for α-synuclein aggregation, contributing to neurodegeneration. Purified α-synuclein binds RNA G-quadruplexes directly through the N terminus. RNA G-quadruplexes undergo Ca2+-induced phase separation and assembly, accelerating α-synuclein sol-gel phase transition. In α-synuclein preformed fibril-treated neurons, RNA G-quadruplex assembly comprising synaptic mRNAs co-aggregates with α-synuclein upon excess cytoplasmic Ca2+ influx, eliciting synaptic dysfunction. Forced RNA G-quadruplex assembly using an optogenetic approach evokes α-synuclein aggregation, causing neuronal dysfunction and neurodegeneration. The administration of 5-aminolevulinic acid, a protoporphyrin IX prodrug, prevents RNA G-quadruplex phase separation, thereby attenuating α-synuclein aggregation, neurodegeneration, and progressive motor deficits in α-synuclein preformed fibril-injected synucleinopathic mice. Therefore, Ca2+ influx-induced RNA G-quadruplex assembly accelerates α-synuclein phase transition and aggregation, potentially contributing to synucleinopathies.

Abstract: Mitochondria provide cells with energy and regulate the cellular metabolism. Almost all mitochondrial proteins are nuclear-encoded, translated on ribosomes in the cytoplasm, and subsequently transferred to the different subcellular compartments of mitochondria. Here, we developed OptoMitoImport, an optogenetic tool to control the import of proteins into the mitochondrial matrix via the presequence pathway on demand. OptoMitoImport is based on a two-step process: first, light-induced cleavage by a TEV protease cuts off a plasma membrane-anchored fusion construct in close proximity to a mitochondrial targeting sequence; second, the mitochondrial targeting sequence preceding the protein of interest recruits to the outer mitochondrial membrane and imports the protein fused to it into mitochondria. Upon reaching the mitochondrial matrix, the matrix processing peptidase cuts off the mitochondrial targeting sequence and releases the protein of interest. OptoMitoImport is available as a two-plasmid system as well as a P2A peptide or IRES sequence-based bicistronic system. Fluorescence studies demonstrate the release of the plasma membrane-anchored protein of interest through light-induced TEV protease cleavage and its localization to mitochondria. Cell fractionation experiments confirm the presence of the peptidase-cleaved protein of interest in the mitochondrial fraction. The processed product is protected from proteinase K treatment. Depletion of the membrane potential across the inner mitochondria membrane prevents the mitochondrial protein import, indicating an import of the protein of interest by the presequence pathway. These data demonstrate the functionality of OptoMitoImport as a generic system with which to control the post-translational mitochondrial import of proteins via the presequence pathway.

Abstract: Centrosomes ensure accurate chromosome segregation during cell division. Although the regulation of centrosome number is well-established, less is known about the suppression of non-centrosomal MTOCs (ncMTOCs). The E3 ligase TRIM37, implicated in Mulibrey nanism and 17q23-amplified cancers, has emerged as a key regulator of both centrosomes and ncMTOCs. Yet, the mechanism by which TRIM37 achieves enzymatic activation to target these mesoscale structures had remained unknown. Here, we elucidate TRIM37’s activation process, beginning with TRAF domain-directed substrate recognition, progressing through B-box domain-mediated oligomerization, and culminating in RING domain dimerization. Using optogenetics, we demonstrate that TRIM37’s E3 activity is directly coupled to the assembly state of its substrates, activating only when centrosomal proteins cluster into higher-order assemblies resembling MTOCs. This regulatory framework provides a mechanistic basis for understanding TRIM37-driven pathologies and, by echoing TRIM5’s restriction of the HIV capsid, unveils a conserved activation blueprint among TRIM proteins for controlling mesoscale assembly turnover.

Abstract: The human nucleoporin RanBP2/Nup358 interacts with SUMO1-modified RanGAP1 and the SUMO E2 Ubc9 at the nuclear pore complex (NPC) to promote export and disassembly of exportin Crm1/Ran(GTP)/cargo complexes. In mitosis, RanBP2/SUMO1-RanGAP1/Ubc9 remains intact after NPC disassembly and is recruited to kinetochores and mitotic spindles by Crm1 where it contributes to mitotic progression. Interestingly, RanBP2 binds SUMO1-RanGAP1/Ubc9 with motifs that also catalyze SUMO E3 ligase activity. Here, we resolve cryo-EM structures of a RanBP2 C-terminal fragment bound to Crm1, SUMO1-RanGAP1/Ubc9, and two molecules of Ran(GTP), one bound to Crm1 and the other bound to RanGAP1 and RanBP2. These structures reveal several unanticipated interactions with Crm1 including a nuclear export signal (NES) for RanGAP1, the deletion of which mislocalizes RanGAP1 and the Ran GTPase in cells. Our structural and biochemical results support models in which RanBP2 E3 ligase activity is dependent on Crm1, the RanGAP1 NES and Ran GTPase cycling.

Abstract: In migrating cells, the GTPase Rac organizes a protrusive front, whereas Rho organizes a contractile back. How these GTPases are appropriately positioned at the opposite poles of a migrating cell is unknown. Here we leverage optogenetics, manipulation of cell mechanics, and mathematical modeling to reveal a surprising long-range mutual activation of the front and back polarity programs that complements their well-known local mutual inhibition. This long-range activation is rooted in two distinct modes of mechanochemical crosstalk. Local Rac-based protrusion stimulates Rho activation at the opposite side of the cell via membrane tension-based activation of mTORC2. Conversely, local Rho-based contraction induces cortical-flow-based remodeling of membrane-to-cortex interactions leading to PIP2 release, PIP3 generation, and Rac activation at the opposite side of the cell. We develop a minimal unifying mechanochemical model of the cell to explain how this long-range mechanical facilitation complements local biochemical inhibition to enable robust global Rho and Rac partitioning. Finally, we validate the importance of this long-range facilitation in the context of chemoattractant-based cell polarization and migration in primary human lymphocytes. Our findings demonstrate that the actin cortex and plasma membrane function as an integrated mechanochemical system for long-range partitioning of Rac and Rho during cell migration and likely other cellular contexts.