Curated Optogenetic Publication Database

Abstract: Cancer cell invasion relies on dynamic cell shape changes, which originate from protrusive and contractile intracellular forces. Previous studies revealed that contractile forces are controlled by positive-feedback amplification of the contraction regulator Rho by Lbc GEFs. These GEFs were previously linked to tumor progression, however, the underlying mechanisms are poorly understood. Here, we generated a mouse melanoma model, in which cytosolic levels of the Lbc GEF GEF-H1 are controlled by light. Using this model, we found that increased GEF-H1 levels strongly stimulate cell contraction dynamics. Interestingly, increased contraction dynamics rapidly induced expansion of tumor spheroids via a focal adhesion kinase-dependent mechanism. Furthermore, long-term stimulation led to the escape of individual cells from spheroids. These findings reveal new insights into the oncogenic roles of Lbc GEFs, and how they might promote tumor cell invasion. We propose a mechanism, in which increased cell contraction dynamics results in asymmetric pulling forces at the tumor border, promoting the detachment and escape of individual cells.

Abstract: Cell migration is crucial for development and tissue homeostasis, while its dysregulation leads to severe pathologies. Cell migration is driven by the extension of actin-based lamellipodia protrusions, powered by actin polymerization, which is tightly regulated by signaling pathways, including Rho GTPases and Ca2+ signaling. While the importance of Ca2+ signaling in lamellipodia protrusions has been established, the molecular mechanisms linking Ca2+ to lamellipodia assembly are unknown. Here, we identify a novel Ca2+ signaling axis involving the mechano-gated channel TRPV4, which regulates lamellipodia protrusions in various cell types. Using Ca2+ and FRET imaging, we demonstrate that TRPV4-mediated Ca2+ influx upregulates RhoA activity within lamellipodia, which then facilitates formin-mediated actin assembly. Mechanistically, we identify CaMKII and TEM4 as key mediators relaying the TRPV4-mediated Ca2+ signal to RhoA. These data define a molecular pathway by which Ca2+ influx regulates small GTPase activity within a specific cellular domain – lamellipodia - and demonstrate the critical role in organizing the actin machinery and promoting cell migration in diverse biological contexts.

Abstract: Cortical positioning of the meiotic spindle within an oocyte is required to expel chromosomes into polar bodies to generate a zygote with the correct number of chromosomes. In C. elegans, yolk granules and mitochondria are packed inward, away from the cortex, while the spindle moves outward, both in a kinesin-dependent manner. The kinesin-dependent inward packing of yolk granules suggests the existence of microtubules with minus ends at the cortex and plus ends extending inward, making it unclear how kinesin moves the spindle outward. We hypothesize that the inward packing of organelles might indirectly force the spindle outward by volume exclusion. To test this hypothesis, we generate a strain in which the only kinesin consists of motor domains with no cargo-binding tail optogenetically attached to mitochondria. This mitochondria-only kinesin packs mitochondria into a tight ball and efficiently moves the meiotic spindle to the cortex, supporting the volume exclusion hypothesis.

Abstract: While optogenetic tools have recently opened new avenues for controlling and understanding cellular behavior, Suh et al.1 present an effective strategy to regulate tissue densification and outgrowth through optogenetic control of EGFR. Their work ultimately uncovers fundamental principles that pave the way for improved tissue engineering approaches.

Abstract: Motile cilia perform crucial functions during embryonic development and in adult tissues. They are anchored by an apical actin network that forms microridge-like structures on the surface of multiciliated cells. Using Xenopus as a model system to investigate the mechanisms underlying the formation of these specialized actin structures, we observed stochastic bursts of intracellular calcium concentration in developing multiciliated cells. Through optogenetic manipulation of calcium signaling, we found that individual calcium bursts triggered the fusion and extension of actin structures by activating non-muscle myosin. Repeated cycles of calcium activation promoted assembly and coherence of the maturing apical actin network. Inhibition of the endogenous inositol triphosphate-calcium pathway disrupted the formation of apical actin/microridge-like structures by reducing local centriolar RhoA signaling. This disruption was rescued by transient expression of constitutively active RhoA in multiciliated cells. Our findings identify repetitive calcium bursts as a driving force that promotes the self-organization of the highly specialized actin cytoskeleton of multiciliated cells.

Abstract: Receptor tyrosine kinases (RTKs) play key roles in coordinating cell movement at both single-cell and tissue scales. The recent development of optogenetic tools for controlling RTKs and their downstream signaling pathways suggests that these responses may be amenable to engineering-based control for sculpting tissue shape and function. Here, we report that a light-controlled epidermal growth factor (EGF) receptor (OptoEGFR) can be deployed in epithelial cells for precise, programmable control of long-range tissue movements. We show that in OptoEGFR-expressing tissues, light can drive millimeter-scale cell rearrangements to densify interior regions or produce rapid outgrowth at tissue edges. Light-controlled tissue movements are driven primarily by phosphoinositide 3-kinase (PI3K) signaling, rather than diffusible ligands, tissue contractility, or ERK kinase signaling as seen in other RTK-driven migration contexts. Our study suggests that synthetic, light-controlled RTKs could serve as a powerful platform for controlling cell positions and densities for diverse applications, including wound healing and tissue morphogenesis.

Abstract: Talin regulates the adhesion and migration of cells in part by promoting the affinity of integrins for extracellular matrix proteins, a process that in cells such as endothelial cells and platelets requires the direct interaction of talin with both the small GTPase Rap1 bound to GTP (Rap1-GTP) and the integrin β3 cytoplasmic tail. To study this process in more detail, we employed an optogenetic approach in living, immortalized endothelial cells to be able to regulate the interaction of talin with the plasma membrane. Previous studies identified talin as the Rap1-GTP effector for β3 integrin activation. Surprisingly, optogenetic recruitment of talin-1 (TLN1; herein referred to as talin) to the plasma membrane also led to the localized activation of Rap1 itself, apparently by talin competing for Rap1-GTP with SHANK3, a protein known to sequester Rap1-GTP and to block integrin activation. Rap1 activation by talin was localized to the cell periphery in suspension cells and within lamellipodia and pseudopodia in cells adherent to fibronectin. Thus, membrane-associated talin can play a dual role in regulating integrin function in endothelial cells: first, by releasing Rap1-GTP from its sequestration by SHANK3, and second, by serving as the relevant Rap1 effector for integrin activation.

Abstract: Ras has traditionally been regarded as a positive regulator and therapeutic target due to its role in cell proliferation, but recent findings indicate a more nuanced role in cell migration, where suppressed Ras activity can unexpectedly promote migration. To clarify this complexity, we systematically modulate Ras activity using various RasGEF and RasGAP proteins and assess their effects on migration dynamics. Leveraging optogenetics, we assess the immediate, non-transcriptional effects of Ras signaling on migration. Local RasGEF recruitment to the plasma membrane induces protrusions and new fronts to effectively guide migration, even in the absence of GPCR/G-protein signaling whereas global recruitment causes immediate cell spreading halting cell migration. Local RasGAP recruitment suppresses protrusions, generates new backs, and repels cells whereas global relocation either eliminates all protrusions to inhibit migration or preserves a single protrusion to maintain polarity. Consistent local and global increases or decreases in signal transduction and cytoskeletal activities accompany these morphological changes. Additionally, we performed cortical tension measurements and found that RasGEFs generally increase cortical tension while RasGAPs decrease it. Our results reveal a biphasic relationship between Ras activity and cellular dynamics, reinforcing our previous findings that optimal Ras activity and cortical tension are critical for efficient migration.

Abstract: The actin cytoskeleton and its nanoscale organization are central to all eukaryotic cells-powering diverse cellular functions including morphology, motility, and cell division-and is dysregulated in multiple diseases. Historically studied largely with purified proteins or in isolated cells, tools to study cell type-specific roles of actin in multicellular contexts are greatly needed. DeActs are recently created, first-in-class genetic tools for perturbing actin nanostructures and dynamics in specific cell types across diverse eukaryotic model organisms. Here, ChiActs are introduced, the next generation of actin-perturbing genetic tools that can be rapidly activated in cells and optogenetically targeted to distinct subcellular locations using light. ChiActs are composed of split halves of DeAct-SpvB, whose potent actin disassembly-promoting activity is restored by chemical-induced dimerization or allosteric switching. It is shown that ChiActs function to rapidly induce actin disassembly in several model cell types and are able to perturb actin-dependent nano-assembly and cellular functions, including inhibiting lamellipodial protrusions and membrane ruffling, remodeling mitochondrial morphology, and reorganizing chromatin by locally constraining actin disassembly to specific subcellular compartments. ChiActs thus expand the toolbox of genetically-encoded tools for perturbing actin in living cells, unlocking studies of the many roles of actin nano-assembly and dynamics in complex multicellular systems.

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: Most bacteria lack membrane-enclosed organelles and rely on macromolecular scaffolds at different subcellular locations to recruit proteins for specific functions. Here, we demonstrate that the optogenetic CRY2-CIB1 system from Arabidopsis thaliana can be used to rapidly direct proteins to different subcellular locations with varying efficiencies in live Escherichia coli cells, including the nucleoid, the cell pole, the membrane, and the midcell division plane. Such light-induced re-localization can be used to rapidly inhibit cytokinesis in actively dividing E. coli cells. We further show that CRY2-CIBN binding kinetics can be modulated by green light, adding a new dimension of control to the system. Finally, we test this optogenetic system in three additional bacterial species, Bacillus subtilis, Caulobacter crescentus, and Streptococcus pneumoniae, providing important considerations for this system's applicability in bacterial cell biology.

Abstract: The actin cytoskeleton forms a mesh-like network that drives cellular deformations. The network property is defined by the network density and the species of the actin-binding proteins. However, the relationship between the actin network density, the penetration ability of actin-binding proteins into the network, and resulting network dynamics remains elusive. Here, we report an in vitro optogenetic system, named OptoVCA, which induces Arp2/3-mediated actin network assembly on a lipid membrane. By changing the illumination power, duration, and pattern, the OptoVCA flexibly manipulates the density, thickness, and shape of the actin network. Taking these advantages, we examine the effects of the network density on the two representative actin-binding proteins, myosin and ADF/cofilin. We find that the penetration of myosin filaments into the network is strictly inhibited by only a several-fold increase in network density due to the steric hindrance. Furthermore, penetrated myosin filaments induce directional actin flow when the network has a density gradient. On the other hand, ADF/cofilin penetrates into the network regardless of network density, however, network disassembly is dramatically inhibited by only a several-fold increase in network density. Thus, the OptoVCA contributes to understanding cell mechanics through the examination of the network density-dependent effect on the actin-binding proteins.

Abstract: Development of neuronal connections is spatially and temporally controlled by extracellular cues which often activate their cognate cell surface receptors and elicit localized cellular responses. Here, we demonstrate the use of an optogenetic tool to activate receptor signaling locally to induce actin-mediated growth cone remodeling in neurons. Based on the light-induced interaction between Cryptochrome 2 (CRY2) and CIB1, we generated a bicistronic vector to co-expresses CRY2 fused to the intracellular domain of a guidance receptor and a membrane-anchored CIB1. When expressed in primary neurons, activation of the growth inhibitory PlexA4 receptor induced growth cone collapse, while activation of the growth stimulating TrkA receptor increased growth cone size. Moreover, local activation of either receptor not only elicited the predicted response in light-activated growth cones but also an opposite response in neighboring no-light-exposed growth cones of the same neuron. Finally, this tool was used to reorient growth cones toward or away from the site of light activation and to stimulate local actin polymerization for branch initiation along axonal shafts. These studies demonstrate the use of an optogenetic tool for precise spatial and temporal control of receptor signaling in neurons and support its future application in investigating cellular mechanisms of neuronal development and plasticity.

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: Intermediate filaments (IFs) are a key component of the cytoskeleton, essential for regulating cell mechanics, maintaining nuclear integrity, positioning organelles, and modulating cell signaling. Unlike actin filaments and microtubules, IFs have slower dynamics, and current insights into IF function primarily come from studies using long-term perturbations, such as protein depletion or mutation. Here, we present tools that allow rapid manipulation of vimentin IFs in the whole cytoplasm or within specific subcellular regions by inducibly coupling them to microtubule motors, either pharmacologically or using light. Perinuclear clustering of vimentin had no strong effect on the actin or microtubule organization, cell spreading, and focal adhesions, but reduced cell stiffness. Mitochondria and endoplasmic reticulum sheets were repositioned together with vimentin, whereas lysosomes were only briefly repositioned and rapidly regained their normal distribution. Keratin was displaced along with vimentin in some cell lines but remained intact in others. Our tools help to study the immediate effects of vimentin perturbation and identify direct links of vimentin to other cellular structures.

Abstract: Epithelia are polarized layers of cells that line the outer and inner surfaces of organs. At the basal side, the epithelial cell layer is supported by a basement membrane, which is a thin polymeric layer of self-assembled extracellular matrix (ECM) that tightly adheres to the basal cell surface. Proper shaping of epithelial layers is an important prerequisite for the development of healthy organs during the morphogenesis of an organism. Experimental evidence suggests that local degradation of the basement membrane is one of the mechanisms that can drive epithelial folding. However, how folding emerges in the absence of tissue growth remains elusive. Here, we present a coarse-grained plate theory model of the basement membrane that assumes force balance between i) cell-transduced active forces and ii) deformation-induced elastic forces. We verify key assumptions of this model through experiments in the Drosophila wing disc epithelium and demonstrate that the model can explain the emergence of outward epithelial folds upon local plate degradation. The model accounts for local degradation of the basement membrane as a mechanism for the generation of epithelial folds in the absence of epithelial growth.

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: Cells use traction forces to sense mechanical cues in their environment. While the molecular clutch model effectively explains how cells exert more forces on stiffer substrates, it falls short in addressing their adaptation to dynamic mechanical fluctuations prevalent in tissues and organs. Here, using hydrogel with photo-responsive rigidity, we show that cells' response to rigidity changes is frequency dependent. Strikingly, at certain frequencies, cellular traction forces exceed those on static substrates 4-fold stiffer, challenging the established molecular clutch model. We discover that the discrepancy between the rapid adaptation of traction forces and the slower deactivation of mechanotransduction signaling proteins results in their accumulation, thereby enhancing long-term cellular traction in dynamic settings. Consequently, we propose a new model that melds immediate mechanosensing with extended mechanical signaling. Our study underscores the significance of dynamic rigidity in the development of synthetic biomaterials, emphasizing the importance of considering both immediate and prolonged cellular responses.

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: Lamellipodia are sheet-like protrusions essential for migration and endocytosis, yet the ultrastructure of the actin cytoskeleton during lamellipodia formation remains underexplored. Here, we combined the optogenetic tool PA-Rac1 with cryo-ET to enable ultrastructural analysis of newly formed lamellipodia. We successfully visualized lamellipodia at various extension stages, representing phases of their formation. In minor extensions, several unbundled actin filaments formed “Minor protrusions” at the leading edge. For moderately extended lamellipodia, cross-linked actin filaments formed small filopodia-like structures, termed “mini filopodia.” In fully extended lamellipodia, filopodia matured at multiple points, and cross-linked actin filaments running nearly parallel to the leading edge increased throughout the lamellipodia. These observations suggest that actin polymerization begins in specific plasma membrane regions, forming mini filopodia that either mature into full filopodia or detach from the leading edge to form parallel filaments. This actin turnover likely drives lamellipodial protrusion, providing new insights into actin dynamics and cell migration.

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: Cell contacts in epithelia are remodeled to regulate paracellular permeability and to control passage of migrating cells, but how barrier function is modulated while preserving epithelial integrity is not clear. In the follicular epithelium of Drosophila ovaries, tricellular junctions (TCJs) open transiently in a process termed patency to allow passage of externally produced yolk proteins for uptake by the oocyte. Here we show that modulation of actomyosin contractility at cell vertices controls TCJ permeability. Before patency, circumferential actomyosin bundles are anchored at apical follicle cell vertices, where tension-sensing junctional proteins, Rho-associated kinase (Rok), and active Myosin II accumulate and maintain vertices closed. TCJ opening is initiated by redistribution of Myosin II from circumferential bundles to a medial pool, accompanied by decreasing tension on vertices. This transition requires activation of Cofilin-dependent F-actin disassembly by the phosphatase Slingshot and Myosin II inactivation by Myosin light chain phosphatase, and is counteracted by Rok. Accordingly, constitutive activation of Myosin or of Rho signaling prevent vertex opening, whereas reduced Myosin II or Rok activity cause excessive and premature vertex opening. Thus, opening of intercellular gaps in the follicular epithelium does not require actomyosin-based forces, but relies on a reduction of actomyosin contractility. Conversely, F-actin assembly is required for closing intercellular gaps after patency. Our findings are consistent with a force transduction model in which TCJ integrity is maintained by vertex-anchored contractile actomyosin. We propose that the cell-type-specific organization of actomyosin at cell vertices determines the mode of contractility-dependent regulation of epithelial permeability.

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.

Abstract: Apical cell-cell junctions, including adherens junctions and tight junctions, adhere epithelial cells to one another and regulate selective permeability at both bicellular junctions and tricellular junctions (TCJs). Although several specialized proteins are known to localize at TCJs, it remains unclear how actomyosin-mediated tension transmission at TCJs contributes to the maintenance of junction integrity and barrier function at these sites. Here, utilizing the embryonic epithelium of gastrula-stage Xenopus laevis embryos, we define a mechanism by which the mechanosensitive protein Vinculin helps anchor the actomyosin network at TCJs, thus maintaining TCJ integrity and barrier function. Using an optogenetic approach to acutely increase junctional tension, we find that Vinculin is mechanosensitively recruited to apical junctions immediately surrounding TCJs. In Vinculin knockdown (KD) embryos, junctional actomyosin intensity is decreased and becomes disorganized at TCJs. Using fluorescence recovery after photobleaching (FRAP), we show that Vinculin KD reduces actin stability at TCJs and destabilizes Angulin-1, a key tricellular tight junction protein involved in regulating barrier function at TCJs. When Vinculin KD embryos are subjected to increased tension, TCJ integrity is not maintained, filamentous actin (F-actin) morphology at TCJs is disrupted, and breaks in the signal of the tight junction protein ZO-1 signal are detected. Finally, using a live imaging barrier assay, we detect increased barrier leaks at TCJs in Vinculin KD embryos. Together, our findings show that Vinculin-mediated actomyosin organization is required to maintain junction integrity and barrier function at TCJs and reveal new information about the interplay between adhesion and barrier function at TCJs.

Abstract: Cortical positioning of the meiotic spindle within an oocyte is required to expel chromosomes into polar bodies to generate a zygote with the correct number of chromosomes. In C. elegans, yolk granules and mitochondria are packed inward, away from the cortex while the spindle moves outward, both in a kinesin-dependent manner. The kinesin-dependent inward packing of yolk granules suggests the existence of microtubules with minus ends at the cortex and plus ends extending inward, making it unclear how kinesin moves the spindle outward. We hypothesized that inward packing of organelles might indirectly force the spindle outward by volume exclusion. To test this hypothesis, we generated a strain in which the only kinesin consists of motor domains with no cargo-binding tail optogenetically attached to mitochondria. This mitochondria-only kinesin packed mitochondria into a tight ball and efficiently moved the meiotic spindle to the cortex, supporting the volume exclusion hypothesis.