Showing 1 - 25 of 30 results
The effect of substrate stiffness on tensile force transduction in the epithelial monolayers.
In recent years, the importance of mechanical signaling and the cellular mechanical microenvironment in affecting cellular behavior has been widely accepted. Cells in epithelial monolayers are mechanically connected to each other and the underlying extracellular matrix (ECM), forming a highly connected mechanical system subjected to various mechanical cues from their environment, such as the ECM stiffness. Changes in the ECM stiffness have been linked to many pathologies, including tumor formation. However, our understanding of how ECM stiffness and its heterogeneities affect the transduction of mechanical forces in epithelial monolayers is lacking. To investigate this, we used a combination of experimental and computational methods. The experiments were conducted using epithelial cells cultured on an elastic substrate and applying a mechanical stimulus by moving a single cell by micromanipulation. To replicate our experiments computationally and quantify the forces transduced in the epithelium, we developed a new model that described the mechanics of both the cells and the substrate. Our model further enabled the simulations with local stiffness heterogeneities. We found the substrate stiffness to distinctly affect the force transduction as well as the cellular movement and deformation following an external force. Also, we found that local changes in the stiffness can alter the cells’ response to external forces over long distances. Our results suggest that this long-range signaling of the substrate stiffness depends on the cells’ ability to resist deformation. Furthermore, we found that the cell’s elasticity in the apico-basal direction provides a level of detachment between the apical cell-cell junctions and the basal focal adhesions. Our simulation results show potential for increased ECM stiffness, e.g. due to a tumor, to modulate mechanical signaling between cells also outside the stiff region. Furthermore, the developed model provides a good platform for future studies on the interactions between epithelial monolayers and elastic substrates.
Light-inducible deformation of mitochondria in live cells.
Mitochondria, the powerhouse of the cell, are dynamic organelles that undergo constant morphological changes. Increasing evidence indicates that mitochondria morphologies and functions can be modulated by mechanical cues. However, the mechano-sensing and -responding properties of mitochondria and the relation between mitochondrial morphologies and functions are unclear due to the lack of methods to precisely exert mechano-stimulation on and deform mitochondria inside live cells. Here, we present an optogenetic approach that uses light to induce deformation of mitochondria by recruiting molecular motors to the outer mitochondrial membrane via light-activated protein-protein hetero-dimerization. Mechanical forces generated by motor proteins distort the outer membrane, during which the inner mitochondrial membrane can also be deformed. Moreover, this optical method can achieve subcellular spatial precision and be combined with different optical dimerizers and molecular motors. This method presents a mitochondria-specific mechano-stimulator for studying mitochondria mechanobiology and the interplay between mitochondria shapes and functions.
Optical regulation of endogenous RhoA reveals selection of cellular responses by signal amplitude.
How protein signaling networks respond to different input strengths is an important but poorly understood problem in cell biology. For example, the small GTPase RhoA regulates both focal adhesion (FA) growth or disassembly, but whether RhoA serves as a switch selecting between cellular outcomes, or if outcomes are simply modulated by additional factors in the cell, is not clear. Here, we develop a photoswitchable RhoA guanine exchange factor, psRhoGEF, to precisely control endogenous RhoA activity. We also develop a FRET-based biosensor to allow visualization of RhoA activity together with psRhoGEF control. Using these new optical tools, we discover that low levels of RhoA activation preferentially induce FA disassembly in a Src-dependent manner, while high levels induce both FA growth and disassembly in a ROCK-dependent manner. Thus, rheostatic control of RhoA activation with photoswitchable RhoGEF reveals that cells can use signal amplitude to produce multiple responses to a single biochemical signal.
Asymmetric Contraction of Adherens Junctions arises through RhoA and E-cadherin feedback.
Tissue morphogenesis often arises from the culmination of discrete changes in cell-cell junction behaviors, namely ratcheted junction contractions that lead to collective cellular rearrangements. Mechanochemical signaling in the form of RhoA underlies these ratcheted contractions, which occur asymmetrically as one highly motile vertex contracts toward a relatively less motile tricellular vertex. The underlying mechanisms driving asymmetric vertex movement remains unknown. Here, we use optogenetically controlled RhoA in model epithelia together with biophysical modeling to uncover the mechanism lending to asymmetric vertex motion. We find that both local and global RhoA activation leads to increases in junctional tension, thereby facilitating vertex motion. RhoA activation occurs in discrete regions along the junction and is skewed towards the less-motile vertex. At these less-motile vertices, E-cadherin acts as an opposing factor to limit vertex motion through increased frictional drag. Surprisingly, we uncover a feedback loop between RhoA and E-cadherin, as regional optogenetic activation of specified junctional zones pools E-cadherin to the location of RhoA activation. Incorporating this circuit into a mathematical model, we find that a positive feedback between RhoA-mediated tension and E-cadherin-induced frictional drag on tricellular vertices recapitulates experimental data. As such, the location of RhoA determines which vertex is under high tension, pooling E-cadherin and increasing the frictional load at the tricellular vertex to limit its motion. This feedback drives a tension-dependent intercellular “clutch” at tricellular vertices which stabilizes vertex motion upon tensional load.
Physically asymmetric division of the C. elegans zygote ensures invariably successful embryogenesis.
Asymmetric divisions that yield daughter cells of different sizes are frequent during early embryogenesis, but the importance of such a physical difference for successful development remains poorly understood. Here, we investigated this question using the first division of Caenorhabditis elegans embryos, which yields a large AB cell and a small P1 cell. We equalized AB and P1 sizes using acute genetic inactivation or optogenetic manipulation of the spindle positioning protein LIN-5. We uncovered that only some embryos tolerated equalization, and that there was a size asymmetry threshold for viability. Cell lineage analysis of equalized embryos revealed an array of defects, including faster cell cycle progression in P1 descendants, as well as defects in cell positioning, division orientation, and cell fate. Moreover, equalized embryos were more susceptible to external compression. Overall, we conclude that unequal first cleavage is essential for invariably successful embryonic development of C. elegans.
Blue Light‐Operated CRISPR/Cas13b‐Mediated mRNA Knockdown (Lockdown).
The introduction of optogenetics into cell biology has furnished systems to control gene expression at the transcriptional and protein stability level, with a high degree of spatial, temporal, and dynamic light‐regulation capabilities. Strategies to downregulate RNA currently rely on RNA interference and CRISPR/Cas‐related methods. However, these approaches lack the key characteristics and advantages provided by optical control. “Lockdown” introduces optical control of RNA levels utilizing a blue light‐dependent switch to induce expression of CRISPR/Cas13b, which mediates sequence‐specific mRNA knockdown. Combining Lockdown with optogenetic tools to repress gene‐expression and induce protein destabilization with blue light yields efficient triple‐controlled downregulation of target proteins. Implementing Lockdown to degrade endogenous mRNA levels of the cyclin‐dependent kinase 1 (hCdk1) leads to blue light‐induced G2/M cell cycle arrest and inhibition of cell growth in mammalian cells.
Correction to Lancet Infectious Diseases 2020; published online April 29. https://doi.org/10.1016/ S1473-3099(20)30064-5.
Abstract not available.
Nano-positioning and tubuline conformation determine transport of mitochondria along microtubules.
Correct spatiotemporal distribution of organelles and vesicles is crucial for healthy cell functioning and is regulated by intracellular transport mechanisms. Controlled transport of bulky mitochondria is especially important in polarized cells such as neurons that rely on these organelles to locally produce energy and buffer calcium. Mitochondrial transport requires and depends on microtubules which fill much of the available axonal space. How mitochondrial transport is affected by their position within the microtubule bundles is not known. Here, we found that anterograde transport, driven by kinesin motors, is susceptible to the molecular conformation of tubulin both in vitro and in vivo. Anterograde velocities negatively correlate with the density of elongated tubulin dimers, similar to GTP-tubulin, that are more straight and rigid. The impact of the tubulin conformation depends primarily on where a mitochondrion is positioned, either within or at the rim of microtubule bundle. Increasing elongated tubulin levels lowers the number of motile anterograde mitochondria within the microtubule bundle and increases anterograde transport speed at the microtubule bundle rim. We demonstrate that the increased kinesin step processivity on microtubules consisting of elongated dimers underlies increased mitochondrial dynamics. Our work indicates that the molecular conformation of tubulin controls mitochondrial motility and as such locally regulates the distribution of mitochondria along axons.
Optogenetic Control of RhoA to Probe Subcellular Mechanochemical Circuitry.
Spatiotemporal localization of protein function is essential for physiological processes from subcellular to tissue scales. Genetic and pharmacological approaches have played instrumental roles in isolating molecular components necessary for subcellular machinery. However, these approaches have limited capabilities to reveal the nature of the spatiotemporal regulation of subcellular machineries like those of cytoskeletal organelles. With the recent advancement of optogenetic probes, the field now has a powerful tool to localize cytoskeletal stimuli in both space and time. Here, we detail the use of tunable light-controlled interacting protein tags (TULIPs) to manipulate RhoA signaling in vivo. This is an optogenetic dimerization system that rapidly, reversibly, and efficiently directs a cytoplasmic RhoGEF to the plasma membrane for activation of RhoA using light. We first compare this probe to other available optogenetic systems and outline the engineering logic for the chosen recruitable RhoGEFs. We also describe how to generate the cell line, spatially control illumination, confirm optogenetic control of RhoA, and mechanically induce cell-cell junction deformation in cultured tissues. Together, these protocols detail how to probe the mechanochemical circuitry downstream of RhoA signaling. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of a stable cell line expressing TULIP constructs Basic Protocol 2: Preparation of collagen substrate for imaging Basic Protocol 3: Transient transfection for visualization of downstream effectors Basic Protocol 4: Calibration of spatial illumination Basic Protocol 5: Optogenetic activation of a region of interest.
RhoA Mediates Epithelial Cell Shape Changes via Mechanosensitive Endocytosis.
Epithelial remodeling involves ratcheting behavior whereby periodic contractility produces transient changes in cell-cell contact lengths, which stabilize to produce lasting morphogenetic changes. Pulsatile RhoA activity is thought to underlie morphogenetic ratchets, but how RhoA governs transient changes in junction length, and how these changes are rectified to produce irreversible deformation, remains poorly understood. Here, we use optogenetics to characterize responses to pulsatile RhoA in model epithelium. Short RhoA pulses drive reversible junction contractions, while longer pulses produce irreversible junction length changes that saturate with prolonged pulse durations. Using an enhanced vertex model, we show this is explained by two effects: thresholded tension remodeling and continuous strain relaxation. Our model predicts that structuring RhoA into multiple pulses overcomes the saturation of contractility and confirms this experimentally. Junction remodeling also requires formin-mediated E-cadherin clustering and dynamin-dependent endocytosis. Thus, irreversible junction deformations are regulated by RhoA-mediated contractility, membrane trafficking, and adhesion receptor remodeling.
SapTrap Assembly of Caenorhabditis elegans MosSCI Transgene Vectors.
The Mos1-mediated Single-Copy Insertion (MosSCI) method is widely used to establish stable Caenorhabditis elegans transgenic strains. Cloning MosSCI targeting plasmids can be cumbersome because it requires assembling multiple genetic elements including a promoter, a 3'UTR and gene fragments. Recently, Schwartz and Jorgensen developed the SapTrap method for the one-step assembly of plasmids containing components of the CRISPR/Cas9 system for C. elegans Here, we report on the adaptation of the SapTrap method for the efficient and modular assembly of a promoter, 3'UTR and either 2 or 3 gene fragments in a MosSCI targeting vector in a single reaction. We generated a toolkit that includes several fluorescent tags, components of the ePDZ/LOV optogenetic system and regulatory elements that control gene expression in the C. elegans germline. As a proof of principle, we generated a collection of strains that fluorescently label the endoplasmic reticulum and mitochondria in the hermaphrodite germline and that enable the light-stimulated recruitment of mitochondria to centrosomes in the one-cell worm embryo. The method described here offers a flexible and efficient method for assembly of custom MosSCI targeting vectors.
Mechanosensitive junction remodelling promotes robust epithelial morphogenesis.
Morphogenesis of epithelial tissues requires tight spatiotemporal coordination of cell shape changes. In vivo, many tissue-scale shape changes are driven by pulsatile contractions of intercellular junctions, which are rectified to produce irreversible deformations. The functional role of this pulsatory ratchet and its mechanistic basis remain unknown. Here we combine theory and biophysical experiments to show that mechanosensitive tension remodelling of epithelial cell junctions promotes robust epithelial shape changes via ratcheting. Using optogenetic control of actomyosin contractility, we find that epithelial junctions show elastic behaviour under low contractile stress, returning to their original lengths after contraction, but undergo irreversible deformation under higher magnitudes of contractile stress. Existing vertex-based models for the epithelium are unable to capture these results, with cell junctions displaying purely elastic or fluid-like behaviours, depending on the choice of model parameters. To describe the experimental results, we propose a modified vertex model with two essential ingredients for junction mechanics: thresholded tension remodelling and continuous strain relaxation. First, a critical strain threshold for tension remodelling triggers irreversible junction length changes for sufficiently strong contractions, making the system robust to small fluctuations in contractile activity. Second, continuous strain relaxation allows for mechanical memory removal, enabling frequency-dependent modulation of cell shape changes via mechanical ratcheting. Taken together, the combination of mechanosensitive tension remodelling and junctional strain relaxation provides a robust mechanism for large-scale morphogenesis.
Neurotrophin receptor tyrosine kinases regulated with near-infrared light.
Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.
Guided by light: optogenetic control of microtubule gliding assays.
Force generation by molecular motors drives biological processes such as asymmetric cell division and cell migration. Microtubule gliding assays, in which surface-immobilized motor proteins drive microtubule propulsion, are widely used to study basic motor properties as well as the collective behavior of active self-organized systems. Additionally, these assays can be employed for nanotechnological applications such as analyte detection, bio-computation and mechanical sensing. While such assays allow tight control over the experimental conditions, spatiotemporal control of force generation has remained underdeveloped. Here we use light-inducible protein-protein interactions to recruit molecular motors to the surface to control microtubule gliding activity in vitro. We show that using these light-inducible interactions, proteins can be recruited to the surface in patterns, reaching a ~5-fold enrichment within 6 seconds upon illumination. Subsequently, proteins are released with a half-life of 13 seconds when the illumination is stopped. We furthermore demonstrate that light-controlled kinesin recruitment results in reversible activation of microtubule gliding along the surface, enabling efficient control over local microtubule motility. Our approach to locally control force generation offers a way to study the effects of non-uniform pulling forces on different microtubule arrays and also provides novel strategies for local control in nanotechnological applications.
Adherens junction-associated pores mediate the intercellular transport of endosomes and cytoplasmic proteins.
Intercellular endosomes (IEs) are endocytosed vesicles shuttled through the adherens junctions (AJs) between two neighboring epidermal cells during Drosophila dorsal closure. The cell-to-cell transport of IEs requires DE-cadherin (DE-cad), microtubules (MTs) and kinesin. However, the mechanisms by which IEs can be transported through the AJs are unknown. Here, we demonstrate the presence of AJ-associated pores with MTs traversing through the pores. Live imaging allows direct visualization of IEs being transported through the AJ-associated pores. By using an optogenetic dimerization system, we observe that the dimerized IE-kinesin complexes move across AJs into the neighboring cell. The AJ-associated pores also allow intercellular movement of soluble proteins. Importantly, most epidermal cells form dorsoventral-oriented two-cell syncytia. Together, we present a model in which an AJ-associated pore mediates the intercellular transport of IEs and proteins between two cells in direct contact.
Optogenetic dissection of mitotic spindle positioning in vivo.
The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα-GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα-GDP, Gα-GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP-GPR-1/2 Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.
An Optogenetic approach to control protein localization during embryogenesis of the sea urchin.
Light inducible protein-protein interactions have been used to manipulate protein localization and function in the cell with utmost spatial and temporal precision. In this technical report, we use a recently developed optogenetic approach to manipulate protein localization in the developing sea urchin embryo. A photosensitive LOV domain from Avena sativa phototropin1 cages a small peptide that binds the engineered PDZ domain (ePDZ) upon blue light irradiation. Using this system, mCherry tagged proteins fused with the LOV domain were recruited to ectopic sub-cellular regions such as the membrane, microtubules, or actin by GFP tagged proteins fused with the ePDZ domain upon blue light irradiation within 1~3 minutes in the sea urchin embryo. The efficiency and speed of recruitment of each protein to its respective subcellular region appeared to be dependent on the power and duration of laser irradiation, as well as the respective level of affinity to the tagged location. Controlled laser irradiation allowed partial recruitment of the spindle to the membrane, and resulted in cell blebbing. Vasa, a cell cycle and germline factor that localizes on the spindle and enriches in the micromeres at 8-16 cell stage was recruited to ectopic sites, preventing normal enrichment. Continuous blue light activation with a regular blue aquarium light over two days of culture successfully induced LOV-ePDZ binding in the developing embryos, resulting in continued ectopic recruitment of Vasa and failure in gastrulation at Day 2. Although some cytotoxicity was observed with prolonged blue light irradiation, this optogenetic system provides a promising approach to test the sub-cellular activities of developmental factors, as well as to alter protein localization and development during embryogenesis.
Engaging myosin VI tunes motility, morphology, and identity in endocytosis.
While unconventional myosins interact with different stages of the endocytic pathway, they are ascribed a transport function that is secondary to the protein complexes that control organelle identity. Endosomes are subject to a dynamic, continuous flux of proteins that control their characteristic properties, including their motility within the cell. Efforts to describe the changes in identity of this compartment have largely focused on the adaptors present on the compartment and not on the motile properties of the compartment itself. In this study, we use a combination of optogenetic and chemical-dimerization strategies to target exogenous myosin VI to early endosomes, and probe its influence on organelle motility, morphology, and identity. Our analysis across time scales suggests a model wherein the artificial engagement of myosin VI motility on early endosomes restricts microtubule-based motion, followed by morphological changes characterized by the rapid condensation and disintegration of organelles, ultimately leading to the enhanced overlap of markers that demarcate endosomal compartments. Together, our findings show that synthetic engagement of myosin VI motility is sufficient to alter organelle homeostasis in the endocytic pathway. This article is protected by copyright. All rights reserved.
Optogenetic interrogation of integrin αVβ3 function in endothelial cells.
αVβ3 is reported to promote angiogenesis in some model systems but not in others. Here we used optogenetics to study effects of αVβ3 interaction with the intracellular adapter, kindlin-2, on endothelial cell functions potentially relevant to angiogenesis. Since interaction of kindlin-2 with αVβ3 requires the C-terminal three residues of the β3 cytoplasmic tail (Arg-Gly-Thr; RGT), optogenetic probes LOVpep and ePDZ1 were fused to β3ΔRGT-GFP and mCherry-kindlin2, respectively, and expressed in β3-null microvascular endothelial cells. Exposure of the cells to 450 nm (blue) light caused rapid and specific interaction of kindlin-2 with αVβ3 as assessed by immunofluorescence and TIRF microscopy, and it led to increased endothelial cell migration, podosome formation and angiogenic sprouting. Analyses of kindlin-2 mutants indicated that interaction of kindlin-2 with other kindlin-2 binding partners, including c-Src, actin, integrin-linked kinase and phosphoinositides, were also likely necessary for these endothelial cell responses. Thus, kindlin-2 promotes αVβ3-dependent angiogenic functions of endothelial cells through its simultaneous interactions with β3 and several other binding partners. Optogenetic approaches should find further use in clarifying spatiotemporal aspects of vascular cell biology.
Cell cycle entry triggers a switch between two modes of Cdc42 activation during yeast polarization.
Cell polarization underlies many cellular and organismal functions. The GTPase Cdc42 orchestrates polarization in many contexts. In budding yeast, polarization is associated with a focus of Cdc42•GTP which is thought to self sustain by recruiting a complex containing Cla4, a Cdc42-binding effector, Bem1, a scaffold, and Cdc24, a Cdc42 GEF. Using optogenetics, we probe yeast polarization and find that local recruitment of Cdc24 or Bem1 is sufficient to induce polarization by triggering self-sustaining Cdc42 activity. However, the response to these perturbations depends on the recruited molecule, the cell cycle stage, and existing polarization sites. Before cell cycle entry, recruitment of Cdc24, but not Bem1, induces a metastable pool of Cdc42 that is sustained by positive feedback. Upon Cdk1 activation, recruitment of either Cdc24 or Bem1 creates a stable site of polarization that induces budding and inhibits formation of competing sites. Local perturbations have therefore revealed unexpected features of polarity establishment.
Optogenetic control of RhoA reveals zyxin-mediated elasticity of stress fibres.
Cytoskeletal mechanics regulates cell morphodynamics and many physiological processes. While contractility is known to be largely RhoA-dependent, the process by which localized biochemical signals are translated into cell-level responses is poorly understood. Here we combine optogenetic control of RhoA, live-cell imaging and traction force microscopy to investigate the dynamics of actomyosin-based force generation. Local activation of RhoA not only stimulates local recruitment of actin and myosin but also increased traction forces that rapidly propagate across the cell via stress fibres and drive increased actin flow. Surprisingly, this flow reverses direction when local RhoA activation stops. We identify zyxin as a regulator of stress fibre mechanics, as stress fibres are fluid-like without flow reversal in its absence. Using a physical model, we demonstrate that stress fibres behave elastic-like, even at timescales exceeding turnover of constituent proteins. Such molecular control of actin mechanics likely plays critical roles in regulating morphodynamic events.
A Phytochrome-Derived Photoswitch for Intracellular Transport.
Cells depend on the proper positioning of their organelles, suggesting that active manipulation of organelle positions can be used to explore spatial cell biology and to restore cellular defects caused by organelle misplacement. Recently, blue-light dependent recruitment of specific motors to selected organelles has been shown to alter organelle motility and positioning, but these approaches lack rapid and active reversibility. The light-dependent interaction of phytochrome B with its interacting factors has been shown to function as a photoswitch, dimerizing under red light and dissociating under far-red light. Here we engineer phytochrome domains into photoswitches for intracellular transport that enable the reversible interaction between organelles and motor proteins. Using patterned illumination and live-cell imaging, we demonstrate that this system provides unprecedented spatiotemporal control. We also demonstrate that it can be used in combination with a blue-light dependent system to independently control the positioning of two different organelles. Precise optogenetic control of organelle motility and positioning will provide a better understanding of and control over the spatial biology of cells.
Transcription activator-like effector-mediated regulation of gene expression based on the inducible packaging and delivery via designed extracellular vesicles.
Transcription activator-like effector (TALE) proteins present a powerful tool for genome editing and engineering, enabling introduction of site-specific mutations, gene knockouts or regulation of the transcription levels of selected genes. TALE nucleases or TALE-based transcription regulators are introduced into mammalian cells mainly via delivery of the coding genes. Here we report an extracellular vesicle-mediated delivery of TALE transcription regulators and their ability to upregulate the reporter gene in target cells. Designed transcriptional activator TALE-VP16 fused to the appropriate dimerization domain was enriched as a cargo protein within extracellular vesicles produced by mammalian HEK293 cells stimulated by Ca-ionophore and using blue light- or rapamycin-inducible dimerization systems. Blue light illumination or rapamycin increased the amount of the TALE-VP16 activator in extracellular vesicles and their addition to the target cells resulted in an increased expression of the reporter gene upon addition of extracellular vesicles to the target cells. This technology therefore represents an efficient delivery for the TALE-based transcriptional regulators.
Local RhoA activation induces cytokinetic furrows independent of spindle position and cell cycle stage.
The GTPase RhoA promotes contractile ring assembly and furrow ingression during cytokinesis. Although many factors that regulate RhoA during cytokinesis have been characterized, the spatiotemporal regulatory logic remains undefined. We have developed an optogenetic probe to gain tight spatial and temporal control of RhoA activity in mammalian cells and demonstrate that cytokinetic furrowing is primarily regulated at the level of RhoA activation. Light-mediated recruitment of a RhoGEF domain to the plasma membrane leads to rapid induction of RhoA activity, leading to assembly of cytokinetic furrows that partially ingress. Furthermore, furrow formation in response to RhoA activation is not temporally or spatially restricted. RhoA activation is sufficient to generate furrows at both the cell equator and cell poles, in both metaphase and anaphase. Remarkably, furrow formation can be initiated in rounded interphase cells, but not adherent cells. These results indicate that RhoA activation is sufficient to induce assembly of functional contractile rings and that cell rounding facilitates furrow formation.
Light-controlled intracellular transport in Caenorhabditis elegans.
To establish and maintain their complex morphology and function, neurons and other polarized cells exploit cytoskeletal motor proteins to distribute cargoes to specific compartments. Recent studies in cultured cells have used inducible motor protein recruitment to explore how different motors contribute to polarized transport and to control the subcellular positioning of organelles. Such approaches also seem promising avenues for studying motor activity and organelle positioning within more complex cellular assemblies, but their applicability to multicellular in vivo systems has so far remained unexplored. Here, we report the development of an optogenetic organelle transport strategy in the in vivo model system Caenorhabditis elegans. We demonstrate that movement and pausing of various organelles can be achieved by recruiting the proper cytoskeletal motor protein with light. In neurons, we find that kinesin and dynein exclusively target the axon and dendrite, respectively, revealing the basic principles for polarized transport. In vivo control of motor attachment and organelle distributions will be widely useful in exploring the mechanisms that govern the dynamic morphogenesis of cells and tissues, within the context of a developing animal.