Curated Optogenetic Publication Database

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: Lipids are a major class of biological molecules, the primary components of cellular membranes, and critical signaling molecules that regulate cell biology and physiology. Due to their dynamic behavior within membranes, rapid transport between organelles, and complex and often redundant metabolic pathways, lipids have traditionally been considered among the most challenging biological molecules to study. In recent years, a plethora of tools bridging the chemistry-biology interface has emerged for studying different aspects of lipid biology. Here, we provide an overview of these approaches. We discuss methods for lipid detection, including genetically encoded biosensors, synthetic lipid analogs, and metabolic labeling probes. For targeted manipulation of lipids, we describe pharmacological agents and controllable enzymes, termed membrane editors, that harness optogenetics and chemogenetics. To conclude, we survey techniques for elucidating lipid-protein interactions, including photoaffinity labeling and proximity labeling. Collectively, these strategies are revealing new insights into the regulation, dynamics, and functions of lipids in cell biology.

Abstract: The binding of photoswitchable molecules to partners forms the basis of many naturally occurring light-dependent signaling pathways and various photopharmacological and optogenetic tools. A critical parameter affecting the function of these molecules is the thermal half-life of the light state. Reports in the literature indicate that, in some cases, a binding partner can significantly influence the thermal half-life, while in other cases it has no effect. Here, we present a unifying framework for quantitatively analyzing the effects of binding partners on thermal reversion rates. We focus on photoswitchable protein/binder interactions involving LOV domains, photoactive yellow protein, and CBCR GAF domains with partners that bind either the light or the dark state of the photoswitchable domain. We show that the effect of a binding partner depends on the extent to which the transition state for reversion resembles the dark state or the light state. We quantify this resemblance with a ϕswitching value, where ϕswitching = 1 if the conformation of the part of the photoswitchable molecule that interacts with the binding partner closely resembles its dark state conformation and ϕswitching = 0 if it resembles its light state. In addition to providing information on the transition state for switching, this analysis can guide the design of photoswitchable systems that retain useful thermal half-lives in practice. The analysis also provides a basis for the use of simple kinetic measurements to determine effective changes in affinity even in complex milieu.

Abstract: Light-based immunotherapy uses specific wavelengths of light to activate or modulate immune responses. It primarily employs two mechanisms: direct activation of immune cells and indirect modulation of the tumor microenvironment (TME). Several light-based technologies are under investigation or clinical use in immunotherapy, including photodynamic immunotherapy (PDIT) and photothermal therapy (PTT). Optogenetic tools have the potential to precisely control T-cell receptor activation, cytokine release, or the activity of other immune effector cells. Light-based technologies present innovative opportunities within the realm of immunotherapy. The ability to precisely regulate immune cell activation via optogenetics, alongside the improved targeting of cancer cells through photoimmunotherapy, signifies a transformative shift in our strategies for immune modulation. Although many of these technologies remain in the experimental stage for various applications, initial findings are encouraging, especially concerning cancer treatment and immune modulation. Continued research and clinical trials are essential to fully harness the capabilities of light technology in the context of immune cell therapy.

Abstract: Optogenetics has substantially enhanced our understanding of biological processes by enabling high-precision tracking and manipulation of individual cells. It relies on photosensitive proteins to monitor and control cellular activities, thereby paving the way for significant advancements in complex system research. Photosensitive proteins play a vital role in the development of optogenetics, facilitating the establishment of cutting-edge methods. Recent breakthroughs in protein design have opened up opportunities to develop protein-based tools that can precisely manipulate and monitor cellular activities. These advancements will significantly accelerate the development and application of optogenetic tools. This article emphasizes the pivotal role of protein design in the development of optogenetic tools, offering insights into potential future directions. We begin by providing an introduction to the historical development and fundamental principles of optogenetics, followed by an exploration of the operational mechanisms of key photosensitive domains, which includes clarifying the conformational changes they undergo in response to light, such as allosteric modulation and dimerization processes. Building on this foundation, we reveal the development of protein design tools that will enable the creation of even more sophisticated optogenetic techniques.

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: Cells contain thousands of different lipids. Their rapid and redundant metabolism, dynamic movement, and many interactions with other biomolecules have justly earned lipids a reputation as a vexing class of molecules to understand. Further, as the cell’s hydrophobic metabolites, lipids assemble into supramolecular structures─most commonly bilayers, or membranes─from which they carry out myriad biological functions. Motivated by this daunting complexity, researchers across disciplines are bringing order to the seeming chaos of biological lipids and membranes. Here, we formalize these efforts as “synthetic lipid biology”. Inspired by the idea, central to synthetic biology, that our abilities to understand and build biological systems are intimately connected, we organize studies and approaches across numerous fields to create, manipulate, and analyze lipids and biomembranes. These include construction of lipids and membranes from scratch using chemical and chemoenzymatic synthesis, editing of pre-existing membranes using optogenetics and protein engineering, detection of lipid metabolism and transport using bioorthogonal chemistry, and probing of lipid–protein interactions and membrane biophysical properties. What emerges is a portrait of an incipient field where chemists, biologists, physicists, and engineers work together in proximity─like lipids themselves─to build a clearer description of the properties, behaviors, and functions of lipids and membranes.

Abstract: Live imaging techniques have revolutionized our understanding of paracrine signaling, a crucial form of cell-to-cell communication in biological processes. This review examines recent advances in visualizing and tracking paracrine factors through four key stages: secretion from producing cells, diffusion through extracellular space, binding to target cells, and activation of intracellular signaling within target cells. Paracrine factor secretion can be directly visualized by fluorescent protein tagging to ligand, or indirectly by visualizing the cleavage of the transmembrane pro-ligands or plasma membrane fusion of endosomes comprising the paracrine factors. Diffusion of paracrine factors has been studied using techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), fluorescence decay after photoactivation (FDAP), and single-molecule tracking. Binding of paracrine factors to target cells has been visualized through various biosensors, including GPCR-activation-based (GRAB) sensors and Förster resonance energy transfer (FRET) probes for receptor tyrosine kinases. Finally, activation of intracellular signaling is monitored within the target cells by biosensors for second messengers, transcription factors, and so on. In addition to the imaging tools, the review also highlights emerging optogenetic and chemogenetic tools for triggering the release of paracrine factors, which is essential for associating the paracrine factor secretion to biological outcomes during the bioimaging of paracrine factor signaling.Key words: paracrine signaling, live imaging, biosensors, optogenetics, chemogenetics.

Abstract: Collective cell migration and tissue morphogenesis play a variety of important roles in the development of many species. Tissue morphogenesis often generates mechanical forces that alter cell shapes and arrangements, resembling collective cell migration-like behaviors. Genetic methods have been widely used to study collective cell migration and its like behavior, advancing our understanding of these processes during development. However, a growing body of research shows that collective cell migration during development is not a simple behavior but is often combined with other cellular and tissue processes. In addition, different surrounding environments can also influence migrating cells, further complicating collective cell migration during development. Due to the complexity of developmental processes and tissues, traditional genetic approaches often encounter challenges and limitations. Thus, some methods with spatiotemporal control become urgent in dissecting collective cell migration and tissue morphogenesis during development. Optogenetics is a method that combines optics and genetics, providing a perfect strategy for spatiotemporally controlling corresponding protein activity in subcellular, cellular or tissue levels. In this review, we introduce the basic mechanisms underlying different optogenetic tools. Then, we demonstrate how optogenetic methods have been applied in vivo to dissect collective cell migration and tissue morphogenesis during development. Additionally, we describe some promising optogenetic approaches for advancing this field. Together, this review will guide and facilitate future studies of collective cell migration in vivo and tissue morphogenesis by optogenetics.

Abstract: Immunotherapy, the medicinal modulation of a host's immune response to better combat a pathogen or disease, has transformed cancer treatments in recent decades. T-cells, an important component of the adaptive immune system, are further paramount for therapy success. Recent immunotherapeutic modalities have therefore more frequently targeted T-cells for cancer treatments and other pathologies and are termed adoptive T-cell (ATC) therapies. ATC therapies characterize various types of immunotherapies but predominantly fall into three established techniques: tumour-infiltrating lymphocyte, chimeric antigen receptor T-cell, and engineered T-cell receptor therapies. Despite promising clinical results, all ATC therapy types fall short in providing long-term sustained tumour clearance while being particularly ineffective against solid tumours, with substantial developments aiming to understand and prevent the typical drawbacks of ATC therapy. Optogenetics is a relatively recent development, incorporating light-sensitive protein domains into cells or tissues of interest to optically tune specific biological processes. Optogenetic manipulation of immunological functions is rapidly becoming an investigative tool in immunology, with light-sensitive systems now being used to optimize many cellular therapeutic modalities and ATC therapies. This review focuses on how optogenetic approaches are currently utilized to improve ATC therapy in clinical settings by deepening our understanding of the molecular rationale behind therapy success. Moreover, this review further critiques current immuno-optogenetic systems and speculates on the expansion of recent developments, enhancing current ATC-based therapeutic modalities to pave the way for clinical progress.

Abstract: As synthetic biology advances, the necessity for robust biocontainment strategies for genetically engineered organisms (GEOs) grows increasingly critical to mitigate biosafety risks related to their potential environmental release. This paper aims to evaluate environment signal-dependent biocontainment systems for engineered organisms, focusing specifically on leveraging triggered responses and combinatorial systems. There are different types of triggers—chemical, light, temperature, and pH—this review illustrates how these systems can be designed to respond to environmental signals, ensuring a higher safety profile. It also focuses on combinatorial biocontainment to avoid consequences of unintended GEO release into an external environment. Case studies are discussed to demonstrate the practical applications of these systems in real-world scenarios.

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: Biomolecular condensates (BMCs), play significant roles in organizing cellular functions in the absence of membranes through phase separation events involving RNA, proteins, and RNA-protein complexes. These membrane-less organelles form dynamic multivalent weak interactions, often involving intrinsically disordered proteins or regions (IDPs/IDRs). However, the nature of these crucial interactions, how most of these organelles are organized and are functional, remains unknown. Aberrant condensates have been implicated in neurodegenerative diseases and various cancers, presenting novel therapeutic opportunities for small molecule condensate modulators. Recent advancements in optogenetic technologies, particularly Corelet, enable precise manipulation of BMC dynamics within living cells, facilitating high-throughput screening for small molecules that target these complex structures. By elucidating the molecular mechanisms governing BMC formation and function, this innovative approach holds promise to unlock therapeutic strategies against previously "undruggable" protein targets, paving the way for effective interventions in disease.

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: Cell signaling through direct physical cell–cell contacts plays vital roles in biology during development, angiogenesis, and immune response. Intercellular communication mechanisms between synthetic cells constructed from the bottom up are majorly reliant on diffusible chemical signals, thus limiting the range of responses in receiver cells. Engineering contact-dependent signaling between synthetic cells promises to unlock more complicated signaling schemes with spatial responses. Herein, a light-activated contact-dependent communication scheme for synthetic cells is designed and demonstrated. A split luminescent protein is utilized to limit signal generation exclusively to contact interfaces of synthetic cells, driving the recruitment of a photoswitchable protein in receiver cells, akin to juxtacrine signaling in living cells. The modular design not only demonstrates contact-dependent communication between synthetic cells but also provides a platform for engineering orthogonal contact-dependent signaling mechanisms.

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: 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: 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: Symmetry breaking, polarity establishment, and spontaneous cell protrusion formation are fundamental but poorly explained cell behaviors. Here, we demonstrate that a biochemical network, where the mutually inhibitory localization of PIP5K and Ras activities plays a central role, governs these processes. First, in resting cells devoid of cytoskeletal activity, PIP5K is uniformly elevated on the plasma membrane, while Ras activity remains minimal. Symmetry is broken by spontaneous local displacements of PIP5K, coupled with simultaneous activations of Ras and downstream signaling events, including PI3K activation. Second, knockout of PIP5K dramatically increases both the incidence and size of Ras-PI3K activation patches, accompanied by branched F-actin assembly. This leads to enhanced cortical wave formation, increased protrusive activity, and a shift in migration mode. Third, high inducible overexpression of PIP5K virtually eliminates Ras-PI3K signaling, cytoskeletal activity, and cell migration, while acute recruitment of cytosolic PIP5K to the membrane induces contraction and blebs in cancer cells. These arrested phenotypes are reversed by reducing myosin II activity, indicating myosin’s involvement in the PIP5K-Ras-centered regulatory network. Remarkably, low inducible overexpression of PIP5K unexpectedly facilitates polarity establishment, highlighting PIP5K as a highly sensitive master regulator of these processes. Simulations of a computational model combining an excitable system, cytoskeletal loops, and dynamic partitioning of PIP5K recreates the experimental observations. Taken together, our results reveal that a bistable, mutually exclusive localization of PIP5K and active Ras on the plasma membrane triggers the initial symmetry breaking. Coupled actomyosin reduction and increased actin polymerization lead to intermittently extended protrusions and, with feedback from the cytoskeleton, self-organizing, complementary gradients of PIP5K versus Ras steepen, raising the threshold of the networks at the rear and lowering it at the front to generate polarity for cell migration.

Abstract: Kinesin and dynein-dynactin motors move endosomes and other vesicles bidirectionally along microtubules, a process mainly studied under in vitro conditions. Here, we provide a physiological bidirectional transport model following color-coded, endogenously tagged transport-related proteins as they move through a crowded cellular environment. Late endosomes (LEs) surf bidirectionally on Protrudin-enriched endoplasmic reticulum (ER) membrane contact sites, while hopping and gliding along microtubules and bypassing cellular obstacles, such as mitochondria. During bidirectional transport, late endosomes do not switch between opposing Rab7 GTPase effectors, RILP and FYCO1, or their associated dynein and KIF5B motor proteins, respectively. In the endogenous setting, far fewer motors associate with endosomal membranes relative to effectors, implying coordination of transport with other aspects of endosome physiology through GTPase-regulated mechanisms. We find that directionality of transport is provided in part by various microtubule-associated proteins (MAPs), including MID1, EB1, and CEP169, which recruit Lis1-activated dynein motors to microtubule plus ends for transport of early and late endosomal populations. At these microtubule plus ends, activated dynein motors encounter the dynactin subunit p150glued and become competent for endosomal capture and minus-end movement in collaboration with membrane-associated Rab7-RILP. We show that endosomes surf over the ER through the crowded cell and move bidirectionally under the control of MAPs for motor activation and through motor replacement and capture by endosomal anchors.

Abstract: Extracellular-signal-regulated kinase (ERK) signaling controls development and homeostasis and is genetically deregulated in human diseases, including neurocognitive disorders and cancers. Although the list of ERK functions is vast and steadily growing, the full spectrum of processes controlled by any specific ERK activation event remains unknown. Here, we show how ERK functions can be systematically identified using targeted perturbations and global readouts of ERK activation. Our experimental model is the Drosophila embryo, where ERK signaling at the embryonic poles has thus far only been associated with the transcriptional patterning of the future larva. Through a combination of live imaging and phosphoproteomics, we demonstrated that ERK activation at the poles is also critical for maintaining the speed and synchrony of embryonic cleavages. The presented approach to interrogating phosphorylation networks identifies a hidden function of a well-studied signaling event and sets the stage for similar studies in other organisms.

Abstract: In recent decades, the field of synthetic biology has witnessed remarkable progress, driving advances in both research and practical applications. One pivotal area of development involves the design of transgene switches capable of precisely regulating specified outputs and controlling cell behaviors in response to physical cues, which encompass light, magnetic fields, temperature, mechanical forces, ultrasound, and electricity. In this review, we delve into the cutting-edge progress made in the field of physically controlled protein expression in engineered mammalian cells, exploring the diverse genetic tools and synthetic strategies available for engineering targeting cells to sense these physical cues and generate the desired outputs accordingly. We discuss the precision and efficiency limitations inherent in these tools, while also highlighting their immense potential for therapeutic applications.

Abstract: Biomolecular condensates are broadly implicated in both normal cellular regulation and disease. Consequently, several chemical biology and optogenetic approaches have been developed to induce phase separation of a protein of interest. However, few tools are available to perform the converse function - dissolving a condensate of interest on demand. Such a tool would aid in testing whether the condensate plays specific functional roles. Here we show that light-gated recruitment of a solubilizing domain, maltose-binding protein (MBP), results in rapid and controlled dissolution of condensates formed from proteins of interest. Our optogenetic MBP-based dissolution strategy (OptoMBP) is rapid, reversible, and can be spatially controlled with subcellular precision. We also provide a proof-of-principle application of OptoMBP by disrupting condensation of the oncogenic fusion protein FUS-CHOP and reverting FUS-CHOP driven transcriptional changes. We envision that the OptoMBP system could be broadly useful for disrupting constitutive protein condensates to probe their biological functions.

Abstract: TANGO1, TANGO1-Short, and cTAGE5 form stable complexes at the endoplasmic reticulum exit sites (ERES) to preferably export bulky cargoes. Their C-terminal proline-rich domain (PRD) binds Sec23A and affects COPII assembly. The PRD in TANGO1-Short was replaced with light-responsive domains to control its binding to Sec23A in U2OS cells (human osteosarcoma). TANGO1-ShortΔPRD was dispersed in the ER membrane but relocated rapidly, reversibly, to pre-existing ERES by binding to Sec23A upon light activation. Prolonged binding between the two, concentrated ERES in the juxtanuclear region, blocked cargo export and relocated ERGIC53 into the ER, minimally impacting the Golgi complex organization. Bulky collagen VII and endogenous collagen I were collected at less than 47% of the stalled ERES, whereas small cargo molecules were retained uniformly at almost all the ERES. We suggest that ERES are segregated to handle cargoes based on their size, permitting cells to traffic them simultaneously for optimal secretion.