RESUMEN
DEAD- and DExH-box ATPases (DDX/DHXs) are abundant and highly conserved cellular enzymes ubiquitously involved in RNA processing. By remodeling RNA-RNA and RNA-protein interactions, they often function as gatekeepers that control the progression of diverse RNA maturation steps. Intriguingly, most DDX/DHXs localize to membraneless organelles (MLOs) such as nucleoli, nuclear speckles, stress granules, or processing bodies. Recent findings suggest not only that localization to MLOs can promote interaction between DDX/DHXs and their targets but also that DDX/DHXs are key regulators of MLO formation and turnover through their condensation and ATPase activity.In this review, we describe the molecular function of DDX/DHXs in ribosome biogenesis, messenger RNA splicing, export, translation, and storage or decay as well as their association with prominent MLOs. We discuss how the enzymatic function of DDX/DHXs in RNA processing is linked to DDX/DHX condensation, the accumulation of ribonucleoprotein particles and MLO dynamics. Future research will reveal how these processes orchestrate the RNA life cycle in MLO space and DDX/DHX time.
Asunto(s)
ARN Helicasas DEAD-box , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/química , Humanos , Animales , ARN/metabolismo , ARN/genética , ARN/química , Empalme del ARN , Orgánulos/metabolismo , Orgánulos/genética , Ribosomas/metabolismo , Ribosomas/genética , Pliegue del ARN , Procesamiento Postranscripcional del ARN , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Nucléolo Celular/metabolismo , Nucléolo Celular/genética , ARN Mensajero/metabolismo , ARN Mensajero/genéticaRESUMEN
Oocytes are among the longest-lived cells in the body and need to preserve their cytoplasm to support proper embryonic development. Protein aggregation is a major threat for intracellular homeostasis in long-lived cells. How oocytes cope with protein aggregation during their extended life is unknown. Here, we find that mouse oocytes accumulate protein aggregates in specialized compartments that we named endolysosomal vesicular assemblies (ELVAs). Combining live-cell imaging, electron microscopy, and proteomics, we found that ELVAs are non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes held together by a protein matrix formed by RUFY1. Functional assays revealed that in immature oocytes, ELVAs sequester aggregated proteins, including TDP-43, and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to the accumulation of protein aggregates in the embryo and is detrimental for embryo survival. Thus, ELVAs represent a strategy to safeguard protein homeostasis in long-lived cells.
Asunto(s)
Vesículas Citoplasmáticas , Oocitos , Agregado de Proteínas , Animales , Femenino , Ratones , Autofagosomas , Vesículas Citoplasmáticas/metabolismo , Lisosomas/metabolismo , Oocitos/citología , Oocitos/metabolismo , Complejo de la Endopetidasa Proteasomal , ProteolisisRESUMEN
The ability to map trafficking for thousands of endogenous proteins at once in living cells would reveal biology currently invisible to both microscopy and mass spectrometry. Here, we report TransitID, a method for unbiased mapping of endogenous proteome trafficking with nanometer spatial resolution in living cells. Two proximity labeling (PL) enzymes, TurboID and APEX, are targeted to source and destination compartments, and PL with each enzyme is performed in tandem via sequential addition of their small-molecule substrates. Mass spectrometry identifies the proteins tagged by both enzymes. Using TransitID, we mapped proteome trafficking between cytosol and mitochondria, cytosol and nucleus, and nucleolus and stress granules (SGs), uncovering a role for SGs in protecting the transcription factor JUN from oxidative stress. TransitID also identifies proteins that signal intercellularly between macrophages and cancer cells. TransitID offers a powerful approach for distinguishing protein populations based on compartment or cell type of origin.
Asunto(s)
Mitocondrias , Proteoma , Proteoma/metabolismo , Mitocondrias/metabolismo , Nucléolo Celular/metabolismo , Espectrometría de Masas/métodos , Regulación de la Expresión GénicaRESUMEN
DEAD-box ATPases constitute a very large protein family present in all cells, often in great abundance. From bacteria to humans, they play critical roles in many aspects of RNA metabolism, and due to their widespread importance in RNA biology, they have been characterized in great detail at both the structural and biochemical levels. DEAD-box proteins function as RNA-dependent ATPases that can unwind short duplexes of RNA, remodel ribonucleoprotein (RNP) complexes, or act as clamps to promote RNP assembly. Yet, it often remains enigmatic how individual DEAD-box proteins mechanistically contribute to specific RNA-processing steps. Here, we review the role of DEAD-box ATPases in the regulation of gene expression and propose that one common function of these enzymes is in the regulation of liquid-liquid phase separation of RNP condensates.
Asunto(s)
ARN Helicasas DEAD-box , ARN , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , ARN Helicasas DEAD-box/química , Expresión Génica , Humanos , ARN/metabolismoRESUMEN
Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here, we demonstrate synthetic membraneless organelles in Escherichia coli termed transcriptionally engineered addressable RNA solvent droplets (TEARS). TEARS are assembled from RNA-binding protein recruiting domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures with composition and reaction robustness governed by non-equilibrium dynamics. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates, and scaffolding protein-protein interactions. We anticipate TEARS to be a simple and versatile tool for spatially controlling E. coli biochemistry. Particularly, the modular design of TEARS enables applications without expression fine-tuning, simplifying the design-build-test cycle of bioengineering.
Asunto(s)
Escherichia coli , Orgánulos , Escherichia coli/genética , Orgánulos/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Solventes/análisis , Solventes/metabolismoRESUMEN
Cellular stress leads to reprogramming of mRNA translation and formation of stress granules (SGs), membraneless organelles consisting of mRNA and RNA-binding proteins. Although the function of SGs remains largely unknown, it is widely assumed they contain exclusively non-translating mRNA. Here, we re-examine this hypothesis using single-molecule imaging of mRNA translation in living cells. Although we observe non-translating mRNAs are preferentially recruited to SGs, we find unequivocal evidence that mRNAs localized to SGs can undergo translation. Our data indicate that SG-associated translation is not rare, and the entire translation cycle (initiation, elongation, and termination) can occur on SG-localized transcripts. Furthermore, translating mRNAs can be observed transitioning between the cytosol and SGs without changing their translational status. Together, these results demonstrate that mRNA localization to SGs is compatible with translation and argue against a direct role for SGs in inhibition of protein synthesis.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Biosíntesis de Proteínas/genética , Transporte de ARN/genética , Imagen Individual de Molécula , Estrés Fisiológico , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Citosol/metabolismo , Células HeLa , Humanos , Sistemas de Lectura Abierta/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Liquid-liquid phase separation (LLPS) mediates formation of membraneless condensates such as those associated with RNA processing, but the rules that dictate their assembly, substructure, and coexistence with other liquid-like compartments remain elusive. Here, we address the biophysical mechanism of this multiphase organization using quantitative reconstitution of cytoplasmic stress granules (SGs) with attached P-bodies in human cells. Protein-interaction networks can be viewed as interconnected complexes (nodes) of RNA-binding domains (RBDs), whose integrated RNA-binding capacity determines whether LLPS occurs upon RNA influx. Surprisingly, both RBD-RNA specificity and disordered segments of key proteins are non-essential, but modulate multiphase condensation. Instead, stoichiometry-dependent competition between protein networks for connecting nodes determines SG and P-body composition and miscibility, while competitive binding of unconnected proteins disengages networks and prevents LLPS. Inspired by patchy colloid theory, we propose a general framework by which competing networks give rise to compositionally specific and tunable condensates, while relative linkage between nodes underlies multiphase organization.
Asunto(s)
Gránulos Citoplasmáticos/fisiología , Estructuras Citoplasmáticas/fisiología , Mapas de Interacción de Proteínas/fisiología , Fenómenos Biofísicos , Línea Celular Tumoral , Citoplasma/metabolismo , Humanos , Proteínas Intrínsecamente Desordenadas/genética , Extracción Líquido-Líquido/métodos , Orgánulos/química , ARN/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/fisiologíaRESUMEN
Microbes gain access to eukaryotic cells as food for bacteria-grazing protists, for host protection by microbe-killing immune cells, or for microbial benefit when pathogens enter host cells to replicate. But microbes can also gain access to a host cell and become an important-often required-beneficial partner. The oldest beneficial microbial infections are the ancient eukaryotic organelles now called the mitochondrion and plastid. But numerous other host-beneficial intracellular infections occur throughout eukaryotes. Here I review the genomics and cell biology of these interactions with a focus on intracellular bacteria. The genomes of host-beneficial intracellular bacteria have features that span a previously unfilled gap between pathogens and organelles. Host cell adaptations to allow the intracellular persistence of beneficial bacteria are found along with evidence for the microbial manipulation of host cells, but the cellular mechanisms of beneficial bacterial infections are not well understood.
Asunto(s)
Bacterias , Orgánulos , Bacterias/genética , Eucariontes , Células Eucariotas , Genómica , Interacciones Huésped-Patógeno/genéticaRESUMEN
Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.
Asunto(s)
Anexinas/metabolismo , Transporte Axonal/fisiología , Gránulos Citoplasmáticos/metabolismo , Lisosomas/metabolismo , ARN/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Anexinas/genética , Axones/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Mutación , Unión Proteica , Ratas/embriología , Ratas Sprague-Dawley , Transfección , Pez CebraRESUMEN
Liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here, we introduce an oligomerizing biomimetic system, "Corelets," and utilize its rapid and quantitative light-controlled tunability to map full intracellular phase diagrams, which dictate the concentrations at which phase separation occurs and the transition mechanism, in a protein sequence dependent manner. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes. VIDEO ABSTRACT.
Asunto(s)
Materiales Biomiméticos , Citoplasma/metabolismo , Animales , Materiales Biomiméticos/farmacocinética , Materiales Biomiméticos/farmacología , Células HEK293 , Células HeLa , Humanos , Ratones , Microscopía Fluorescente/métodos , Células 3T3 NIHRESUMEN
Approximately 30%-40% of global CO2 fixation occurs inside a non-membrane-bound organelle called the pyrenoid, which is found within the chloroplasts of most eukaryotic algae. The pyrenoid matrix is densely packed with the CO2-fixing enzyme Rubisco and is thought to be a crystalline or amorphous solid. Here, we show that the pyrenoid matrix of the unicellular alga Chlamydomonas reinhardtii is not crystalline but behaves as a liquid that dissolves and condenses during cell division. Furthermore, we show that new pyrenoids are formed both by fission and de novo assembly. Our modeling predicts the existence of a "magic number" effect associated with special, highly stable heterocomplexes that influences phase separation in liquid-like organelles. This view of the pyrenoid matrix as a phase-separated compartment provides a paradigm for understanding its structure, biogenesis, and regulation. More broadly, our findings expand our understanding of the principles that govern the architecture and inheritance of liquid-like organelles.
Asunto(s)
Chlamydomonas reinhardtii/citología , Cloroplastos/ultraestructura , Proteínas Algáceas/metabolismo , Dióxido de Carbono/metabolismo , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/química , Cloroplastos/metabolismo , Microscopía por Crioelectrón , Biogénesis de Organelos , Ribulosa-Bifosfato Carboxilasa/metabolismoRESUMEN
Phase transitions driven by intrinsically disordered protein regions (IDRs) have emerged as a ubiquitous mechanism for assembling liquid-like RNA/protein (RNP) bodies and other membrane-less organelles. However, a lack of tools to control intracellular phase transitions limits our ability to understand their role in cell physiology and disease. Here, we introduce an optogenetic platform that uses light to activate IDR-mediated phase transitions in living cells. We use this "optoDroplet" system to study condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1. Above a concentration threshold, these constructs undergo light-activated phase separation, forming spatiotemporally definable liquid optoDroplets. FUS optoDroplet assembly is fully reversible even after multiple activation cycles. However, cells driven deep within the phase boundary form solid-like gels that undergo aging into irreversible aggregates. This system can thus elucidate not only physiological phase transitions but also their link to pathological aggregates.
Asunto(s)
Imagen Molecular/métodos , Transición de Fase , Proteínas/química , Animales , Proteínas de Arabidopsis , Criptocromos , Proteínas Intrínsecamente Desordenadas , Cinética , Luz , Ratones , Modelos Químicos , Células 3T3 NIH , Optogenética , Mapas de Interacción de Proteínas , Proteínas/metabolismoRESUMEN
Cell diversification is at the base of increasing multicellular organism complexity in phylogeny achieved during ontogeny. However, there are also functions common to all cells, such as cell division, cell migration, translation, endocytosis, exocytosis, etc. Here we revisit the organelles involved in such common functions, reviewing recent evidence of unexpected differences of proteins at these organelles. For instance, centrosomes or mitochondria differ significantly in their protein composition in different, sometimes closely related, cell types. This has relevance for development and disease. Particularly striking is the high amount and diversity of RNA-binding proteins at these and other organelles, which brings us to review the evidence for RNA at different organelles and suborganelles. We include a discussion about (sub)organelles involved in translation, such as the nucleolus and ribosomes, for which unexpected cell type-specific diversity has also been reported. We propose here that the heterogeneity of these organelles and compartments represents a novel mechanism for regulating cell diversity. One reason is that protein functions can be multiplied by their different contributions in distinct organelles, as also exemplified by proteins with moonlighting function. The specialized organelles still perform pan-cellular functions but in a cell type-specific mode, as discussed here for centrosomes, mitochondria, vesicles, and other organelles. These can serve as regulatory hubs for the storage and transport of specific and functionally important regulators. In this way, they can control cell differentiation, plasticity, and survival. We further include examples highlighting the relevance for disease and propose to examine organelles in many more cell types for their possible differences with functional relevance.
Asunto(s)
Mitocondrias , Orgánulos , Orgánulos/metabolismo , Mitocondrias/metabolismo , División Celular , Ribosomas/metabolismo , Diferenciación CelularRESUMEN
Tethering of synaptic vesicles (SVs) to the active zone determines synaptic strength, although the molecular basis governing SV tethering is elusive. Here, we discover that small unilamellar vesicles (SUVs) and SVs from rat brains coat on the surface of condensed liquid droplets formed by active zone proteins RIM, RIM-BP, and ELKS via phase separation. Remarkably, SUV-coated RIM/RIM-BP condensates are encapsulated by synapsin/SUV condensates, forming two distinct SUV pools reminiscent of the reserve and tethered SV pools that exist in presynaptic boutons. The SUV-coated RIM/RIM-BP condensates can further cluster Ca2+ channels anchored on membranes. Thus, we reconstitute a presynaptic bouton-like structure mimicking the SV-tethered active zone with its one side attached to the presynaptic membrane and the other side connected to the synapsin-clustered SV condensates. The distinct interaction modes between membraneless protein condensates and membrane-based organelles revealed here have general implications in cellular processes, including vesicular formation and trafficking, organelle biogenesis, and autophagy.
Asunto(s)
Encéfalo/metabolismo , Canales de Calcio/metabolismo , Terminales Presinápticos/metabolismo , Sinapsinas/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Canales de Calcio/genética , Humanos , Ratones , Ratas , Sinapsinas/genética , Vesículas Sinápticas/genéticaRESUMEN
Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/metabolismo , Gránulos Citoplasmáticos/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Proteína C9orf72/genética , Línea Celular Tumoral , Gránulos Citoplasmáticos/genética , Gránulos Citoplasmáticos/patología , Dipéptidos/genética , Dipéptidos/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Ratones , Proteómica , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genéticaRESUMEN
Enzymes or enzyme complexes can be concentrated in different cellular loci to modulate distinct functional processes in response to specific signals. How cells condense and compartmentalize enzyme complexes for spatiotemporally distinct cellular events is not well understood. Here we discover that specific and tight association of GIT1 and ß-Pix, a pair of GTPase regulatory enzymes, leads to phase separation of the complex without additional scaffolding molecules. GIT1/ß-Pix condensates are modular in nature and can be positioned at distinct cellular compartments, such as neuronal synapses, focal adhesions, and cell-cell junctions, by upstream adaptors. Guided by the structure of the GIT/PIX complex, we specifically probed the role of phase separation of the enzyme complex in cell migration and synapse formation. Our study suggests that formation of modular enzyme complex condensates via phase separation can dynamically concentrate limited quantities of enzymes to distinct cellular compartments for specific and optimal signaling.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Transducción de Señal , Animales , Proteínas de Ciclo Celular/química , Proteínas Activadoras de GTPasa/química , Células HEK293 , Células HeLa , Humanos , Ratones , Modelos Moleculares , Paxillin/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismoRESUMEN
Intrinsic and innate immune responses are essential lines of defense in the body's constant surveillance of pathogens. The discovery of liquid-liquid phase separation (LLPS) as a key regulator of this primal response to infection brings an updated perspective to our understanding of cellular defense mechanisms. Here, we review the emerging multifaceted role of LLPS in diverse aspects of mammalian innate immunity, including DNA and RNA sensing and inflammasome activity. We discuss the intricate regulation of LLPS by post-translational modifications (PTMs), and the subversive tactics used by viruses to antagonize LLPS. This Review, therefore, underscores the significance of LLPS as a regulatory node that offers rapid and plastic control over host immune signaling, representing a promising target for future therapeutic strategies.
Asunto(s)
Inmunidad Innata , Inflamasomas , Humanos , Animales , Inflamasomas/metabolismo , Inflamasomas/inmunología , Procesamiento Proteico-Postraduccional , Transducción de Señal , Extracción Líquido-Líquido , Separación de FasesRESUMEN
Stress granules and P-bodies are cytosolic biomolecular condensates that dynamically form by the phase separation of RNAs and proteins. They participate in translational control and buffer the proteome. Upon stress, global translation halts and mRNAs bound to the translational machinery and other proteins coalesce to form stress granules (SGs). Similarly, translationally stalled mRNAs devoid of translation initiation factors shuttle to P-bodies (PBs). Here, we review the cumulative progress made in defining the protein components that associate with mammalian SGs and PBs. We discuss the composition of SG and PB proteomes, supported by a new user-friendly database (http://rnagranuledb.lunenfeld.ca/) that curates current literature evidence for genes or proteins associated with SGs or PBs. As previously observed, the SG and PB proteomes are biased toward intrinsically disordered regions and have a high propensity to contain primary sequence features favoring phase separation. We also provide an outlook on how the various components of SGs and PBs may cooperate to organize and form membraneless organelles.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Proteoma/metabolismo , ARN Mensajero/metabolismo , Animales , HumanosRESUMEN
During cell division, precise and regulated distribution of cellular material between daughter cells is a critical step and is governed by complex biochemical and biophysical mechanisms. To achieve this, membraneless organelles and condensates often require complete disassembly during mitosis. The biophysical principles governing the disassembly of condensates remain poorly understood. Here, we used a physical biology approach to study how physical and material properties of the nucleolus, a prominent nuclear membraneless organelle in eukaryotic cells, change during mitosis and across different scales. We found that nucleolus disassembly proceeds continuously through two distinct phases with a slow and reversible preparatory phase followed by a rapid irreversible phase that was concurrent with the nuclear envelope breakdown. We measured microscopic properties of nucleolar material including effective diffusion rates and binding affinities as well as key macroscopic properties of surface tension and bending rigidity. By incorporating these measurements into the framework of critical phenomena, we found evidence that near mitosis surface tension displays a power-law behavior as a function of biochemically modulated interaction strength. This two-step disassembly mechanism maintains structural and functional stability of nucleolus while enabling its rapid and efficient disassembly in response to cell cycle cues.
Asunto(s)
Nucléolo Celular , Mitosis , Nucléolo Celular/metabolismoRESUMEN
The endoplasmic reticulum (ER) forms an interconnected network of tubules stretching throughout the cell. Understanding how ER functionality relies on its structural organization is crucial for elucidating cellular vulnerability to ER perturbations, which have been implicated in several neuronal pathologies. One of the key functions of the ER is enabling Ca[Formula: see text] signaling by storing large quantities of this ion and releasing it into the cytoplasm in a spatiotemporally controlled manner. Through a combination of physical modeling and live-cell imaging, we demonstrate that alterations in ER shape significantly impact its ability to support efficient local Ca[Formula: see text] releases, due to hindered transport of luminal content within the ER. Our model reveals that rapid Ca[Formula: see text] release necessitates mobile luminal buffer proteins with moderate binding strength, moving through a well-connected network of ER tubules. These findings provide insight into the functional advantages of normal ER architecture, emphasizing its importance as a kinetically efficient intracellular Ca[Formula: see text] delivery system.