RESUMEN
Some protein components of intracellular non-membrane-bound entities, such as RNA granules, are known to form hydrogels in vitro. The physico-chemical properties and functional role of these intracellular hydrogels are difficult to study, primarily due to technical challenges in probing these materials in situ. Here, we present iPOLYMER, a strategy for a rapid induction of protein-based hydrogels inside living cells that explores the chemically inducible dimerization paradigm. Biochemical and biophysical characterizations aided by computational modelling show that the polymer network formed in the cytosol resembles a physiological hydrogel-like entity that acts as a size-dependent molecular sieve. We functionalize these polymers with RNA-binding motifs that sequester polyadenine-containing nucleotides to synthetically mimic RNA granules. These results show that iPOLYMER can be used to synthetically reconstitute the nucleation of biologically functional entities, including RNA granules in intact cells.
Asunto(s)
Hidrogeles/metabolismo , Polímeros/metabolismo , ARN/metabolismo , Animales , Materiales Biocompatibles , Células COS , Chlorocebus aethiopsRESUMEN
In most bacteria, the tubulin-like GTPase FtsZ forms an annulus at midcell (the Z-ring) which recruits the division machinery and regulates cell wall remodeling. Although both activities require membrane attachment of FtsZ, few membrane anchors have been characterized. FtsA is considered to be the primary membrane tether for FtsZ in bacteria, however in Caulobacter crescentus, FtsA arrives at midcell after stable Z-ring assembly and early FtsZ-directed cell wall synthesis. We hypothesized that additional proteins tether FtsZ to the membrane and demonstrate that in C. crescentus, FzlC is one such membrane anchor. FzlC associates with membranes directly in vivo and in vitro and recruits FtsZ to membranes in vitro. As for most known membrane anchors, the C-terminal peptide of FtsZ is required for its recruitment to membranes by FzlC in vitro and midcell recruitment of FzlC in cells. In vivo, overproduction of FzlC causes cytokinesis defects whereas deletion of fzlC causes synthetic defects with dipM, ftsE and amiC mutants, implicating FzlC in cell wall hydrolysis. Our characterization of FzlC as a novel membrane anchor for FtsZ expands our understanding of FtsZ regulators and establishes a role for membrane-anchored FtsZ in the regulation of cell wall hydrolysis.
Asunto(s)
Proteínas Bacterianas/metabolismo , Caulobacter crescentus/metabolismo , Proteínas del Citoesqueleto/metabolismo , División Celular/fisiología , Pared Celular/metabolismo , Citocinesis/fisiología , GTP Fosfohidrolasas/metabolismo , Hidrólisis , Proteínas de la Membrana/metabolismo , Unión ProteicaRESUMEN
Nonlinear biomolecular interactions on membranes drive membrane remodeling crucial for biological processes including chemotaxis, cytokinesis, and endocytosis. The complexity of biomolecular interactions, their redundancy, and the importance of spatiotemporal context in membrane organization impede understanding of the physical principles governing membrane mechanics. Developing a minimal in vitro system that mimics molecular signaling and membrane remodeling while maintaining physiological fidelity poses a major challenge. Inspired by chemotaxis, we reconstructed chemically regulated actin polymerization inside vesicles, guiding membrane self-organization. An external, undirected chemical input induced directed actin polymerization and membrane deformation uncorrelated with upstream biochemical cues, suggesting symmetry breaking. A biophysical model incorporating actin dynamics and membrane mechanics proposes that uneven actin distributions cause nonlinear membrane deformations, consistent with experimental findings. This protocellular system illuminates the interplay between actin dynamics and membrane shape during symmetry breaking, offering insights into chemotaxis and other cell biological processes.
Asunto(s)
Actinas , Células Artificiales , Membrana Celular , Polimerizacion , Actinas/metabolismo , Células Artificiales/metabolismo , Células Artificiales/química , Membrana Celular/metabolismo , Quimiotaxis , Modelos BiológicosRESUMEN
Genetic circuits that control transgene expression in response to pre-defined transcriptional cues would enable the development of smart therapeutics. To this end, here we engineer programmable single-transcript RNA sensors in which adenosine deaminases acting on RNA (ADARs) autocatalytically convert target hybridization into a translational output. Dubbed DART VADAR (Detection and Amplification of RNA Triggers via ADAR), our system amplifies the signal from editing by endogenous ADAR through a positive feedback loop. Amplification is mediated by the expression of a hyperactive, minimal ADAR variant and its recruitment to the edit site via an orthogonal RNA targeting mechanism. This topology confers high dynamic range, low background, minimal off-target effects, and a small genetic footprint. We leverage DART VADAR to detect single nucleotide polymorphisms and modulate translation in response to endogenous transcript levels in mammalian cells.
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Edición Génica , Edición de ARN , Animales , Regulación de la Expresión Génica , ARN/metabolismo , Redes Reguladoras de Genes , Adenosina Desaminasa/genética , Mamíferos/genéticaRESUMEN
Non-linear biomolecular interactions on the membranes drive membrane remodeling that underlies fundamental biological processes including chemotaxis, cytokinesis, and endocytosis. The multitude of biomolecules, the redundancy in their interactions, and the importance of spatiotemporal context in membrane organization hampers understanding the physical principles governing membrane mechanics. A minimal, in vitro system that models the functional interactions between molecular signaling and membrane remodeling, while remaining faithful to cellular physiology and geometry is powerful yet remains unachieved. Here, inspired by the biophysical processes underpinning chemotaxis, we reconstituted externally-controlled actin polymerization inside giant unilamellar vesicles, guiding self-organization on the membrane. We show that applying undirected external chemical inputs to this system results in directed actin polymerization and membrane deformation that are uncorrelated with upstream biochemical cues, indicating symmetry breaking. A biophysical model of the dynamics and mechanics of both actin polymerization and membrane shape suggests that inhomogeneous distributions of actin generate membrane shape deformations in a non-linear fashion, a prediction consistent with experimental measurements and subsequent local perturbations. The active protocellular system demonstrates the interplay between actin dynamics and membrane shape in a symmetry breaking context that is relevant to chemotaxis and a suite of other biological processes.
RESUMEN
Form and function are often interdependent throughout biology. Inside cells, mitochondria have particularly attracted attention since both their morphology and functionality are altered under pathophysiological conditions. However, directly assessing their causal relationship has been beyond reach due to the limitations of manipulating mitochondrial morphology in a physiologically relevant manner. By engineering a bacterial actin regulator, ActA, we developed tools termed "ActuAtor" that inducibly trigger actin polymerization at arbitrary subcellular locations. The ActuAtor-mediated actin polymerization drives striking deformation and/or movement of target organelles, including mitochondria, Golgi apparatus, and nucleus. Notably, ActuAtor operation also disperses non-membrane-bound entities such as stress granules. We then implemented ActuAtor in functional assays, uncovering the physically fragmented mitochondria being slightly more susceptible to degradation, while none of the organelle functions tested are morphology dependent. The modular and genetically encoded features of ActuAtor should enable its application in studies of the form-function interplay in various intracellular contexts.
Asunto(s)
Listeria monocytogenes , Listeria , Actinas/metabolismo , Listeria/metabolismo , Listeria monocytogenes/fisiología , Polimerizacion , Orgánulos/metabolismo , Proteínas Bacterianas/metabolismoRESUMEN
CRISPR-Cas systems provide versatile tools for programmable genome editing. Here, we developed a caged RNA strategy that allows Cas9 to bind DNA but not cleave until light-induced activation. This approach, referred to as very fast CRISPR (vfCRISPR), creates double-strand breaks (DSBs) at the submicrometer and second scales. Synchronized cleavage improved kinetic analysis of DNA repair, revealing that cells respond to Cas9-induced DSBs within minutes and can retain MRE11 after DNA ligation. Phosphorylation of H2AX after DNA damage propagated more than 100 kilobases per minute, reaching up to 30 megabases. Using single-cell fluorescence imaging, we characterized multiple cycles of 53BP1 repair foci formation and dissolution, with the first cycle taking longer than subsequent cycles and its duration modulated by inhibition of repair. Imaging-guided subcellular Cas9 activation further facilitated genomic manipulation with single-allele resolution. vfCRISPR enables DNA-repair studies at high resolution in space, time, and genomic coordinates.
Asunto(s)
Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , División del ADN/efectos de la radiación , Reparación del ADN/genética , Edición Génica/métodos , Análisis de la Célula Individual/métodos , Roturas del ADN de Doble Cadena , Células HEK293 , Histonas/metabolismo , Humanos , Luz , Proteína Homóloga de MRE11/genética , Imagen Óptica/métodos , FosforilaciónRESUMEN
A negating functionality is fundamental to information processing of logic circuits within cells and computers. Aiming to adapt unutilized electronic concepts to the interrogation of signaling circuits in cells, we first took a bottom-up strategy whereby we created protein-based devices that perform negating Boolean logic operations such as NOT, NOR, NAND, and N-IMPLY. These devices function in living cells within a minute by precisely commanding the localization of an activator molecule among three subcellular spaces. We networked these synthetic gates to an endogenous signaling circuit and devised a physiological output. In search of logic functions in signal transduction, we next took a top-down approach and computationally screened 108 signaling pathways to identify commonalities and differences between these biological pathways and electronic circuits. This combination of synthetic and systems approaches will guide us in developing foundations for deconstruction of intricate cell signaling, as well as construction of biomolecular computers.
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Algoritmos , Modelos Teóricos , Animales , Antibacterianos/farmacología , Células COS , Chlorocebus aethiops , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo , Transfección , Proteína Fluorescente RojaRESUMEN
One fascinating recent avenue of study in the field of synthetic biology is the creation of biomolecule-based computers. The main components of a computing device consist of an arithmetic logic unit, the control unit, memory, and the input and output devices. Boolean logic gates are at the core of the operational machinery of these parts, and hence to make biocomputers a reality, biomolecular logic gates become a necessity. Indeed, with the advent of more sophisticated biological tools, both nucleic acid- and protein-based logic systems have been generated. These devices function in the context of either test tubes or living cells and yield highly specific outputs given a set of inputs. In this review, we discuss various types of biomolecular logic gates that have been synthesized, with particular emphasis on recent developments that promise increased complexity of logic gate circuitry, improved computational speed, and potential clinical applications.
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Computadores Moleculares , Biología Sintética/métodos , Humanos , Lógica , Ácidos Nucleicos/química , Ácidos Nucleicos/genética , Proteínas/química , Proteínas/genéticaRESUMEN
Physiological Ca(2+) signaling in T lymphocytes and other cells depends on the STIM-ORAI pathway of store-operated Ca(2+) entry. STIM1 and STIM2 are Ca(2+) sensors in the endoplasmic reticulum (ER) membrane, with ER-luminal domains that monitor cellular Ca(2+) stores and cytoplasmic domains that gate ORAI channels in the plasma membrane. The STIM ER-luminal domain dimerizes or oligomerizes upon dissociation of Ca(2+), but the mechanism transmitting activation to the STIM cytoplasmic domain was previously undefined. Using Tb(3+)-acceptor energy transfer, we show that dimerization of STIM1 ER-luminal domains causes an extensive conformational change in mouse STIM1 cytoplasmic domains. The conformational change, triggered by apposition of the predicted coiled-coil 1 (CC1) regions, releases the ORAI-activating domains from their interaction with the CC1 regions and allows physical extension of the STIM1 cytoplasmic domain across the gap between ER and plasma membrane and communication with ORAI channels.