RESUMO
Volume control is a fundamental challenge for all cells, the mechanisms of which have been long debated. In this issue of Cell, Boyd-Shiwarski et al. find that increased molecular crowding drives condensation of WNK kinase, allowing cells to sense and respond to cell volume loss.
Assuntos
Tamanho CelularRESUMO
Cell size varies greatly between cell types, yet within a specific cell type and growth condition, cell size is narrowly distributed. Why maintenance of a cell-type specific cell size is important remains poorly understood. Here we show that growing budding yeast and primary mammalian cells beyond a certain size impairs gene induction, cell-cycle progression, and cell signaling. These defects are due to the inability of large cells to scale nucleic acid and protein biosynthesis in accordance with cell volume increase, which effectively leads to cytoplasm dilution. We further show that loss of scaling beyond a certain critical size is due to DNA becoming limiting. Based on the observation that senescent cells are large and exhibit many of the phenotypes of large cells, we propose that the range of DNA:cytoplasm ratio that supports optimal cell function is limited and that ratios outside these bounds contribute to aging.
Assuntos
Crescimento Celular , Senescência Celular/fisiologia , Citoplasma/metabolismo , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Ciclo Celular , Proliferação de Células , Tamanho Celular , Senescência Celular/genética , Fibroblastos/metabolismo , Células HEK293 , Humanos , Cultura Primária de Células , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomycetales/genética , Saccharomycetales/crescimento & desenvolvimento , Saccharomycetales/metabolismo , Transdução de SinaisRESUMO
The cell interior is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cellular physiology. Cellular stress responses almost universally lead to inhibition of translation, resulting in polysome collapse and release of mRNA. The released mRNA molecules condense with RNA-binding proteins to form ribonucleoprotein (RNP) condensates known as processing bodies and stress granules. Here, we show that polysome collapse and condensation of RNA transiently fluidize the cytoplasm, and coarse-grained molecular dynamic simulations support this as a minimal mechanism for the observed biophysical changes. Increased mesoscale diffusivity correlates with the efficient formation of quality control bodies (Q-bodies), membraneless organelles that compartmentalize misfolded peptides during stress. Synthetic, light-induced RNA condensation also fluidizes the cytoplasm. Together, our study reveals a functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to enable efficient response of cells to stress conditions.
Assuntos
Citoplasma , Polirribossomos , Ribonucleoproteínas , Polirribossomos/metabolismo , Citoplasma/metabolismo , Humanos , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Simulação de Dinâmica Molecular , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Condensados Biomoleculares/metabolismo , Grânulos de Estresse/metabolismo , Grânulos de Estresse/genéticaRESUMO
Phase separation can concentrate biomolecules and accelerate reactions. However, the mechanisms and principles connecting this mesoscale organization to signaling dynamics are difficult to dissect because of the pleiotropic effects associated with disrupting endogenous condensates. To address this limitation, we engineered new phosphorylation reactions within synthetic condensates. We generally found increased activity and broadened kinase specificity. Phosphorylation dynamics within condensates were rapid and could drive cell-cycle-dependent localization changes. High client concentration within condensates was important but not the main factor for efficient phosphorylation. Rather, the availability of many excess client-binding sites together with a flexible scaffold was crucial. Phosphorylation within condensates was also modulated by changes in macromolecular crowding. Finally, the phosphorylation of the Alzheimer's-disease-associated protein Tau by cyclin-dependent kinase 2 was accelerated within condensates. Thus, condensates enable new signaling connections and can create sensors that respond to the biophysical properties of the cytoplasm.
Assuntos
Transdução de Sinais , Proteínas tau , Quinase 2 Dependente de Ciclina/metabolismo , Citoplasma/metabolismo , Humanos , Substâncias Macromoleculares/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
Gene expression in eukaryotes requires the effective separation of nuclear transcription and RNA processing from cytosolic translation1. This separation is achieved by the nuclear envelope, which controls the exchange of macromolecules through nuclear pores2. During mitosis, however, the nuclear envelope in animal and plant cells disassembles, allowing cytoplasmic and nuclear components to intermix3. When the nuclear envelope is reformed, cytoplasmic components are removed from the nucleus by receptor-mediated transport through nuclear pores2. These pores have a size limit of 39 nanometres4-7, which raises the question of how larger cytoplasmic molecules are cleared from the nucleus. Here we show in HeLa cells that large cytoplasmic components are displaced before nuclear envelope assembly by the movement of chromosomes to a dense cluster. This clustering occurs when chromosomes approach the poles of anaphase spindles, and is mediated by a microtubule-independent mechanism that involves the surfactant-like protein Ki-67. Ki-67 forms repulsive molecular brushes during the early stages of mitosis8, but during mitotic exit the brushes collapse and Ki-67 promotes chromosome clustering. We show that the exclusion of mature ribosomes from the nucleus after mitosis depends on Ki-67-regulated chromosome clustering. Thus, our study reveals that chromosome mechanics help to re-establish the compartmentalization of eukaryotic cells after open mitosis.
Assuntos
Posicionamento Cromossômico , Cromossomos Humanos/metabolismo , Citoplasma/metabolismo , Antígeno Ki-67/metabolismo , Membrana Nuclear/metabolismo , Transporte Biológico , Células HeLa , Humanos , Mitose , Ribossomos/metabolismo , Fuso AcromáticoRESUMO
Cells that proliferate within a confined environment build up mechanical compressive stress. For example, mechanical pressure emerges in the naturally space-limited tumor environment. However, little is known about how cells sense and respond to mechanical compression. We developed microfluidic bioreactors to enable the investigation of the effects of compressive stress on the growth of the genetically tractable model organism Saccharomyces cerevisiae We used this system to determine that compressive stress is partly sensed through a module consisting of the mucin Msb2 and the cell wall protein Sho1, which act together as a sensor module in one of the two major osmosensing pathways in budding yeast. This signal is transmitted via the MAPKKK kinase Ste11. Thus, we term this mechanosensitive pathway the "SMuSh" pathway, for Ste11 through Mucin/Sho1 pathway. The SMuSh pathway delays cells in the G1 phase of the cell cycle and improves cell survival in response to growth-induced pressure. We also found that the cell wall integrity (CWI) pathway contributes to the response to mechanical compressive stress. These latter results are confirmed in complimentary experiments in Mishra et al. [Mishra R, et al. (2017) Proc Natl Acad Sci USA, 10.1073/pnas.1709079114]. When both the SMuSh and the CWI pathways are deleted, cells fail to adapt to compressive stress, and all cells lyse at relatively low pressure when grown in confinement. Thus, we define a network that is essential for cell survival during growth under pressure. We term this mechanosensory system the SCWISh (survival through the CWI and SMuSh) network.
Assuntos
Parede Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Mecânico , Citoesqueleto de Actina/metabolismo , Ciclo Celular , Peptídeos e Proteínas de Sinalização Intracelular/genética , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Mecanotransdução Celular , Proteínas de Membrana/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Assembly of appropriately oriented actin cables nucleated by formin proteins is necessary for many biological processes in diverse eukaryotes. However, compared with knowledge of how nucleation of dendritic actin filament arrays by the actin-related protein-2/3 complex is regulated, the in vivo regulatory mechanisms for actin cable formation are less clear. To gain insights into mechanisms for regulating actin cable assembly, we reconstituted the assembly process in vitro by introducing microspheres functionalized with the C terminus of the budding yeast formin Bni1 into extracts prepared from yeast cells at different cell-cycle stages. EM studies showed that unbranched actin filament bundles were reconstituted successfully in the yeast extracts. Only extracts enriched in the mitotic cyclin Clb2 were competent for actin cable assembly, and cyclin-dependent kinase 1 activity was indispensible. Cyclin-dependent kinase 1 activity also was found to regulate cable assembly in vivo. Here we present evidence that formin cell-cycle regulation is conserved in vertebrates. The use of the cable-reconstitution system to test roles for the key actin-binding proteins tropomyosin, capping protein, and cofilin provided important insights into assembly regulation. Furthermore, using mass spectrometry, we identified components of the actin cables formed in yeast extracts, providing the basis for comprehensive understanding of cable assembly and regulation.
Assuntos
Citoesqueleto de Actina/metabolismo , Ciclo Celular/fisiologia , Proteínas dos Microfilamentos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/ultraestrutura , Western Blotting , Proteína Quinase CDC2/metabolismo , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Espectrometria de Massas , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Microesferas , Polimerização , Saccharomyces cerevisiaeRESUMO
Most cell cycle regulation research has been conducted in model organisms representing a very small part of the eukaryotic domain. The highly divergent human pathogen Giardia intestinalis is ideal for studying the conservation of eukaryotic pathways. Although Giardia has many cell cycle regulatory components, its genome lacks all anaphase-promoting complex (APC) components. In the present study, we show that a single mitotic cyclin in Giardia is essential for progression into mitosis. Strikingly, Giardia cyclin B lacks the conserved N-terminal motif required for timely degradation mediated by the APC and ubiquitin conjugation. Expression of Giardia cyclin B in fission yeast is toxic, leading to a prophase arrest, and this toxicity is suppressed by the addition of a fission yeast degradation motif. Cyclin B is degraded during mitosis in Giardia cells, but this degradation appears to be independent of the ubiquitination pathway. Other putative APC substrates, aurora and polo-like kinases, also show no evidence of ubiquitination. This is the first example of mitosis not regulated by the APC and might reflect an evolutionary ancient form of cell cycle regulation.
Assuntos
Ciclina B/metabolismo , Giardia lamblia/fisiologia , Proteínas de Protozoários/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Evolução Biológica , Ciclo Celular/genética , Sequência Conservada/genética , Ciclina B/genética , Morfolinos/genética , Fosforilação , Estrutura Terciária de Proteína/genética , Proteólise , Proteínas de Protozoários/genética , Schizosaccharomyces , Proteínas de Schizosaccharomyces pombe/genética , Especificidade da Espécie , UbiquitinaçãoRESUMO
Crystalline biominerals do not resemble faceted crystals. Current explanations for this property involve formation via amorphous phases. Using X-ray absorption near-edge structure (XANES) spectroscopy and photoelectron emission microscopy (PEEM), here we examine forming spicules in embryos of Strongylocentrotus purpuratus sea urchins, and observe a sequence of three mineral phases: hydrated amorphous calcium carbonate (ACC · H(2)O) â dehydrated amorphous calcium carbonate (ACC) â calcite. Unexpectedly, we find ACC · H(2)O-rich nanoparticles that persist after the surrounding mineral has dehydrated and crystallized. Protein matrix components occluded within the mineral must inhibit ACC · H(2)O dehydration. We devised an in vitro, also using XANES-PEEM, assay to identify spicule proteins that may play a role in stabilizing various mineral phases, and found that the most abundant occluded matrix protein in the sea urchin spicules, SM50, stabilizes ACC · H(2)O in vitro.
Assuntos
Materiais Biocompatíveis/química , Calcificação Fisiológica , Carbonato de Cálcio/química , Transição de Fase , Animais , Materiais Biocompatíveis/metabolismo , Carbonato de Cálcio/metabolismo , Cristalização , Embrião não Mamífero/química , Embrião não Mamífero/metabolismo , Embrião não Mamífero/ultraestrutura , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Microscopia Eletrônica/métodos , Minerais/química , Minerais/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Strongylocentrotus purpuratus/química , Strongylocentrotus purpuratus/embriologia , Strongylocentrotus purpuratus/metabolismo , Água/química , Espectroscopia por Absorção de Raios X/métodosRESUMO
At the onset of anaphase, sister-chromatid cohesion is dissolved abruptly and irreversibly, ensuring that all chromosome pairs disjoin almost simultaneously. The regulatory mechanisms that generate this switch-like behaviour are unclear. Anaphase is initiated when a ubiquitin ligase, the anaphase-promoting complex (APC), triggers the destruction of securin, thereby allowing separase, a protease, to disrupt sister-chromatid cohesion. Here we demonstrate that the cyclin-dependent kinase 1 (Cdk1)-dependent phosphorylation of securin near its destruction-box motif inhibits securin ubiquitination by the APC. The phosphatase Cdc14 reverses securin phosphorylation, thereby increasing the rate of securin ubiquitination. Because separase is known to activate Cdc14 (refs 5 and 6), our results support the existence of a positive feedback loop that increases the abruptness of anaphase. Consistent with this model, we show that mutations that disrupt securin phosphoregulation decrease the synchrony of chromosome segregation. Our results also suggest that coupling securin degradation with changes in Cdk1 and Cdc14 activities helps coordinate the initiation of sister-chromatid separation with changes in spindle dynamics.
Assuntos
Anáfase/fisiologia , Retroalimentação Fisiológica/fisiologia , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Retroalimentação Fisiológica/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Securina , Deleção de Sequência , Fuso Acromático/metabolismo , Ubiquitinação/fisiologiaRESUMO
The mechanisms that regulate the physical properties of the cell interior remain poorly understood, especially at the mesoscale (10nm-100nm). Changes in these properties have been suggested to be crucial for both normal physiology and disease. Many crucial macromolecules and molecular assemblies such as ribosomes, RNA polymerase, and biomolecular condensates span the mesoscale size range. Therefore, we need better tools to study the cellular environment at this scale. A recent approach has been to use genetically encoded multimeric nanoparticles (GEMs), which consist of self-assembling scaffold proteins fused to fluorescent tags. After translation of the fusion protein, the monomers self-assemble into bright and stable nanoparticles of defined geometry that can be visualized by fluorescence microscopy. Physical properties of the cell can then be inferred through analysis of the motion of these particles, an approach called nanorheology. Previously, 40nm-GEMs elucidated TORC1 kinase as a regulator of cytoplasmic crowding. However, extremely sensitive microscopes were required. Here, we describe the development and characterization of a 50 nm diameter GEM that is brighter and probes a larger length scale. 50nm-GEMs will make high-throughput nanorheology accessible to a broader range of researchers and reveal new insights into the biophysical properties of cells.
RESUMO
The crowded bacterial cytoplasm is comprised of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial Genetically Encoded Multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (-2160 to +1800 e) in live Escherichia coli cells. To probe intermolecular details at spatial and temporal resolutions beyond experimental limits, we also developed a colloidal whole-cell model that explicitly represents the size and charge of cytoplasmic macromolecules and the porous structure of the bacterial nucleoid. Combining these techniques, we show that bGEMs spatially segregate by size, with small 20-nm particles enriched inside the nucleoid, and larger and/or positively charged particles excluded from this region. Localization is driven by entropic and electrostatic forces arising from cytoplasmic polydispersity, nucleoid structure, geometrical confinement, and interactions with other biomolecules including ribosomes and DNA. We observe that at the timescales of traditional single molecule tracking experiments, motion appears sub-diffusive for all particle sizes and charges. However, using computer simulations with higher temporal resolution, we find that the apparent anomalous exponents are governed by the region of the cell in which bGEMs are located. Molecular motion does not display anomalous diffusion on short time scales and the apparent sub-diffusion arises from geometrical confinement within the nucleoid and by the cell boundary.
RESUMO
Macromolecular crowding is an important parameter that impacts multiple biological processes. Passive microrheology using single particle tracking is a powerful means of studying macromolecular crowding. Here we monitored the diffusivity of self-assembling fluorescent nanoparticles (µNS) in response to acute glucose starvation. mRNP diffusivity was reduced upon glucose starvation as previously reported. In contrast, we observed increased diffusivity of µNS particles. Our results suggest that, upon glucose starvation, mRNP granule diffusivity may be reduced due to changes in physical interactions, while global crowding in the cytoplasm may be reduced.
RESUMO
All living cells are crowded with macromolecules. Crowding can directly modulate biochemical reactions to various degrees depending on the sizes, shapes, and binding affinities of the reactants. Here, we explore the possibility that cells can sense and adapt to changes in crowding through the widespread modulation of biochemical reactions without the need for a dedicated sensor. Additionally, we explore phase separation as a general physicochemical response to changes in crowding, and a mechanism to both transduce information and physically restore crowding homeostasis.
Assuntos
Fenômenos Fisiológicos Celulares , Substâncias Macromoleculares/metabolismoRESUMO
Macromolecular crowding is an important property of cells that impacts multiple biological processes. Passive microrheology using single particle tracking is a powerful means of studying macromolecular crowding. Here we monitored the diffusivity of self-assembling fluorescent nanoparticles (µNS) and mRNPs ( GFA1 -PP7) in response to acute glucose starvation. mRNP diffusivity was reduced upon glucose starvation as previously reported. By contrast, we observed increased diffusivity of µNS particles. Our results suggest that, upon glucose starvation, mRNP granule diffusivity may be reduced due to increased physical interactions, whereas macromolecular crowding in the cytoplasm may be globally reduced.
RESUMO
The intracellular environment is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cell physiology. When exposed to stress, mRNAs released after translational arrest condense with RNA binding proteins, resulting in the formation of membraneless RNA protein (RNP) condensates known as processing bodies (P-bodies) and stress granules (SGs). However, the impact of the assembly of these condensates on the biophysical properties of the crowded cytoplasmic environment remains unclear. Here, we find that upon exposure to stress, polysome collapse and condensation of mRNAs increases mesoscale particle diffusivity in the cytoplasm. Increased mesoscale diffusivity is required for the efficient formation of Q-bodies, membraneless organelles that coordinate degradation of misfolded peptides that accumulate during stress. Additionally, we demonstrate that polysome collapse and stress granule formation has a similar effect in mammalian cells, fluidizing the cytoplasm at the mesoscale. We find that synthetic, light-induced RNA condensation is sufficient to fluidize the cytoplasm, demonstrating a causal effect of RNA condensation. Together, our work reveals a new functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to effectively respond to stressful conditions.
RESUMO
Life emerges from thousands of biochemical processes occurring within a shared intracellular environment. We have gained deep insights from in vitro reconstitution of isolated biochemical reactions. However, the reaction medium in test tubes is typically simple and diluted. The cell interior is far more complex: macromolecules occupy more than a third of the space, and energy-consuming processes agitate the cell interior. Here, we review how this crowded, active environment impacts the motion and assembly of macromolecules, with an emphasis on mesoscale particles (10-1000 nm diameter). We describe methods to probe and analyze the biophysical properties of cells and highlight how changes in these properties can impact physiology and signaling, and potentially contribute to aging, and diseases, including cancer and neurodegeneration.
RESUMO
Cell proliferation is a central process in tissue development, homeostasis, and disease, yet how proliferation is regulated in the tissue context remains poorly understood. Here, we introduce a quantitative framework to elucidate how tissue growth dynamics regulate cell proliferation. Using MDCK epithelial monolayers, we show that a limiting rate of tissue expansion creates confinement that suppresses cell growth; however, this confinement does not directly affect the cell cycle. This leads to uncoupling between rates of cell growth and division in epithelia and, thereby, reduces cell volume. Division becomes arrested at a minimal cell volume, which is consistent across diverse epithelia in vivo. Here, the nucleus approaches the minimum volume capable of packaging the genome. Loss of cyclin D1-dependent cell-volume regulation results in an abnormally high nuclear-to-cytoplasmic volume ratio and DNA damage. Overall, we demonstrate how epithelial proliferation is regulated by the interplay between tissue confinement and cell-volume regulation.
Assuntos
Células Epiteliais , Células Epiteliais/metabolismo , Ciclo Celular/fisiologia , Divisão Celular , Epitélio , Proliferação de CélulasRESUMO
LINE-1 (L1) is the only autonomously active retrotransposon in the human genome, and accounts for 17% of the human genome. The L1 mRNA encodes two proteins, ORF1p and ORF2p, both essential for retrotransposition. ORF2p has reverse transcriptase and endonuclease activities, while ORF1p is a homotrimeric RNA-binding protein with poorly understood function. Here, we show that condensation of ORF1p is critical for L1 retrotransposition. Using a combination of biochemical reconstitution and live-cell imaging, we demonstrate that electrostatic interactions and trimer conformational dynamics together tune the properties of ORF1p assemblies to allow for efficient L1 ribonucleoprotein (RNP) complex formation in cells. Furthermore, we relate the dynamics of ORF1p assembly and RNP condensate material properties to the ability to complete the entire retrotransposon life-cycle. Mutations that prevented ORF1p condensation led to loss of retrotransposition activity, while orthogonal restoration of coiled-coil conformational flexibility rescued both condensation and retrotransposition. Based on these observations, we propose that dynamic ORF1p oligomerization on L1 RNA drives the formation of an L1 RNP condensate that is essential for retrotransposition.