RESUMO
Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier1 that defines cell shape2 and allows the cell to sustain large mechanical loads such as turgor pressure3. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope4,5. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.
Assuntos
Membrana Celular/metabolismo , Parede Celular/metabolismo , Bactérias Gram-Negativas/citologia , Bactérias Gram-Negativas/metabolismo , Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Detergentes/farmacologia , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Suporte de CargaRESUMO
Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli's inner membrane from the cell wall. Shrinkage was accompanied by an â¼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic.
Assuntos
Citoplasma/fisiologia , Escherichia coli/fisiologia , Carbono/deficiência , Carbono/farmacologia , Citoplasma/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular/efeitos dos fármacos , Nitrogênio/análise , Fósforo/análiseRESUMO
Chromatin recruitment of effector proteins involved in gene regulation depends on multivalent interaction with histone post-translational modifications (PTMs) and structural features of the chromatin fiber. Due to the complex interactions involved, it is currently not understood how effectors dynamically sample the chromatin landscape. Here, we dissect the dynamic chromatin interactions of a family of multivalent effectors, heterochromatin protein 1 (HP1) proteins, using single-molecule fluorescence imaging and computational modeling. We show that the three human HP1 isoforms are recruited and retained on chromatin by a dynamic exchange between histone PTM and DNA bound states. These interactions depend on local chromatin structure, the HP1 isoforms as well as on PTMs on HP1 itself. Of the HP1 isoforms, HP1α exhibits the longest residence times and fastest binding rates due to DNA interactions in addition to PTM binding. HP1α phosphorylation further increases chromatin retention through strengthening of multivalency while reducing DNA binding. As DNA binding in combination with specific PTM recognition is found in many chromatin effectors, we propose a general dynamic capture mechanism for effector recruitment. Multiple weak protein and DNA interactions result in a multivalent interaction network that targets effectors to a specific chromatin modification state, where their activity is required.
Assuntos
Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Código das Histonas/fisiologia , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Homólogo 5 da Proteína Cromobox , Epigênese Genética , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Cinética , Camundongos , Células NIH 3T3 , Fosforilação , Ligação Proteica , Imagem Individual de MoléculaRESUMO
Nanoscale characterization of living samples has become essential for modern biology. Atomic force microscopy (AFM) creates topological images of fragile biological structures from biomolecules to living cells in aqueous environments. However, correlating nanoscale structure to biological function of specific proteins can be challenging. To this end we have built and characterized a correlated single molecule localization microscope (SMLM)/AFM that allows localizing specific, labeled proteins within high-resolution AFM images in a biologically relevant context. Using direct stochastic optical reconstruction microscopy (dSTORM)/AFM, we directly correlate and quantify the density of localizations with the 3D topography using both imaging modalities along (F-)actin cytoskeletal filaments. In addition, using photo activated light microscopy (PALM)/AFM, we provide correlative images of bacterial cells in aqueous conditions. Moreover, we report the first correlated AFM/PALM imaging of live mammalian cells. The complementary information provided by the two techniques opens a new dimension for structural and functional nanoscale biology.
RESUMO
Multicellular communities of contiguous cells attached to solid surfaces called biofilms represent a common microbial strategy to improve resilience in adverse environments.1,2,3 While bacterial biofilms have been under intense investigation, whether archaeal biofilms follow similar assembly rules remains unknown.4,5Haloferax volcanii is an extremely halophilic euryarchaeon that commonly colonizes salt crust surfaces. H. volcanii produces long and thin appendages called type IV pili (T4Ps). These play a role in surface attachment and biofilm formation in both archaea and bacteria. In this study, we employed biophysical experiments to identify the function of T4Ps in H. volcanii biofilm morphogenesis. H. volcanii expresses not one but six types of major pilin subunits that are predicted to compose T4Ps. Non-invasive imaging of T4Ps in live cells using interferometric scattering (iSCAT) microscopy reveals that piliation varies across mutants expressing single major pilin isoforms. T4Ps are necessary to secure attachment of single cells to surfaces, and the adhesive strength of pilin mutants correlates with their level of piliation. In flow, H. volcanii forms clonal biofilms that extend in three dimensions. Notably, the expression of PilA2, a single pilin isoform, is sufficient to maintain levels of piliation, surface attachment, and biofilm formation that are indistinguishable from the wild type. Furthermore, we discovered that fluid flow stabilizes biofilm integrity; as in the absence of flow, biofilms tend to lose cohesion and disperse in a density-dependent manner. Overall, our results demonstrate that T4P-surface and possibly T4P-T4P interactions promote biofilm formation and integrity and that flow is a key factor regulating archaeal biofilm formation.
Assuntos
Proteínas de Fímbrias , Haloferax volcanii , Proteínas de Fímbrias/metabolismo , Haloferax volcanii/fisiologia , Fímbrias Bacterianas/metabolismo , BiofilmesRESUMO
We report the coencapsulation of glutathione reductase and disulfide-linked polymer-oligopeptide conjugates into capsosomes, polymer carrier capsules containing liposomal subcompartments. The architecture of the capsosomes enables a temperature-triggered conversion of oxidized glutathione to its reduced sulfhydryl form by the encapsulated glutathione reductase. The reduced glutathione subsequently induces the release of the encapsulated oligopeptides from the capsosomes by reducing the disulfide linkages of the conjugates. This study highlights the potential of capsosomes to continuously generate a potent antioxidant while simultaneously releasing small molecule therapeutics.
Assuntos
Preparações de Ação Retardada/química , Glutationa Redutase/química , Lipossomos/química , Nanocápsulas/química , Catálise , Difusão , TemperaturaRESUMO
The steady-state size of bacterial cells correlates with nutrient-determined growth rate. Here, we explore how rod-shaped bacterial cells regulate their morphology during rapid environmental changes. We quantify cellular dimensions throughout passage cycles of stationary-phase cells diluted into fresh medium and grown back to saturation. We find that cells exhibit characteristic dynamics in surface area to volume ratio (SA/V), which are conserved across genetic and chemical perturbations as well as across species and growth temperatures. A mathematical model with a single fitting parameter (the time delay between surface and volume synthesis) is quantitatively consistent with our SA/V experimental observations. The model supports that this time delay is due to differential expression of volume and surface-related genes, and that the first division after dilution occurs at a tightly controlled SA/V. Our minimal model thus provides insight into the connections between bacterial growth rate and cell shape in dynamic environments.
Assuntos
Bactérias/genética , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica , Proteômica/métodos , Algoritmos , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Proteínas de Bactérias/genética , Divisão Celular/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Cinética , Modelos Teóricos , Propriedades de SuperfícieRESUMO
Intracellular density impacts the physical nature of the cytoplasm and can globally affect cellular processes, yet density regulation remains poorly understood. Here, using a new quantitative phase imaging method, we determined that dry-mass density in fission yeast is maintained in a narrow distribution and exhibits homeostatic behavior. However, density varied during the cell cycle, decreasing during G2, increasing in mitosis and cytokinesis, and dropping rapidly at cell birth. These density variations were explained by a constant rate of biomass synthesis, coupled to slowdown of volume growth during cell division and rapid expansion post-cytokinesis. Arrest at specific cell-cycle stages exacerbated density changes. Spatially heterogeneous patterns of density suggested links between density regulation, tip growth, and intracellular osmotic pressure. Our results demonstrate that systematic density variations during the cell cycle are predominantly due to modulation of volume expansion, and reveal functional consequences of density gradients and cell-cycle arrests.
Assuntos
Ciclo Celular/fisiologia , Espaço Intracelular/fisiologia , Schizosaccharomyces/citologia , Schizosaccharomyces/crescimento & desenvolvimento , Tamanho Celular , Citocinese/fisiologia , Espaço Intracelular/química , Imagem com Lapso de TempoRESUMO
We report the synthesis of poly(methacrylic acid)-co-(oleyl methacrylate) with three different amounts of oleyl methacrylate and compare the ability of these polymers with that of poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMA(c)) to noncovalently anchor liposomes to polymer layers. We subsequently assembled â¼1 µm diameter PMA(c)-based capsosomes, polymer hydrogel capsules that contain up to â¼2000 liposomal subcompartments, and investigate the potential of these carriers to deliver water-insoluble drugs by encapsulating two different antitumor compounds, thiocoraline or paclitaxel, into the liposomes. The viability of lung cancer cells is used to substantiate the cargo concentration-dependent activity of the capsosomes. These findings cover several crucial aspects for the application of capsosomes as potential drug delivery vehicles.
Assuntos
Cápsulas/química , Sistemas de Liberação de Medicamentos/métodos , Lipossomos/química , Neoplasias Pulmonares/tratamento farmacológico , Ácidos Polimetacrílicos/uso terapêutico , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Depsipeptídeos/administração & dosagem , Portadores de Fármacos/química , Humanos , Hidrogéis , Neoplasias Pulmonares/patologia , Miniaturização , Paclitaxel/administração & dosagemRESUMO
Mechanisms to control cell division are essential for cell proliferation and survival 1. Bacterial cell growth and division require the coordinated activity of peptidoglycan synthases and hydrolytic enzymes 2-4 to maintain mechanical integrity of the cell wall 5. Recent studies suggest that cell separation is governed by mechanical forces 6,7. How mechanical forces interact with molecular mechanisms to control bacterial cell division in space and time is poorly understood. Here, we use a combination of atomic force microscope (AFM) imaging, nanomechanical mapping, and nanomanipulation to show that enzymatic activity and mechanical forces serve overlapping and essential roles in mycobacterial cell division. We find that mechanical stress gradually accumulates in the cell wall concentrated at the future division site, culminating in rapid (millisecond) cleavage of nascent sibling cells. Inhibiting cell wall hydrolysis delays cleavage; conversely, locally increasing cell wall stress causes instantaneous and premature cleavage. Cells deficient in peptidoglycan hydrolytic activity fail to locally decrease their cell wall strength and undergo natural cleavage, instead forming chains of non-growing cells. Cleavage of these cells can be mechanically induced by local application of stress with AFM. These findings establish a direct link between actively controlled molecular mechanisms and passively controlled mechanical forces in bacterial cell division.
RESUMO
Cell growth is a complex process in which cells synthesize cellular components while they increase in size. It is generally assumed that the rate of biosynthesis must somehow be coordinated with the rate of growth in order to maintain intracellular concentrations. However, little is known about potential feedback mechanisms that could achieve proteome homeostasis or the consequences when this homeostasis is perturbed. Here, we identify conditions in which fission yeast cells are prevented from volume expansion but nevertheless continue to synthesize biomass, leading to general accumulation of proteins and increased cytoplasmic density. Upon removal of these perturbations, this biomass accumulation drove cells to undergo a multi-generational period of "supergrowth" wherein rapid volume growth outpaced biosynthesis, returning proteome concentrations back to normal within hours. These findings demonstrate a mechanism for global proteome homeostasis based on modulation of volume growth and dilution.
Assuntos
Proliferação de Células/fisiologia , Proteostase/fisiologia , Schizosaccharomyces/crescimento & desenvolvimento , Ciclo Celular , Proliferação de Células/genética , Homeostase , Biossíntese de Proteínas , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
A central question in mechanobiology is how cellular-scale structures are established and regulated. In bacteria, the cell envelope is essential for mechanical integrity, protecting against environmental stresses and bearing the load from high turgor pressures. Trivedi et al. (mBio 9:e01340-18, 2018, https://doi.org/10.1128/mBio.01340-18) screened a Pseudomonas aeruginosa transposon library and identified genes that influence cell stiffness by measuring cell growth while cells were embedded in an agarose gel. Their findings provide a broad knowledge base for how biochemical pathways regulate cellular mechanical properties in this pathogen. Dozens of genes across diverse functional categories were implicated, suggesting that cellular mechanics is a systems-level emergent property. Furthermore, changes in d-alanine levels in a dadA (d-alanine dehydrogenase) mutant resulted in decreases in the expression of cell wall enzymes, cross-linking density, and cell stiffness. These insights into the biochemical and mechanical roles of dadA highlight the importance of systems-level investigations into the physical properties of cells.
Assuntos
Genoma Bacteriano , Pseudomonas aeruginosa/genética , Alanina , Parede Celular , GenômicaRESUMO
Cell division is tightly controlled in space and time to maintain cell size and ploidy within narrow bounds. In bacteria, the canonical Minicell (Min) and nucleoid occlusion (Noc) systems together ensure that division is restricted to midcell after completion of chromosome segregation1. It is unknown how division site selection is controlled in bacteria that lack homologues of the Min and Noc proteins, including mycobacteria responsible for tuberculosis and other chronic infections2. Here, we use correlated optical and atomic-force microscopy3,4 to demonstrate that morphological landmarks (waveform troughs) on the undulating surface of mycobacterial cells correspond to future sites of cell division. Newborn cells inherit wave troughs from the (grand)mother cell and ultimately divide at the centre-most wave trough, making these morphological features the earliest known landmark of future division sites. In cells lacking the chromosome partitioning (Par) system, missegregation of chromosomes is accompanied by asymmetric cell division at off-centre wave troughs, resulting in the formation of anucleate cells. These results demonstrate that inherited morphological landmarks and chromosome positioning together restrict mycobacterial division to the midcell position.
Assuntos
Divisão Celular/genética , Cromossomos Bacterianos/genética , Mycobacterium/fisiologia , Mycobacterium/ultraestrutura , Divisão Celular Assimétrica/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Segregação de Cromossomos , Microscopia , Microscopia de Força Atômica , Mycobacterium/genéticaRESUMO
Chronic inflammation is associated with a variety of pathological conditions in epithelial tissues, including cancer, metaplasia and aberrant wound healing. In relation to this, a significant body of evidence suggests that aberration of epithelial stem and progenitor cell function is a contributing factor in inflammation-related disease, although the underlying cellular and molecular mechanisms remain to be fully elucidated. In this study, we have delineated the effect of chronic inflammation on epithelial stem/progenitor cells using the corneal epithelium as a model tissue. Using a combination of mouse genetics, pharmacological approaches and in vitro assays, we demonstrate that chronic inflammation elicits aberrant mechanotransduction in the regenerating corneal epithelium. As a consequence, a YAP-TAZ/ß-catenin cascade is triggered, resulting in the induction of epidermal differentiation on the ocular surface. Collectively, the results of this study demonstrate that chronic inflammation and mechanotransduction are linked and act to elicit pathological responses in regenerating epithelia.
Assuntos
Diferenciação Celular , Lesões da Córnea/metabolismo , Células Epiteliais/metabolismo , Epitélio Corneano/metabolismo , Ceratite/metabolismo , Mecanotransdução Celular , Regeneração , Células-Tronco/metabolismo , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Administração Oftálmica , Animais , Anti-Inflamatórios/administração & dosagem , Proteínas de Ciclo Celular , Diferenciação Celular/efeitos dos fármacos , Doença Crônica , Lesões da Córnea/genética , Lesões da Córnea/patologia , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Epitélio Corneano/efeitos dos fármacos , Epitélio Corneano/lesões , Epitélio Corneano/patologia , Matriz Extracelular/metabolismo , Células HEK293 , Humanos , Mediadores da Inflamação/metabolismo , Ceratite/genética , Ceratite/patologia , Ceratite/prevenção & controle , Mecanotransdução Celular/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptor Notch1/deficiência , Receptor Notch1/genética , Regeneração/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Células-Tronco/patologia , Suínos , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Via de Sinalização Wnt , Proteínas de Sinalização YAP , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Multifrequency atomic force microscopy imaging has been recently demonstrated as a powerful technique for quickly obtaining information about the mechanical properties of a sample. Combining this development with recent gains in imaging speed through small cantilevers holds the promise of a convenient, high-speed method for obtaining nanoscale topography as well as mechanical properties. Nevertheless, instrument bandwidth limitations on cantilever excitation and readout have restricted the ability of multifrequency techniques to fully benefit from small cantilevers. We present an approach for cantilever excitation and deflection readout with a bandwidth of 20 MHz, enabling multifrequency techniques extended beyond 2 MHz for obtaining materials contrast in liquid and air, as well as soft imaging of delicate biological samples.