RESUMO
How complex 3D tissue shape emerges during animal development remains an important open question in biology and biophysics. Here, we discover a mechanism for 3D epithelial shape change based on active, in-plane cellular events that is analogous to inanimate "shape programmable" materials, which undergo blueprinted 3D shape transformations from in-plane gradients of spontaneous strains. We study eversion of the Drosophila wing disc pouch, when the epithelium transforms from a dome into a curved fold, quantifying 3D tissue shape changes and mapping spatial patterns of cellular behaviors on the evolving geometry using cellular topology. Using a physical model inspired by shape programming, we find that active cell rearrangements are the major contributor to pouch eversion and validate this conclusion using a knockdown of MyoVI, which reduces rearrangements and disrupts morphogenesis. This work shows that shape programming is a mechanism for animal tissue morphogenesis and suggests that patterns in nature could present design strategies for shape-programmable materials.
Assuntos
Morfogênese , Asas de Animais , Animais , Asas de Animais/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila , Modelos Biológicos , Discos Imaginais/metabolismo , Discos Imaginais/crescimento & desenvolvimentoRESUMO
How morphogenetic movements are robustly coordinated in space and time is a fundamental open question in biology. We study this question using the wing of Drosophila melanogaster, an epithelial tissue that undergoes large-scale tissue flows during pupal stages. Previously, we showed that pupal wing morphogenesis involves both cellular behaviors that allow relaxation of mechanical tissue stress, as well as cellular behaviors that appear to be actively patterned (Etournay et al., 2015). Here, we show that these active cellular behaviors are not guided by the core planar cell polarity (PCP) pathway, a conserved signaling system that guides tissue development in many other contexts. We find no significant phenotype on the cellular dynamics underlying pupal morphogenesis in mutants of core PCP. Furthermore, using laser ablation experiments, coupled with a rheological model to describe the dynamics of the response to laser ablation, we conclude that while core PCP mutations affect the fast timescale response to laser ablation they do not significantly affect overall tissue mechanics. In conclusion, our work shows that cellular dynamics and tissue shape changes during Drosophila pupal wing morphogenesis do not require core PCP as an orientational guiding cue.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Pupa/genética , Asas de Animais/fisiologia , Morfogênese/genética , Polaridade Celular , MutaçãoRESUMO
Adenosine triphosphate (ATP) production and utilization is critically important for animal development. How these processes are regulated in space and time during tissue growth remains largely unclear. We used a FRET-based sensor to dynamically monitor ATP levels across a growing tissue, using the Drosophila larval wing disc. Although steady-state levels of ATP are spatially uniform across the wing pouch, inhibiting oxidative phosphorylation reveals spatial differences in metabolic behavior, whereby signaling centers at compartment boundaries produce more ATP from glycolysis than the rest of the tissue. Genetic perturbations indicate that the conserved Hedgehog signaling pathway can enhance ATP production by glycolysis. Collectively, our work suggests the existence of a homeostatic feedback loop between Hh signaling and glycolysis, advancing our understanding of the connection between conserved developmental patterning genes and ATP production during animal tissue development.
Assuntos
Proteínas de Drosophila , Drosophila , Animais , Drosophila/genética , Drosophila/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Asas de Animais/metabolismo , Glicólise , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMO
In this chapter, I present a method for the ex vivo cultivation and live imaging of Drosophila imaginal disc explants using low concentrations of the steroid hormone 20-hydroxyecdysone (20E). This method has been optimized for analyzing cellular dynamics during wing disc growth and leverages recent insights from in vivo experiments demonstrating that 20E is required for growth and patterning of the imaginal tissues. Using this protocol, we directly observe wing disc proliferation at a rapid rate for at least 13 h during live imaging. The orientation of tissue growth is also consistent with that inferred from indirect in vivo techniques. Thus, this method provides an improved way of studying dynamic cellular processes and tissue movements during imaginal disc development. I first describe the preparation of the growth medium and the dissection, and then I include a protocol for mounting and live imaging of the explants.
Assuntos
Proteínas de Drosophila , Discos Imaginais , Animais , Meios de Cultura , Drosophila , Drosophila melanogaster , Larva , Asas de AnimaisRESUMO
Organ and tissue growth result from an integration of biophysical communication across biological scales, both in time and space. In this review, we highlight new insight into the dynamic connections between control mechanisms operating at different length scales. First, we consider how the dynamics of chemical and electrical signaling in the shape of gradients or waves affect spatiotemporal signal interpretation. Then, we discuss the mechanics underlying dynamic cell behavior during oriented tissue growth, followed by the connections between signaling at the tissue and organismal levels.
Assuntos
Modelos Biológicos , Transdução de Sinais , MorfogêneseRESUMO
Tissue organization is often characterized by specific patterns of cell morphology. How such patterns emerge in developing tissues is a fundamental open question. Here, we investigate the emergence of tissue-scale patterns of cell shape and mechanical tissue stress in the Drosophila wing imaginal disc during larval development. Using quantitative analysis of the cellular dynamics, we reveal a pattern of radially oriented cell rearrangements that is coupled to the buildup of tangential cell elongation. Developing a laser ablation method, we map tissue stresses and extract key parameters of tissue mechanics. We present a continuum theory showing that this pattern of cell morphology and tissue stress can arise via self-organization of a mechanical feedback that couples cell polarity to active cell rearrangements. The predictions of this model are supported by knockdown of MyoVI, a component of mechanosensitive feedback. Our work reveals a mechanism for the emergence of cellular patterns in morphogenesis.
During development, carefully choreographed cell movements ensure the creation of a healthy organism. To determine their identity and place across a tissue, cells can read gradients of far-reaching signaling molecules called morphogens; in addition, physical forces can play a part in helping cells acquire the right size and shape. Indeed, cells are tightly attached to their neighbors through connections linked to internal components. Structures or proteins inside the cells can pull on these junctions to generate forces that change the physical features of a cell. However, it is poorly understood how these forces create patterns of cell size and shape across a tissue. Here, Dye, Popovic et al. combined experiments with physical models to examine how cells acquire these physical characteristics across the developing wing of fruit fly larvae. This revealed that cells pushing and pulling on one another create forces that trigger internal biochemical reorganization for instance, force-generating structures become asymmetrical. In turn, the cells exert additional forces on their neighbors, setting up a positive feedback loop which results in cells adopting the right size and shape across the organ. As such, cells in the fly wing can spontaneously self-organize through the interplay of mechanical and biochemical signals, without the need for pre-existing morphogen gradients. A refined understanding of how physical forces shape cells and organs would help to grasp how defects can emerge during development. This knowledge would also allow scientists to better grow tissues and organs in the laboratory, both for theoretical research and regenerative medicine.
Assuntos
Forma Celular , Drosophila melanogaster/fisiologia , Discos Imaginais/fisiologia , Mecanotransdução Celular , Asas de Animais/fisiologia , Animais , Padronização Corporal , Divisão Celular , Polaridade Celular , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Retroalimentação Fisiológica , Feminino , Discos Imaginais/embriologia , Masculino , Modelos Biológicos , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Estresse Mecânico , Fatores de Tempo , Asas de Animais/embriologiaRESUMO
The levels of nuclear protein Lamin A/C are crucial for nuclear mechanotransduction. Lamin A/C levels are known to scale with tissue stiffness and extracellular matrix levels in mesenchymal tissues. But in epithelial tissues, where cells lack a strong interaction with the extracellular matrix, it is unclear how Lamin A/C is regulated. Here, we show in epithelial tissues that Lamin A/C levels scale with apico-basal cell compression, independent of tissue stiffness. Using genetic perturbations in Drosophila epithelial tissues, we show that apico-basal cell compression regulates the levels of Lamin A/C by deforming the nucleus. Further, in mammalian epithelial cells, we show that nuclear deformation regulates Lamin A/C levels by modulating the levels of phosphorylation of Lamin A/C at Serine 22, a target for Lamin A/C degradation. Taken together, our results reveal a mechanism of Lamin A/C regulation which could provide key insights for understanding nuclear mechanotransduction in epithelial tissues.
Assuntos
Núcleo Celular/fisiologia , Proteínas de Drosophila/metabolismo , Lamina Tipo A/metabolismo , Laminas/metabolismo , Mecanotransdução Celular/fisiologia , Estresse Mecânico , Animais , Linhagem Celular , Cães , Drosophila , Proteínas de Drosophila/genética , Epitélio/metabolismo , Lamina Tipo A/genética , Laminas/genética , Células Madin Darby de Rim Canino , FosforilaçãoRESUMO
Changes in cell metabolism and plasma membrane potential have been linked to shifts between tissue growth and differentiation, and to developmental patterning. How such changes mediate these effects is poorly understood. Here, we use the developing wing of Drosophila to investigate the interplay between cell metabolism and a key developmental regulator-the Hedgehog (Hh) signalling pathway. We show that reducing glycolysis both lowers steady-state levels of ATP and stabilizes Smoothened (Smo), the 7-pass transmembrane protein that transduces the Hh signal. As a result, the transcription factor Cubitus interruptus accumulates in its full-length, transcription activating form. We show that glycolysis is required to maintain the plasma membrane potential and that plasma membrane depolarization blocks cellular uptake of N-acylethanolamides-lipoprotein-borne Hh pathway inhibitors required for Smo destabilization. Similarly, pharmacological inhibition of glycolysis in mammalian cells induces ciliary translocation of Smo-a key step in pathway activation-in the absence of Hh. Thus, changes in cell metabolism alter Hh signalling through their effects on plasma membrane potential.
Assuntos
Membrana Celular/metabolismo , Glicólise/genética , Glicólise/fisiologia , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Potenciais da Membrana/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Animais , Animais Geneticamente Modificados , Transporte Biológico , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Gramicidina/uso terapêutico , Lipoproteínas , Proteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Receptor Smoothened/metabolismo , Fatores de Transcrição/metabolismo , Asas de Animais/crescimento & desenvolvimento , Asas de Animais/patologia , Asas de Animais/fisiologiaRESUMO
The signaling protein Sonic Hedgehog (SHH) is crucial for the development and function of many vertebrate tissues. It remains largely unclear, however, what defines the range and specificity of pathway activation. The adrenal gland represents a useful model to address this question, where the SHH pathway is activated in a very specific subset of cells lying near the SHH-producing cells, even though there is an abundance of lipoproteins that would allow SHH to travel and signal long-range. We determine that, whereas adrenal cells can secrete SHH on lipoproteins, this form of SHH is inactive due to the presence of cosecreted inhibitors, potentially explaining the absence of long-range signaling. Instead, we find that SHH-producing cells signal at short range via membrane-bound SHH, only to receiving cells with primary cilia. Finally, our data from NCI-H295R adrenocortical carcinoma cells suggest that adrenocortical tumors may evade these regulatory control mechanisms by acquiring the ability to activate SHH target genes in response to TGF-ß.
Assuntos
Glândulas Suprarrenais/metabolismo , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Cílios/genética , Proteínas Hedgehog/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
Epithelial folding transforms simple sheets of cells into complex three-dimensional tissues and organs during animal development. Epithelial folding has mainly been attributed to mechanical forces generated by an apically localized actomyosin network, however, contributions of forces generated at basal and lateral cell surfaces remain largely unknown. Here we show that a local decrease of basal tension and an increased lateral tension, but not apical constriction, drive the formation of two neighboring folds in developing Drosophila wing imaginal discs. Spatially defined reduction of extracellular matrix density results in local decrease of basal tension in the first fold; fluctuations in F-actin lead to increased lateral tension in the second fold. Simulations using a 3D vertex model show that the two distinct mechanisms can drive epithelial folding. Our combination of lateral and basal tension measurements with a mechanical tissue model reveals how simple modulations of surface and edge tension drive complex three-dimensional morphological changes.
Assuntos
Drosophila/crescimento & desenvolvimento , Células Epiteliais/citologia , Epitélio/anatomia & histologia , Epitélio/embriologia , Morfogênese , Estresse Mecânico , Actinas/metabolismo , Actomiosina , Amidas/antagonistas & inibidores , Animais , Fenômenos Biomecânicos , Padronização Corporal/genética , Divisão Celular , Proliferação de Células , Forma Celular , Tamanho Celular , Drosophila/anatomia & histologia , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células Epiteliais/efeitos dos fármacos , Epitélio/efeitos dos fármacos , Matriz Extracelular , Discos Imaginais/crescimento & desenvolvimento , Larva/citologia , Larva/metabolismo , Terapia a Laser , Modelos Anatômicos , Modelos Biológicos , Piridinas/antagonistas & inibidoresRESUMO
Quantitative analysis of the dynamic cellular mechanisms shaping the Drosophila wing during its larval growth phase has been limited, impeding our ability to understand how morphogen patterns regulate tissue shape. Such analysis requires explants to be imaged under conditions that maintain both growth and patterning, as well as methods to quantify how much cellular behaviors change tissue shape. Here, we demonstrate a key requirement for the steroid hormone 20-hydroxyecdysone (20E) in the maintenance of numerous patterning systems in vivo and in explant culture. We find that low concentrations of 20E support prolonged proliferation in explanted wing discs in the absence of insulin, incidentally providing novel insight into the hormonal regulation of imaginal growth. We use 20E-containing media to observe growth directly and to apply recently developed methods for quantitatively decomposing tissue shape changes into cellular contributions. We discover that whereas cell divisions drive tissue expansion along one axis, their contribution to expansion along the orthogonal axis is cancelled by cell rearrangements and cell shape changes. This finding raises the possibility that anisotropic mechanical constraints contribute to growth orientation in the wing disc.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/crescimento & desenvolvimento , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento , Animais , Animais Geneticamente Modificados , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Padronização Corporal/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Drosophila melanogaster/genética , Ecdisterona/farmacologia , Ecdisterona/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genes de Insetos , Discos Imaginais/citologia , Discos Imaginais/efeitos dos fármacos , Discos Imaginais/crescimento & desenvolvimento , Insulina/farmacologia , Insulina/fisiologia , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Morfogênese/fisiologia , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacos , Asas de Animais/efeitos dos fármacosRESUMO
Segmentation and tracking of cells in long-term time-lapse experiments has emerged as a powerful method to understand how tissue shape changes emerge from the complex choreography of constituent cells. However, methods to store and interrogate the large datasets produced by these experiments are not widely available. Furthermore, recently developed methods for relating tissue shape changes to cell dynamics have not yet been widely applied by biologists because of their technical complexity. We therefore developed a database format that stores cellular connectivity and geometry information of deforming epithelial tissues, and computational tools to interrogate it and perform multi-scale analysis of morphogenesis. We provide tutorials for this computational framework, called TissueMiner, and demonstrate its capabilities by comparing cell and tissue dynamics in vein and inter-vein subregions of the Drosophila pupal wing. These analyses reveal an unexpected role for convergent extension in shaping wing veins.
Assuntos
Biologia Computacional/métodos , Bases de Dados Factuais , Epitélio/fisiologia , Morfogênese , Animais , Drosophila/fisiologia , Processamento de Imagem Assistida por Computador/métodos , Imagem com Lapso de TempoRESUMO
Ecdysteroids are the hormones regulating development, physiology and fertility in arthropods, which synthesize them exclusively from dietary sterols. But how dietary sterol diversity influences the ecdysteroid profile, how animals ensure the production of desired hormones and whether there are functional differences between different ecdysteroids produced in vivo remains unknown. This is because currently there is no analytical technology for unbiased, comprehensive and quantitative assessment of the full complement of endogenous ecdysteroids. We developed a new LC-MS/MS method to screen the entire chemical space of ecdysteroid-related structures and to quantify known and newly discovered hormones and their catabolites. We quantified the ecdysteroidome in Drosophila melanogaster and investigated how the ecdysteroid profile varies with diet and development. We show that Drosophila can produce four different classes of ecdysteroids, which are obligatorily derived from four types of dietary sterol precursors. Drosophila makes makisterone A from plant sterols and epi-makisterone A from ergosterol, the major yeast sterol. However, they prefer to selectively utilize scarce ergosterol precursors to make a novel hormone 24,28-dehydromakisterone A and trace cholesterol to synthesize 20-hydroxyecdysone. Interestingly, epi-makisterone A supports only larval development, whereas all other ecdysteroids allow full adult development. We suggest that evolutionary pressure against producing epi-C-24 ecdysteroids might explain selective utilization of ergosterol precursors and the puzzling preference for cholesterol.
Assuntos
Drosophila melanogaster/química , Ecdisteroides/análise , Ração Animal , Animais , Cromatografia Líquida , Dieta , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/fisiologia , Ecdisona/análogos & derivados , Ecdisona/metabolismo , Ecdisteroides/classificação , Ecdisteroides/metabolismo , Larva/química , Larva/fisiologia , Esteróis/metabolismo , Espectrometria de Massas em TandemRESUMO
Rod-shaped bacteria typically elongate at a uniform width. To investigate the genetic and physiological determinants involved in this process, we studied a mutation in the morphogenetic protein MreB in Caulobacter crescentus that gives rise to cells with a variable-width phenotype, where cells have regions that are both thinner and wider than wild-type. During growth, individual cells develop a balance of wide and thin regions, and mutant MreB dynamically localizes to poles and thin regions. Surprisingly, the surface area to volume ratio of these irregularly shaped cells is, on average, very similar to wild-type. We propose that, while mutant MreB localizes to thin regions and promotes rod-like growth there, wide regions develop as a compensatory mechanism, allowing cells to maintain a wild-type-like surface area to volume ratio. To support this model, we have shown that cell widening is abrogated in growth conditions that promote higher surface area to volume ratios, and we have observed individual cells with high ratios return to wild-type levels over several hours by developing wide regions, suggesting that compensation can take place at the level of individual cells.
RESUMO
We studied the effect of pre-incubation with NU7441, a specific inhibitor of DNA-dependent protein kinase (DNA-PK), on molecular mechanisms triggered by ionizing radiation (IR). The experimental design involved four groups of human T-lymphocyte leukaemic MOLT-4 cells: control, NU7441-treated (1 µM), IR-treated (1 Gy), and combination of NU7441 and IR. We used flow cytometry for apoptosis assessment, Western blotting and ELISA for detection of proteins involved in DNA repair signalling and epifluorescence microscopy for detection of IR-induced phosphorylation of histone H2A.X. We did not observe any major changes in the amount of DNA-PK subunits Ku70/80 caused by the combination of NU7441 and radiation. Their combination led to an increased phosphorylation of H2A.X, a hallmark of DNA damage. However, it did not prevent up-regulation of neither p53 (and its phosphorylation at Ser 15 and 392) nor p21. We observed a decrease in the levels of anti-apoptotic Mcl-1, cdc25A phosphatase, cleavage of PARP and a significant increase in apoptosis in the group treated with combination. In conclusion, the combination of NU7441 with IR caused increased phosphorylation of H2A.X early after irradiation and subsequent induction of apoptosis. It was efficient in MOLT-4 cells in 10× lower concentration than the inhibitor NU7026. NU7441 proved as a potent radio-sensitizing agent, and it might provide a platform for development of new radio-sensitizers in radiotherapy.
Assuntos
Cromonas/farmacologia , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Leucemia/patologia , Morfolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Histonas/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Fatores de TempoRESUMO
The synthesis of the peptidoglycan cell wall is carefully regulated in time and space. In nature, this essential process occurs in cells that live in fluctuating environments. Here we show that the spatial distributions of specific cell wall proteins in Caulobacter crescentus are sensitive to small external osmotic upshifts. The penicillin-binding protein PBP2, which is commonly branded as an essential cell elongation-specific transpeptidase, switches its localization from a dispersed, patchy pattern to an accumulation at the FtsZ ring location in response to osmotic upshifts as low as 40 mosmol/kg. This osmolality-dependent relocation to the division apparatus is initiated within less than a minute, while restoration to the patchy localization pattern is dependent on cell growth and takes 1 to 2 generations. Cell wall morphogenetic protein RodA and penicillin-binding protein PBP1a also change their spatial distribution by accumulating at the division site in response to external osmotic upshifts. Consistent with its ecological distribution, C. crescentus displays a narrow range of osmotolerance, with an upper limit of 225 mosmol/kg in minimal medium. Collectively, our findings reveal an unsuspected level of environmental regulation of cell wall protein behavior that is likely linked to an ecological adaptation.
Assuntos
Caulobacter crescentus/metabolismo , Concentração Osmolar , Proteínas de Ligação às Penicilinas/metabolismo , Estresse Fisiológico , Caulobacter crescentus/efeitos dos fármacos , Caulobacter crescentus/crescimento & desenvolvimento , Parede Celular/metabolismoRESUMO
The maintenance of cell shape in Caulobacter crescentus requires the essential gene mreB, which encodes a member of the actin superfamily and the target of the antibiotic, A22. We isolated 35 unique A22-resistant Caulobacter strains with single amino acid substitutions near the nucleotide binding site of MreB. Mutations that alter cell curvature and mislocalize the intermediate filament crescentin cluster on the back surface of MreB's structure. Another subset have variable cell widths, with wide cell bodies and actively growing thin extensions of the cell poles that concentrate fluorescent MreB. We found that the extent to which MreB localization is perturbed is linearly correlated with the development of pointed cell poles and variable cell widths. Further, we find that a mutation to glycine of two conserved aspartic acid residues that are important for nucleotide hydrolysis in other members of the actin superfamily abolishes robust midcell recruitment of MreB but supports a normal rate of growth. These mutant strains provide novel insight into how MreB's protein structure, subcellular localization, and activity contribute to its function in bacterial cell shape.
Assuntos
Caulobacter crescentus/citologia , Caulobacter crescentus/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Mutação de Sentido Incorreto , Nucleotídeos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Antibacterianos/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/efeitos dos fármacos , Proteínas do Citoesqueleto/antagonistas & inibidores , Análise Mutacional de DNA , Microscopia , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação ProteicaRESUMO
FtsZ is an essential bacterial GTPase that polymerizes at midcell, recruits the division machinery, and may generate constrictive forces necessary for cytokinesis. However, many of the mechanistic details underlying these functions are unknown. We sought to identify FtsZ-binding proteins that influence FtsZ function in Caulobacter crescentus. Here, we present a microscopy-based screen through which we discovered two FtsZ-binding proteins, FzlA and FzlC. FzlA is conserved in α-proteobacteria and was found to be functionally critical for cell division in Caulobacter. FzlA altered FtsZ structure both in vivo and in vitro, forming stable higher-order structures that were resistant to depolymerization by MipZ, a spatial determinant of FtsZ assembly. Electron microscopy revealed that FzlA organizes FtsZ protofilaments into striking helical bundles. The degree of curvature induced by FzlA depended on the nucleotide bound to FtsZ. Induction of FtsZ curvature by FzlA carries implications for regulating FtsZ function by modulating its superstructure.
Assuntos
Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , Proteínas do Citoesqueleto/metabolismo , Estrutura Quaternária de Proteína/fisiologia , Adenosina Trifosfatases/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/ultraestrutura , Caulobacter crescentus/genética , Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Forma Celular/genética , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/ultraestrutura , Expressão Gênica/genética , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/metabolismo , Microscopia Eletrônica de Transmissão , Microscopia de Interferência , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Polimerização , Ligação Proteica/fisiologia , Mapeamento de Interação de Proteínas/métodos , Multimerização Proteica/fisiologiaRESUMO
We have analyzed the spontaneous symmetry breaking and initiation of actin-based motility in keratocytes (fish epithelial cells). In stationary keratocytes, the actin network flow was inwards and radially symmetric. Immediately before motility initiation, the actin network flow increased at the prospective cell rear and reoriented in the perinuclear region, aligning with the prospective axis of movement. Changes in actin network flow at the cell front were detectable only after cell polarization. Inhibition of myosin II or Rho kinase disrupted actin network organization and flow in the perinuclear region and decreased the motility initiation frequency, whereas increasing myosin II activity with calyculin A increased the motility initiation frequency. Local stimulation of myosin activity in stationary cells by the local application of calyculin A induced directed motility initiation away from the site of stimulation. Together, these results indicate that large-scale actin-myosin network reorganization and contractility at the cell rear initiate spontaneous symmetry breaking and polarized motility of keratocytes.