RESUMO
Change history: In Fig. 1b and c of this Letter, the inset times in the DIC and GFP microscopy images should be in minutes ('min') instead of seconds ('s'). This has not been corrected online.
RESUMO
During mitosis, adherent animal cells undergo a drastic shape change, from essentially flat to round. Mitotic cell rounding is thought to facilitate organization within the mitotic cell and be necessary for the geometric requirements of division. However, the forces that drive this shape change remain poorly understood in the presence of external impediments, such as a tissue environment. Here we use cantilevers to track cell rounding force and volume. We show that cells have an outward rounding force, which increases as cells enter mitosis. We find that this mitotic rounding force depends both on the actomyosin cytoskeleton and the cells' ability to regulate osmolarity. The rounding force itself is generated by an osmotic pressure. However, the actomyosin cortex is required to maintain this rounding force against external impediments. Instantaneous disruption of the actomyosin cortex leads to volume increase, and stimulation of actomyosin contraction leads to volume decrease. These results show that in cells, osmotic pressure is balanced by inwardly directed actomyosin cortex contraction. Thus, by locally modulating actomyosin-cortex-dependent surface tension and globally regulating osmotic pressure, cells can control their volume, shape and mechanical properties.
Assuntos
Actomiosina/metabolismo , Forma Celular/fisiologia , Citoesqueleto/metabolismo , Mitose , Animais , Forma Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Células HeLa , Humanos , Pressão Hidrostática , Microscopia de Força Atômica , Modelos Biológicos , Concentração Osmolar , Pressão Osmótica , PrófaseRESUMO
Cleavage of viral DNA by the bacterial Type III Restriction-Modification enzymes requires the ATP-dependent long-range communication between a distant pair of DNA recognition sequences. The classical view is that Type III endonuclease activity is only activated by a pair of asymmetric sites in a specific head-to-head inverted repeat. Based on this assumption and due to the presence of helicase domains in Type III enzymes, various motor-driven DNA translocation models for communication have been suggested. Using both single-molecule and ensemble assays we demonstrate that Type III enzymes can also cleave DNA with sites in tail-to-tail repeat with high efficiency. The ability to distinguish both inverted repeat substrates from direct repeat substrates in a manner independent of DNA topology or accessory proteins can only be reconciled with an alternative sliding mode of communication.
Assuntos
DNA Viral/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Sequências Repetidas Invertidas , Conformação de Ácido Nucleico , Sítios de Ligação/genética , Sítios de Ligação/fisiologia , Modelos Moleculares , Oligonucleotídeos , Especificidade por SubstratoRESUMO
To cleave DNA, Type III restriction enzymes must communicate the relative orientation of two asymmetric recognition sites over hundreds of base pairs. The basis of this long-distance communication, for which ATP hydrolysis by their helicase domains is required, is poorly understood. Several conflicting DNA-looping mechanisms have been proposed, driven either by active DNA translocation or passive 3D diffusion. Using single-molecule DNA stretching in combination with bulk-solution assays, we provide evidence that looping is both highly unlikely and unnecessary, and that communication is strictly confined to a 1D route. Integrating our results with previous data, a simple communication scheme is concluded based on 1D diffusion along DNA.
Assuntos
DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/metabolismo , Modelos Químicos , Sítios de Ligação , DNA/química , Difusão , Hidrólise , Conformação de Ácido Nucleico , Pinças Ópticas , Especificidade por SubstratoRESUMO
As epithelial tissues develop, groups of cells related by descent tend to associate in clonal populations rather than dispersing within the cell layer. While this is frequently assumed to be a result of differential adhesion, precise mechanisms controlling clonal cohesiveness remain unknown. Here we employ computational simulations to modulate epithelial cell size in silico and show that junctions between small cells frequently collapse, resulting in clone-cell dispersal among larger neighbors. Consistent with similar dynamics in vivo, we further demonstrate that mosaic disruption of Drosophila Tor generates small cells and results in aberrant clone dispersal in developing wing disc epithelia. We propose a geometric basis for this phenomenon, supported in part by the observation that soap-foam cells exhibit similar size-dependent junctional rearrangements. Combined, these results establish a link between cell-size pleomorphism and the control of epithelial cell packing, with potential implications for understanding tumor cell dispersal in human disease.
Assuntos
Tamanho Celular , Drosophila melanogaster/embriologia , Epitélio/embriologia , Animais , Apoptose , Adesão Celular , Divisão Celular , Proliferação de Células , Simulação por Computador , Proteínas de Drosophila/metabolismo , Células Epiteliais/citologia , Feminino , Células Espumosas/citologia , Masculino , Morfogênese , Receptores Proteína Tirosina Quinases/metabolismo , Asas de Animais/embriologiaRESUMO
Actomyosin-dependent mitotic rounding occurs in both cell culture and tissue, where it is involved in cell positioning and epithelial organization. How actomyosin is regulated to mediate mitotic rounding is not well understood. Here we characterize the mechanics of single mitotic cells while imaging actomyosin recruitment to the cell cortex. At mitotic onset, the assembly of a uniform DIAPH1-dependent F-actin cortex coincides with initial rounding. Thereafter, cortical enrichment of F-actin remains stable while myosin II progressively accumulates at the cortex, and the amount of myosin at the cortex correlates with intracellular pressure. Whereas F-actin provides only short-term (<10 s) resistance to mechanical deformation, myosin sustains intracellular pressure for a longer duration (>60 s). Our data suggest that progressive accumulation of myosin II to the mitotic cell cortex probably requires the Cdk1 activation of both p21-activated kinases, which inhibit myosin recruitment, and of Rho kinase, which stimulates myosin recruitment to the cortex.
Assuntos
Proteína Quinase CDC2/metabolismo , Mitose , Miosina Tipo II/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Cisteína/análogos & derivados , Cisteína/farmacologia , Proteínas Fetais/metabolismo , Forminas , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microscopia de Força Atômica , Mitose/efeitos dos fármacos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Pressão , Fatores de Tempo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Single-cell force spectroscopy (SCFS) is becoming a widely used method to quantify the adhesion of a living cell to a substrate, another cell or tissue. The high sensitivity of SCFS permits determining the contributions of individual cell adhesion molecules (CAMs) to the adhesion force of an entire cell. However, to prepare adherent cells for SCFS, they must first be detached from tissue-culture flasks or plates. EDTA and trypsin are often applied for this purpose. Because cellular properties can be affected by this treatment, cells need to recover before being further characterized by SCFS. Here we introduce atomic force microscopy (AFM)-based SCFS to measure the mechanical and adhesive properties of HeLa cells and mouse embryonic kidney fibroblasts while they are recovering after detachment from tissue-culture. We find that mechanical and adhesive properties of both cell lines recover quickly (<10 min) after detachment using EDTA, while trypsin-detached fibroblasts require >60 min to fully recover. Our assay introduced to characterize the recovery of mammalian cells after detachment can in future be used to estimate the recovery behavior of other adherent cell types.