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1.
Soft Matter ; 15(45): 9310-9317, 2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31674621

RESUMO

The mechanisms by which mammalian nuclear shape and size are established in cells, and become abnormal in disease states are not understood. Here, we tracked motile cells that underwent systematic changes in cell morphology as they moved from 1-D to 2-D micro-patterned adhesive domains. Motion of the cell boundaries during cell motility caused a dynamic and systematic change in nuclear volume. Short time scales (∼1 h) distinguished the dilation of the nucleus from the familiar increase that occurs during the cell cycle. Nuclear volume was systematically different between cells cultured in 3-D, 2-D and 1-D environments. Dilation of the nuclear volume was accompanied by dilation of chromatin, a decrease in the number of folds in the nuclear lamina, and an increase in nucleolar volume. Treatment of 2-D cells with non-muscle myosin-II inhibitors decreased cell volume, and proportionately caused a decrease in nuclear volume. These data suggest that nuclear size changes during cell migration may potentially impact gene expression through the modulation of intranuclear structure.

2.
Ann Biomed Eng ; 2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31745676

RESUMO

Studying a cell's ability to sense and respond to mechanical cues has emerged as a field unto itself over the last several decades, and this research area is now populated by engineers and biologists alike. As just one example of this cell mechanosensing, fibroblasts on soft substrates have slower growth rates, smaller spread areas, lower traction forces, and slower migration speeds compared to cells on stiff substrates. This phenomenon is not unique to fibroblasts, as these behaviors, and others, on soft substrates has been shown across a variety of cell types, and reproduced in many different labs. Thus far, the field has focused on discerning the mechanisms of cell mechanosensing through ion channels, focal adhesions and integrin-binding sites to the ECM, and the cell cytoskeleton. A relatively new concept in the field is that of mechanical memory, which refers to persistent effects of mechanical stimuli long after they have been removed from said stimulus. Here, we review this literature, provide an overview of emerging substrate fabrication approaches likely to be helpful for the field, and suggest the adaption of genetic tools for studying mechanical memory.

3.
J Mol Biol ; 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31629771

RESUMO

Surface sensing in bacteria is a precursor to the colonization of biotic and abiotic surfaces, and an important cause of drug resistance and virulence. As a motile bacterium approaches and adheres to a surface from the bulk fluid, the mechanical forces that act on it change. Bacteria are able to sense these changes in the mechanical load through a process termed mechanosensing. Bacterial mechanosensing has featured prominently in recent literature as playing a key role in surface sensing. However, the changes in mechanical loads on different parts of the cell at a surface vary in magnitudes as well as in signs. This confounds the determination of a causal relationship between the activation of specific mechanosensors and surface sensing. Here, we explain how contrasting mechanical stimuli arise on a surface-adherent cell and how known mechanosensors respond to these stimuli. The evidence for mechanosensing in select bacterial species is re-interpreted, with a focus on mechanosensitive molecular motors. We conclude with proposed criteria that bacterial mechanosensors must satisfy to successfully mediate surface sensing.

4.
Curr Biol ; 29(17): 2826-2839.e4, 2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31402305

RESUMO

The nucleoskeleton and cytoskeleton are important protein networks that govern cellular behavior and are connected together by the linker of nucleoskeleton and cytoskeleton (LINC) complex. Mutations in LINC complex components may be relevant to cancer, but how cell-level changes might translate into tissue-level malignancy is unclear. We used glandular epithelial cells in a three-dimensional culture model to investigate the effect of perturbations of the LINC complex on higher order cellular architecture. We show that inducible LINC complex disruption in human mammary epithelial MCF-10A cells and canine kidney epithelial MDCK II cells mechanically destabilizes the acinus. Lumenal collapse occurs because the acinus is unstable to increased mechanical tension that is caused by upregulation of Rho-kinase-dependent non-muscle myosin II motor activity. These findings provide a potential mechanistic explanation for how disruption of LINC complex may contribute to a loss of tissue structure in glandular epithelia.

5.
J Cell Sci ; 132(14)2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31308244

RESUMO

Cellular nuclei are bound by two uniformly separated lipid membranes that are fused with each other at numerous donut-shaped pores. These membranes are structurally supported by an array of distinct proteins with distinct mechanical functions. As a result, the nuclear envelope possesses unique mechanical properties, which enables it to resist cytoskeletal forces. Here, we review studies that are beginning to provide quantitative insights into nuclear membrane mechanics. We discuss how the mechanical properties of the fused nuclear membranes mediate their response to mechanical forces exerted on the nucleus and how structural reinforcement by different nuclear proteins protects the nuclear membranes against rupture. We also highlight some open questions in nuclear envelope mechanics, and discuss their relevance in the context of health and disease.

6.
Cancer Discov ; 9(10): 1438-1451, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31337617

RESUMO

By examination of the cancer genomics database, we identified a new set of mutations in core histones that frequently recur in cancer patient samples and are predicted to disrupt nucleosome stability. In support of this idea, we characterized a glutamate to lysine mutation of histone H2B at amino acid 76 (H2B-E76K), found particularly in bladder and head and neck cancers, that disrupts the interaction between H2B and H4. Although H2B-E76K forms dimers with H2A, it does not form stable histone octamers with H3 and H4 in vitro, and when reconstituted with DNA forms unstable nucleosomes with increased sensitivity to nuclease. Expression of the equivalent H2B mutant in yeast restricted growth at high temperature and led to defective nucleosome-mediated gene repression. Significantly, H2B-E76K expression in the normal mammary epithelial cell line MCF10A increased cellular proliferation, cooperated with mutant PIK3CA to promote colony formation, and caused a significant drift in gene expression and fundamental changes in chromatin accessibility, particularly at gene regulatory elements. Taken together, these data demonstrate that mutations in the globular domains of core histones may give rise to an oncogenic program due to nucleosome dysfunction and deregulation of gene expression. SIGNIFICANCE: Mutations in the core histones frequently occur in cancer and represent a new mechanism of epigenetic dysfunction that involves destabilization of the nucleosome, deregulation of chromatin accessibility, and alteration of gene expression to drive cellular transformation.See related commentary by Sarthy and Henikoff, p. 1346.This article is highlighted in the In This Issue feature, p. 1325.

7.
J Cell Biol ; 218(7): 2136-2149, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31147383

RESUMO

Cell nuclei rupture following exposure to mechanical force and/or upon weakening of nuclear integrity, but nuclear ruptures are repairable. Barrier-to-autointegration factor (BAF), a small DNA-binding protein, rapidly localizes to nuclear ruptures; however, its role at these rupture sites is unknown. Here, we show that it is predominantly a nonphosphorylated cytoplasmic population of BAF that binds nuclear DNA to rapidly and transiently localize to the sites of nuclear rupture, resulting in BAF accumulation in the nucleus. BAF subsequently recruits transmembrane LEM-domain proteins, causing their accumulation at rupture sites. Loss of BAF impairs recruitment of LEM-domain proteins and nuclear envelope membranes to nuclear rupture sites and prevents nuclear envelope barrier function restoration. Simultaneous depletion of multiple LEM-domain proteins similarly inhibits rupture repair. LEMD2 is required for recruitment of the ESCRT-III membrane repair machinery to ruptures; however, neither LEMD2 nor ESCRT-III is required to repair ruptures. These results reveal a new role for BAF in the response to and repair of nuclear ruptures.

8.
J Cell Physiol ; 234(11): 20675-20684, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31006858

RESUMO

Breast cancer nuclei have highly irregular shapes, which are diagnostic and prognostic markers of breast cancer progression. The mechanisms by which irregular cancer nuclear shapes develop are not well understood. Here we report the existence of vertical, apical cell protrusions in cultured MDA-MB-231 breast cancer cells. Once formed, these protrusions persist over time scales of hours and are associated with vertically upward nuclear deformations. They are absent in normal mammary epithelial cells (MCF-10A cells). Microtubule disruption enriched these protrusions preferentially in MDA-MB-231 cells compared with MCF-10A cells, whereas inhibition of nonmuscle myosin II (NMMII) abolished this enrichment. Dynamic confocal imaging of the vertical cell and nuclear shape revealed that the apical cell protrusions form first, and in response, the nucleus deforms and/or subsequently gets vertically extruded into the apical protrusion. Overexpression of lamin A/C in MDA-MB-231 cells reduced nuclear deformation in apical protrusions. These data highlight the role of mechanical stresses generated by moving boundaries, as well as abnormal nuclear mechanics in the development of abnormal nuclear shapes in breast cancer cells.

9.
Mol Biol Cell ; 30(7): 899-906, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30566037

RESUMO

Cancer cell migration through narrow constrictions generates compressive stresses on the nucleus that deform it and cause rupture of nuclear membranes. Nuclear membrane rupture allows uncontrolled exchange between nuclear and cytoplasmic contents. Local tensile stresses can also cause nuclear deformations, but whether such deformations are accompanied by nuclear membrane rupture is unknown. Here we used a direct force probe to locally deform the nucleus by applying a transient tensile stress to the nuclear membrane. We found that a transient (∼0.2 s) deformation (∼1% projected area strain) in normal mammary epithelial cells (MCF-10A cells) was sufficient to cause rupture of the nuclear membrane. Nuclear membrane rupture scaled with the magnitude of nuclear deformation and the magnitude of applied tensile stress. Comparison of diffusive fluxes of nuclear probes between wild-type and lamin-depleted MCF-10A cells revealed that lamin A/C, but not lamin B2, protects the nuclear membranes against rupture from tensile stress. Our results suggest that transient nuclear deformations typically caused by local tensile stresses are sufficient to cause nuclear membrane rupture.


Assuntos
Lamina Tipo A/metabolismo , Lamina Tipo B/metabolismo , Membrana Nuclear/fisiologia , Animais , Linhagem Celular , Movimento Celular/fisiologia , Núcleo Celular/metabolismo , Citosol/metabolismo , Células Epiteliais/fisiologia , Estresse Mecânico , Resistência à Tração/fisiologia
10.
J Cell Biol ; 217(10): 3330-3342, 2018 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-30194270

RESUMO

Positioning and shaping the nucleus represents a mechanical challenge for the migrating cell because of its large size and resistance to deformation. Cells shape and position the nucleus by transmitting forces from the cytoskeleton onto the nuclear surface. This force transfer can occur through specialized linkages between the nuclear envelope and the cytoskeleton. In response, the nucleus can deform and/or it can move. Nuclear movement will occur when there is a net differential in mechanical force across the nucleus, while nuclear deformation will occur when mechanical forces overcome the mechanical resistance of the various structures that comprise the nucleus. In this perspective, we review current literature on the sources and magnitude of cellular forces exerted on the nucleus, the nuclear envelope proteins involved in transferring cellular forces, and the contribution of different nuclear structural components to the mechanical response of the nucleus to these forces.

11.
Open Biol ; 8(9)2018 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-30209038

RESUMO

The periodontium is a structurally and functionally complex tissue that facilitates the anchorage of teeth in jaws. The periodontium consists of various cell types including stem cells, fibroblasts and epithelial cells. Cells of the periodontium are constantly exposed to mechanical stresses generated by biological processes such as the chewing motions of teeth, by flows generated by tongue motions and by forces generated by implants. Mechanical stresses modulate the function of cells in the periodontium, and may play a significant role in the development of periodontal disease. Here, we review the literature on the effect of mechanical forces on periodontal cells in health and disease with an emphasis on molecular and cellular mechanisms.

12.
Methods Mol Biol ; 1840: 81-90, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30141040

RESUMO

We describe a recently reported method for directly applying a known, nanonewton-scale force to the nucleus in a living, intact cell. First, a suction seal is applied on the nuclear surface using a micropipette. Then, the micropipette is translated away from the nucleus. The nucleus deforms and translates with the moving micropipette and then eventually detaches from the micropipette and recovers (roughly) its original shape and position. At the point of detachment, the resisting force (from the deformed nucleus and connected cytoskeleton) balances the suction force. Because the suction force is precisely known and reproducibly applied, this method therefore allows comparisons of nuclear response across disruptions to the cytoskeleton, nucleus, or cell. This method is useful for quantifying nuclear elastic properties in its native, integrated environment.

13.
J Vis Exp ; (137)2018 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-30102282

RESUMO

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous with the cytoskeleton, methods are needed to probe its mechanical behavior in adherent cells. Here, we discuss the direct force probe (DFP) as a tool to apply force directly to the nucleus in a living adherent cell. We attach a narrow micropipette to the nuclear surface with suction. The micropipette is translated away from the nucleus, which causes the nucleus to deform and translate. When the restoring force is equal to the suction force, the nucleus detaches and elastically relaxes. Because the suction pressure is precisely known, the force on the nuclear surface is known. This method has revealed that nano-scale forces are sufficient to deform and translate the nucleus in adherent cells, and identified cytoskeletal elements that enable the nucleus to resist forces. The DFP can be used to dissect the contributions of cellular and nuclear components to nuclear mechanical properties in living cells.


Assuntos
Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Humanos , Fenômenos Mecânicos , Estresse Mecânico
14.
J Cell Physiol ; 233(2): 1446-1454, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28542912

RESUMO

Actomyosin stress fibers impinge on the nucleus and can exert compressive forces on it. These compressive forces have been proposed to elongate nuclei in fibroblasts, and lead to abnormally shaped nuclei in cancer cells. In these models, the elongated or flattened nuclear shape is proposed to store elastic energy. However, we found that deformed shapes of nuclei are unchanged even after removal of the cell with micro-dissection, both for smooth, elongated nuclei in fibroblasts and abnormally shaped nuclei in breast cancer cells. The lack of shape relaxation implies that the nuclear shape in spread cells does not store any elastic energy, and the cellular stresses that deform the nucleus are dissipative, not static. During cell spreading, the deviation of the nucleus from a convex shape increased in MDA-MB-231 cancer cells, but decreased in MCF-10A cells. Tracking changes of nuclear and cellular shape on micropatterned substrata revealed that fibroblast nuclei deform only during deformations in cell shape and only in the direction of nearby moving cell boundaries. We propose that motion of cell boundaries exert a stress on the nucleus, which allows the nucleus to mimic cell shape. The lack of elastic energy in the nuclear shape suggests that nuclear shape changes in cells occur at constant surface area and volume.


Assuntos
Neoplasias da Mama/patologia , Movimento Celular , Forma do Núcleo Celular , Núcleo Celular/patologia , Forma Celular , Fibroblastos/citologia , Fibras de Estresse/patologia , Animais , Linhagem Celular Tumoral , Transferência de Energia , Feminino , Humanos , Mecanotransdução Celular , Camundongos , Células NIH 3T3 , Estresse Mecânico , Fatores de Tempo
15.
Artigo em Inglês | MEDLINE | ID: mdl-27562344

RESUMO

Microtubules are vital to many important cell processes, such as cell division, transport of cellular cargo, organelle positioning, and cell migration. Owing to their diverse functions, understanding microtubule function is an important part of cell biological research that can help in combating various diseases. For example, microtubules are an important target of chemotherapeutic drugs such as paclitaxel because of their pivotal role in cell division. Many functions of microtubules relate to the generation of mechanical forces. These forces are generally either a direct result of microtubule polymerization/depolymerization or generated by motor proteins that move processively along microtubules. In this review, we summarize recent efforts to quantify and model force generation by microtubules in the context of microtubule function. WIREs Nanomed Nanobiotechnol 2017, 9:e1428. doi: 10.1002/wnan.1428 For further resources related to this article, please visit the WIREs website.


Assuntos
Microtúbulos , Modelos Biológicos , Animais , Pesquisa Biomédica , Humanos , Camundongos , Nanotecnologia
16.
Sci Rep ; 6: 38063, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27905489

RESUMO

Mechanical integration of the nucleus with the extracellular matrix (ECM) is established by linkage between the cytoskeleton and the nucleus. This integration is hypothesized to mediate sensing of ECM rigidity, but parsing the function of nucleus-cytoskeleton linkage from other mechanisms has remained a central challenge. Here we took advantage of the fact that the LINC (linker of nucleoskeleton and cytoskeleton) complex is a known molecular linker of the nucleus to the cytoskeleton, and asked how it regulates the sensitivity of genome-wide transcription to substratum rigidity. We show that gene mechanosensitivity is preserved after LINC disruption, but reversed in direction. Combined with myosin inhibition studies, we identify genes that depend on nuclear tension for their regulation. We also show that LINC disruption does not attenuate nuclear shape sensitivity to substrate rigidity. Our results show for the first time that the LINC complex facilitates mechano-regulation of expression across the genome.


Assuntos
Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , RNA Mensageiro/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Camundongos , Células NIH 3T3 , Análise de Sequência de RNA , Transcrição Genética
17.
Proc Natl Acad Sci U S A ; 113(40): 11094-11099, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27647910

RESUMO

The nuclear envelope is a unique topological structure formed by lipid membranes in eukaryotic cells. Unlike other membrane structures, the nuclear envelope comprises two concentric membrane shells fused at numerous sites with toroid-shaped pores that impart a "geometric" genus on the order of thousands. Despite the intriguing architecture and vital biological functions of the nuclear membranes, how they achieve and maintain such a unique arrangement remains unknown. Here, we used the theory of elasticity and differential geometry to analyze the equilibrium shape and stability of this structure. Our results show that modest in- and out-of-plane stresses present in the membranes not only can define the pore geometry, but also provide a mechanism for destabilizing membranes beyond a critical size and set the stage for the formation of new pores. Our results suggest a mechanism wherein nanoscale buckling instabilities can define the global topology of a nuclear envelope-like structure.


Assuntos
Bicamadas Lipídicas/química , Modelos Teóricos , Membrana Nuclear/química , Poro Nuclear/química , Núcleo Celular/química , Núcleo Celular/ultraestrutura , Elasticidade , Membrana Nuclear/ultraestrutura , Poro Nuclear/ultraestrutura
18.
Cell Mol Bioeng ; 9(2): 252-257, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27330571

RESUMO

The nuclear envelope segregates the nucleoplasm from the cytoplasm and is a key feature of eukaryotic cells. Nuclear envelope architecture is comprised of two concentric membrane shells which fuse at multiple sites and yet maintain a uniform separation of 30-50 nm over the rest of the membrane. Studies have revealed the roles for numerous nuclear proteins in forming and maintaining the architecture of the nuclear envelope. However, there is a lack of consensus on the fundamental forces and physical mechanisms that establish the geometry. The objective of this review is to discuss recent findings in the context of membrane mechanics in an effort to define open questions and possible answers.

19.
PLoS One ; 11(3): e0151322, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26974838

RESUMO

Microtubules have a persistence length of the order of millimeters in vitro, but inside cells they bend over length scales of microns. It has been proposed that polymerization forces bend microtubules in the vicinity of the cell boundary or other obstacles, yet bends develop even when microtubules are polymerizing freely, unaffected by obstacles and cell boundaries. How these bends are formed remains unclear. By tracking the motions of microtubules marked by photobleaching, we found that in LLC-PK1 epithelial cells local bends develop primarily by plus-end directed transport of portions of the microtubule contour towards stationary locations (termed pinning points) along the length of the microtubule. The pinning points were transient in nature, and their eventual release allowed the bends to relax. The directionality of the transport as well as the overall incidence of local bends decreased when dynein was inhibited, while myosin inhibition had no observable effect. This suggests that dynein generates a tangential force that bends microtubules against stationary pinning points. Simulations of microtubule motion and polymerization accounting for filament mechanics and dynein forces predict the development of bends of size and shape similar to those observed in cells. Furthermore, simulations show that dynein-generated bends at a pinning point near the plus end can cause a persistent rotation of the tip consistent with the observation that bend formation near the tip can change the direction of microtubule growth. Collectively, these results suggest a simple physical mechanism for the bending of growing microtubules by dynein forces accumulating at pinning points.


Assuntos
Microtúbulos/metabolismo , Animais , Transporte Biológico , Fenômenos Biomecânicos , Núcleo Celular/metabolismo , Simulação por Computador , Dineínas/metabolismo , Células LLC-PK1 , Modelos Biológicos , Miosinas/metabolismo , Rotação , Suínos
20.
PLoS One ; 11(2): e0149213, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26872214

RESUMO

During development of the vertebrate neuroepithelium, the nucleus in neural progenitor cells (NPCs) moves from the apex toward the base and returns to the apex (called interkinetic nuclear migration) at which point the cell divides. The fate of the resulting daughter cells is thought to depend on the sampling by the moving nucleus of a spatial concentration profile of the cytoplasmic Notch intracellular domain (NICD). However, the nucleus executes complex stochastic motions including random waiting and back and forth motions, which can expose the nucleus to randomly varying levels of cytoplasmic NICD. How nuclear position can determine daughter cell fate despite the stochastic nature of nuclear migration is not clear. Here we derived a mathematical model for reaction, diffusion, and nuclear accumulation of NICD in NPCs during interkinetic nuclear migration (INM). Using experimentally measured trajectory-dependent probabilities of nuclear turning, nuclear waiting times and average nuclear speeds in NPCs in the developing zebrafish retina, we performed stochastic simulations to compute the nuclear trajectory-dependent probabilities of NPC differentiation. Comparison with experimentally measured nuclear NICD concentrations and trajectory-dependent probabilities of differentiation allowed estimation of the NICD cytoplasmic gradient. Spatially polarized production of NICD, rapid NICD cytoplasmic consumption and the time-averaging effect of nuclear import/export kinetics are sufficient to explain the experimentally observed differentiation probabilities. Our computational studies lend quantitative support to the feasibility of the nuclear concentration-sensing mechanism for NPC fate determination in zebrafish retina.


Assuntos
Núcleo Celular/metabolismo , Células-Tronco Neurais/citologia , Células Neuroepiteliais/citologia , Retina/embriologia , Peixe-Zebra/embriologia , Animais , Diferenciação Celular , Simulação por Computador , Modelos Biológicos , Células-Tronco Neurais/metabolismo , Células Neuroepiteliais/metabolismo , Receptores Notch/análise , Receptores Notch/metabolismo , Retina/citologia , Processos Estocásticos , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/análise , Proteínas de Peixe-Zebra/metabolismo
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