Rho activation drives luminal collapse and eversion in epithelial acini.

Epithelial cells lining a gland and cells grown in a soft extracellular matrix polarize with apical proteins exposed to the lumen and basal proteins in contact with the extracellular matrix. Alterations to polarity, including an apical-out polarity, occur in human cancers. Although some aberrant polarity states may result from altered protein trafficking, recent observations of an extraordinary tissue-level inside-out unfolding suggest an alternative pathway for altered polarity. Because mechanical alterations are common in human cancer, including an upregulation of RhoA-mediated actomyosin tension in acinar epithelia, we explored whether perturbing mechanical homeostasis could cause apical-out eversion. Acinar eversion was robustly induced by direct activation of RhoA in normal and tumor epithelial acini, or indirect activation of RhoA through blockage of ß1-integrins, disruption of the LINC complex, oncogenic Ras activation, or Rac1 inhibition. Furthermore, laser ablation of a portion of the untreated acinus was sufficient to induce eversion. Analyses of acini revealed high curvature and low phosphorylated myosin in the apical cell surfaces relative to the basal surfaces. A vertex-based mathematical model that balances tension at cell-cell interfaces revealed a fivefold greater basal cell surface tension relative to the apical cell surface tension. The model suggests that the difference in surface energy between the apical and basal surfaces is the driving force for acinar eversion. Our findings raise the possibility that a loss of mechanical homeostasis may cause apical-out polarity states in human cancers.

Distinct VASP tetramers synergize in the processive elongation of individual actin filaments from clustered arrays.

Ena/VASP proteins act as actin polymerases that drive the processive elongation of filament barbed ends in membrane protrusions or at the surface of bacterial pathogens. Based on previous analyses of fast and slow elongating VASP proteins by in vitro total internal reflection fluorescence microscopy (TIRFM) and kinetic and thermodynamic measurements, we established a kinetic model of Ena/VASP-mediated actin filament elongation. At steady state, it entails that tetrameric VASP uses one of its arms to processively track growing filament barbed ends while three G-actin-binding sites (GABs) on other arms are available to recruit and deliver monomers to the filament tip, suggesting that VASP operates as a single tetramer in solution or when clustered on a surface, albeit processivity and resistance toward capping protein (CP) differ dramatically between both conditions. Here, we tested the model by variation of the oligomerization state and by increase of the number of GABs on individual polypeptide chains. In excellent agreement with model predictions, we show that in solution the rates of filament elongation directly correlate with the number of free GABs. Strikingly, however, irrespective of the oligomerization state or presence of additional GABs, filament elongation on a surface invariably proceeded with the same rate as with the VASP tetramer, demonstrating that adjacent VASP molecules synergize in the elongation of a single filament. Additionally, we reveal that actin ATP hydrolysis is not required for VASP-mediated filament assembly. Finally, we show evidence for the requirement of VASP to form tetramers and provide an amended model of processive VASP-mediated actin assembly in clustered arrays.

Apical cell protrusions cause vertical deformation of the soft cancer nucleus.

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.

Nuclear size changes caused by local motion of cell boundaries unfold the nuclear lamina and dilate chromatin and intranuclear bodies.

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.

The nucleus is irreversibly shaped by motion of cell boundaries in cancer and non-cancer cells.

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.

Direct force probe reveals the mechanics of nuclear homeostasis in the mammalian cell.

How cells maintain nuclear shape and position against various intracellular and extracellular forces is not well understood, although defects in nuclear mechanical homeostasis are associated with a variety of human diseases. We estimated the force required to displace and deform the nucleus in adherent living cells with a technique to locally pull the nuclear surface. A minimum pulling force of a few nanonewtons--far greater than typical intracellular motor forces--was required to significantly displace and deform the nucleus. Upon force removal, the original shape and position were restored quickly within a few seconds. This stiff, elastic response required the presence of vimentin, lamin A/C, and SUN (Sad1p, UNC-84)-domain protein linkages, but not F-actin or microtubules. Although F-actin and microtubules are known to exert mechanical forces on the nuclear surface through molecular motor activity, we conclude that the intermediate filament networks maintain nuclear mechanical homeostasis against localized forces.

The nucleus is an intracellular propagator of tensile forces in NIH 3T3 fibroblasts.

Nuclear positioning is a crucial cell function, but how a migrating cell positions its nucleus is not understood. Using traction-force microscopy, we found that the position of the nucleus in migrating fibroblasts closely coincided with the center point of the traction-force balance, called the point of maximum tension (PMT). Positioning of the nucleus close to the PMT required nucleus-cytoskeleton connections through linker of nucleoskeleton-to-cytoskeleton (LINC) complexes. Although the nucleus briefly lagged behind the PMT following spontaneous detachment of the uropod during migration, the nucleus quickly repositioned to the PMT within a few minutes. Moreover, traction-generating spontaneous protrusions deformed the nearby nucleus surface to pull the nuclear centroid toward the new PMT, and subsequent retraction of these protrusions relaxed the nuclear deformation and restored the nucleus to its original position. We propose that the protruding or retracting cell boundary transmits a force to the surface of the nucleus through the intervening cytoskeletal network connected by the LINC complexes, and that these forces help to position the nucleus centrally and allow the nucleus to efficiently propagate traction forces across the length of the cell during migration.

New approaches for understanding the nuclear force balance in living, adherent cells.

Cytoskeletal forces are transmitted to the nucleus to position and shape it. Linkages mediated by the LINC (linker of nucleoskeleton and cytoskeleton) complex transfer these forces to the nuclear envelope. Nuclear position and shape can be thought to be determined by a balance of cytoskeletal forces generated by microtubule motors that shear the nuclear surface, actomyosin forces that can pull, push and shear the nucleus, and intermediate filaments that may passively resist nuclear decentering and deformation. Parsing contributions of these different forces to nuclear mechanics is a very challenging task. Here we review new approaches that can be used in living cells to probe and understand the nuclear force balance.

Coordinated Dynamics of RNA Splicing Speckles in the Nucleus.

Despite being densely packed with chromatin, nuclear bodies and a nucleoskeletal network, the nucleus is a remarkably dynamic organelle. Chromatin loops form and relax, RNA transcripts and transcription factors move diffusively, and nuclear bodies move. We show here that RNA splicing speckled domains (splicing speckles) fluctuate in constrained nuclear volumes and remodel their shapes. Small speckles move in a directed way toward larger speckles with which they fuse. This directed movement is reduced upon decreasing cellular ATP levels or inhibiting RNA polymerase II activity. The random movement of speckles is reduced upon decreasing cellular ATP levels, moderately reduced after inhibition of SWI/SNF chromatin remodeling and modestly increased upon inhibiting RNA polymerase II activity. To define the paths through which speckles can translocate in the nucleus, we generated a pressure gradient to create flows in the nucleus. In response to the pressure gradient, speckles moved along curvilinear paths in the nucleus. Collectively, our results demonstrate a new type of ATP-dependent motion in the nucleus. We present a model where recycling splicing factors return as part of small sub-speckles from distal sites of RNA processing to larger splicing speckles by a directed ATP-driven mechanism through interchromatin spaces.

Moving Cell Boundaries Drive Nuclear Shaping during Cell Spreading.

The nucleus has a smooth, regular appearance in normal cells, and its shape is greatly altered in human pathologies. Yet, how the cell establishes nuclear shape is not well understood. We imaged the dynamics of nuclear shaping in NIH3T3 fibroblasts. Nuclei translated toward the substratum and began flattening during the early stages of cell spreading. Initially, nuclear height and width correlated with the degree of cell spreading, but over time, reached steady-state values even as the cell continued to spread. Actomyosin activity, actomyosin bundles, microtubules, and intermediate filaments, as well as the LINC complex, were all dispensable for nuclear flattening as long as the cell could spread. Inhibition of actin polymerization as well as myosin light chain kinase with the drug ML7 limited both the initial spreading of cells and flattening of nuclei, and for well-spread cells, inhibition of myosin-II ATPase with the drug blebbistatin decreased cell spreading with associated nuclear rounding. Together, these results show that cell spreading is necessary and sufficient to drive nuclear flattening under a wide range of conditions, including in the presence or absence of myosin activity. To explain this observation, we propose a computational model for nuclear and cell mechanics that shows how frictional transmission of stress from the moving cell boundaries to the nuclear surface shapes the nucleus during early cell spreading. Our results point to a surprisingly simple mechanical system in cells for establishing nuclear shapes.

Molecular mechanism of Ena/VASP-mediated actin-filament elongation.

Ena/VASP proteins are implicated in a variety of fundamental cellular processes including axon guidance and cell migration. In vitro, they enhance elongation of actin filaments, but at rates differing in nearly an order of magnitude according to species, raising questions about the molecular determinants of rate control. Chimeras from fast and slow elongating VASP proteins were generated and their ability to promote actin polymerization and to bind G-actin was assessed. By in vitro TIRF microscopy as well as thermodynamic and kinetic analyses, we show that the velocity of VASP-mediated filament elongation depends on G-actin recruitment by the WASP homology 2 motif. Comparison of the experimentally observed elongation rates with a quantitative mathematical model moreover revealed that Ena/VASP-mediated filament elongation displays a saturation dependence on the actin monomer concentration, implying that Ena/VASP proteins, independent of species, are fully saturated with actin in vivo and generally act as potent filament elongators. Moreover, our data showed that spontaneous addition of monomers does not occur during processive VASP-mediated filament elongation on surfaces, suggesting that most filament formation in cells is actively controlled.

Actomyosin pulls to advance the nucleus in a migrating tissue cell.

The cytoskeletal forces involved in translocating the nucleus in a migrating tissue cell remain unresolved. Previous studies have variously implicated actomyosin-generated pushing or pulling forces on the nucleus, as well as pulling by nucleus-bound microtubule motors. We found that the nucleus in an isolated migrating cell can move forward without any trailing-edge detachment. When a new lamellipodium was triggered with photoactivation of Rac1, the nucleus moved toward the new lamellipodium. This forward motion required both nuclear-cytoskeletal linkages and myosin activity. Apical or basal actomyosin bundles were found not to translate with the nucleus. Although microtubules dampen fluctuations in nuclear position, they are not required for forward translocation of the nucleus during cell migration. Trailing-edge detachment and pulling with a microneedle produced motion and deformation of the nucleus suggestive of a mechanical coupling between the nucleus and the trailing edge. Significantly, decoupling the nucleus from the cytoskeleton with KASH overexpression greatly decreased the frequency of trailing-edge detachment. Collectively, these results explain how the nucleus is moved in a crawling fibroblast and raise the possibility that forces could be transmitted from the front to the back of the cell through the nucleus.

Nuclear shapes are geometrically determined by the excess surface area of the nuclear lamina.

Introduction: Nuclei have characteristic shapes dependent on cell type, which are critical for proper cell function, and nuclei lose their distinct shapes in multiple diseases including cancer, laminopathies, and progeria. Nuclear shapes result from deformations of the sub-nuclear components-nuclear lamina and chromatin. How these structures respond to cytoskeletal forces to form the nuclear shape remains unresolved. Although the mechanisms regulating nuclear shape in human tissues are not fully understood, it is known that different nuclear shapes arise from cumulative nuclear deformations post-mitosis, ranging from the rounded morphologies that develop immediately after mitosis to the various nuclear shapes that roughly correspond to cell shape (e.g., elongated nuclei in elongated cells, flat nuclei in flat cells). Methods: We formulated a mathematical model to predict nuclear shapes of cells in various contexts under the geometric constraints of fixed cell volume, nuclear volume and lamina surface area. Nuclear shapes were predicted and compared to experiments for cells in various geometries, including isolated on a flat surface, on patterned rectangles and lines, within a monolayer, isolated in a well, or when the nucleus is impinging against a slender obstacle. Results and Discussion: The close agreement between predicted and experimental shapes demonstrates a simple geometric principle of nuclear shaping: the excess surface area of the nuclear lamina (relative to that of a sphere of the same volume) permits a wide range of highly deformed nuclear shapes under the constraints of constant surface area and constant volume. When the lamina is smooth (tensed), the nuclear shape can be predicted entirely from these geometric constraints alone for a given cell shape. This principle explains why flattened nuclear shapes in fully spread cells are insensitive to the magnitude of the cytoskeletal forces. Also, the surface tension in the nuclear lamina and nuclear pressure can be estimated from the predicted cell and nuclear shapes when the cell cortical tension is known, and the predictions are consistent with measured forces. These results show that excess surface area of the nuclear lamina is the key determinant of nuclear shapes. When the lamina is smooth (tensed), the nuclear shape can be determined purely by the geometric constraints of constant (but excess) nuclear surface area, nuclear volume, and cell volume, for a given cell adhesion footprint, independent of the magnitude of the cytoskeletal forces involved.

A new function for nuclear lamins: providing surface tension to the nuclear drop.

The nuclear lamina, a conserved structure in metazoans, provides mechanical rigidity to the nuclear envelope. A decrease in lamin levels and/or lamin mutations are associated with a host of human diseases. Despite being only about 15 nm thick, perturbation of components of the nuclear lamina dramatically impacts the deformation response of the entire nucleus through mechanisms that are not well understood. Here we discuss evidence for the recently proposed 'nuclear drop' model that explains the role of A-type lamins in nuclear deformation in migrating cells. In this model, the nuclear lamina acts as an inextensible surface, supporting a surface tension when fully unfolded, that balances nuclear interior pressure. Much like a liquid drop surface where the molecularly thin interface governs surface tension and drop shape under external forces, the thin nuclear lamina imparts a surface tension on the nuclear drop to resist nuclear deformation as well as to establish nuclear shape. We discuss implications of the nuclear drop model for the function of this crucially important eukaryotic organelle.

Viscous shaping of the compliant cell nucleus.

The cell nucleus is commonly considered to be a stiff organelle that mechanically resists changes in shape, and this resistance is thought to limit the ability of cells to migrate through pores or spread on surfaces. Generation of stresses on the cell nucleus during migration and nuclear response to these stresses is fundamental to cell migration and mechano-transduction. In this Perspective, we discuss our previous experimental and computational evidence that supports a dynamic model, in which the soft nucleus is irreversibly shaped by viscous stresses generated by the motion of cell boundaries and transmitted through the intervening cytoskeletal network. While the nucleus is commonly modeled as a stiff elastic body, we review how nuclear shape changes on the timescale of migration can be explained by simple geometric constraints of constant nuclear volume and constant surface area of the nuclear lamina. Because the lamina surface area is in excess of that of a sphere of the same volume, these constraints permit dynamic transitions between a wide range of shapes during spreading and migration. The excess surface area allows the nuclear shape changes to mirror those of the cell with little mechanical resistance. Thus, the nucleus can be easily shaped by the moving cell boundaries over a wide range of shape changes and only becomes stiff to more extreme deformations that would require the lamina to stretch or the volume to compress. This model explains how nuclei can easily flatten on surfaces during cell spreading or elongate as cells move through pores until the lamina smooths out and becomes tense.

The Nucleus Bypasses Obstacles by Deforming Like a Drop with Surface Tension Mediated by Lamin A/C.

Migrating cells must deform their stiff cell nucleus to move through pores and fibers in tissue. Lamin A/C is known to hinder cell migration by limiting nuclear deformation and passage through confining channels, but its role in nuclear deformation and passage through fibrous environments is less clear. Cell and nuclear migration through discrete, closely spaced, slender obstacles which mimic the mechanical properties of collagen fibers are studied. Nuclei bypass slender obstacles while preserving their overall morphology by deforming around them with deep local invaginations of little resisting force. The obstacles do not impede the nuclear trajectory and do not cause rupture of the nuclear envelope. Nuclei likewise deform around single collagen fibers in cells migrating in 3D collagen gels. In contrast to its limiting role in nuclear passage through confining channels, lamin A/C facilitates nuclear deformation and passage through fibrous environments; nuclei in lamin-null (Lmna-/- ) cells lose their overall morphology and become entangled on the obstacles. Analogous to surface tension-mediated deformation of a liquid drop, lamin A/C imparts a surface tension on the nucleus that allows nuclear invaginations with little mechanical resistance, preventing nuclear entanglement and allowing nuclear passage through fibrous environments.

How dynein and microtubules rotate the nucleus.

In living cells, a fluctuating torque is exerted on the nuclear surface but the origin of the torque is unclear. In this study, we found that the nuclear rotation angle is directionally persistent on a time scale of tens of minutes, but rotationally diffusive on longer time scales. Rotation required the activity of the microtubule motor dynein. We formulated a model based on microtubules undergoing dynamic instability, with tensional forces between a stationary centrosome and the nuclear surface mediated by dynein. Model simulations suggest that the persistence in rotation angle is due to the transient asymmetric configuration of microtubules exerting a net torque in one direction until the configuration is again randomized by dynamic instability. The model predicts that the rotational magnitude must depend on the distance between the nucleus and the centrosome. To test this prediction, rotation was quantified in patterned cells in which the cell's centrosome was close to the projected nuclear centroid. Consistent with the prediction, the angular displacement was found to decrease in these cells relative to unpatterned cells. This work provides the first mechanistic explanation for how nuclear dynein interactions with discrete microtubules emanating from a stationary centrosome cause rotational torque on the nucleus.

Mechanics of Vorticella contraction.

Vorticella convallaria is one of a class of fast-moving organisms that can traverse its body size in less than a millisecond by rapidly coiling a slender stalk anchoring it to a nearby surface. The stalk houses a fiber called the spasmoneme, which winds helically within the stalk and rapidly contracts in response to calcium signaling. We have developed a coupled mechanical-chemical model of the coiling process, accounting for the coiling of the elastic stalk and the binding of calcium to the protein spasmin. Simulations of the model describe the contraction and recovery processes quantitatively. The stalk-spasmoneme system is shown to satisfy geometric constraints, which explains why the cell body sometimes rotates during contraction. The shape of the collapsing and recovering stalk bounds its effective bending stiffness. Simulations suggest that recovery from the contracted state is driven by the stalk at a rate controlled by dissociation of calcium from spasmin.

Sarcomere mechanics in capillary endothelial cells.

Tension generation in endothelial cells of the aorta, spleen, and eye occurs in actin stress fibers, and is necessary for normal cell function. Sarcomeres are the tension-generating units of actin stress fibers in endothelial cells. How sarcomeres generate and maintain tension in stress fibers is not well understood. Using femtosecond laser ablation, we severed living stress fibers and measured sarcomere contraction under zero tension. The length of the sarcomere decreased in two phases: an instantaneous initial response, followed by a slower change in length attributed to myosin activity. The latter phase ceased abruptly after a minimum sarcomere length was reached, suggesting a rigid resistance that prevents further contraction. Furthermore, severed, contracted stress fibers did not relax when treated with myosin inhibitors, indicating that contracted stress fibers do not store elastic potential energy. These novel measurements combined with modeling suggest that myosin-generated forces in adjacent sarcomeres are directly in balance, and argue against sarcomere models with springlike elements in parallel with myosin contractile elements. We propose a new model for tension generation in the sarcomere, which provides a mechanistic interpretation for our observations and previous observations of inhomogeneous sarcomere contraction and apparent stress fiber viscoelastic behavior.