Modulation of Nuclear Shape by Substrate Rigidity

Lovett, David B.; Shekhar, Nandini; Nickerson, Jeffrey A.; Roux, Kyle J.; Lele, Tanmay P.

doi:10.1007/s12195-013-0270-2

Cited by 134 publications

(144 citation statements)

References 65 publications

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“…5). In previous studies on flat substrates, a similar trend is observed, where the nucleus height and shape index both change with increasing substrate elasticity, leading to a shorter nucleus with a rounder footprint (15,58). The NSI (Eq.…”

Section: Nsisupporting

confidence: 73%

Role of Suspended Fiber Structural Stiffness and Curvature on Single-Cell Migration, Nucleus Shape, and Focal-Adhesion-Cluster Length

Meehan

Nain

2014

Biophysical Journal

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It has been shown that cellular migration, persistence, and associated cytoskeletal arrangement are highly dependent on substrate stiffness (modulus: N/m(2) and independent of geometry), but little is known on how cells respond to subtle changes in local geometry and structural stiffness (N/m). Here, using fibers of varying diameter (400, 700, and 1200 nm) and length (1 and 2 mm) deposited over hollow substrates, we demonstrate that single mouse C2C12 cells attached to single suspended fibers form spindle morphologies that are sensitive to fiber mechanical properties. Over a wide range of increasing structural stiffness (2 to 100+ mN/m), cells exhibited decreases in migration speed and average nucleus shape index of ∼57% (from 58 to 25 μm/h) and ∼26% (from 0.78 to 0.58), respectively, whereas the average paxillin focal-adhesion-cluster (FAC, formed at poles) length increased by ∼38% (from 8 to 11 μm). Furthermore, the increase in structural stiffness directly correlates with cellular persistence, with 60% of cells moving in the direction of increasing structural stiffness. At similar average structural stiffness (25 ± 5 mN/m), cells put out longer FAC lengths on smaller diameters, suggesting a conservation of FAC area, and also exhibited higher nucleus shape index and migration speeds on larger-diameter fibers. Interestingly, cells were observed to deform fibers locally or globally through forces applied through the FAC sites and cells undergoing mitosis were found to be attached to the FAC sites by single filamentous tethers. These varied reactions have implications in developmental and disease biology models as they describe a strong dependence of cellular behavior on the cell's immediate mechanistic environment arising from alignment and geometry of fibers.

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Section: Nsisupporting

confidence: 73%

Role of Suspended Fiber Structural Stiffness and Curvature on Single-Cell Migration, Nucleus Shape, and Focal-Adhesion-Cluster Length

Meehan

Nain

2014

Biophysical Journal

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“…1A). Because the mechanical behavior of the nucleus has been shown to depend on tissue stiffness (Swift et al, 2013), and we have shown that the rigidity of the substratum alters nuclear shape (Lovett et al, 2013), we performed these experiments on polyacrylamide gels with sufficiently different rigidities. Remarkably, in control cells, the nuclear position closely coincided with the PMT (Fig.…”

Section: Nuclear Position Coincides With the Point Of Maximum Tensionmentioning

confidence: 99%

“…Fibronectin-coated (5 µg/ml, BD Biosciences) polyacrylamide (PAA) gels were prepared on glass-bottomed dishes (WPI, Sarasota, FL) as described previously (Lovett et al, 2013). Fluorescent microspheres of 0.5-µm diameter (Invitrogen) were suspended in the hydrogel before polymerization and used as fiduciary markers.…”

Section: Traction-force Microscopymentioning

confidence: 99%

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

Alam

Lovett

Kim

et al. 2015

Journal of Cell Science

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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, tractiongenerating 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.

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“…This strategy allows for the visualization of the NK IS en face in a single z-stack plane. Due to their morphology and adherence, NIH3T3 cells produce a monolayer of target cells and a large surface area for NK cell interaction [41][42][43]. The use of a monolayer of NIH3T3 cells is a novel experimental approach to study the NK IS.…”

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confidence: 99%

Nanoscale colocalization of NK cell activating and inhibitory receptors controls signal integration

Tomaz

Pereira

Guerra

et al. 2018

Preprint

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NK cell responses depend on the balance of signals from inhibitory and activating receptors. However, how the integration of antagonistic signals occurs upon NK cell-target cell interaction is not fully understood. Here, we provide evidence that NK cell inhibition via the inhibitory receptor Ly49A is dependent on its relative colocalization at nanometer-scale with the activating receptor NKG2D upon immune synapse (IS) formation. NKG2D and Ly49A signal integration and colocalization was studied using NKG2D-GFP and Ly49A-RFP-expressing primary NK cells, forming ISs with NIH3T3 target cells, with or without expression of single chain trimer (SCT) H2-D d and an extended form of SCT H2-D d -CD4 MHC-I molecules. Nanoscale colocalization was assessed by Förster resonance energy transfer (FRET) between NKG2D-GFP and Ly49A-RFP and measured for each synapse. In the presence of their respective cognate ligands, NKG2D and Ly49A colocalize at a nanometer scale leading to NK cell inhibition. However, increasing the size of the Ly49A ligand reduced the nanoscale colocalization with NKG2D consequently impairing Ly49A-mediated inhibition. Thus, our data shows NK cell signal integration is critically dependent on the dimensions of NK cell ligand-receptor pairs by affecting their relative nanometer-scale colocalization at the IS. Together, our results suggest the balance of NK cell signals, and NK cell responses, are determined by the relative nanoscale colocalization of activating and inhibitory receptors in the immune synapse. NK cell signal integration | FRET nanoscale localization | NKG2D | Ly49ACorrespondence:david.tomaz@kcl.ac.uk, r.henriques@ucl.ac.uk, k.gould@imperial.ac.uk Introduction NK cell activation is dependent on a different array of germline-encoded receptors capable of triggering effector responses against infection [1][2][3] and cellular transformation [4,5], and yet maintaining tolerance towards healthy cells and tissues [6,7]. NK cell functionality is widely characterized by a balance between activating and inhibitory receptors [8,9]. However, upon immune synapse formation, how NK cells integrate signals from functionally antagonistic receptors, is not yet fully understood [10]. One major hypothesis to explain immune signal integration is based on the kinetic segregation (K-S) model [11]. This model states that the balance of antagonistic signals is mediated by the equilibrium of phosphorylation (kinases) and dephosphorylation (phosphatases), which depends on the relative colocalization of receptors and their associated signaling molecules upon synapse formation. This hypothesis has been validated using T cells, in the context of TCR triggering [12,13] and, more recently, NK cells [14,15]. The K-S model suggests immune inhibition, upon immune synapse formation, is maintained by the dephosphorylation of tyrosines in stimulatory receptors (e.g ITAMs-associated receptors such as TCR-CD3 or NKG2D-DAP12), due to a nanoscale colocalization with receptors bearing phosphatase activity (e.g. CD45, CD148) or with t...

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Modulation of Nuclear Shape by Substrate Rigidity

Cited by 134 publications

References 65 publications

Role of Suspended Fiber Structural Stiffness and Curvature on Single-Cell Migration, Nucleus Shape, and Focal-Adhesion-Cluster Length

Role of Suspended Fiber Structural Stiffness and Curvature on Single-Cell Migration, Nucleus Shape, and Focal-Adhesion-Cluster Length

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

Nanoscale colocalization of NK cell activating and inhibitory receptors controls signal integration

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