Experimental techniques for study of chromatin mechanics in intact nuclei and living cells

Abstract

While the structure of chromatin and its physical properties have been well studied on isolated chromatin fibres and DNA strands in vitro, its organization and function in the intact interphase nucleus is less clear. Chromatin organization is critical for transcriptional regulation and DNA replication, and mounting evidence suggests that cells respond to changes in the mechanical environment with alterations in nuclear architecture that are accompanied by modifications in gene expression. Since the nucleus forms part of a continuous physical network spanning the extracellular matrix, the cytoskeleton and the nuclear envelope, environmentally mediated forces can be transmitted to the nucleus and induce deformations of the chromatin.Here, we describe a subset of techniques that can be applied to probe nuclear mechanics, ranging from micropipette aspiration to strain experiments on living cells. These experiments probe the physical properties of the nuclear envelope, the nucleoplasm, and the chromatin. We discuss the advantages and disadvantages of each technique and elaborate on their use to examine specific aspects of chromatin. In the end, a combination of these technologies can provide important insights into the delicate relationship between form and function of chromatin organization in the living cell.

Nuclear Mechanics and Stem Cell Differentiation

“…Defects in A-type lamins in fibroblasts result in the loss of emerin and nesprin-3 from the nuclear envelope, leading to an abnormal connection between the nucleus and cytoskeleton [13]. Delayed wound healing has also been found, possibly due to the deformed orientation of the nucleus and microtubule-organizing center and the loss of nuclear oscillatory rotation [24,28,29]. LMNA−/− mouse embryonic fibroblasts exhibit increased nuclear fragility and sensitivity to mechanical strain [28].…”

“…Lamin A/C Lamin A/C is not found in ESCs and HSCs; lamin A/C is selectively expressed in various differentiated cells [18,19]; lamin A/C is the most significant factor in controlling nuclear stiffness, facilitating heterochromatin stability and regulating gene expression [11,12,16,20,21] Lamin B1/B2 Lamin B1/B2 is ubiquitously expressed in mammalian cells; lamin B1/B2 has little effect on nuclear stiffness; defects or over-expression of lamin B1/B2 cause nuclear blebs or nuclear lobulation [20,22] Chromatin configuration Nuclei with loose chromatin configurations exhibit fluid-like mechanical properties; condensed chromatin is associated with decreased nuclear plasticity and increased nuclear stiffness; heterochromatin modified by lamin A/C and histones at the periphery of the nucleus is stiffer than euchromatin in the nuclear interior [19,23,24] Lamin B receptor (LBR) LBR is a membrane protein that binds lamin B; over-expression of LBR causes nuclear lobulation and excess nuclear envelope formation; defects in LBR are associated with bone and cartilage disorders and developmental delays [20,22,23] Linkers of the nucleoskeleton to the cytoskeleton (LINC) complexes LINC complexes are comprised of SUN and nesprin proteins; LINC complexes connect the lamina with the cytoskeleton, making a bridge between the nucleus and the cytoplasm; LINC complexes arebeneficial to the stability of nuclear mechanics and mechanotransduction [15,19,25] summary, lamins, particularly lamin A/C, are indispensable and predominant contributors to nuclear mechanics. Lamins are critical proteins that support the nucleus and the cell; cells lacking lamins cannot mechanotransduce effectively or respond to mechanical signals.…”

“…Generally, euchromatin is located in the nuclear interior and is abundant in active genes. Conversely, heterochromatin is a densely packed chromatin that binds with lamins and histones at the periphery of nucleus and contains a large number of genes that have been silenced due to their altered chromatin configuration [13,14,24]. ESCs are the best candidates for investigating the role of chromatin on nuclear mechanics due to their lack of lamin A/ C in the nucleus.…”

“…This finding may at least partially explain the lower Young's modulus of stem cells compared with differentiated cells. To further test this hypothesis, Dahl et al knocked down lamin A/C expression in human epithelial cells and found that the deformability of these cells was similar to HSCs [24]. Therefore, a lower expression of lamin A/C can explain the observation that the nuclei of stem cells are softer and more plastic.…”

The nuclear envelope as a mechanostat: a central cog in the machinery of cell and tissue regulation?

“…13 The model of mechanically regulated nuclear stiffness and viscoelasticity proposed by Swift and colleagues is suggested to regulate downstream cell behaviors via modulation of nuclear deformations. Nuclear stretch has been implicated in locally regulating the spatial organization of chromatin, 14 a fact that may have potentially important consequences for transcriptional regulation. 15 In this sense, a stretch-regulated stiffening of the nuclear envelope represents a potentially central mechanical 'switch' with a degree of physical and temporal stability.…”