Cellular mechanosensing: getting to the nucleus of it all

Abstract

Cells respond to mechanical forces by activating specific genes and signaling pathways that allow the cells to adapt to their physical environment. Examples include muscle growth in response to exercise, bone remodeling based on their mechanical load, or endothelial cells aligning under fluid shear stress. While the involved downstream signaling pathways and mechanoresponsive genes are generally well characterized, many of the molecular mechanisms of the initiating 'mechanosensing' remain still elusive. In this review, we discuss recent findings and accumulating evidence suggesting that the cell nucleus plays a crucial role in cellular mechanotransduction, including processing incoming mechanoresponsive signals and even directly responding to mechanical forces. Consequently, mutations in the involved proteins or changes in nuclear envelope composition can directly impact mechanotransduction signaling and contribute to the development and progression of a variety of human diseases, including muscular dystrophy, cancer, and the focus of this review, dilated cardiomyopathy. Improved insights into the molecular mechanisms underlying nuclear mechanotransduction, brought in part by the emergence of new technologies to study intracellular mechanics at high spatial and temporal resolution, will not only result in a better understanding of cellular mechanosensing in normal cells but may also lead to the development of novel therapies in the many diseases linked to defects in nuclear envelope proteins.

Keywords: Cell signaling; Lamins; Mechanics; Mechanotransduction; Nesprins; Nuclear envelope.

Figure 1. Nuclear deformation during cardiac myocytes contraction

Time-lapse sequence of mouse neonatal cardiac myocytes spontaneous contracting in culture; the cytoskeleton exerts substantial forces on the cell nucleus (blue), resulting in reversible large nuclear deformations (white arrow). Mitochondria are shown in red.

Figure 2. A ‘direct connection’ from the extracellular matric (ECM) to the genome

Schematic illustration highlighting the (protein) elements that maintain the structural integrity of the cell, as well as some of the important signaling molecules and transcriptional regulators (ERK 1/2, β-catenin, nuclear, pRb) and intranuclear domains (nucleolus, Cajal bodies). Integrins at the plasma membrane connect the ECM to cytoskeletal filaments, which bind the nucleus through LINC complex proteins (nesprins and SUN proteins) and the nuclear lamina. The outer nuclear membrane (ONM), along with the 30-50 nm wide luminal space enclosed between the inner nuclear membrane (INM) and ONM, is continuous with the endoplasmic reticulum. Bridging the nuclear membranes are nuclear pores that allow for transport of large molecules, such as transcription factors or RNA, between the nucleus and the cytoplasm. Composed of both A- and B-type lamins, the lamina helps to tether heterochromatin, transcription factors, and nuclear membrane proteins to the nuclear periphery (Ho and Lammerding, 2012; Lin et al., 2000; Wilson and Foisner, 2010; Zuleger et al., 2012). This physical connectivity between nesprins, SUN-proteins, and the nuclear interior allows a direct route for mechanical signals to reach the nucleus and also impacts how biochemical signals traverse the cytoplasm and the nuclear envelope or interact with nuclear proteins once inside the nucleus. It is important to note that emerin and some nesprin isoforms can be found on both the INM and ONM (Salpingidou et al., 2007; Zhang et al., 2002); however, it remains unclear whether they fulfill distinct functions depending on their localization. Within the nesprin families, there are wide variations in size due to alternative splicing and transcriptional initiation, with the largest isoforms, referred to as nesprin-1/2 giant, respectively, reaching ~800 – 1,000 kD in size, which may enable them to reach as far as 1 μm into the cytoplasm (Rajgor et al., 2012).

Figure 3. Potential mechanisms of nuclear mechanosensing

The nucleus receives a vast amount of mechanical and biochemical signals from the surrounding cytoplasm. Incoming signals may trigger various responses at the nuclear envelope or within the interior that can result in changes in gene expression. (1) Nuclear deformations may alter the interactions (via change in proximity or other means) between chromatin and the nuclear lamina, causing transcriptional activation or repression. Force-induced changes in chromatin organization may also alter accessibility of chromatin to transcriptional regulators. (2) Mechanically-induced damage to the nuclear membranes, or changes in the permeability of nuclear pores or stretch-sensitive ion channels can results in altered nuclear import/export. (3) Ca²⁺ or other ions may enter the nucleus or be release from perinuclear stores in response to osmotic stress or other mechanical stimuli. (4) Forces transmitted across the LINC complex may lead to force-induced conformational changes in nuclear envelope proteins, resulting in their phosphorylation or altered interaction with binding partners. (5) Nuclear actin serves as both a mechanical scaffold and a signaling moiety; nuclear actin polymerization can modulate activity and import/export of MKL1/SRF. (6) Changes in membrane fluidity or curvature may alter the conformational state of bound proteins and cause assembly/disassembly of multi-protein structures at the nuclear envelope. (7) Nuclear envelope proteins can sequester transcriptional regulators at the nuclear periphery or form active/inactive complexes, thereby regulating their activity. (8) Nuclear actin (along with other structural elements such as spectrin IIα may also serve as a scaffold for transcriptional regulation by positioning DNA near transcriptional machinery or specific DNA-regulatory elements. In addition to the depicted mechanisms, additional mechanisms, such as osmotic changes in the nucleus caused by volume changes, may contribute to nuclear mechanosensing.

Figure 4. Avenues for new technologies to investigate nuclear mechanosensing

Schematic cartoon of the typical experimental workflow. Most experimental approaches begin with cells or tissues derived from animal models or human patients. Animal models can provide cells and tissues with a specific genetic background or mutation of interest. Mice with several deletions (DKO – double knock-out) can help assess synergies and redundancies between different genes. The development of advanced in vitro models, including cells cultured in 3-D environments and decellularized tissues, can provide improved accessibility of cells for experimental purposes while mimicking physiological conditions. Tissue samples, cells, or isolated nuclei can then be manipulated with an array of experimental techniques to apply either controlled mechanical forces or deformations. The biomechanical properties can be inferred from the measured force-displacement relationships. Advanced imaging modalities and emerging novel technologies that enable measuring the intracellular forces at the molecular level provide improved insights into force-induced changes of nuclear structures at higher and higher temporal and spatial resolution. Examples include genetically encoded FRET-based tension sensors or other methods such as microfabricated pressure sensors. Alternatively (or in addition), researchers may wish to employ a method of detecting molecular events or structural changes (e.g. chromatin/protein unfolding, gene translocation, etc.). Ultimately, observed mechanically induced changes in nuclear structure must be linked to readouts of cellular function to ensure their biological relevance. Such readouts can include changes in transcriptional activity (gene expression), epigenetic modifications, or kinase activation. The quantitative experimental data can provide input for computational models, ranging from molecular dynamics to systems biology analysis. These models are then validated by experimentally testing generated predictions, or refined to reflect new experimental insights.