The nuclear lamina is mechano-responsive to ECM elasticity in mature tissue

Abstract

How cells respond to physical cues in order to meet and withstand the physical demands of their immediate surroundings has been of great interest for many years, with current research efforts focused on mechanisms that transduce signals into gene expression. Pathways that mechano-regulate the entry of transcription factors into the cell nucleus are emerging, and our most recent studies show that the mechanical properties of the nucleus itself are actively controlled in response to the elasticity of the extracellular matrix (ECM) in both mature and developing tissue. In this Commentary, we review the mechano-responsive properties of nuclei as determined by the intermediate filament lamin proteins that line the inside of the nuclear envelope and that also impact upon transcription factor entry and broader epigenetic mechanisms. We summarize the signaling pathways that regulate lamin levels and cell-fate decisions in response to a combination of ECM mechanics and molecular cues. We will also discuss recent work that highlights the importance of nuclear mechanics in niche anchorage and cell motility during development, hematopoietic differentiation and cancer metastasis, as well as emphasizing a role for nuclear mechanics in protecting chromatin from stress-induced damage.

Keywords: Cell mechanics; Extra-cellular matrix; Mechanotransduction; Nucleoskeleton; Nucleus; Proteostasis.

Fig. 1.

Relationship between the ECM and lamin in mature tissue and during development. (A) A-type and B-type lamins (red and blue, respectively) form juxtaposed networks on the inside of the nuclear envelope; they are effectively located at an interface between chromatin and the cytoskeleton, the latter of which is attached to the lamina through the LINC complex (shown in the magnified view). The A-type lamins, lamin-A and lamin-C, are alternative spliceoform products of the LMNA gene; the B-type lamins, lamin-B1 and lamin-B2, are protein products of LMNB1 and LMNB2, respectively (adapted from Buxboim et al., 2010a, originally published in The Journal of Cell Science). (B) Left: the quantity of type I collagen present in tissues is proportional to tissue micro-elasticity (Swift et al., 2013b). As collagen is one of the most prevalent proteins in the body, it is, perhaps, expected that it defines mechanical properties. Right: the ratio of A-type lamin to B-type lamin in the nuclear lamina is proportional to tissue microelasticity. The lamina contains predominantly A-type lamins in stiff tissue, whereas B-type lamins are prevalent in the nuclear lamina in soft tissue (Swift et al., 2013b). (C) Left: observations made in adult tissue are consistent with results from the developing chick; the embryonic disc was initially soft, but divergent tissues either remained soft (e.g. brain, blue) or became increasingly stiff (e.g. heart, red). Inset: developing chick hearts were probed by micropipette aspiration to determine micro-elasticity. Middle: tissue stiffening during development is accompanied by increased levels of collagen and A-type lamins (Lehner et al., 1987; Majkut et al., 2013). Right: embryonic stem cells initially express negligible quantities of A-type lamin proteins, but these levels increase as the nucleus stiffens during lineage commitment (Pajerowski et al., 2007).

Fig. 2.

The mechanical role of lamin in the nucleus. (A) Deformations applied by micropipette aspiration were used to quantify nuclear compliance (effectively a measure of ‘softness’, the inverse of stiffness) in a range of nuclei with experimentally altered lamina compositions (achieved, for example, by overexpressing a GFP–lamin-A fusion construct). Compliance can be calculated over a range of deformation timescales as a function of the micropipette diameter, the extent of deformation (L) and the applied pressure (ΔP). (B) When a constant deforming pressure was delivered by micropipette over timescales on the order of seconds, nuclei with low expression of lamin-A (LMNA) were found to be more compliant than those with high expression of lamin-A. (C) The mechanical properties of the lamina can be considered as a combination of elastic (spring-like) and viscous (flowing) properties, which together define the ‘deformation response time’, the timescale over which the nuclear shape deforms under force. Nuclei with greater quantities of A-type lamins relative to B-type lamins were found to deform more slowly under stress (Swift et al., 2013b).

Fig. 3.

Protein regulation as a function of stress. (A) Schematic showing the factors that can regulate the levels of A-type lamin in the cell. LMNA transcription is promoted by retinoic-acid-binding factors (Okumura et al., 2004a; Olins et al., 2001). The transcript (gray) is alternatively spliced to give rise to lamin-A and lamin-C forms. In some tissues, such as brain, the lamin-A form is suppressed through miRNA (Jung et al., 2012). Mature lamin-A (following post-translational processing) and lamin-C (both shown in red) assemble into the nuclear lamina, although some protein remains mobile in the nucleoplasm (Shimi et al., 2008). Phosphorylation leads to increased solubility, and might precede enzymatic protein turnover. Further stress-dependent pathways have been reported; stress on the nucleus causes unfolding of the Ig-domain of A-type lamin, and phosphorylation is suppressed under tension (Swift et al., 2013b). Laminopathic nuclei have been shown to have transient membrane defects that allow ingress of transcription factors (De Vos et al., 2011). (B) Left: the nuclei of MSCs cultured on soft substrate are wrinkled, whereas those in cells on stiff substrate have a smooth stretched appearance, suggestive of greater tension. We have shown that A-type lamin is less phosphorylated under tension – we hypothesize that this might be because interaction with kinases is altered by stress-induced changes in the organization of lamin protein filaments (Swift et al., 2013b). A lower level of phosphorylation reduces the solubility and mobility of lamin and thus drives a ‘stress-strengthening’ of the lamina (Swift et al., 2013b). Right: collagen fibrils under tension have been shown to be less susceptible to enzymatic digestion; when a film of fibrillar collagen is subjected to localized stretching, subsequent treatment with a matrix metalloproteinase degrades the collagen but the fibrils that are under tension remain intact (Flynn et al., 2010). This experiment is another example of multimeric coiled-coil protein assembly being enzymatically regulated when subjected to stress, suggesting some generality for the mechanism. Arrowheads indicate the direction of tension.

Fig. 4.

Decisions of cell fate downstream of the regulation of A-type lamin. (A) MSCs cultured on soft and stiff substrates take on different phenotypes and are biased towards alternative cell fates (Engler et al., 2006). On soft substrate, MSCs are rounded and exhibit small nuclear and cellular areas, and the nuclear lamina is thinned (thin red nuclear outline) by a stress-sensitive phosphorylation-feedback mechanism (Fig. 3B, left panel; Swift et al., 2013b). The transcription factors RARG and YAP1 (Dupont et al., 2011) remain in the cytoplasm, and adipogenic cell fate is preferred. On stiff substrate, cells spread extensively, and the nuclei are pinned down by well-developed stress fibers (gray). A-type lamin is less phosphorylated under strain, thus strengthening the lamina (thick red nuclear outline). RARG also translocates to the nucleus, increasing LMNA transcription. Activity of the transcription factor SRF (downstream of A-type lamin) increases the expression of cytoskeletal components (Ho et al., 2013). Under these conditions, YAP1 translocates to the nucleus and cells are more likely to undergo osteogenesis. On both soft and stiff substrates, the effects of ECM elasticity and the levels of lamin cooperate to enhance differentiation; knockdown of A-type lamin on soft substrate leads to more adipogenesis, and lamin-A overexpression on stiff substrate leads to more osteogenesis. (B) Transcriptional activity is believed to be regulated by conserved interchromatin contacts that give rise to the spatial ordering of chromosomes (chromosome territories shown here in different colors, Cremer and Cremer, 2001). A-type lamin can interact with DNA directly (lamina–chromatin contacts) or through protein intermediaries (Simon and Wilson, 2011), but could have additional regulatory roles by mechanically determining the extent and rate that the nucleus deforms under tension, a process that could lead to the formation of altered interchromatin and lamina–chromatin contacts. Arrowheads indicate the direction of tension.

Fig. 5.

The influence of the mechanical properties of the nucleus on cell migration. (A) Left: as the largest and stiffest organelle in the cell, the nucleus can act as an ‘anchor’, preventing cell movement through the ECM or into the surrounding vasculature. Middle: to model migration through the ECM, cells are induced to pass through 3-µm pores, a diameter sufficiently small to require deformation of the nucleus (inset). Lamin-A overexpression (OE) inhibits migration, whereas knockdown (KD) increases migration to five times that of wild-type (WT) cells; however, highly effective knockdown leads to substantial apoptosis. Thus, extremely low or high levels of A-type lamin are unfavorable for cell migration, an observation that potentially impacts upon the understanding of processes such as cell migration during development and cancer metastasis. Right: lamin-A-rich nuclei (upper panel) show a persistently elongated morphology upon emerging from the pores (yellow arrowheads), whereas lamin-B-rich nuclei (lower panel) rapidly recover their shape (Fig. 5A adapted from Harada et al., 2014. Originally published in The Journal of Cell Biology, doi: 10.1083/jcb.201308029). (B) The effect of lamina composition on nuclear deformability during hematopoiesis. Stem cells that are retained in the marrow niche have higher lamin levels than differentiated blood lineages (Shin et al., 2013). A downregulation of nuclear cytoskeletal components in granulocytes, for example, ostensibly makes the cells better suited for passage through narrow blood vessels, but the lack of nuclear stability might contribute to their relatively short circulation times (Olins et al., 2009).