Jan Lammerding

Mechanotransduction gone awry

Cells sense their physical surroundings through mechanotransduction — that is, by translating mechanical forces and deformations into biochemical signals such as changes in intracellular calcium concentration or by activating diverse signalling pathways. In turn, these signals can adjust cellular and extracellular structure. This mechanosensitive feedback modulates cellular functions as diverse as migration, proliferation, differentiation and apoptosis, and is crucial for organ development and homeostasis. Consequently, defects in mechanotransduction — often caused by mutations or misregulation of proteins that disturb cellular or extracellular mechanics — are implicated in the development of various diseases, ranging from muscular dystrophies and cardiomyopathies to cancer progression and metastasis.

Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction

Mutations in the lamin A/C gene (LMNA) cause a variety of human diseases including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. The tissue-specific effects of lamin mutations are unclear, in part because the function of lamin A/C is incompletely defined, but the many muscle-specific phenotypes suggest that defective lamin A/C could increase cellular mechanical sensitivity. To investigate the role of lamin A/C in mechanotransduction, we subjected lamin A/C-deficient mouse embryo fibroblasts to mechanical strain and measured nuclear mechanical properties and strain-induced signaling. We found that Lmna-/- cells have increased nuclear deformation, defective mechanotransduction, and impaired viability under mechanical strain. NF-kappaB-regulated transcription in response to mechanical or cytokine stimulation was attenuated in Lmna-/- cells despite increased transcription factor binding. Lamin A/C deficiency is thus associated with both defective nuclear mechanics and impaired mechanically activated gene transcription. These findings suggest that the tissue-specific effects of lamin A/C mutations observed in the laminopathies may arise from varying degrees of impaired nuclear mechanics and transcriptional activation.

Nuclear Shape, Mechanics, and Mechanotransduction

In eukaryotic cells, the nucleus contains the genome and is the site of transcriptional regulation. The nucleus is the largest and stiffest organelle and is exposed to mechanical forces transmitted through the cytoskeleton from outside the cell and from force generation within the cell. Here, we discuss the effect of intra- and extracellular forces on nuclear shape and structure and how these force-induced changes could be implicated in nuclear mechanotransduction, ie, force-induced changes in cell signaling and gene transcription. We review mechanical studies of the nucleus and nuclear structural proteins, such as lamins. Dramatic changes in nuclear shape, organization, and stiffness are seen in cells where lamin proteins are mutated or absent, as in genetically engineered mice, RNA interference studies, or human disease. We examine the different mechanical pathways from the force-responsive cytoskeleton to the nucleus. We also highlight studies that link changes in nuclear shape with cell function during developmental, physiological, and pathological modifications. Together, these studies suggest that the nucleus itself may play an important role in the response of the cell to force.

Lamins A and C but Not Lamin B1 Regulate Nuclear Mechanics

Mutations in the nuclear envelope proteins lamins A and C cause a broad variety of human diseases, including Emery-Dreifuss muscular dystrophy, dilated cardiomyopathy, and Hutchinson-Gilford progeria syndrome. Cells lacking lamins A and C have reduced nuclear stiffness and increased nuclear fragility, leading to increased cell death under mechanical strain and suggesting a potential mechanism for disease. Here, we investigated the contribution of major lamin subtypes (lamins A, C, and B1) to nuclear mechanics by analyzing nuclear shape, nuclear dynamics over time, nuclear deformations under strain, and cell viability under prolonged mechanical stimulation in cells lacking both lamins A and C, cells lacking only lamin A (i.e. “lamin C-only” cells), cells lacking wild-type lamin B1, and wild-type cells. Lamin A/C-deficient cells exhibited increased numbers of misshapen nuclei and had severely reduced nuclear stiffness and decreased cell viability under strain. Lamin C-only cells had slightly abnormal nuclear shape and mildly reduced nuclear stiffness but no decrease in cell viability under strain. Interestingly, lamin B1-deficient cells exhibited normal nuclear mechanics despite having a significantly increased frequency of nuclear blebs. Our study indicates that lamins A and C are important contributors to the mechanical stiffness of nuclei, whereas lamin B1 contributes to nuclear integrity but not stiffness.

Nuclear envelope rupture and repair during cancer cell migration

Repairing tears in the nuclear envelope The nuclear envelope segregates genomic DNA from the cytoplasm and regulates protein trafficking between the cytosol and the nucleus. Maintaining nuclear envelope integrity during interphase is considered crucial. However, Raab et al. and Denais et al. show that migrating immune and cancer cells experience frequent and transitory nuclear envelope ruptures when they move through tight spaces (see the Perspective by Burke). The nuclear envelope reseals rapidly during interphase, assisted by components of the ESCRT III membrane-remodeling machinery. Science, this issue pp. 359 and 353; see also p. 295 When cells migrate, they stress their nuclear envelopes and need to guard against leakage of the nuclear contents. [Also see Perspective by Burke] During cancer metastasis, tumor cells penetrate tissues through tight interstitial spaces, which requires extensive deformation of the cell and its nucleus. Here, we investigated mammalian tumor cell migration in confining microenvironments in vitro and in vivo. Nuclear deformation caused localized loss of nuclear envelope (NE) integrity, which led to the uncontrolled exchange of nucleo-cytoplasmic content, herniation of chromatin across the NE, and DNA damage. The incidence of NE rupture increased with cell confinement and with depletion of nuclear lamins, NE proteins that structurally support the nucleus. Cells restored NE integrity using components of the endosomal sorting complexes required for transport III (ESCRT III) machinery. Our findings indicate that cell migration incurs substantial physical stress on the NE and its content and requires efficient NE and DNA damage repair for cell survival.

Abnormal nuclear shape and impaired mechanotransduction in emerin-deficient cells

Emery-Dreifuss muscular dystrophy can be caused by mutations in the nuclear envelope proteins lamin A/C and emerin. We recently demonstrated that A-type lamin-deficient cells have impaired nuclear mechanics and altered mechanotransduction, suggesting two potential disease mechanisms (Lammerding, J., P.C. Schulze, T. Takahashi, S. Kozlov, T. Sullivan, R.D. Kamm, C.L. Stewart, and R.T. Lee. 2004. J. Clin. Invest. 113:370–378). Here, we examined the function of emerin on nuclear mechanics and strain-induced signaling. Emerin-deficient mouse embryo fibroblasts have abnormal nuclear shape, but in contrast to A-type lamin-deficient cells, exhibit nuclear deformations comparable to wild-type cells in cellular strain experiments, and the integrity of emerin-deficient nuclear envelopes appeared normal in a nuclear microinjection assay. Interestingly, expression of mechanosensitive genes in response to mechanical strain was impaired in emerin-deficient cells, and prolonged mechanical stimulation increased apoptosis in emerin-deficient cells. Thus, emerin-deficient mouse embryo fibroblasts have apparently normal nuclear mechanics but impaired expression of mechanosensitive genes in response to strain, suggesting that emerin mutations may act through altered transcriptional regulation and not by increasing nuclear fragility.

Nuclear mechanics during cell migration

During cell migration, the movement of the nucleus must be coordinated with the cytoskeletal dynamics at the leading edge and trailing end, and, as a result, undergoes complex changes in position and shape, which in turn affects cell polarity, shape, and migration efficiency. We here describe the steps of nuclear positioning and deformation during cell polarization and migration, focusing on migration through three-dimensional matrices. We discuss molecular components that govern nuclear shape and stiffness, and review how nuclear dynamics are connected to and controlled by the actin, tubulin and intermediate cytoskeleton-based migration machinery and how this regulation is altered in pathological conditions. Understanding the regulation of nuclear biomechanics has important implications for cell migration during tissue regeneration, immune defence and cancer.

The Interaction between Nesprins and Sun Proteins at the Nuclear Envelope Is Critical for Force Transmission between the Nucleus and Cytoskeleton

Maintaining physical connections between the nucleus and the cytoskeleton is important for many cellular processes that require coordinated movement and positioning of the nucleus. Nucleo-cytoskeletal coupling is also necessary to transmit extracellular mechanical stimuli across the cytoskeleton to the nucleus, where they may initiate mechanotransduction events. The LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, formed by the interaction of nesprins and SUN proteins at the nuclear envelope, can bind to nuclear and cytoskeletal elements; however, its functional importance in transmitting intracellular forces has never been directly tested. This question is particularly relevant since recent findings have linked nesprin mutations to muscular dystrophy and dilated cardiomyopathy. Using biophysical assays to assess intracellular force transmission and associated cellular functions, we identified the LINC complex as a critical component for nucleo-cytoskeletal force transmission. Disruption of the LINC complex caused impaired propagation of intracellular forces and disturbed organization of the perinuclear actin and intermediate filament networks. Although mechanically induced activation of mechanosensitive genes was normal (suggesting that nuclear deformation is not required for mechanotransduction signaling) cells exhibited other severe functional defects after LINC complex disruption; nuclear positioning and cell polarization were impaired in migrating cells and in cells plated on micropatterned substrates, and cell migration speed and persistence time were significantly reduced. Taken together, our findings suggest that the LINC complex is critical for nucleo-cytoskeletal force transmission and that LINC complex disruption can result in defects in cellular structure and function that may contribute to the development of muscular dystrophies and cardiomyopathies.

Lamin A/C and emerin regulate MKL1-SRF activity by modulating actin dynamics

Laminopathies, caused by mutations in the LMNA gene encoding the nuclear envelope proteins lamins A and C, represent a diverse group of diseases that include Emery–Dreifuss muscular dystrophy (EDMD), dilated cardiomyopathy (DCM), limb-girdle muscular dystrophy, and Hutchison–Gilford progeria syndrome. Most LMNA mutations affect skeletal and cardiac muscle by mechanisms that remain incompletely understood. Loss of structural function and altered interaction of mutant lamins with (tissue-specific) transcription factors have been proposed to explain the tissue-specific phenotypes. Here we report in mice that lamin-A/C-deficient (Lmna−/−) and LmnaN195K/N195K mutant cells have impaired nuclear translocation and downstream signalling of the mechanosensitive transcription factor megakaryoblastic leukaemia 1 (MKL1), a myocardin family member that is pivotal in cardiac development and function. Altered nucleo-cytoplasmic shuttling of MKL1 was caused by altered actin dynamics in Lmna−/− and LmnaN195K/N195K mutant cells. Ectopic expression of the nuclear envelope protein emerin, which is mislocalized in Lmna mutant cells and also linked to EDMD and DCM, restored MKL1 nuclear translocation and rescued actin dynamics in mutant cells. These findings present a novel mechanism that could provide insight into the disease aetiology for the cardiac phenotype in many laminopathies, whereby lamin A/C and emerin regulate gene expression through modulation of nuclear and cytoskeletal actin polymerization.

Prelamin A and lamin A appear to be dispensable in the nuclear lamina

Lamin A and lamin C, both products of Lmna, are key components of the nuclear lamina. In the mouse, a deficiency in both lamin A and lamin C leads to slow growth, muscle weakness, and death by 6 weeks of age. Fibroblasts deficient in lamins A and C contain misshapen and structurally weakened nuclei, and emerin is mislocalized away from the nuclear envelope. The physiologic rationale for the existence of the 2 different Lmna products lamin A and lamin C is unclear, although several reports have suggested that lamin A may have particularly important functions, for example in the targeting of emerin and lamin C to the nuclear envelope. Here we report the development of lamin C-only mice (Lmna(LCO/LCO)), which produce lamin C but no lamin A or prelamin A (the precursor to lamin A). Lmna(LCO/LCO) mice were entirely healthy, and Lmna(LCO/LCO) cells displayed normal emerin targeting and exhibited only very minimal alterations in nuclear shape and nuclear deformability. Thus, at least in the mouse, prelamin A and lamin A appear to be dispensable. Nevertheless, an accumulation of farnesyl-prelamin A (as occurs with a deficiency in the prelamin A processing enzyme Zmpste24) caused dramatically misshapen nuclei and progeria-like disease phenotypes. The apparent dispensability of prelamin A suggested that lamin A-related progeroid syndromes might be treated with impunity by reducing prelamin A synthesis. Remarkably, the presence of a single Lmna(LCO) allele eliminated the nuclear shape abnormalities and progeria-like disease phenotypes in Zmpste24-/- mice. Moreover, treating Zmpste24-/- cells with a prelamin A-specific antisense oligonucleotide reduced prelamin A levels and significantly reduced the frequency of misshapen nuclei. These studies suggest a new therapeutic strategy for treating progeria and other lamin A diseases.