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Abstract Lamins are nuclear intermediate filament proteins with diverse functions, ranging from organizing chromatin and regulating gene expression to providing structural support to the nucleus. Mammalian cells express two types of lamins, A-type and B-type, which, despite their similar structure and biochemical properties, exhibit distinct differences in expression, interaction partners, and function. One major difference is that A-type lamins have a significantly larger effect on the mechanical properties of the nucleus, which are crucial for protecting the nucleus from cytoskeletal forces, enabling cell migration through confined spaces, and contributing to cellular mechanotransduction. The molecular mechanism underlying this difference has remained unresolved. Here, we applied custom-developed biophysical and proteomic assays to lamin-deficient cell lines engineered to express specific full-length lamin proteins, lamin truncations, or chimeras combining domains from A- and B-type lamins, to systematically determine their contributions to nuclear mechanics. We found that although all expressed lamins contribute to the biophysical properties of the nuclear interior and confer some mechanical stability to the nuclear envelope, which is sufficient to protect the nuclear envelope from small cell-intrinsic forces and ensure proper positioning of nuclear pores, A-type lamins endow cells with a unique ability to resist large forces on the nucleus. Surprisingly, this effect was conferred through the A-type lamin rod domain, rather than the head or tail domains, which diverge more substantially between A- and B-type lamins and play important roles in lamin network formation. Collectively, our work provides an improved understanding of the distinct functions of different lamins in mammalian cells and may also explain why mutations in the A-type lamin rod domain cause more severe muscle defects in mouse models than other mutations.
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N-terminal tags impair the ability of lamin A to provide structural support to the nucleus
ABSTRACT Lamins are intermediate filament proteins that contribute to numerous cellular functions, including nuclear morphology and mechanical stability. The N-terminal head domain of lamin is crucial for higher order filament assembly and function, yet the effects of commonly used N-terminal tags on lamin function remain largely unexplored. Here, we systematically studied the effect of two differently sized tags on lamin A (LaA) function in a mammalian cell model engineered to allow for precise control of expression of tagged lamin proteins. Untagged, FLAG-tagged and GFP-tagged LaA completely rescued nuclear shape defects when expressed at similar levels in lamin A/C-deficient (Lmna–/–) MEFs, and all LaA constructs prevented increased nuclear envelope ruptures in these cells. N-terminal tags, however, altered the nuclear localization of LaA and impaired the ability of LaA to restore nuclear deformability and to recruit emerin to the nuclear membrane in Lmna–/– MEFs. Our finding that tags impede some LaA functions but not others might explain the partial loss of function phenotypes when tagged lamins are expressed in model organisms and should caution researchers using tagged lamins to study the nucleus.
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- Award ID(s):
- 2022048
- PAR ID:
- 10646197
- Publisher / Repository:
- The Company of Biologists Ltd
- Date Published:
- Journal Name:
- Journal of Cell Science
- Volume:
- 137
- Issue:
- 16
- ISSN:
- 0021-9533
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Discher, Dennis (Ed.)The LMNA gene encodes the nuclear envelope proteins Lamins A and C, which comprise a major part of the nuclear lamina, provide mechanical support to the nucleus, and participate in diverse-intracellular signaling. LMNA mutations give rise to a collection of diseases called laminopathies, including dilated cardiomyopathy ( LMNA-DCM) and muscular dystrophies. Although nuclear deformities are a hallmark of LMNA-DCM, the role of nuclear abnormalities in the pathogenesis of -DCM remains incompletely understood. Using induced-pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LMNA-mutant patients and healthy controls, we show that LMNA mutant iPSC-CM nuclei have altered shape or increased size compared with healthy control iPSC-CM nuclei. The LMNA mutation exhibiting the most severe nuclear deformities, R249Q, additionally caused reduced nuclear stiffness and increased nuclear fragility. Importantly, for all cell lines, the degree of nuclear abnormalities corresponded to the degree of Lamin A/C and Lamin B1 mislocalization from the nuclear envelope. The mislocalization was likely due to altered assembly of Lamin A/C. Collectively, these results point to the importance of correct lamin assembly at the nuclear envelope in providing mechanical stability to the nucleus and suggests that defects in nuclear lamina organization may contribute to the nuclear and cellular dysfunction in LMNA-DCM.more » « less
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Discher, Dennis (Ed.)Lamins are nuclear intermediate filament proteins that are ubiquitously found in metazoan cells, where they contribute to nuclear morphology, stability, and gene expression. Lamin-like sequences have recently been identified in distantly related eukaryotes, but it remains unclear whether these proteins share conserved functions with the lamins found in metazoans. Here, we investigate conserved features between metazoan and amoebozoan lamins using a genetic complementation system to express the Dictyostelium discoideum lamin-like protein NE81 in mammalian cells lacking either specific lamins or all endogenous lamins. We report that NE81 localizes to the nucleus in cells lacking Lamin A/C, and that NE81 expression improves nuclear circularity, reduces nuclear deformability, and prevents nuclear envelope rupture in these cells. However, NE81 did not completely rescue loss of Lamin A/C, and was unable to restore normal distribution of metazoan lamin interactors, such as emerin and nuclear pore complexes, which are frequently displaced in Lamin A/C deficient cells. Collectively, our results indicate that the ability of lamins to modulate the morphology and mechanical properties of nuclei may have been a feature present in the common ancestor of Dictyostelium and animals, whereas other, more specialized interactions may have evolved more recently in metazoan lineages.more » « less
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Abstract Lamins A and C, encoded by theLMNAgene, are nuclear intermediate filaments that provide structural support to the nucleus and contribute to chromatin organization and transcriptional regulation.LMNAmutations cause muscular dystrophies, dilated cardiomyopathy, and other diseases. The mechanisms by which manyLMNAmutations result in muscle-specific diseases have remained elusive, presenting a major hurdle in the development of effective treatments. Previous studies using striated muscle laminopathy mouse models found that cytoskeletal forces acting on mechanically fragileLmna-mutant nuclei led to transient nuclear envelope rupture, extensive DNA damage, and activation of DNA damage response (DDR) pathways in skeletal muscle cells in vitro and in vivo. Furthermore, hearts ofLmnamutant mice have elevated activation of the tumor suppressor protein p53, a central regulator of DDR signaling. We hypothesized that elevated p53 activation could present a pathogenic mechanism in striated muscle laminopathies, and that eliminating p53 activation could improve muscle function and survival in laminopathy mouse models. Supporting a pathogenic function of p53 activation in muscle, stabilization of p53 was sufficient to reduce contractility and viability in wild-type muscle cells in vitro. Using three laminopathy models, we found that increased p53 activity inLmna-mutant muscle cells primarily resulted from mechanically induced damage to the myonuclei, and not from altered transcriptional regulation due to loss of lamin A/C expression. However, global deletion of p53 in a severe muscle laminopathy model did not reduce the disease phenotype or increase survival, indicating that additional drivers of disease must contribute to the disease pathogenesis.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1242/jcs.262207
