Dataset Information

Nuclear mechanical resilience but not stiffness is modulated by ?II-spectrin.

ABSTRACT: Spectrins are multi-domain, elastic proteins that provide elasticity to the plasma membrane of erythrocytes and select nucleated cells. Spectrins have also been found in the nucleus of non-erythrocytes, but their function remains to be uncovered. It has been hypothesized that a spring-like spectrin network exists within the lamina nucleoskeleton, however, experiments testing a spectrin network?s mechanical impact on the nucleus are lacking. Here, we knock-down levels of nuclear ?II-spectrin with the goal of disrupting this nucleoskeletal spectrin network. We mechanically test live cells with intranuclear particle tracking and compression assays to probe changes in nuclear mechanics with decreases in ?II-spectrin. We show no changes in chromatin mechanics or in the stiffness of nuclei under compression. However, we do observe a reduction in the ability of nuclei with decreased ?II-spectrin to recover after compression. These results establish spectrin as a nucleoskeletal component that specifically contributes to elastic recovery after compression.

SUBMITTER: Armiger TJ

PROVIDER: S-EPMC5164977 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Nuclear mechanical resilience but not stiffness is modulated by αII-spectrin.

Armiger Travis J TJ Spagnol Stephen T ST Dahl Kris Noel KN

Journal of biomechanics 20161027 16

Spectrins are multi-domain, elastic proteins that provide elasticity to the plasma membrane of erythrocytes and select nucleated cells. Spectrins have also been found in the nucleus of non-erythrocytes, but their function remains to be uncovered. It has been hypothesized that a spring-like spectrin network exists within the lamina nucleoskeleton, however, experiments testing a spectrin network׳s mechanical impact on the nucleus are lacking. Here, we knock-down levels of nuclear αII-spectrin with ...[more]

PMID: 27836504

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Project description:Background: Palmdelphin (PALMD) belongs to the family of Paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms (SNPs) in the PALMD locus that result in reduced expression are strong risk factors for development of calcific aortic valve stenosis (CAVS) and predict severity of the disease. Methods: Immunodetection and public database screening showed dominant expression of PALMD in endothelial cells (ECs) in brain and cardiovascular tissues including aortic valves. Mass spectrometry, co-immunoprecipitation and immunofluorescent staining allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in siRNA-treated EC cultures, in knockout mice, and human valve samples. RNA sequencing of ECs and transcript arrays on valve samples from an aortic valve study cohort including patients with the SNP rs7543130, informed about gene regulatory changes. Results: ECs express the cytosolic PALMD-KKVI splice variant, which associated with RAN GTPase activating protein1 (RANGAP1). RANGAP1 regulates the activity of the GTPase RAN and thereby, nucleocytoplasmic shuttling via Exportin1 (XPO1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, and nuclear arrest of the XPO1 cargoes p53 and p21. This indicates an important role for PALMD in nucleocytoplasmic transport and consequently, in gene regulation due to the impact on localization of transcriptional regulators. Changes in EC responsiveness upon loss of PALMD expression included failure to form a perinuclear actin cap when exposed to flow, indicating lack of protection against mechanical stress. Loss of the actin cap correlated with misalignment of the nuclear long axis relative to the cell body, observed in PALMD-deficient ECs, Palmd-/- mouse aorta and human aortic valve samples derived from CAVS patients. In agreement with these changes in EC behavior, gene ontology analysis showed enrichment of nuclear- and cytoskeleton-related terms in PALMD-silenced ECs. Conclusions: We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex alters the subcellular localization of RANGAP1 and XPO1, and leads to nuclear arrest of the XPO1 cargoes p53 and p21, accompanied by gene regulatory changes and loss of actin-dependent nuclear resilience. Combined, these consequences of reduced PALMD expression provide a mechanistic underpinning for PALMD's contribution to CAVS pathology.

Dataset Information

Nuclear mechanical resilience but not stiffness is modulated by ?II-spectrin.

Publications

Nuclear mechanical resilience but not stiffness is modulated by αII-spectrin.

Similar Datasets

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