Influence of Lamin A E145K progeria mutation on nuclear mechanics

Abstract

Lamins are intermediate filament proteins. They are structural components of the nuclear lamina, a filamentous meshwork beneath the inner nuclear membrane, which confers mechanical stability to the nucleus. Mutations in the human LMNA gene cause a wide range of diseases called laminopathies. Amongst these is the Hutchinson-Gilford progeria syndrome (HGPS), a rare premature aging disorder. One of the mutations causing HGPS is a heterozygous point mutation E145K within the central domain of the lamin A protein. The E145K mutation affects lamin filament assembly and induces profound changes in the composition and architecture of the patient cell nuclei. In vitro analyses of purified E145K lamin A reveal severe assembly defects into higher order lamin structures, indicating an abnormal lateral association of lamin protofilaments. Ex vivo expression of the wild-type and E145K lamin A in Xenopus oocytes showed influence of the mutant lamin A on the mechanical properties of the lamina as revealed by atomic force microscopy (AFM). Nuclear laminae made up of overexpressed E145K lamin A were stiffer than those harboring wild-type lamin A. In this work, mechanical properties of somatic cell nuclei were studied using AFM. In pilot experiments with HeLa cells AFM data acquisition and analysis was optimized. Next, the mechanical properties of the dermal fibroblasts of a four years old progeria patient bearing the E145K lamin A mutation were analyzed using AFM. The abnormal shape of nuclei expressing E145K lamin A and alterations in the cellular actin network were revealed by fluorescence microscopy. Lamina thickness was assessed by transmission electron microscopy. AFM probing of entire dermal fibroblasts revealed minor differences in the elastic moduli of nuclear and cytoplasmic cell regions. Thus, isolation of the nuclei was done to directly measure their mechanical properties by AFM. Isolated nuclei of the progeria patient (age 4) and the old person (age 61) were significantly stiffer than those of a young person (age 10). These results indicated that lamin A E145K alters the mechanical properties of the nuclei of the dermal fibroblasts obtained from a progeria patient. Thus, it was shown that the process of aging, be it natural or abnormal, increases the stiffness of nuclei.