These differentiation defects are associated with a lack of proper cell cycle exit and Rb remaining hyper-phosphorylated (Favreau et al., 2004), as well as a failure of Lamin A/C to relocate from the nucleoplasm to the nuclear lamina (Markiewicz et al., 2005). in K32del, R249W and L35P HIDEMs. (A) Distribution of Lamin B1 fluorescence intensity values across the nucleus (dotted line) in control and comparisons, *< 0.05, **< 0.01, ***< 0.001. Data are shown as box a whiskers plots based on all values from three repeats combined (C, E, G), or the proportion of nuclei per imaging field (D, F, H), whiskers: min and max values, +: mean of all values plotted, numbers on graph show average of three repeats. Image_3.JPEG (1.0M) GUID:?EDE5EED0-A37D-45C0-A40D-5C1B991FB804 Supplementary Figure 4: are Lamin A and C, which together with Lamin B1 and B2, form the nuclear lamina: a mesh-like structure located underneath the inner nuclear membrane. Laminopathies show striking tissue specificity, with subtypes affecting striated muscle, peripheral nerve, and adipose tissue, Rabbit Polyclonal to ARF6 while others cause multisystem disease with accelerated aging. Nilotinib monohydrochloride monohydrate Although several pathogenic mechanisms have been proposed, the exact pathophysiology of laminopathies remains unclear, compounded by the rarity of these disorders and lack of easily accessible cell types to study. To overcome this limitation, we used induced pluripotent stem cells (iPSCs) from patients with skeletal muscle laminopathies such as gene encodes two major protein isoforms: Lamin A and C; these nuclear intermediate filament proteins are expressed in most somatic cells, but absent from undifferentiated cells such as embryonic, germ and pluripotent cells (Dechat et al., 2010a; Worman, 2012). At the nuclear periphery, Lamin A/C, together with Lamin B1 and B2, forms the nuclear lamina, a protein meshwork that underlies the nuclear membrane. The nuclear lamina provides structural support to the nucleus, and participates in mechanotransduction, heterochromatin tethering and regulation of transcription (Azibani et al., 2014; Gruenbaum and Foisner, 2015). Lamin A/C is also present in the nucleoplasm, where it is thought to be involved in regulation of cell proliferation, differentiation, chromatin organization and DNA replication (Dechat et al., 2010b). Mutations in cause at least 16 rare disorders, collectively known as laminopathies (Scharner et al., 2010; Worman, Nilotinib monohydrochloride monohydrate 2012). Laminopathies demonstrate tissue-specific phenotypes and can be grouped into those affecting striated cardiac and/or skeletal muscle (the most common group), peripheral nerve or adipose tissue, and those causing multisystem disease with accelerated aging. Striated muscle laminopathies are usually caused by missense mutations, typically having a dominant inheritance and include dilated cardiomyopathy (DCM), Emery-Dreifuss muscular dystrophy (EDMD), limb-girdle muscular dystrophy type 1B (LGMD1B) and mutation causing different disorders (Scharner et al., 2010, 2014; Bertrand et al., 2011). Two non-mutually exclusive theories have been proposed to explain the pathological causes of striated muscle laminopathies. In the mechanical stress hypothesis, mutations in Lamin A/C lead to a nucleus that is more vulnerable to damage from mechanical force during muscle contraction. The gene expression and stem cell differentiation hypothesis suggests that mutant Lamin A/C deregulates expression of certain genes, which causes defective cell differentiation and function (Azibani et al., 2014; Gruenbaum and Foisner, 2015). A typical cellular hallmark of mutations is abnormal nuclear morphology, as observed in muscle biopsies of EDMD patients (Park et al., 2009). Such nuclear abnormalities have been modeled in primary fibroblasts and C2C12 myoblasts ectopically expressing pathogenic mutations. Fibroblasts from patients with LGMD1B (Muchir et al., 2003), autosomal dominant EDMD (Muchir et al., 2004), L-CMD (Tan et al., 2015), DCM (Muchir et al., 2004), familial partial lipodystrophy (FPLD) (Vigouroux et al., 2001; Verstraeten et al., 2009), Mandibuloacral dysplasia (MAD) (Novelli et al., 2002), Hutchinson-Gilford progeria syndrome (HGPS) (Eriksson et al., 2003), and Werner syndrome 2 (WRN2) (Chen et al., 2003) all have nuclear abnormalities, such as abnormal nuclear shape and mislocalization of lamina proteins. These can be characterized by: (1) mislocalization of Lamin A/C protein (e.g., into structures with a honeycomb-like appearance), (2) nucleoplasmic foci and/or increased nucleoplasmic/lamina ratios, (3) areas Nilotinib monohydrochloride monohydrate with no lamin B1 (capping), and (4) Emerin mislocalization. C2C12 mouse myoblasts expressing EDMD/L-CMD-causing mutations exhibit similar defects (Ostlund et al., 2001; Favreau et al., 2003; Markiewicz et al., 2005; Scharner.
