The centrosome-nucleus attachment is a prerequisite for faithful chromosome segregation during mitosis. We addressed the function of the nuclear envelope (NE) protein Sun-1 in centrosome-nucleus connection and the maintenance of genome stability in Dictyostelium discoideum. We provide evidence that Sun-1 requires direct chromatin binding for its inner nuclear membrane targeting. Truncation of the cryptic N-terminal chromatinbinding domain of Sun-1 induces dramatic separation of the inner from the outer nuclear membrane and deformations in nuclear morphology, which are also observed using a Sun-1 RNAi construct. Thus, chromatin binding of Sun-1 defines the integrity of the nuclear architecture. In addition to its role as a NE scaffold, we find that abrogation of the chromatin binding of Sun-1 dissociates the centrosome-nucleus connection, demonstrating that Sun-1 provides an essential link between the chromatin and the centrosome. Moreover, loss of the centrosomenucleus connection causes severe centrosome hyperamplification and defective spindle formation, which enhances aneuploidy and cell death significantly. We highlight an important new aspect for Sun-1 in coupling the centrosome and nuclear division during mitosis to ensure faithful chromosome segregation.Key words: aneuploidy, centrosome hyperamplification, nuclear envelope architecture, spindle formation defects, Unc-84 The nuclear envelope (NE) separates the nuclear compartment from the cytoplasm. It is composed of two membranes: the outer nuclear membrane (ONM) and the inner nuclear membrane (INM). The lumen between the two membranes is the perinuclear space (PNS). The ONM is continuous with the endoplasmic reticulum (ER), whereas the INM harbors a unique set of proteins. INM and ONM proteins can interact within the PNS. Underneath the INM, the nuclear lamina is located, which is formed by intermediate filament (IF) proteins and associated proteins. The lamina forms the nucleoskeleton and associates with the INM, chromatin and nuclear pore complexes. Proteins of the NE have important roles. They are involved in nuclear migration and positioning and are essential for many processes such as mitosis, meiosis, differentiation and cell migration. Furthermore, several of the NE proteins have been associated with inherited diseases (1,2). Research in mammalian cells and in Caenorhabditis eleganshas identified conserved components of the NE that link the nucleoskeleton to the cytoskeleton. In C. elegans, two putative INM proteins, matefin/SUN-1 and UNC-84, bind to the nuclear lamina and extend their C-terminus into the PNS where they interact with the C-termini of KASH domain proteins (Klarsicht/Anc-1/Syne homology, designated KASH domain). Matefin/SUN-1 and UNC-84 belong to the SUN family of proteins based on the presence of the conserved SUN (Sad1/UNC-84 homology) domain at their C-terminus. KASH domain proteins are type II transmembrane proteins of the NE and have been identified as molecular linkers connecting the nucleus to actin filaments [filamentous acti...
We analyzed the influence of lamins on nuclear envelope growth in cultured Xenopus A6 cells by the overexpression of human lamin A, Xenopus and zebrafish lamins B2 and Drosophila lamins Dm0 and C as GFP fusion proteins. Lamins containing a CxxM motif in their primary sequence (lamins A, B2, Dm0) induced the formation of lobulated nuclei with multi-membrane-layered, highly folded nuclear membranes and intranuclear membrane assemblies, as observed by electron microscopy. Such morphological alterations were not observed with Drosophila lamin C, a lamin without this motif or with a lamin B2 mutant (B2-SxxM) where the cysteine of the CxxM motif is replaced by a serine. Drosophila lamin C mutants containing a CxxM motif behaved like B-type lamins thus confirming that this tetrapeptide is directly involved in the morphological changes we observed. Nuclear membrane proliferation could also be induced by lamin B2 in COS-7 cells and in zebrafish embryos but not by human lamin A in COS-7 cells. We speculate that the human lamin A is incompletely processed in Xenopus A6 cells and therefore behaves in this cell line like a B-type lamin. Our results indicate that the CxxM motif of B-type lamins has a dual function: it mediates lamin targeting to the inner nuclear membrane thereby promoting nuclear membrane growth.
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