The role of emerin and LEM domain proteins in nuclear envelope assembly and cytoskeleton organisation

The nuclear envelope (NE) plays a fundamental role in the cell by separating nuclear from cytoplasmic activities, and mutations in NE proteins have been associated with a diverse array of diseases. In the present study the Xenopus cell-free system was used to investigate the function of the inner nuclear membrane protein, emerin, which is associated with the Emery-Dreifuss muscular dystrophy (X-EDMD).Initially, the order and dynamics of NE assembly in Xenopus egg extracts have been investigated. Using a panel of antibodies it was shown that NE assembly proceeds by the ordered recruitment of two membrane populations, Nuclear Envelope Precursor vesicles -A and -B (NEP-A and NEP-B), to chromatin. As shown by immunofluorescence NEP-B vesicles, together with nucleoporins (Nups), appear first around chromatin at about ten minutes after initiation of NE assembly while NEP-A vesicles appear at a later stage, at about twenty minutes. To investigate the role of different emerin domains in this process, four human emerin peptides consisting of amino acids (aa) 1-70, 1-176, 1-220 and 73-180 were added individually to Xenopus nuclear assembly reactions at different concentrations and the effect on nuclear vesicle recruitment and NPC formation was monitored. Immunofluorescence analysis showed that peptides containing the LEM domain of emerin interfere with a correct NE assembly by inhibiting chromatin decondensation and recruitment of membranes to chromatin. This inhibitory effect was shown to be exerted mainly on NEP-A membranes and on Nup62 and Nupl53. By the use of two antibodies, raised against the LEM domain of human emerin and LAP2ß, two proteins of 30 and 36 kD, respectively, were identified in Xenopus. Both proteins were shown to reside in the NEP-A membrane population providing an explanation for the preferential inhibition of NEP-A recruitment to chromatin by exogenously added LEM domain containing emerin peptides. To further investigate whether the domain specific inhibitory effects of emerin on nuclear assembly correlate with specific interacting proteins, co-precipitation experiments were performed to identify emerin binding proteins in the Xenopus cytosol. From these experiments ß -tubulin was identified as a protein able to interact with emerin peptides 1-70 and 73-180. Staining of X-EDMD cells, which lack emerin, with a ß -tubulin antibody revealed no alterations in the organisation of the microtubule (MT) network. The most prominent effect of emerin mutations regarding MTs was the position of the Microtubule Organising Centre (MTOC) relative to the NE. Staining for the centrosomal protein pericentrin revealed a mis-localisation of the MTOC away from the NE in X-EDMD cell lines at distances at least double compared to control cells

A Nup133-dependent NPC-anchored network tethers centrosomes to the nuclear envelope in prophase

Nup133 links CENP-F, NudE/EL, and the dynein/dynactin complex to anchor centrosomes to the nuclear membrane

Embryonic and adult isoforms of XLAP2 form microdomains associated with chromatin and the nuclear envelope

Laminin-associated polypeptide 2 (LAP2) proteins are alternatively spliced products of a single gene; they belong to the LEM domain family and, in mammals, locate to the nuclear envelope (NE) and nuclear lamina. Isoforms lacking the transmembrane domain also locate to the nucleoplasm. We used new specific antibodies against the N-terminal domain of Xenopus LAP2 to perform immunoprecipitation, identification and localization studies during Xenopus development. By immunoprecipitation and mass spectrometry (LC/MS/MS), we identified the embryonic isoform XLAP2γ, which was downregulated during development similarly to XLAP2ω. Embryonic isoforms XLAP2ω and XLAP2γ were located in close association with chromatin up to the blastula stage. Later in development, both embryonic isoforms and the adult isoform XLAP2β were localized in a similar way at the NE. All isoforms colocalized with lamin B2/B3 during development, whereas XLAP2β was colocalized with lamin B2 and apparently with the F/G repeat nucleoporins throughout the cell cycle in adult tissues and culture cells. XLAP2β was localized in clusters on chromatin, both at the NE and inside the nucleus. Embryonic isoforms were also localized in clusters at the NE of oocytes. Our results suggest that XLAP2 isoforms participate in the maintenance and anchoring of chromatin domains to the NE and in the formation of lamin B microdomains

Common themes in centriole and centrosome movements.

addresses: School of Life Sciences, Oxford Brookes University, Gipsy Lane, Oxford, OX3 0BP, UK.Copyright © 2011 Elsevier. NOTICE: this is the author’s version of a work that was accepted for publication in Trends in Cell Biology. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Trends in Cell Biology, 2011, Vol. 21, Issue 1, pp. 57 – 66 DOI: 10.1016/j.tcb.2010.09.004Centrioles are found in nearly all eukaryotic cells and are required for growth and maintenance of the radial array of microtubules, the mitotic spindle, and cilia and flagella. Different types of microtubule structures are often required at different places in a given cell; centrioles must move around to nucleate these varied structures. Here, we draw together recent data on diverse centriole movements to decipher common themes in how centrioles move. Par proteins establish and maintain the required cellular asymmetry. The actin cytoskeleton facilitates movement of multiple basal bodies. Microtubule forces acting on the cell cortex, and nuclear-cytoskeletal links, are important for positioning individual centrosomes, and during cell division. Knowledge of these common mechanisms can inform the study of centriole movements across biology

Centriole movements in mammalian epithelial cells during cytokinesis

<p>Abstract</p> <p>Background</p> <p>In cytokinesis, when the cleavage furrow has been formed, the two centrioles in each daughter cell separate. It has been suggested that the centrioles facilitate and regulate cytokinesis to some extent. It has been postulated that termination of cytokinesis (abscission) depends on the migration of a centriole to the intercellular bridge and then back to the cell center. To investigate the involvement of centrioles in cytokinesis, we monitored the movements of centrioles in three mammalian epithelial cell lines, HeLa, MCF 10A, and the p53-deficient mouse mammary tumor cell line KP-7.7, by time-lapse imaging. Centrin1-EGFP and α-Tubulin-mCherry were co-expressed in the cells to visualize respectively the centrioles and microtubules.</p> <p>Results</p> <p>Here we report that separated centrioles that migrate from the cell pole are very mobile during cytokinesis and their movements can be characterized as 1) along the nuclear envelope, 2) irregular, and 3) along microtubules forming the spindle axis. Centriole movement towards the intercellular bridge was only seen occasionally and was highly cell-line dependent.</p> <p>Conclusions</p> <p>These findings show that centrioles are highly mobile during cytokinesis and suggest that the repositioning of a centriole to the intercellular bridge is not essential for controlling abscission. We suggest that centriole movements are microtubule dependent and that abscission is more dependent on other mechanisms than positioning of centrioles.</p

The Different Function of Single Phosphorylation Sites of Drosophila melanogaster Lamin Dm and Lamin C

Lamins' functions are regulated by phosphorylation at specific sites but our understanding of the role of such modifications is practically limited to the function of cdc 2 (cdk1) kinase sites in depolymerization of the nuclear lamina during mitosis. In our study we used Drosophila lamin Dm (B-type) to examine the function of particular phosphorylation sites using pseudophosphorylated mutants mimicking single phosphorylation at experimentally confirmed in vivo phosphosites (S25E, S45E, T435E, S595E). We also analyzed lamin C (A-type) and its mutant S37E representing the N-terminal cdc2 (mitotic) site as well as lamin Dm R64H mutant as a control, non-polymerizing lamin. In the polymerization assay we could observe different effects of N-terminal cdc2 site pseudophosphorylation on A- and B-type lamins: lamin Dm S45E mutant was insoluble, in contrast to lamin C S37E. Lamin Dm T435E (C-terminal cdc2 site) and R64H were soluble in vitro. We also confirmed that none of the single phosphorylation site modifications affected the chromatin binding of lamin Dm, in contrast to the lamin C N-terminal cdc2 site. In vivo, all lamin Dm mutants were incorporated efficiently into the nuclear lamina in transfected Drosophila S2 and HeLa cells, although significant amounts of S45E and T435E were also located in cytoplasm. When farnesylation incompetent mutants were expressed in HeLa cells, lamin Dm T435E was cytoplasmic and showed higher mobility in FRAP assay

Abnormal proliferation and spontaneous differentiation of myoblasts from a symptomatic female carrier of X-linked Emery-Dreifuss muscular dystrophy

AbstractEmery–Dreifuss muscular dystrophy (EDMD) is a neuromuscular disease characterized by early contractures, slowly progressive muscular weakness and life-threatening cardiac arrhythmia that can develop into cardiomyopathy. In X-linked EDMD (EDMD1), female carriers are usually unaffected. Here we present a clinical description and in vitro characterization of a mildly affected EDMD1 female carrying the heterozygous EMD mutation c.174_175delTT; p.Y59* that yields loss of protein. Muscle tissue sections and cultured patient myoblasts exhibited a mixed population of emerin-positive and -negative cells; thus uneven X-inactivation was excluded as causative. Patient blood cells were predominantly emerin-positive, but considerable nuclear lobulation was observed in non-granulocyte cells – a novel phenotype in EDMD. Both emerin-positive and emerin-negative myoblasts exhibited spontaneous differentiation in tissue culture, though emerin-negative myoblasts were more proliferative than emerin-positive cells. The preferential proliferation of emerin-negative myoblasts together with the high rate of spontaneous differentiation in both populations suggests that loss of functional satellite cells might be one underlying mechanism for disease pathology. This could also account for the slowly developing muscle phenotype