Immunolocalization of detergent‐susceptible nucleoplasmic lamin A/C foci by a novel monoclonal antibody

Muralikrishna, Bh; Thanumalayan, Subramonian; Jagatheesan, Ganapathy; Rangaraj, Nandini; Karande, Anjali A.; Parnaik, Veena K.

doi:10.1002/jcb.10765

Cited by 9 publications

(12 citation statements)

References 25 publications

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“…Although most prevalent in early G1, nucleoplasmic lamins A/C are present throughout interphase (Goldman et al 1992;Broers et al 1999;Moir et al 2000c) and are thought to play a role in retinoblastomamediated cell proliferation (Johnson et al 2004;Pekovic et al 2007), initiation of DNA replication (Kennedy et al 2000), and RNA splicing (Jagatheesan et al 1999;Kumaran et al 2002), although the latter function is controversial (Vecerova et al 2004). Lamin A/C structures present in the nucleoplasm appear to be more dynamic and less tightly bound to the nucleoskeleton than those associated with the lamina, based on detergent extractability and FRAP analyses (Broers et al 1999;Moir et al 2000c;Muralikrishna et al 2004). Lamin B is also present in the nucleoplasm but as a more stable structure compared with A-type lamins (Moir et al 2000c), and intranuclear lamin B foci are associated with sites of mid-late-S-phase replication (Moir et al 1994).…”

Section: Lamins As Structural Components Of the Nucleusmentioning

confidence: 99%

Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin

Dechat

Pfleghaar²,

Sengupta³

et al. 2008

Genes Dev.

880

879

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Over the past few years it has become evident that the intermediate filament proteins, the types A and B nuclear lamins, not only provide a structural framework for the nucleus, but are also essential for many aspects of normal nuclear function. Insights into lamin-related functions have been derived from studies of the remarkably large number of disease-causing mutations in the human lamin A gene. This review provides an up-to-date overview of the functions of nuclear lamins, emphasizing their roles in epigenetics, chromatin organization, DNA replication, transcription, and DNA repair. In addition, we discuss recent evidence supporting the importance of lamins in viral infections.In eukaryotic cells, chromatin is tightly packed in a highly organized fashion within a nucleus that is composed of two main compartments: the nucleoplasm and the nuclear envelope (NE). There are also subcompartments in the nucleus containing factors involved in essential nuclear functions such as DNA replication, transcription, and RNA splicing (Prasanth and Spector 2006;Spector 2006). The NE separates nuclear functions from cytoplasmic functions and at its inner surface it provides a docking site for chromatin. The major structural elements of the NE are the inner nuclear membrane (INM), the outer nuclear membrane (ONM), the nuclear pore complexes (NPCs), and the nuclear lamina. The lamina is comprised of a complex meshwork of proteins closely associated with the INM and attached to the periphery of NPCs and to chromatin (Fawcett 1966;Patrizi and Poger 1967;Aaronson and Blobel 1975). The main constituents of the lamina are the type V intermediate filament (IF) proteins, the nuclear lamins. Lamins are also found, in lower concentrations, distributed throughout the nucleoplasm. The organization of lamins at the nuclear periphery as well as within the nucleoplasm is influenced by numerous lamin-binding proteins (Dorner et al. 2007;Schirmer and Foisner 2007;Wagner and Krohne 2007).This review focuses on the role of nuclear lamins in the organization and regulation of chromatin in the interphase nucleus-specifically, the involvement of lamins in essential processes such as transcription, DNA replication, DNA repair, and various epigenetic phenomena involved in the regulation of euchromatin-heterochromatin transitions. Emphasis is also placed on the remarkable array of disease-causing mutations in the human lamin A gene, from which many of the most recent insights into lamin functions have been derived. In addition, we also discuss emerging ideas regarding the roles of lamins in viral infections. General properties of the nuclear laminsNuclear lamins were initially described as the major protein components of detergent-high salt resistant "lamina" fractions of rat liver and chicken erythrocyte nuclei (Aaronson and Blobel 1975;Gerace et al. 1978). Subsequently it was shown that they are members of the IF protein family (Aebi et al. 1986;Goldman et al. 1986;McKeon et al. 1986). Lamin genes are found in all metazoa examined to date, but are...

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Section: Lamins As Structural Components Of the Nucleusmentioning

confidence: 99%

Nuclear lamins: major factors in the structural organization and function of the nucleus and chromatin

Dechat

Pfleghaar²,

Sengupta³

et al. 2008

Genes Dev.

880

879

View full text Add to dashboard Cite

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“…Scale bars, 5 micron. lamin network is visualized by immunostaining only after the removal of chromatin from the nucleus (22).…”

Section: Figmentioning

confidence: 99%

“…NuMat and its associated proteins are likely to be involved in the nuclear transport and may also play a vital role by providing a scaffold for the transport machinery. In addition, post-translational modifications appear to be important for NuMat proteins (21,22). The presence of protein kinases in the NuMat may be relevant in this context.…”

Section: Figmentioning

confidence: 99%

Nuclear Matrix Proteome Analysis of Drosophila melanogaster

Kallappagoudar

Varma

Pathak

et al. 2010

Molecular & Cellular Proteomics

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The nucleus is a highly structured organelle and contains many functional compartments. Although the structural basis for this complex spatial organization of compartments is unknown, a major component of this organization is likely to be the non-chromatin scaffolding called nuclear matrix (NuMat). Experimental evidence over the past decades indicates that most of the nuclear functions are at least transiently associated with the NuMat, although the components of NuMat itself are poorly known. Here, we report NuMat proteome analysis from Drosophila melanogaster embryos and discuss its links with nuclear architecture and functions. In the NuMat proteome, we found structural proteins, chaperones, DNA/RNAbinding proteins, chromatin remodeling and transcription factors. This complexity of NuMat proteome is an indicator of its structural and functional significance. The eukaryotic cell nucleus contains a number of structural features like heterochromatin, nucleolus, nuclear lamina, and nuclear territories and functional features like transcription foci, replication foci, Cajal bodies, stress-induced foci, etc.(1-7). Packaging of the genome on the other hand also results in non-random distribution of a variety of structural features like chromatin domains that help to regulate expression of genes (8). How these complex structural and functional domains are established and maintained is not known. It is likely that the non-chromatin scaffolding called nuclear matrix (9) plays an important role in this process. Nuclear matrix (NuMat) 1 mainly consists of the nuclear lamina, the nucleolar remnants, and an internal nuclear meshwork of fibers of unknown constitution that can be seen by electron microscopy (10). These fibers have ultrastructural features reminiscent of intermediate filaments of the cytosol (11). The protein component of nucleoplasmic filaments, however, is largely unknown. Earlier studies on a few proteins isolated from nuclear matrix from different organisms have shown them to be DNA-and RNA-binding and structural components of the nuclear pore complex (12), enzymes (13), and nuclear membrane proteins (14). This diversity among the small number of identified NuMat constituents reflects the link of nuclear architecture with the variety of nuclear functions. The importance of NuMat constituents in nuclear processes has been apparent for a long time, and several studies have been carried out to identify the proteins of this complex structure in different organisms (15-18). Major advancements in the field of proteomics now provide further scope for extensive analysis of NuMat in this context.Here, we present a comprehensive analysis of the NuMat proteome of Drosophila melanogaster. NuMat preparations from D. melanogaster embryos were separated by one-dimensional gel electrophoresis, and the proteins were identified by LC-MS/MS. We also report a remarkable variation in the NuMat proteome depending on developmental stages, indicating a link between embryonic development and nuclear architecture. Finally, immunos...

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“…65 Immunoprecipitations (right) with an antibody directed against lamin A/C, using mild lysis conditions (for example 0.1% NP-40, 250 mM NaCl 12 ), preferentially dissolve and precipitate nucleosoluble A-type lamins and protein interactors. 35 For a lamin A OST pull-down assay, 46 cross-linking, indicated by crosses, captures protein-protein and protein-chromatin interactions and allows solubilization of the total lamin A/C pool while preserving interactions.…”

Section: Protein Interactionsmentioning

confidence: 99%

“…Low amounts of detergents and salt preferentially solubilize nucleoplasmic pools of proteins (Fig. 1), as described to exist for lamin A, 35 and were mainly applied to study easy extractable, weakly bound NE interactors (Smad2, PP2A, Rb, Ubc9, hnRNP1, EGF1, SREBP1; See Table 2). 5,12,[36][37][38] Increasing amounts of detergents, salts and the solvent glycerol 39 successfully solubilize protein complexes of well-anchored NE and lamina components (LAP2β, Emerin, Nesprin2, Lamin B; Table 2), although sometimes at the cost of disrupting interactions (LaminA/B1/B2-LAP2β) 40 ( Table 2).…”

Section: Nucleusmentioning

confidence: 99%