We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximity-dependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximity-dependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment.

/pdf/a-promiscuous-biotin-ligase-fusion-protein-identifies-4w2yhxu77k.pdf

A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells

http://sdevoto.web.wesleyan.edu/343/readings/emery2002.pdf

The muscular dystrophies.

"Laminopathies": a wide spectrum of human diseases.

Nuclear lamins were initially identified as the major components of the nuclear lamina, a proteinaceous layer found at the interface between chromatin and the inner nuclear membrane (Fawcett 1966). Due to their position at the periphery of the nucleus, lamins were originally proposed to support the nuclear envelope and provide anchorage sites for chromatin. Recently, the nuclear lamins have also been found in the nucleoplasm (Fig. 1). In addition, experimental and genetic evidence suggest that nuclear lamins are involved in a number of other functions including nuclear envelope assembly, DNA synthesis, transcription, and apoptosis. Interestingly, mutations in nuclear lamins have been linked to human diseases. In this review, we describe what is known of the structure and function of lamins and their associated proteins. Further, we speculate about possible mechanisms through which mutations in nuclear lamins give rise to disease. Immunological and structural data originally suggested that the lamins were related to cytoplasmic intermediate filaments (IF) (Aebi et al. 1986; Goldman et al. 1986). The cloning and sequencing of lamin cDNAs confirmed that lamins have the typical domain structure of IF including an -helical coiled-coil domain flanked by nonhelical domains (Fisher et al. 1986; McKeon et al. 1986). Interestingly, analyses of lamin and cytoplasmic IF genomic sequences indicate that nuclear lamins are the progenitors of all IF, with cytoplasmic IF arising through gene duplication (Riemer et al. 2000).

/pdf/nuclear-lamins-building-blocks-of-nuclear-architecture-58pcfdrj66.pdf

Nuclear lamins: building blocks of nuclear architecture

It has been demonstrated that nuclear lamins are important proteins in maintaining cellular as well as nuclear integrity, and in maintaining chromatin organization in the nucleus. Moreover, there is growing evidence that lamins play a prominent role in transcriptional control. The family of laminopathies is a fast-growing group of diseases caused by abnormalities in the structure or processing of the lamin A/C (LMNA) gene. Mutations or incorrect processing cause more than a dozen different inherited diseases, ranging from striated muscular diseases, via fat- and peripheral nerve cell diseases, to progeria. This broad spectrum of diseases can only be explained if the responsible A-type lamin proteins perform multiple functions in normal cells. This review gives an overview of current knowledge on lamin structure and function and all known diseases associated with LMNA abnormalities. Based on the knowledge of the different functions of A-type lamins and associated proteins, explanations for the observed phenotypes are postulated. It is concluded that lamins seem to be key players in, among others, controlling the process of cellular ageing, since disturbance in lamin protein structure gives rise to several forms of premature ageing.

/pdf/nuclear-lamins-laminopathies-and-their-role-in-premature-1s8z0veypo.pdf

Nuclear lamins: laminopathies and their role in premature ageing

Emery-Dreifuss muscular dystrophy (EDMD) is characterized by early contractures of the elbows and Achilles tendons, slowly progressive muscle wasting and weakness, and life-threatening cardiomyopathy with conduction blocks. We recently identified LMNA encoding two nuclear envelope proteins, lamins A and C, to be implicated in the autosomal dominant form of EDMD. Here, we report on the variability of the phenotype and spectrum of LMNA mutations in 53 autosomal dominant EDMD patients (36 members of 6 families and 17 sporadic cases). Twelve of the 53 patients showed cardiac involvement exclusively, although the remaining 41 all showed muscle weakness and contractures. We were able to identify a common phenotype among the patients with skeletal muscle involvement, consisting of humeroperoneal wasting and weakness, scapular winging, rigidity of the spine, and elbow and Achilles tendon contractures. The disease course was generally slow, but we observed either a milder phenotype characterized by late onset and a mild degree of weakness and contractures or a more severe phenotype with early presentation and a rapidly progressive course in a few cases. Mutation analysis identified 18 mutations in LMNA (ie, 1 nonsense mutation, 2 deletions of a codon, and 15 missense mutations). All the mutations were distributed between exons 1 and 9 in the region of LMNA that is common to lamins A and C. LMNA mutations arose de novo in 76% of the cases; 2 of these de novo mutations were typical hot spots, and 2 others were identified in 2 unrelated cases. There was no clear correlation between the phenotype and type or localization of the mutations within the gene. Moreover, a marked inter- and intra-familial variability in the clinical expression of LMNA mutations exists, ranging from patients expressing the full clinical picture of EDMD to those characterized only by cardiac involvement, which points toward a significant role of possible modifier genes in the course of this disease. In conclusion, the high proportion of de novo mutations together with the large spectrum of both LMNA mutations and the expression of the disease should now prompt screening for LMNA in familial and sporadic cases of both EDMD and dilated cardiomyopathy associated with conduction system disease. Ann Neurol 2000;48:170–180

Wicklein Em

Papers

Clinical and molecular genetic spectrum of autosomal dominant Emery‐Dreifuss muscular dystrophy due to mutations of the lamin A/C gene