Dataset Information

Structural basis for assembly of lamin at the molecular level

ABSTRACT: Nuclear structure and function are governed by lamins, which are intermediate filaments mostly consisting of alpha-helices. Different lamin assembly models have been proposed based on low resolution or fragmented structures. However, their assembly mechanisms at the molecular level are poorly understood. Here, we present a crystal structure of a long human lamin fragment at 3.2 Å resolution, which visualized the full-length features. The structure presented the anti-parallel arrangement of two coiled-coil dimers, which was important for the assembly process. We further discovered a new interaction between the lamin dimers by using chemical cross-linking and mass analysis. Based on these two interactions we proposed a molecular mechanism of lamin assembly, which agreed well with the recent model representing the native state, and could explain pathological mutations. Our findings provide the structural information to understand molecular mechanisms of assembly of intermediate filaments, and molecular insights into nuclear functions and aging process.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture, Cell Line Cell

DISEASE(S): Muscular Dystrophy

SUBMITTER: Yong-Hak Kim

LAB HEAD: Yong-Hak Kim

PROVIDER: PXD013144 | Pride | 2019-08-26

REPOSITORIES: Pride

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Publications

Structural basis for lamin assembly at the molecular level.

Ahn Jinsook J Jo Inseong I Kang So-Mi SM Hong Seokho S Kim Suhyeon S Jeong Soyeon S Kim Yong-Hak YH Park Bum-Joon BJ Ha Nam-Chul NC

Nature communications 20190821 1

Nuclear structure and function are governed by lamins, which are intermediate filaments that mostly consist of α-helices. Different lamin assembly models have been proposed based on low resolution and fragmented structures. However, their assembly mechanisms are still poorly understood at the molecular level. Here, we present the crystal structure of a long human lamin fragment at 3.2 Å resolution that allows the visualization of the features of the full-length protein. The structure shows an an ...[more]

PMID: 31434876

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Project description:B-type lamins, the major structural component of the nuclear lamina, is thought to play a repressive role in the expression of many of its bound genes whose silencing are known to be critical for cell differentiation during early development. Using global mapping of lamin-B-chromatin interaction, we find that the binding to lamin-B indeed correlated with gene silencing in mouse embryonic stem cells (ESC) and ESC-derived trophectoderm (TE) lineage cells. To address whether lamin-B directly suppresses the expression of the bound genes, we have derived ESCs from lamin-B-null mouse blastocysts. Unexpectedly, microarray analyses of ESCs and TE cells show that B-type lamins do not repress the expression of their bound genes. Furthermore, the knockout mice that do not express any B-type lamins can develop to term but exhibit small body size and perinatal lethality. Our studies demonstrate that B-type lamins are not required for cell differentiation during early embryonic development. Instead, they are required for proper late embryonic and perinatal development. The availability of the null ESCs and mice opens the door to further define the functions of B-type lamins. The Dam and Dam-lamin B1 (Lmnb1) lentivirus generated from 293T cells were concentrated by spinning at 25,000 rpm with the SW28Ti rotor (Beckman) for 3 hours. The viral pellets were resuspended in 1x PBS. For ESCs, freshly trypsinized ESCs were plated into a 6-well plate with 2 ml complete GMEM medium containing either Dam or Dam-Lmnb1 lentivirus, spin-infected at 700g for 45 min, and incubated for a total of 48 hours. For TE cells, TSCs were differentiated toward TE cells for 6 days. On day 6, the TE cells were transduced by spin-infection as above with a fresh TS medium containing either Dam or Dam-Lmnb1 lentivirus. The TE cells were incubated for 48 hours after viral transduction. The adenine-methylated DNAs were amplified from the genomic DNA by adaptor-mediated PCR. The amplified DNAs were labeled by the Dual color DNA labeling kit (Roche). The labeled DNAs were hybridized with 2.1M mouse whole-genome tiling arrays (NimbleGen, 05327911001), which covers non-repetitive regions in approximately 250 bp intervals, according to the manufacturer's recommendation. This submission represents the gene expression component of the study.

Dataset Information

Structural basis for assembly of lamin at the molecular level

Publications

Structural basis for lamin assembly at the molecular level.

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