A lamin A/C variant causing striated muscle disease provides insights into filament organization

Abstract

The LMNA gene encodes the A-type lamins that polymerize into ∼3.5 nm thick filaments, and together with B-type lamins and associated proteins form the nuclear lamina. Mutations in LMNA cause a wide variety of pathologies. In this study, we analyzed the nuclear lamina of embryonic fibroblasts from LmnaH222P/H222P mice, which develop cardiomyopathy and muscular dystrophy. Although the organization of the lamina appeared unaltered, there were changes in chromatin and B-type lamin expression. An increase in nuclear size and consequently a relative reduction in heterochromatin near the lamina allowed for a higher resolution structural analysis of lamin filaments using cryo-electron tomography. This was most apparent when visualizing lamin filaments in situ and using a nuclear extraction protocol. Averaging of individual segments of filaments in LmnaH222P/H222P mouse fibroblasts resolved two-polymers that constitute the mature filaments. Our findings provide better views of the organization of lamin filaments and the effect of a striated muscle disease-causing mutation on nuclear structure.

Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy

“…K5/14_1 cells were cultured on cryo-EM grids and subjected to cytoskeleton extraction buffer that permeabilizes the cells and removes soluble cytoplasmic components and nuclear structures, producing IF-enriched ghost cells (Material and Methods section) ( Hu et al, 2019 ; Turgay et al, 2017 ; Kronenberg-Tenga et al, 2021 ; Svitkina and Borisy, 1998 ; Svitkina et al, 1995 ; Sailer et al, 2010 ). The ghost cells were instantly plunge frozen and imaged by cryo-EM and cryo-ET ( Figure 1B–D ).…”

The molecular architecture of vimentin filaments

“…Resolving the structural fluctuations along the VIFs requires the reconstruction of extended polymer stretches 45,53 . Therefore, we extracted smaller segments (38 x 38 nm 2 ) and decreased the distance separating them, in order to increase the number of samples to more accurately track the natural bending of the VIFs.…”

“…5). For this purpose, the transformations calculated for each segment (namely their in-plane rotation angle psi and their xy-translation) were inverted and applied to the respective class averages, so that the inversely transformed class averages match position and orientation of the initial segments in the image frame of the tomograms 53 . As a result of this operation the VIFs are represented by the class averages, which drastically improves their signal-to-noise ratio compared to the raw filaments.…”

Development of a New cryo-S(T)EM Technique Allowing Simultaneous STEM and SEM Imaging and its Application to Biological Samples

“…Therefore, it is necessary to reduce the thickness of frozen samples enough to allow electrons to pass through. In previous studies, frozen samples were thinned by using a cryo-ultramicrotome (CEMOVIS) 7 or focused ion beam (FIB) [19][20][21][22] . In this study, unroo ng 23,25,26 was used instead.…”

“…In fact, cryo-EM of cells has provided high-resolution structural information about cells and organelles such as desmosomes and nuclear lamina, which have never been observed by conventional methods [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23] . In particular, when this technique is combined with tomography, the molecular structure of membranes and several types of laments can be analysed three-dimensionally [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] . Atomic resolution is not always required in cell biology, but it is desirable to have a cryo-TEM instrument available in a laboratory so that frozen samples can be observed frequently, as is possible with conventional TEM.…”