Journal: J Cell Sci / Year: 2021 Title: A lamin A/C variant causing striated muscle disease provides insights into filament organization. Authors: Rafael Kronenberg-Tenga / Meltem Tatli / Matthias Eibauer / Wei Wu / Ji-Yeon Shin / Gisèle Bonne / Howard J Worman / Ohad Medalia / Abstract: The gene encodes the A-type lamins, which polymerize into ∼3.5-nm-thick filaments and, together with B-type lamins and associated proteins, form the nuclear lamina. Mutations in cause a wide ...The gene encodes the A-type lamins, which polymerize into ∼3.5-nm-thick filaments and, together with B-type lamins and associated proteins, form the nuclear lamina. Mutations in cause a wide variety of pathologies. In this study, we analyzed the nuclear lamina of embryonic fibroblasts from 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 and using a nuclear extraction protocol. Averaging of individual segments of filaments in 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.
EMPIAR-10601 (Title: A lamin A/C variant causing striated muscle disease provides insights into filament organization Data size: 2.7 Data #1: Tilt-series of LmnaH222P/H222P revealing lamin meshwork in nuclease treated and FIB-milled nucleus [tilt series])
MEF cell carrying homozygous H222P mutation in Lmna.
Sectioning
Focused ion beam - Instrument: OTHER / Focused ion beam - Ion: OTHER / Focused ion beam - Voltage: 30 kV / Focused ion beam - Current: 0.24 nA / Focused ion beam - Duration: 3600 sec. / Focused ion beam - Temperature: 128 K / Focused ion beam - Initial thickness: 4000 nm / Focused ion beam - Final thickness: 200 nm Focused ion beam - Details: The value given for _emd_sectioning_focused_ion_beam.instrument is Zeiss Auriga. This is not in a list of allowed values {'DB235', 'OTHER'} so OTHER is written into the XML file.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 3.7 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Final reconstruction
Algorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: IMOD / Number images used: 41
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