Purification of Lamins and Soluble Fragments of NETs

Макаров, А. А.; Rizzotto, Andrea; Meinke, Peter; Schirmer, Eric C.

doi:10.1016/bs.mie.2015.09.006

Cited by 4 publications

(5 citation statements)

References 40 publications

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“…Lamin C WT and K311R plasmids with an N-terminal His tag and a TEV protease site in pET24d(+) vectors were used to transform Rosetta(DE3)pLysS bacteria. Lamin C proteins were purified as previously reported 55,56 . Paracrystalline arrays were visualized at a TEM-FEI CM100 100 kV microscope equipped with a digital CCD camera.…”

Section: Articlesmentioning

confidence: 99%

The NSL complex maintains nuclear architecture stability via lamin A/C acetylation

et al. 2019

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While nuclear lamina abnormalities are hallmarks of human diseases, their interplay with epigenetic regulators and precise epigenetic landscape remain poorly understood. Here, we show that loss of the lysine acetyltransferase MOF or its associated NSL-complex members KANSL2 or KANSL3 leads to a stochastic accumulation of nuclear abnormalities with genomic instability patterns including chromothripsis. SILAC-based MOF and KANSL2 acetylomes identified lamin A/C as an acetylation target of MOF. HDAC inhibition or acetylation-mimicking lamin A derivatives rescue nuclear abnormalities observed in MOF-deficient cells. Mechanistically, loss of lamin A/C acetylation resulted in its increased solubility, defective phosphorylation dynamics and impaired nuclear mechanostability. We found that nuclear abnormalities include EZH2-dependent histone H3 Lys 27 trimethylation and loss of nascent transcription. We term this altered epigenetic landscape "heterochromatin enrichment in nuclear abnormalities" (HENA). Collectively, the NSL-complex-dependent lamin A/C acetylation provides a mechanism that maintains nuclear architecture and genome integrity.

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Section: Articlesmentioning

confidence: 99%

The NSL complex maintains nuclear architecture stability via lamin A/C acetylation

et al. 2019

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“…Attempts to purify the recombinant protein under denaturing conditions also did not yield sufficient amounts of intact, soluble protein. Expression of untagged NE81 in Escherichia coli for the production of inclusion bodies, an established strategy for animal lamins [41], failed due to a low expression level. Taken together, we concluded that bacterial expression is not a suitable method to obtain sufficient amounts of NE81 in a functional, correctly folded state.…”

Section: Resultsmentioning

confidence: 99%

Supramolecular Structures of the Dictyostelium Lamin NE81

Grafe

Batsios

Meyer

et al. 2019

Cells

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Nuclear lamins are nucleus-specific intermediate filaments (IF) found at the inner nuclear membrane (INM) of the nuclear envelope (NE). Together with nuclear envelope transmembrane proteins, they form the nuclear lamina and are crucial for gene regulation and mechanical robustness of the nucleus and the whole cell. Recently, we characterized Dictyostelium NE81 as an evolutionarily conserved lamin-like protein, both on the sequence and functional level. Here, we show on the structural level that the Dictyostelium NE81 is also capable of assembling into filaments, just as metazoan lamin filament assemblies. Using field-emission scanning electron microscopy, we show that NE81 expressed in Xenopous oocytes forms filamentous structures with an overall appearance highly reminiscent of Xenopus lamin B2. The in vitro assembly properties of recombinant His-tagged NE81 purified from Dictyostelium extracts are very similar to those of metazoan lamins. Super-resolution stimulated emission depletion (STED) and expansion microscopy (ExM), as well as transmission electron microscopy of negatively stained purified NE81, demonstrated its capability of forming filamentous structures under low-ionic-strength conditions. These results recommend Dictyostelium as a non-mammalian model organism with a well-characterized nuclear envelope involving all relevant protein components known in animal cells.

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“…Briefly, 1 L of bacteria were collected by centrifugation at 2,000 × g , lysed in 12 ml of 50 mM Hepes, pH 8.0, 3 mM MgCl 2 , 3 mM 2-mercaptoethanol, 1 mM PMSF, 1 μg mL −1 aprotinin, 1 μM leupeptin, and 1 μM pepstatin by repeated sonication in the presence of DNAseI (1 U μl −1 ). Inclusion bodies were then pelleted, washed twice in ddH 2 O with 2% Triton X-100 and resuspended in the buffer with 6 M urea 48 . Protein was equilibrated in either Tris buffer (25 mM Tris, 250 mM NaCl, pH 8.0) or NaPi buffer (100 mM sodium phosphate, 250 mM NaCl, pH = 8.0) supplemented with 6 M urea and 1 mM PMSF.…”

Section: Methodsmentioning

confidence: 99%

“…A total of 1.6 mg of protein per a single slab was run for 8 h at 150 V. The area containing monomerised and mixed-in-the-gel light and heavy lamin A was identified via IntstantBlue (Expedeon, #ISB1L) staining of a thin strip of the gel. Unstained protein was then gel-extracted and desalted 28,29 via precipitation with 400 mM KCl and sequential washes with an 86:7:7 (vol:vol) mix of Acetone, Triethylamine and Acetic Acid and the same solution diluted to 5% in ddH 2 O 48 ; then and reconstituted in NaPi buffer with 6 M urea. Successful refolding of gel-extracted lamin A was confirmed by rotary metal shadowing EM.…”

Section: Methodsmentioning

confidence: 99%

Lamin A molecular compression and sliding as mechanisms behind nucleoskeleton elasticity

et al. 2019

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Lamin A is a nuclear intermediate filament protein critical for nuclear architecture and mechanics and mutated in a wide range of human diseases. Yet little is known about the molecular architecture of lamins and mechanisms of their assembly. Here we use SILAC cross-linking mass spectrometry to determine interactions within lamin dimers and between dimers in higher-order polymers. We find evidence for a compression mechanism where coiled coils in the lamin A rod can slide onto each other to contract rod length, likely driven by a wide range of electrostatic interactions with the flexible linkers between coiled coils. Similar interactions occur with unstructured regions flanking the rod domain during oligomeric assembly. Mutations linked to human disease block these interactions, suggesting that this spring-like contraction can explain in part the dynamic mechanical stretch and flexibility properties of the lamin polymer and other intermediate filament networks.

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Purification of Lamins and Soluble Fragments of NETs

Cited by 4 publications

References 40 publications

The NSL complex maintains nuclear architecture stability via lamin A/C acetylation

The NSL complex maintains nuclear architecture stability via lamin A/C acetylation

Supramolecular Structures of the Dictyostelium Lamin NE81

Lamin A molecular compression and sliding as mechanisms behind nucleoskeleton elasticity

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