Distinct structural and mechanical properties of the nuclear lamina in Hutchinson–Gilford progeria syndrome

Abstract

The nuclear lamina is a network of structural filaments, the A and B type lamins, located at the nuclear envelope and throughout the nucleus. Lamin filaments provide the nucleus with mechanical stability and support many basic activities, including gene regulation. Mutations in LMNA, the gene encoding A type lamins, cause numerous human diseases, including the segmental premature aging disease Hutchinson-Gilford progeria syndrome (HGPS). Here we show that structural and mechanical properties of the lamina are altered in HGPS cells. We demonstrate by live-cell imaging and biochemical analysis that lamins A and C become trapped at the nuclear periphery in HGPS patient cells. Using micropipette aspiration, we show that the lamina in HGPS cells has a significantly reduced ability to rearrange under mechanical stress. Based on polarization microscopy results, we suggest that the lamins are disordered in the healthy nuclei, whereas the lamins in HGPS nuclei form orientationally ordered microdomains. The reduced deformability of the HGPS nuclear lamina possibly could be due to the inability of these orientationally ordered microdomains to dissipate mechanical stress. Surprisingly, intact HGPS cells exhibited a degree of resistance to acute mechanical stress similar to that of cells from healthy individuals. Thus, in contrast to the nuclear fragility seen in lmna null cells, the lamina network in HGPS cells has unique mechanical properties that might contribute to disease phenotypes by affecting responses to mechanical force and misregulation of mechanosensitive gene expression.laminopathy ͉ mechanics ͉ nucleus ͉ photobleaching ͉ micropipette aspiration H utchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease that causes segmental premature aging in children. HGPS patients are mentally normal, but fail to reach full stature and experience hair loss, thin wrinkled skin, and joint stiffness, and usually die in their early teens of cardiovascular disease or stroke (1, 2). Mutations in LMNA, the gene encoding lamins A and C, were recently identified as the cause of HGPS (3, 4). Lamins A and C are major constituents of the nuclear lamina, the meshwork of nuclear intermediate filaments that support the inner nuclear membrane and also extend throughout the nucleus (5). Other major components of the nuclear lamina are lamins B1 and B2, which are encoded by two distinct genes (6). Most HGPS cases are caused by a de novo single-point mutation (G608G; GGCϾGGT) in one allele of LMNA (3, 4). This substitution activates a cryptic splice site in exon 11, which affects only the lamin A protein, and the mutant allele produces an alternatively spliced truncated variant of the lamin A mRNA lacking the 3Ј terminal 150 nt of exon 11. The resulting polypeptide, ⌬50 lamin A, has an internal deletion of 50 residues in the C-terminal tail domain and also lacks an endoproteolytic cleavage site required for normal processing of the lamin A precursor (3, 4, 7). HGPS patient fibroblasts often are characterized by numerous nuclear defe...

Direct actin binding to A- and B-type lamin tails and actin filament bundling by the lamin A tail

“…25,26 Based on this milderthan-expected disruption of binding, and the weak binding of lamin C and ∆90 tails we suggest the Ig-fold may be stabilized by interactions with another region of the tail, specifically residues 564-608 in AB-2. Inefficient actin binding and bundling by the lamin A ∆50 protein ('progerin') might contribute to the structural collapse of the lamina network in HGPS patient nuclei 12 and might also impair dynamic rearrangements of nuclear actin polymers in response to mechanical stress. 12,47 Based on these findings, we propose that prelamin A residues 647-664, which are normally cleaved, carboxymethylated, farnesylated and then proteolytically removed during lamin A maturation in vivo, might act in concert with residues 385-393 52 to auto-inhibit prelamin A binding to actin and potentially other partners that bind the Ig-fold region, 27 including nuclear titin, 19 Sun1, 48 Sun2, 49 emerin 15 and nesprins.…”

“…Inefficient actin binding and bundling by the lamin A ∆50 protein ('progerin') might contribute to the structural collapse of the lamina network in HGPS patient nuclei 12 and might also impair dynamic rearrangements of nuclear actin polymers in response to mechanical stress. 12,47 Based on these findings, we propose that prelamin A residues 647-664, which are normally cleaved, carboxymethylated, farnesylated and then proteolytically removed during lamin A maturation in vivo, might act in concert with residues 385-393 52 to auto-inhibit prelamin A binding to actin and potentially other partners that bind the Ig-fold region, 27 including nuclear titin, 19 Sun1, 48 Sun2, 49 emerin 15 and nesprins. 50,51 We hypothesize that lamin head-to-tail polymerization, proposed to involve lamin A residues 385-393, 52 is also subject to auto-inhibition in prelamin A.…”

“…Other mutations in LMNA that activate cryptic mRNA splice sites can result in the deletion of 35 or 50 residues within the lamin A tail domain (mutations ∆35 and ∆50, respectively); these deletions block proteolytic processing of the farnesylated lamin A precursor protein (prelamin A) and cause Hutchinson-Gilford Progeria Syndrome (HGPS). [9][10][11] In nuclei from HGPS patient cells, all lamins aberrantly accumulate near the nuclear inner membrane; these nuclei have unusual mechanical properties (increased stiffness) when challenged by mechanical force, 12 …”

A progeria mutation reveals functions for lamin A in nuclear assembly, architecture, and chromosome organization

“…Since previous studies showed that LA⌬50/progerin is less soluble than WT-LA (22), the solubility of E145K-LA was also tested. Sequential extraction of FLAG-E145K-LA and FLAG-WT-LA expressing HeLa cells, however, showed no differences in the solubilities of WT-LA and E145K-LA, suggesting that both assembled into stable structures under these conditions (Fig.…”

Prelamin A Farnesylation and Progeroid Syndromes

“…Also, progerin has been shown to affect the mechanical stability of the nucleus (58), and a recent study has indicated that progerin interferes with lamin A and lamin B1 homopolymer formation and results in the production of structurally abnormal progerin-containing heteropolymers (50). In the future, it will be important to define the extent to which these abnormalities are affected by an FTI.…”