Structure and stability of the lamin A tail domain and HGPS mutant

Hutchinson–Gilford progeria syndrome (HGPS) is a premature aging syndrome caused by the expression and accumulation of a mutant form of lamin A, Δ50 lamin A. As a component of the cell’s nucleoskeleton, lamin A plays an important role in the mechanical stabilization of the nuclear envelope and in other nuclear functions. It is largely unknown how the characteristic 50 amino acid deletion affects the conformation of the mostly intrinsically disordered tail domain of lamin A. Here we perform replica exchange molecular dynamics simulations of the tail domain and determine an ensemble of semi-stable structures. Based on these structures we show that the ZMPSTE 24 cleavage site on the precursor form of the lamin A tail domain orients itself in such a way as to facilitate cleavage during the maturation process. We confirm our simulated structures by comparing the thermodynamic properties of the ensemble structures to in vitro stability measurements. Using this combination of experimental and computational techniques, we compare the size, heterogeneity of size, thermodynamic stability of the Ig-fold, as well as the mechanisms of force-induced denaturation. Our data shows that the Δ50 lamin A tail domain is more compact and displays less heterogeneity than the mature lamin A tail domain. Altogether these results suggest that the altered structure and stability of the tail domain can explain changed protein–protein and protein–DNA interactions and may represent an etiology of the disease. Also, this study provides the first molecular structure(s) of the lamin A tail domain, which is confirmed by thermodynamic tests in experiment.United States. Air Force Office of Scientific ResearchUnited States. Dept. of Defense. Presidential Early Career Award for Scientists and Engineer

Physical plasticity of the nucleus in stem cell differentiation

Cell differentiation in embryogenesis involves extensive changes in gene expression structural reorganization within the nucleus, including chromatin condensation and nucleoprotein immobilization. We hypothesized that nuclei in naive stem cells would therefore prove to be physically plastic and also more pliable than nuclei in differentiated cells. Micromanipulation methods indeed show that nuclei in human embryonic stem cells are highly deformable and stiffen 6-fold through terminal differentiation, and that nuclei in human adult stem cells possess an intermediate stiffness and deform irreversibly. Because the nucleo-skeletal component Lamin A/C is not expressed in either type of stem cell, we knocked down Lamin A/C in human epithelial cells and measured a deformability similar to that of adult hematopoietic stem cells. Rheologically, lamin-deficient states prove to be the most fluidlike, especially within the first ≈10 sec of deformation. Nuclear distortions that persist longer than this are irreversible, and fluorescence- imaged microdeformation with photobleaching confirms that chromatin indeed flows, distends, and reorganizes while the lamina stretches. The rheological character of the nucleus is thus set largely by nucleoplasm/chromatin, whereas the extent of deformation is modulated by the lamina

Calcium Causes a Conformational Change in Lamin A Tail Domain that Promotes Farnesyl-Mediated Membrane Association

AbstractLamin proteins contribute to nuclear structure and function, primarily at the inner nuclear membrane. The posttranslational processing pathway of lamin A includes farnesylation of the C-terminus, likely to increase membrane association, and subsequent proteolytic cleavage of the C-terminus. Hutchinson Gilford progeria syndrome is a premature aging disorder wherein a mutant version of lamin A, Δ50 lamin A, retains its farnesylation. We report here that membrane association of farnesylated Δ50 lamin A tail domains requires calcium. Experimental evidence and molecular dynamics simulations collectively suggest that the farnesyl group is sequestered within a hydrophobic region in the tail domain in the absence of calcium. Calcium binds to the tail domain with an affinity KD ≈ 250 μM where it alters the structure of the Ig-fold and increases the solvent accessibility of the C-terminus. In 2 mM CaCl2, the affinity of the farnesylated protein to a synthetic membrane is KD ≈ 2 μM, as measured with surface plasmon resonance, but showed a combination of aggregation and binding. Membrane binding in the absence of calcium could not be detected. We suggest that a conformational change induced in Δ50 lamin A with divalent cations plays a regulatory role in the posttranslational processing of lamin A, which may be important in disease pathogenesis

Interfacial binding and aggregation of lamin A tail domains associated with Hutchinson–Gilford progeria syndrome

Hutchinson–Gilford progeria syndrome is a premature aging disorder associated with the expression of ∆50 lamin A (∆50LA), a mutant form of the nuclear structural protein lamin A (LA). ∆50LA is missing 50 amino acids from the tail domain and retains a C-terminal farnesyl group that is cleaved from the wild-type LA. Many of the cellular pathologies of HGPS are thought to be a consequence of protein–membrane association mediated by the retained farnesyl group. To better characterize the protein–membrane interface, we quantified binding of purified recombinant ∆50LA tail domain (∆50LA-TD) to tethered bilayer membranes composed of phosphatidylserine and phosphocholine using surface plasmon resonance. Farnesylated ∆50LA-TD binds to the membrane interface only in the presence of Ca[superscript 2 +] or Mg[superscript 2 +] at physiological ionic strength. At extremely low ionic strength, both the farnesylated and non-farnesylated forms of ∆50LA-TD bind to the membrane surface in amounts that exceed those expected for a densely packed protein monolayer. Interestingly, the wild-type LA-TD with no farnesylation also associates with membranes at low ionic strength but forms only a single layer. We suggest that electrostatic interactions are mediated by charge clusters with a net positive charge that we calculate on the surface of the LA-TDs. These studies suggest that the accumulation of ∆50LA at the inner nuclear membrane observed in cells is due to a combination of aggregation and membrane association rather than simple membrane binding; electrostatics plays an important role in mediating this association.National Institute of General Medical Sciences (U.S.) (1R01-GM101647)United States. Office of Naval Research. Presidential Early Career Award for Scientists and Engineers (N000141010562)National Institutes of Health (U.S.) (U01 EB014976

Single wall carbon nanotubes enter cells by endocytosis and not membrane penetration

<p>Abstract</p> <p>Background</p> <p>Carbon nanotubes are increasingly being tested for use in cellular applications. Determining the mode of entry is essential to control and regulate specific interactions with cells, to understand toxicological effects of nanotubes, and to develop nanotube-based cellular technologies. We investigated cellular uptake of Pluronic copolymer-stabilized, purified ~145 nm long single wall carbon nanotubes (SWCNTs) through a series of complementary cellular, cell-mimetic, and in vitro model membrane experiments.</p> <p>Results</p> <p>SWCNTs localized within fluorescently labeled endosomes, and confocal Raman spectroscopy showed a dramatic reduction in SWCNT uptake into cells at 4°C compared with 37°C. These data suggest energy-dependent endocytosis, as shown previously. We also examined the possibility for non-specific physical penetration of SWCNTs through the plasma membrane. Electrochemical impedance spectroscopy and Langmuir monolayer film balance measurements showed that Pluronic-stabilized SWCNTs associated with membranes but did not possess sufficient insertion energy to penetrate through the membrane. SWCNTs associated with vesicles made from plasma membranes but did not rupture the vesicles.</p> <p>Conclusions</p> <p>These measurements, combined, demonstrate that Pluronic-stabilized SWCNTs only enter cells via energy-dependent endocytosis, and association of SWCNTs to membrane likely increases uptake.</p

Single wall carbon nanotubes enter cells by endocytosis and not membrane penetration

<p>Abstract</p> <p>Background</p> <p>Carbon nanotubes are increasingly being tested for use in cellular applications. Determining the mode of entry is essential to control and regulate specific interactions with cells, to understand toxicological effects of nanotubes, and to develop nanotube-based cellular technologies. We investigated cellular uptake of Pluronic copolymer-stabilized, purified ~145 nm long single wall carbon nanotubes (SWCNTs) through a series of complementary cellular, cell-mimetic, and in vitro model membrane experiments.</p> <p>Results</p> <p>SWCNTs localized within fluorescently labeled endosomes, and confocal Raman spectroscopy showed a dramatic reduction in SWCNT uptake into cells at 4°C compared with 37°C. These data suggest energy-dependent endocytosis, as shown previously. We also examined the possibility for non-specific physical penetration of SWCNTs through the plasma membrane. Electrochemical impedance spectroscopy and Langmuir monolayer film balance measurements showed that Pluronic-stabilized SWCNTs associated with membranes but did not possess sufficient insertion energy to penetrate through the membrane. SWCNTs associated with vesicles made from plasma membranes but did not rupture the vesicles.</p> <p>Conclusions</p> <p>These measurements, combined, demonstrate that Pluronic-stabilized SWCNTs only enter cells via energy-dependent endocytosis, and association of SWCNTs to membrane likely increases uptake.</p

Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage

Summary Tissue homeostasis requires maintenance of functional integrity under stress. A central source of stress is mechanical force that acts on cells, their nuclei, and chromatin, but how the genome is protected against mechanical stress is unclear. We show that mechanical stretch deforms the nucleus, which cells initially counteract via a calcium-dependent nuclear softening driven by loss of H3K9me3-marked heterochromatin. The resulting changes in chromatin rheology and architecture are required to insulate genetic material from mechanical force. Failure to mount this nuclear mechanoresponse results in DNA damage. Persistent, high-amplitude stretch induces supracellular alignment of tissue to redistribute mechanical energy before it reaches the nucleus. This tissue-scale mechanoadaptation functions through a separate pathway mediated by cell-cell contacts and allows cells/tissues to switch off nuclear mechanotransduction to restore initial chromatin state. Our work identifies an unconventional role of chromatin in altering its own mechanical state to maintain genome integrity in response to deformation.Peer reviewe

From the red cell to the nucleus: Mechanics and architecture of composite membrane systems

Red cells and nuclei are both composite membrane systems: a lipid bilayer or bilayers supported by an underlying membrane skeleton with architectural proteins which connect the lipid membrane to the skeleton. The mechanical properties of red cell bilayers and membrane skeletons have been determined, but the roles of some membrane proteins in red cell architecture are still unknown. In this thesis, numerous biophysical and fluorescence techniques are used to show protein interactions of CD47 and Rh proteins on the red cell surface. Measures of a significant immobile fraction of CD47 suggest protein interactions of CD47 with the red cell membrane skeleton. Further studies suggest this connection is mediated by the cytoplasmic protein 4.2 and is independent of Rh. Biophysical techniques and theories developed for red cells can be applied to nuclear composite membrane systems. Mobility measurements show free diffusion of nuclear pore complexes in yeast, which lack a membrane skeleton. In contrast, metazoan pore complexes are known to be immobile, indicating an important architectural role of the nuclear membrane skeleton in eukaryotic evolution. The nuclear membrane skeleton, known as the lamina network, is known to provide organization and mechanical support to metazoan nuclei. Here, the elasticity of the lamina network is determined by micropipette aspiration to be orders of magnitude stiffer than the red cell membrane skeleton in a model system. Nuclei are able to expand drastically while maintaining their mechanical integrity. Similar analyses with somatic nuclei showed a rigid three-dimensional mechanical character, but the lamina network still provided significant mechanical character

From the red cell to the nucleus: Mechanics and architecture of composite membrane systems

Red cells and nuclei are both composite membrane systems: a lipid bilayer or bilayers supported by an underlying membrane skeleton with architectural proteins which connect the lipid membrane to the skeleton. The mechanical properties of red cell bilayers and membrane skeletons have been determined, but the roles of some membrane proteins in red cell architecture are still unknown. In this thesis, numerous biophysical and fluorescence techniques are used to show protein interactions of CD47 and Rh proteins on the red cell surface. Measures of a significant immobile fraction of CD47 suggest protein interactions of CD47 with the red cell membrane skeleton. Further studies suggest this connection is mediated by the cytoplasmic protein 4.2 and is independent of Rh. Biophysical techniques and theories developed for red cells can be applied to nuclear composite membrane systems. Mobility measurements show free diffusion of nuclear pore complexes in yeast, which lack a membrane skeleton. In contrast, metazoan pore complexes are known to be immobile, indicating an important architectural role of the nuclear membrane skeleton in eukaryotic evolution. The nuclear membrane skeleton, known as the lamina network, is known to provide organization and mechanical support to metazoan nuclei. Here, the elasticity of the lamina network is determined by micropipette aspiration to be orders of magnitude stiffer than the red cell membrane skeleton in a model system. Nuclei are able to expand drastically while maintaining their mechanical integrity. Similar analyses with somatic nuclei showed a rigid three-dimensional mechanical character, but the lamina network still provided significant mechanical character

Correction: Spatially Resolved Quantification of Chromatin Condensation through Differential Local Rheology in Cell Nuclei Fluorescence Lifetime Imaging.

[This corrects the article DOI: 10.1371/journal.pone.0146244.]