Chromatin condensation regulates endothelial cell adaptation to shear stress.

Vascular endothelial cells (ECs) have been shown to be mechanoresponsive to the forces of blood flow, including fluid shear stress (FSS), the frictional force of blood on the vessel wall. Recent reports have shown that FSS induces epigenetic changes in chromatin. Epigenetic changes, such as methylation and acetylation of histones, not only affect gene expression but also affect chromatin condensation, which can alter nuclear stiffness. Thus, we hypothesized that changes in chromatin condensation may be an important component for how ECs adapt to FSS. Using both in vitro and in vivo models of EC adaptation to FSS, we observed an increase in histone acetylation and a decrease in histone methylation in ECs adapted to flow as compared with static. Using small molecule drugs, as well as vascular endothelial growth factor, to change chromatin condensation, we show that decreasing chromatin condensation enables cells to more quickly align to FSS, whereas increasing chromatin condensation inhibited alignment. Additionally, we show data that changes in chromatin condensation can also prevent or increase DNA damage, as measured by phosphorylation of γH2AX. Taken together, these results indicate that chromatin condensation, and potentially by extension nuclear stiffness, is an important aspect of EC adaptation to FSS.

A single stiffened nucleus alters cell dynamics and coherence in a monolayer.

Force transmission throughout a monolayer is the result of complex interactions between cells. Monolayer adaptation to force imbalances such as singular stiffened cells provides insight into the initiation of disease and fibrosis. Here, NRK-52E cells transfected with ∆50LA, which significantly stiffens the nucleus. These stiffened cells were sparsely placed in a monolayer of normal NRK-52E cells. Through morphometric analysis and temporal tracking, the impact of the singular stiffened cells shows a pivotal role in mechanoresponse of the monolayer. A method for a detailed analysis of the spatial aspect and temporal progression of the nuclear boundary was developed and used to achieve a full description of the phenotype and dynamics of the monolayers under study. Our findings reveal that cells are highly sensitive to the presence of mechanically impaired neighbors, leading to generalized loss of coordination in collective cell migration, but without seemingly affecting the potential for nuclear lamina fluctuations of neighboring cells. Reduced translocation in neighboring cells appears to be compensated by an increase in nuclear rotation and dynamic variation of shape, suggesting a "frustration" of cells and maintenance of motor activity. Interestingly, some characteristics of the behavior of these cells appear to be dependent on the distance to a ∆50LA cell, pointing to compensatory behavior in response to force transmission imbalances in a monolayer. These insights may suggest the long-range impacts of single cell defects related to tissue dysfunction.

CRISPR Cas13-Based Tools to Track and Manipulate Endogenous Telomeric Repeat-Containing RNAs in Live Cells.

TERRA, TElomeric Repeat-containing RNA, is a long non-coding RNA transcribed from telomeres. Emerging evidence indicates that TERRA regulates telomere maintenance and chromosome end protection in normal and cancerous cells. However, the mechanism of how TERRA contributes to telomere functions is still unclear, partially owing to the shortage of approaches to track and manipulate endogenous TERRA molecules in live cells. Here, we developed a method to visualize TERRA in live cells via a combination of CRISPR Cas13 RNA labeling and SunTag technology. Single-particle tracking reveals that TERRA foci undergo anomalous diffusion in a manner that depends on the timescale and telomeric localization. Furthermore, we used a chemically-induced protein dimerization system to manipulate TERRA subcellular localization in live cells. Overall, our approaches to monitor and control TERRA locations in live cells provide powerful tools to better understand its roles in telomere maintenance and genomic integrity.

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

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.

Cells with Higher Cortical Membrane Tension Are More Sensitive to Lysis by Biosurfactant Di-rhamnolipids.

Tissue and cellular stiffening is associated with pathologies including fibrosis and cancer. Healthy cells also exhibit a wide range of membrane cortical tensions, which have been studied in the field of mechanobiology. Here, we quantify the mechanosensitivity of the lysis agent the di-rhamnolipid (RHA), which is a bacterially produced biosurfactant. RHA exhibited selective lysis correlated strongly with cortical membrane tension in osteoblasts, smooth muscle cells, fibroblasts, epithelial cells, and erythrocytes. Reducing cortical membrane tension by cytoskeleton inhibitors (cytochalasin D and nocodazole) or osmotic regulators (sucrose, polyethylene glycol, and nonionic surfactants) attenuated the RHA toxicity. The selective toxicity of RHA toward human chronic myeloid leukemia K562 cells over healthy blood cells suggests a potential therapy for blood cancer. Targeted killing of myofibroblasts transformed from either fibroblasts or epithelial cells indicates its antifibrotic effect. Combined, these studies showed the potential for specific targeting of cells with differential mechanical properties rather than chemical or biological pathways.

Piperazine Derivatives Enhance Epithelial Cell Monolayer Permeability by Increased Cell Force Generation and Loss of Cadherin Structures.

A major obstacle for topical and enteral drug delivery is the poor transport of macromolecular drugs through the epithelium. One potential solution is the use of permeation enhancers that alter epithelial structures. Piperazine derivatives are known permeation enhancers that modulate epithelial structures, reduce transepithelial electrical resistance, and augment the absorption of macromolecular drugs. The mechanism by which piperazine derivatives disrupt the structures of epithelial monolayers is not well understood. Here, the effects of 1-phenylpiperazine and 1-methyl-4-phenylpiperazine are modeled in the epithelial cell line NRK-52E. Live-cell imaging reveals a dose-dependent gross reorganization of monolayers at high concentrations, but reorganization differs based on the piperazine molecule. Results show that low concentrations of piperazine derivatives increase myosin force generation within the cells and do not disrupt the cytoskeletal structure. Also, cytoskeletally attached cadherin junctions are disrupted before tight junctions. In summary, piperazines appear to increase myosin-mediated contraction followed by disruption of cell-cell contacts. These results provide new mechanistic insight into how transient epithelial permeation enhancers act and will inform of the development of future generations of transepithelial delivery systems.

Quantifying site-specific chromatin mechanics and DNA damage response.

DNA double-strand breaks pose a direct threat to genomic stability. Studies of DNA damage and chromatin dynamics have yielded opposing results that support either increased or decreased chromatin motion after damage. In this study, we independently measure the dynamics of transcriptionally active or repressed chromatin regions using particle tracking microrheology. We find that the baseline motion of transcriptionally repressed regions of chromatin are significantly less mobile than transcriptionally active chromatin, which is statistically similar to the bulk motion of chromatin within the nucleus. Site specific DNA damage using KillerRed tags induced in loci within repressed chromatin causes an increased motion, while loci within transcriptionally active regions remains unchanged at similar time scales. We also observe a time-dependent response associated with a further increase in chromatin decondensation. Global induction of damage with bleocin displays similar trends of chromatin decondensation and increased mobility only at 53BP1-labeled damage sites but not at non-damaged sites, indicating that chromatin dynamics are tightly regulated locally after damage. These results shed light on the evolution of the local and global DNA damage response associated with chromatin remodeling and dynamics, with direct implications for their role in repair.

Determining mechanical features of modulated epithelial monolayers using subnuclear particle tracking.

Force generation within cells, mediated by motor proteins along cytoskeletal networks, maintains the function of multicellular structures during homeostasis and when generating collective forces. Here, we describe the use of chromatin dynamics to detect cellular force propagation [a technique termed SINK (sensors from intranuclear kinetics)] and investigate the force response of cells to disruption of the monolayer and changes in substrate stiffness. We find that chromatin dynamics change in a substrate stiffness-dependent manner within epithelial monolayers. We also investigate point defects within monolayers to map the impact on the strain field of a heterogeneous monolayer. We find that cell monolayers behave as a colloidal assembly rather than as a continuum since the data fit an exponential decay; the lateral characteristic length of recovery from the mechanical defect is ∼50 µm for cells with a 10 µm spacing. At distances greater than this characteristic length, cells behave similarly to those in a fully intact monolayer. This work demonstrates the power of SINK to investigate diseases including cancer and atherosclerosis that result from single cells or heterogeneities in monolayers.This article has an associated First Person interview with the first author of the paper.

SIRT6 facilitates directional telomere movement upon oxidative damage.

Oxidative damage to telomeres leads to telomere attrition and genomic instability, resulting in poor cell viability. Telomere dynamics contribute to the maintenance of telomere integrity; however, whether oxidative damage induces telomere movement and how telomere mobility is regulated remain poorly understood. Here, we show that oxidative damage at telomeres triggers directional telomere movement. The presence of the human Sir2 homolog, Sirtuin 6 (SIRT6) is required for oxidative damage-induced telomeric movement. SIRT6 knock out (KO) cells show neither damage-induced telomere movement nor chromatin decondensation at damaged telomeres; both are observed in wild type (WT) cells. A deacetylation mutant of SIRT6 increases damage-induced telomeric movement in SIRT6 KO cells as well as WT SIRT6. SIRT6 recruits the chromatin-remodeling protein SNF2H to damaged telomeres, which appears to promote chromatin decondensation independent of its deacetylase activity. Together, our results suggest that SIRT6 plays a role in the regulation of telomere movement upon oxidative damage, shedding new light onto the function of SIRT6 in telomere maintenance.

SSRP1 Cooperates with PARP and XRCC1 to Facilitate Single-Strand DNA Break Repair by Chromatin Priming.

DNA single-strand breaks (SSB) are the most common form of DNA damage, requiring repair processes that to initiate must overcome chromatin barriers. The FACT complex comprised of the SSRP1 and SPT16 proteins is important for maintaining chromatin integrity, with SSRP1 acting as an histone H2A/H2B chaperone in chromatin disassembly during DNA transcription, replication, and repair. In this study, we show that SSRP1, but not SPT16, is critical for cell survival after ionizing radiation or methyl methanesulfonate-induced single-strand DNA damage. SSRP1 is recruited to SSB in a PARP-dependent manner and retained at DNA damage sites by N-terminal interactions with the DNA repair protein XRCC1. Mutational analyses showed how SSRP1 function is essential for chromatin decondensation and histone H2B exchange at sites of DNA strand breaks, which are both critical to prime chromatin for efficient SSB repair and cell survival. By establishing how SSRP1 facilitates SSB repair, our findings provide a mechanistic rationale to target SSRP1 as a general approach to selectively attack cancer cells. Cancer Res; 77(10); 2674-85. ©2017 AACR.

Length-dependent intracellular bundling of single-walled carbon nanotubes influences retention.

Single-walled carbon nanotubes (SWCNTs) are increasingly being investigated for biomedical imaging, sensing, and drug delivery. Cell types, cellular entry mechanisms, and SWCNT lengths dictate SWCNT uptake, subsequent intracellular trafficking, and retention. Specialized immune cells known as macrophages are capable of two size-dependent entry mechanisms: endocytosis of small particles (diameter < 200 nm) and phagocytosis of large particles (diameter > 500 nm). In comparison, fibroblasts uptake particles predominantly through endocytosis. We report dependence of cellular processing including uptake, subcellular distribution, and retention on the SWCNT length and immune cell-specific processes. We chose SWCNTs of three different average lengths: 50 nm (ultrashort, US), 150 nm (short) and 500 nm (long) to encompass two different entry mechanisms, and noncovalently dispersed them in water, cell culture media, and phosphate buffer (pH 5) with bovine serum albumin, which maintains the SWCNT optical properties and promotes their cellular uptake. Using confocal Raman imaging and spectroscopy, we quantified cellular uptake, tracked the intracellular dispersion state (i.e., individualized versus bundled), and monitored recovery as a function of SWCNT lengths in macrophages. Cellular uptake of SWCNTs increases with decreasing SWCNT length. Interestingly, short-SWCNTs become highly bundled in concentrated phase dense regions of macrophages after uptake and most of these SWCNTs are retained for at least 24 h. On the other hand, both US- and long-SWCNTs remain largely individualized after uptake into macrophages and are lost over a similar elapsed time. After uptake into fibroblasts, however, short-SWCNTs remain individualized and are exocytosed over 24 h. We hypothesize that aggregation of SWCNTs within macrophages but not fibroblasts may facilitate the retention of SWCNTs within the former cell type. Furthermore, the differential length-dependent cellular processing suggests potential applications of macrophages as live cell carriers of SWCNTs into tumors and regions of inflammation for therapy and imaging.

Delivering Single-Walled Carbon Nanotubes to the Nucleus Using Engineered Nuclear Protein Domains.

Single-walled carbon nanotubes (SWCNTs) have great potential for cell-based therapies due to their unique intrinsic optical and physical characteristics. Consequently, broad classes of dispersants have been identified that individually suspend SWCNTs in water and cell media in addition to reducing nanotube toxicity to cells. Unambiguous control and verification of the localization and distribution of SWCNTs within cells, particularly to the nucleus, is needed to advance subcellular technologies utilizing nanotubes. Here we report delivery of SWCNTs to the nucleus by noncovalently attaching the tail domain of the nuclear protein lamin B1 (LB1), which we engineer from the full-length LMNB1 cDNA. More than half of this low molecular weight globular protein is intrinsically disordered but has an immunoglobulin-fold composed of a central hydrophobic core, which is highly suitable for associating with SWCNTs, stably suspending SWCNTs in water and cell media. In addition, LB1 has an exposed nuclear localization sequence to promote active nuclear import of SWCNTs. These SWCNTs-LB1 dispersions in water and cell media display near-infrared (NIR) absorption spectra with sharp van Hove peaks and an NIR fluorescence spectra, suggesting that LB1 individually disperses nanotubes. The dispersing capability of SWCNTs by LB1 is similar to that by albumin proteins. The SWCNTs-LB1 dispersions with concentrations ≥150 µg/mL (≥30 µg/mL) in water (cell media) remain stable for ≥75 days (≥3 days) at 4 °C (37 °C). Further, molecular dynamics modeling of association of LB1 with SWCNTs reveal that the exposure of the nuclear localization sequence is independent of LB1 binding conformation. Measurements from confocal Raman spectroscopy and microscopy, NIR fluorescence imaging of SWCNTs, and fluorescence lifetime imaging microscopy show that millions of these SWCNTs-LB1 complexes enter HeLa cells, localize to the nucleus of cells, and interact with DNA. We postulate that the modification of native cellular proteins as noncovalent dispersing agents to provide specific transport will open new possibilities to utilize both SWCNT and protein properties for multifunctional subcellular targeting applications. Specifically, nuclear targeting could allow delivery of anticancer therapies, genetic treatments, or DNA to the nucleus.

Early Passage Dependence of Mesenchymal Stem Cell Mechanics Influences Cellular Invasion and Migration.

The cellular structures and mechanical properties of human mesenchymal stem cells (hMSCs) vary significantly during culture and with differentiation. Previously, studies to measure mechanics have provided divergent results using different quantitative parameters and mechanical models of deformation. Here, we examine hMSCs prepared for clinical use and subject them to mechanical testing conducive to the relevant deformability associated with clinical injection procedures. Micropipette aspiration of hMSCs shows deformation as a viscoelastic fluid, with little variation from cell to cell within a population. After two passages, hMSCs deform as viscoelastic solids. Further, for clinical applicability during stem cell migration in vivo, we investigated the ability of hMSCs to invade into micropillar arrays of increasing confinement from 12 to 8 µm spacing between adjacent micropillars. We find that hMSC samples with reduced deformability and cells that are more solid-like with passage are more easily able to enter the micropillar arrays. Increased cell fluidity is an advantage for injection procedures and optimization of cell selection based on mechanical properties may enhance efficacy of injected hMSC populations. However, the ability to invade and migrate within tight interstitial spaces appears to be increased with a more solidified cytoskeleton, likely from increased force generation and contractility. Thus, there may be a balance between optimal injection survival and in situ tissue invasion.

Nuclear stiffening inhibits migration of invasive melanoma cells.

During metastasis, melanoma cells must be sufficiently deformable to squeeze through extracellular barriers with small pore sizes. We visualize and quantify deformability of single cells using micropipette aspiration and examine the migration potential of a population of melanoma cells using a flow migration apparatus. We artificially stiffen the nucleus with recombinant overexpression of Δ50 lamin A, which is found in patients with Hutchison Gilford progeria syndrome and in aged individuals. Melanoma cells, both WM35 and Lu1205, both show reduced nuclear deformability and reduced cell invasion with the expression of Δ50 lamin A. These studies suggest that cellular aging including expression of Δ50 lamin A and nuclear stiffening may reduce the potential for metastatic cancer migration. Thus, the pathway of cancer metastasis may be kept in check by mechanical factors in addition to known chemical pathway regulation.

Active cytoskeletal force and chromatin condensation independently modulate intranuclear network fluctuations.

Chromatin remodeling, including the movement of genes and regulatory factors, precedes or accompanies stimulated changes in gene expression. Here we quantify chromatin fluctuations in primary human cells using particle-tracking microrheology and determine the physical mechanisms which influence chromatin reorganization. We find that intranuclear movements are enhanced beyond thermal motion by active force generation from cytoskeletal motor activity propagated through the LINC complex; intranuclear movements are also dependent on the viscoelasticity of the DNA-protein polymer network. Chromatin movements were dramatically altered by modulation of chromatin condensation state, which we independently verified using fluorescence lifetime imaging microscopy (FLIM). These findings suggest that chromatin condensation and cytoskeletal force generation play distinct functional roles in regulating intranuclear movements, and these effects are decoupled as measured by particle tracking. We further utilize this approach in identifying the nuclear responsiveness of primary human endothelial cells to vascular endothelial growth factor (VEGF): early in the response chromatin movements increase and are dominated by cytoskeletal force, which transitions at later times to a chromatin decondensation event. Given the hierarchical genome organization in primary cells, our work generally suggests an important role for force generation and chromatin mechanics in altered gene expression kinetics.

Hutchinson-Gilford progeria syndrome alters nuclear shape and reduces cell motility in three dimensional model substrates.

Cell migration through tight interstitial spaces in three dimensional (3D) environments impacts development, wound healing and cancer metastasis and is altered by the aging process. The stiffness of the extracellular matrix (ECM) increases with aging and affects the cells and cytoskeletal processes involved in cell migration. However, the nucleus, which is the largest and densest organelle, has not been widely studied during cell migration through the ECM. Additionally, the nucleus is stiffened during the aging process through the accumulation of a mutant nucleoskeleton protein lamin A, progerin. By using microfabricated substrates to mimic the confined environment of surrounding tissues, we characterized nuclear movements and deformation during cell migration into micropillars where interspacing can be tuned to vary nuclear confinement. Cell motility decreased with decreased micropillar (µP) spacing and correlated with increased dysmorphic shapes of nuclei. We examined the effects of increased nuclear stiffness which correlates with cellular aging by studying Hutchinson-Gilford progeria syndrome cells which are known to accumulate progerin. With the expression of progerin, cells showed a threshold response to decreased µP spacing. Cells became trapped in the close spacing, possibly from visible micro-defects in the nucleoskeleton induced by cell crawling through the µP and from reduced force generation, measured independently. We suggest that ECM changes during aging could be compounded by the increasing stiffness of the nucleus and thus changes in cell migration through 3D tissues.

Mechanical characterization of adult stem cells from bone marrow and perivascular niches.

Therapies using adult stem cells often require mechanical manipulation such as injection or incorporation into scaffolds. However, force-induced rupture and mechanosensitivity of cells during manipulation is largely ignored. Here, we image cell mechanical structures and perform a biophysical characterization of three different types of human adult stem cells: bone marrow CD34+ hematopoietic, bone marrow mesenchymal and perivascular mesenchymal stem cells. We use micropipette aspiration to characterize cell mechanics and quantify deformation of subcellular structures under force and its contribution to global cell deformation. Our results suggest that CD34+ cells are mechanically suitable for injection systems since cells transition from solid- to fluid-like at constant aspiration pressure, probably due to a poorly developed actin cytoskeleton. Conversely, mesenchymal stem cells from the bone marrow and perivascular niches are more suitable for seeding into biomaterial scaffolds since they are mechanically robust and have developed cytoskeletal structures that may allow cellular stable attachment and motility through solid porous environments. Among these, perivascular stem cells cultured in 6% oxygen show a developed cytoskeleton but a more compliant nucleus, which can facilitate the penetration into pores of tissues or scaffolds. We confirm the relevance of our measurements using cell motility and migration assays and measure survival of injected cells. Since different types of adult stem cells can be used for similar applications, we suggest considering mechanical properties of stem cells to match optimal mechanical characteristics of therapies.

Quantification of uptake and localization of bovine serum albumin-stabilized single-wall carbon nanotubes in different human cell types.

Single-wall carbon nanotubes (SWCNTs) possess many unique, inherent properties that make them attractive materials for application in medical and biological technologies. Development of concentrated SWCNT dispersions of isolated nanotubes that retain SWCNTs' inherent properties with minimal negative cellular effects is essential to fully realize the potential of SWCNTs in biotechnology. It is shown that bovine serum albumin (BSA), a common and well-characterized model blood serum protein, can individually disperse SWCNTs at concentrations of up to 0.3 mg mL(-1) while retaining SWCNTs' optical properties. Uptake into human mesenchymal stem cells (hMSC) and HeLa cells is quantified, revealing strikingly high concentrations of 86 ± 33 × 10(6) and 21 ± 13 × 10(6) SWCNTs per cell, respectively, without any apparent acute deleterious cellular effects. Through high-resolution confocal Raman spectroscopy and imaging, it is established that SWCNT-BSAs are preferentially localized intracellularly, especially in the cytoplasm of both hMSCs and HeLa cells. The uptake and localization results demonstrate the efficacy of BSA as a biocompatible dispersant and a mediator of bioactivity. BSA is widely available and inexpensive, which make these concentrated, highly-dispersed, noncovalently modified SWCNT-BSAs suitable for the development of SWCNT-based biotechnologies.

In the middle of it all: mutual mechanical regulation between the nucleus and the cytoskeleton.

The nucleus is typically treated as the large phase-dense or easy-to-label structure at the center of the cell which is manipulated by the governing mechanical machinery inside the cytoplasm. However, recent evidence has suggested that the mechanical properties of the nucleus are important to cell fate. We will discuss many aspects of the structural and functional interconnections between nuclear mechanics and cellular mechanics in this review. There are numerous implications for the progression of many disease states associated with both nuclear structural proteins and cancers. The nucleus itself is a large organelle taking up significant volume within the cell, and most studies agree that nuclei are significantly stiffer than the surrounding cytoplasm. Thus when a cell is exposed to force, the nucleus is exposed to and helps resist that force. The nucleus and nucleoskeleton are interconnected with the cellular cytoskeleton, and these connections may aid in helping disperse forces within tissues and/or with mechanotransduction. During translocation and transmigration the nucleus can act as a resistive element. Understanding the role of mechanical regulation of the nucleus may aid in understanding cellular motility and crawling through confined geometries. Thus the nucleus plays a role in developing mechanical territories and niches, affecting rates of wound healing and allowing cells to transmigrate through tissues for developmental, repair or pathological means.

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 fluid-like, especially within the first approximately 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.