Cell engineering: Biophysical regulation of the nucleus

Journal Pre-proof Cell engineering: Biophysical regulation of the nucleus Yang Song, Jennifer Soto, Binru Chen, Li Yang, Song Li PII:

S0142-9612(19)30861-0

DOI:

https://doi.org/10.1016/j.biomaterials.2019.119743

Reference:

JBMT 119743

To appear in:

Biomaterials

Received Date: 25 July 2019 Revised Date:

2 December 2019

Accepted Date: 25 December 2019

Please cite this article as: Song Y, Soto J, Chen B, Yang L, Li S, Cell engineering: Biophysical regulation of the nucleus, Biomaterials (2020), doi: https://doi.org/10.1016/j.biomaterials.2019.119743. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Cell Engineering: Biophysical Regulation of the Nucleus Yang Song1,3*, Jennifer Soto1*, Binru Chen1, Li Yang3, Song Li1,2# 1

Department of Bioengineering, University of California, Los Angeles, CA, USA Department of Medicine, University of California, Los Angeles, CA, USA 3 School of Bioengineering, Chongqing University, Chongqing, China 400044 2

*Equal contribution #

Correspondence: Song Li, Ph.D. [email protected]

Keywords: nuclear biomechanics, mechanobiology, mechanotransduction, epigenetics

Abstract Cells live in a complex and dynamic microenvironment, and a variety of microenvironmental cues can regulate cell behavior. In addition to biochemical signals, biophysical cues can induce not only immediate intracellular responses, but also long-term effects on phenotypic changes such as stem cell differentiation, immune cell activation and somatic cell reprogramming. Cells respond to mechanical stimuli via an outside-in and inside-out feedback loop, and the cell nucleus plays an important role in this process. The mechanical properties of the nucleus can directly or indirectly modulate mechanotransduction, and the physical coupling of the cell nucleus with the cytoskeleton can affect chromatin structure and regulate the epigenetic state, gene expression and cell function. In this review, we will highlight the recent progress in nuclear biomechanics and mechanobiology in the context of cell engineering, tissue remodeling and disease development.

Introduction Mechanotransduction is the mechanism by which cells convert mechanical stimuli from the extracellular microenvironment into intracellular biochemical signals to induce a variety of cellular responses. This process plays an important role in regulating various cell functions during development, regeneration and disease [1–3]. Recent research has shown that certain cellular structures function as mechanosensors that perceive and respond to changes in mechanical force or mechanotransducers that produce a chemical signal in response to a mechanical stimulus, as a part of mechanotransduction [4–8]. Focal adhesions are large, multiprotein complexes and one of several transmembrane receptors that provide a physical link between the extracellular matrix (ECM) and the cytoskeleton (Figure 1). In response to force, integrins within these complexes undergo conformational changes that lead to the recruitment of distinct focal adhesion proteins to the mechanosensing site [9,10]. In turn, cells also reorganize their cytoskeleton and generate contractile forces through motor proteins, such as myosin, to adjust their internal tension and reach a state of mechanical stability [11,12]. Thus, focal adhesions function not only as mechanosensors since they can be modulated by biochemical and 1

physical cues found in the microenvironment, but also as mechanotransducers to activate signaling pathways that regulate cytoskeletal organization [4,13]. Actin filaments, a major forcebearing component of the cytoskeleton, is believed to function as a major mechanotransmitter as they can transmit mechanical signals to the nucleus via the linker of the nucleoskeleton and cytoskeleton (LINC) complex to regulate cell behavior [6,14]. As such, through fast stress wave propagation, actin filaments behave as the main transmitter for a rapid response to mechanical cues, enabling cells to rapidly adapt to dynamic changes in their surrounding environment [15]. The cell nucleus stores the genetic material and transcriptional machinery of eukaryotic cells and plays an important role in regulating cell fate and behavior. Within the nucleoplasm, DNA is wrapped around histones, forming higher-order structures that occupy distinct locations, which can be categorized as either open, transcriptionally active euchromatin or tightly packed, inactive heterochromatin [16]. The nucleus is enclosed by the nuclear envelope (NE), a dual lipid bilayer that is connected by nuclear pore complexes that mediate nuclear-cytoplasmic transport [17,18]. The nuclear lamina is a dense protein network consisting of integral membrane proteins (e.g. emerin, lamina-associated polypeptide 2 and MAN1) and type V nuclear intermediate filaments (i.e. lamins A, B, and C) that provide mechanical support to the inner nuclear membrane [19,20]. Lamins not only associate with various NE proteins and transcriptional regulators, but also directly interact with chromatin by tethering lamina-associated domains of chromatin to the nuclear periphery [20,21]. Disruption of lamins can cause changes in chromatin assembly, such as the loss of peripheral heterochromatin [22], suggesting that lamins can regulate chromatin organization and gene expression. In addition to providing mechanical support to the nucleus, lamins, in particular lamins A and C, serve to anchor the LINC complex, enabling the transmission of mechanical cues from the ECM and the cytoskeleton to the nucleus [4,6,15,23]. In the past three decades, many studies have shown that mutations in genes encoding for nuclear lamina components can cause numerous diseases such as Hutchinson-Gilford progeria syndrome, Charcot-Marie-Tooth, and Emery-Dreifuss muscular dystrophy [24]. Although the molecular mechanisms of such diseases are not well understood, recent studies have shown that dysfunction of nuclear envelope proteins is linked to altered nuclear mechanics and several diseases, particularly those affecting cardiac and skeletal muscle [23–26]. These findings underscore the importance of the biomechanics and mechanobiology of the cell nucleus. Furthermore, the LINC complex acts as a conserved nuclear envelope-spanning molecular bridge that functions to physically couple the nucleus and the cytoskeleton [23,27,28]. The LINC complex consists of two protein families, Sad1/UNC-84 (SUN) and Klarsicht/ANC1/Syne-1 homology (KASH) domain proteins, which are located in the inner and outer nuclear membranes, respectively, and are connected by transmembrane segments [27]. In mammalian cells, five SUN domain proteins and six KASH domain proteins have been identified [27]. KASH domain proteins extend into both the cytoplasm and perinuclear space between the inner and outer nuclear membranes where they are tethered by SUN domain proteins. The KASH domain proteins that extend into the cytoplasm interact with cytoskeletal elements, including actin, microtubules and intermediate filaments. On the other hand, SUN domains proteins span into the perinuclear space and the nucleus. Thus, SUN domain proteins are able to not only interact with the nuclear lamina, in this case through SUN1, SUN2 and SUN4 , but also with chromatin [29]. Altogether, these intracellular structures can transmit the forces exerted on the cell surface to the nucleus, and thereby potentially impact nuclear structure and function. Recent 2

studies have shown that the nucleus plays a critical role in mechanosensing, mechanotransduction, and disease pathogenesis [6,30,31]. In this review, we will focus on the biomechanics and mechanobiology of the nucleus and their effects on several cellular processes. Mechanical properties of the cell nucleus Mechanical coupling of the cytoskeleton and nucleus In eukaryotic cells, nuclear positioning occurs in an active manner via interactions between the NE and the cytoskeleton and is critical for several cellular processes, such as migration and cell division [29,32,33]. It is becoming clear that the nucleus is always under force, even when stationary, which allows for the nucleus to be stably positioned for mechanohomeostasis [34]. F-actin and microtubule motors generate active forces on the nuclear surface to position the nucleus in a defined location within the cell [29,34]. Similarly, microtubule motor proteins, such as kinesin and dynein, and actomyosin generate fluctuating push and pull forces on the nucleus [35–37]. When an external force is applied to fibroblasts, it significantly displaces and deforms the nucleus [38]. Upon removal of the force, the nucleus rapidly reverts to its initial original shape and position. This behavior is attributed to the nuclear elastic resistance to translation and deformation [38]. The source of resistance primarily originates in cytoplasmic intermediate filaments (i.e. vimentin) and the nuclear proteins, lamin A/C. Recent evidence has provided some new insight into the mechanisms of homeostatic nuclear positioning, demonstrating it is context-dependent and involves distinct cytoskeletal and LINC complex components [39]. Using centrifugal force to displace the nuclei in adherent cells, homeostatic nuclear positioning is found to be dependent on SUN1 and SUN2 engaging with microtubules and actin, respectively, via nesprin-2G [39]. However, whether nuclear deformation is truly elastic is still somewhat controversial as several studies have shown that nuclear deformation persists for far longer than an elastic body, suggesting that the nucleus may not store elastic energy and is, thus, a viscoelastic structure [34,40,41]. In spite of this, all these studies demonstrate that there is a tight mechanical integration between the nucleus and its surroundings, which maintains the mechano-homeostasis of the nucleus. This is important as deregulation of nuclear positioning can result in cell dysfunction and diseases, such as muscular and central nervous disorders [29,42]. In the context of mechano-homeostasis, the mechanical properties of nucleus, such as stiffness, are essential in regulating various physiological and pathological processes, such as cell differentiation, migration, adhesion, and metastasis. The nuclear lamina underlying the NE forms a network of intermediate filaments, which is thought to provide mechanical support to the nucleus and a buffer to cellular forces in differentiated cells [16,20]. In mammalian cells, the lamina is composed of two type of lamins, A-type and B-type lamins [20]. Through alternative splicing, the LMNA gene encodes the proteins lamins A and C. Lamins B1 and B2 are two major B-type lamins that are encoded from the LMNB1 and LMNB2 genes, respectively. While at least one type of B-type lamin is present in all cell types, the expression of A-type lamins is developmentally regulated, suggesting that it may be involved in cell fate determination [16]. Utilizing cryo-electron tomography, Mahamid et al. acquired three-dimensional (3D) snapshots of the nuclear periphery in HeLa cells and further elucidated that lamin A formed a fine isotropic matrix and it is the key player in contributing to metazoan nuclear stiffness [43]. More recently, it has been demonstrated that A-type and B-type lamins form independent, nonoverlapping 3

networks at the nuclear periphery and have distinct roles in the maintenance of nuclear lamina organization [44–46]. There is also accumulating evidence that lamin A/C is the major contributor to nuclear mechanics and levels of lamin A/C expression correlate with nuclear stiffness [47–49]. For example, increases in lamin A/C expression leads to stiffer nuclei [48] whereas decreased levels results in softer, more deformable nuclei [47]. Emerin is another inner nuclear membrane protein that can affect nuclear mechanics. Utilizing magnetic tweezers to apply mechanical stress on Nesprin-1 of isolated nuclei, it was determined that mechanical forces induced nuclear stiffening via tyrosine phosphorylation of emerin [50]. These findings suggest that isolated nuclei can rapidly adapt their mechanical properties in response to forces and emerin plays an important role in regulating nuclear rigidity. Moreover, Schreiner and colleagues demonstrated that in response to cytoskeletal forces, untethering the chromatin from the inner NE resulted in highly deformable nuclei [51]. These findings suggest that the chromatin may contribute to nuclear stiffness [51]. More recent evidence indicates that the chromatin histone modification state can also dictate nuclear rigidity [52]. For instance, increases in euchromatin or decreases in heterochromatin using small molecule compounds that alter the histone modification state resulted in a softer nucleus [52]. In addition to the nuclear lamina, the chromatin, which displays viscoelastic properties, also contributes to nuclear deformation in response to mechanical strain [49,53]. Apart from the nuclear lamina and the degree of chromatin condensation, the physical linkages between the cytoskeleton and nucleus also dictate the mechanical properties of the nucleus. Several studies have reported that mechanical forces can rapidly induce the formation of a perinuclear actin cap through a process that requires nuclear lamina proteins and the LINC complex [54–56]. This actin structure, which is composed of actomyosin filaments that surrounds the nucleus, serves to protect the structural integrity of the nucleus and determine its shape [54,56]. Moreover, perinuclear actin associates with focal adhesions and the nuclear lamina via the LINC complex, providing evidence of a direct connection in which mechanical forces can be transmitted directly from the cell periphery to the nucleus and thus, alter nuclear mechanics, gene expression and cell function [5,57–60]. Nuclear actin filaments also play a role in nuclear mechanics by regulating nuclear structure and various nuclear processes, including chromatin remodeling, gene transcription regulation and DNA damage response [61–63]. Additionally, nuclear actin polymerization can regulate transcription factor activity through modulation of nucleocytoplasmic trafficking, which suggests that nuclear actin dynamics may be involved in mechano-transmission and mechanotransduction signaling [64–67]. Altogether, these studies highlight how the mechanical properties of the nucleus can be modulated by cytoskeletal components on the surface of the NE, the nuclear lamina and chromatin itself. Techniques for the measurement of cell and nuclear mechanical property In addition to biomolecular analysis of cells, there is a growing interest in the mechanical property of cells and nuclei as it can be a disease indicator and thus, potentially used for pathological, diagnostic and therapeutic purposes. As summarized in Figure 2 and Table 1, there are several widely-used methods to study the mechanical properties of living cells, including micropipette aspiration, atomic force microscopy (AFM) and optical and magnetic tweezers; more recently, microfluidic-based systems have emerged as promising approaches for highthroughput cell and nuclear mechanical phenotyping.

4

Micropipette aspiration was first developed by Mitchison and Swann in 1954 to investigate the mechanical property of sea-urchin egg membranes [68]. This method involves applying a negative pressure to partially suck a cell into a micropipette, measuring the length of the cell extruded into the micropipette and using the proper mathematical model to determine the mechanical properties of the cell. By controlling the applied negative pressure, the length of cell extension into the micropipette can be controlled. While a small extension gives the mechanical measurement of the cell surface, a large extension provides the measurement of the cytoskeleton and cytoplasmic components. Micropipette aspiration is typically used for cells in suspension. Additionally, it can be used to measure nuclear mechanical properties upon isolating the nucleus from the cell body using nuclear extraction buffers or ice-cold hypotonic buffers [69,70]. However, custom-made parts, such as the glass micropipette, are required for the setup, and the accuracy of the measurements can be affected by variations in micropipettes, environmental fluctuations and physiological cell deterioration [71]. Table 1 Examples of cellular and nuclear stiffness in different cell types measured using various technologies TECHNOLOGY Micropipette aspiration

NUCLEI/ CELL

REFERENCE

Intact nuclei

[69]

Micropipette aspiration

Isolated nuclei/ Intact nuclei

[72]

0.42 ± 0.12 kPa

Micropipette aspiration

Isolated nuclei

[41]

Human

3.2 ± 1.4 kPa

AFM

Intact cells

[73]

Rat Rat Human

3.1 ± 1.5 kPa 3.3 ± 0.8 kPa 9.29 ± 1.80 kPa

AFM AFM AFM

Intact nuclei Intact nuclei Isolated nuclei

[74] [74] [75]

Human

7.22 ± 0.46 kPa

AFM

Intact nuclei

[76]

Human

6.7±2.2 kPa

AFM

Intact nuclei

[77]

Human

23.2±14.3 kPa

AFM

Intact nuclei

[77]

Human

1.19 ± 0.19 kPa

AFM

Intact nuclei

[78]

Human

287 ± 52 Pa

AFM

Intact cells

[79]

Human

855 ± 670 Pa

AFM

Intact cells

[80]

Human Human

48 ± 35 Pa 156 ± 87 Pa

AFM AFM

Intact cells Intact cells

[80] [80]

Human

~149 Pa

Optical tweezers

Isolated nuclei

[81]

Human

23.5 ± 10.6 Pa

Optical tweezers

Intact cells

[82]

Human

30.2 ± 15.0 Pa

Optical tweezers

Intact cells

[82]

Human

12.6 ± 6.1 Pa

Optical tweezers

Intact cells

[82]

Human

0.53 ± 0.04 kPa

Lab-on-chip

Intact cells

[83]

CELL TYPE

SPECIES

Embryonic fibroblast

Mouse

Hela cell

Human

Endothelial cell

Bovine

Mesenchymal stem cells Epithelial cell Lung fibroblast Valve interstitial cell Umbilical vein endothelial cells Hela cell Vascular smooth muscle cell Schlemm’s canal endothelial cell Prostate cancer cell line (LNCaP) Myeloid leukemia cells (HL60) Jurkat Neutrophil Myeloid leukemia cells (K562) Normal myoepithelial cells (HBL-100) Luminal breast cancer cells (MCF-7) Basal breast cancer cells (MDA-MB-231) Myeloid leukemia cells (HL60)

STIFFNESS α= 0.21 ± 0.01 K/µ= 5.1 ± 1.3 α= 0.41±0.08 α= 0.41±0.04 K/µ= 1.5±0.3

5

Luminal breast cancer cells (MCF-7) Basal breast cancer cells (MDA-MB-231)

Human

2.1 ± 0.1 kPa

Lab-on-chip

Intact cells

[83]

Human

0.80 ± 0.19 kPa

Lab-on-chip

Intact cells

[83]

α: Values of the material parameter K/µ: The ratio of the area expansion to shear moduli

AFM is another method that has been extensively used to study the mechanical behavior of cells attached to a surface [84]. In addition, it has also been applied to mechanically characterize isolated nuclei or intact nuclei within cells by directly probing the nuclear area as this area has been shown to be the most meaningful for nuclear mechanics characterization [82,85,86]. The development of a modified AFM needle that can penetrate the cell membrane, in conjunction with fluorescent confocal imaging, has enabled direct intracellular probing of the nucleus in situ [75]. A typical AFM measurement encompasses performing nanoindentations on cells. An AFM probe with proper geometry (e.g. spherical, cone or pyramidal shape) is used as an indenter. A cantilever with a specific probe and proper spring constant reflects a laser signal, and the deformation of the cantilever is characterized by the recorded laser changes from which the applied force to the cantilever can be calculated. The major advantage of AFM indentation measurement is precision since the lateral position of AFM tip and the load force can be precisely controlled [87]. However, AFM measurement requires a well-controlled microenvironment, and the measured result is location-dependent as the results will vary with indentation sites. Optical tweezers use a highly focused laser beam to trap and manipulate a microscopic dielectric particle, based on the refractive index mismatch between the particle and the surrounding solution [88]. Upon trapping a microbead with the laser, optical tweezers can be used to perform indentation [89] or stretching [90] measurements on cells to determine the mechanical properties. In contrast to micropipette aspiration and AFM, there is no direct cell contact with the instrument except for the trapped microbeads in the sample, providing an isolated and precisely-controlled environment for local measurements [91]. A major limitation of optical tweezers is that it can only apply a relatively small force on cells, typically in the piconewton range, which may not be sufficient enough to deform cells to study whole-cell level properties. Furthermore, by manipulating paramagnetic beads under a controlled, external magnetic field, magnetic tweezers can be utilized to measure the mechanical properties of cells and nuclei [92]. Implementation of this approach in recent studies has provided some important insights into mechanotransduction processes, especially within the nucleus [50,93–95]. This technique offers several unique advantages such as samples do not experience intense irradiation, thereby preventing accelerated cell degradation. Additionally, no compensation for enzyme translocation is necessary as tweezers can generate a nearly homogeneous force field over a large scale. However, this method also has its limitations, including identifying and working with particles that are susceptible to magnetic fields, and the bulky geometry of the magnetic poles, which must be positioned close to the sample, may make it difficult to combine this technique with other applications [96]. Moreover, significant improvements in optimal imaging techniques have provided novel tools to not only analyze the mechanical properties of cells, but also examine cell nanostructures (e.g. chromatin) and their temporal behavior in a non-contact fashion. Several studies have shown that Brillouin spectroscopy can be utilized to measure the mechanical property of cells and tissues in vitro and in vivo [97–100]. Additionally, partial wave spectroscopy (PWS) is a 6

quantitative and non-invasive imaging technique that enables the visualization of nanoscale structures and organization, which are in the 20-200 nanometer range [101,102]. Interestingly, this technique was found to be capable of detecting changes in chromatin structure that are indicative of carcinogenesis, suggesting that it can potentially serve as a new screening tool for diagnostic purposes [101]. More recently, a live-cell PWS technique has been developed that can be used to study the relationship between nanoscale subcellular organization and function in realtime [102]. In order to determine the mechanical properties of cells, mathematical models are critical in all the pre-described methods, with the Hertz model being the most commonly used to determine the elasticity (i.e. Young’s modulus) of cells [103]. In this model, the cell is assumed to be a homogenous, elastic semisphere with a well-defined boundary [103]. Using the Hertz model, a previous study showed that cell rigidity increased at the rate in which forces were applied to the cell [104]. However, this assumption is not accurate for cellular mechanical measurements since cells typically display viscoelastic behavior [103,104]. This led researchers to develop a rate-jump method to take cell viscoelasticity into account, which involved applying a sudden force step change on the sample to analyze the intrinsic elastic modulus independently of the test conditions used [105]. It is important to note that elastic moduli for a single cell type may vary depending on the technique and parameters that are used for the measurement. Thus, combining multiple techniques, such as AFM and optical tweezers, may aid to improve the accuracy of mechanical measurements and identify meaningful differences among tested groups [82]. A common limitation among the techniques discussed above is low throughput, thereby limiting their applications and translation due to their inability to rapidly measure cell mechanical properties within large populations. Advancements in microfluidics has enabled researchers to integrate and develop novel high-throughput systems for cell and nuclear mechanical measurements. Some examples of microfluidic-based methods include micro-constriction arrays [106–108], microchannel resonators [109], and deformability cytometry [83,110,111]. Using fluid-based deformability cytometry systems, it has been shown that mechanical property measurements can be derived from measuring the transit time through constrictions [83] or cell deformation after hydrodynamic stretching [110]. Recent work has integrated Brillouin spectroscopy with microfluidics to use light scattering as a parameter to measure nuclear mechanical properties, providing a non-contact and highly-sensitive method to detect nuclear stiffness [112]. As deformability capacity relates to stiffness and varies among different cell types [113–115], this parameter can potentially be used as a biomarker or predictor of disease status. For example, cancerous cells are softer than non-cancerous cells in several cancer models (e.g. pancreatic and bladder) [79,116,117], and more recently, tumor-initiating cells were found to be more deformable than cancer cells that do not display stem-cell like properties [118,119]. In addition to mechanical characterization, cell deformability can also be used as parameter in microfluidic sorting devices to separate cells for further molecular characterization or drug screening applications [118,120,121]. Thus, these specialized microfluidic devices display great potential for high-throughput cell mechanical measurements that are inherently label-free and noninvasive, providing clinically relevant platforms for diagnostic and therapeutic purposes. Nuclear mechanics in cell differentiation, immune response, migration and disease

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Stem cells and differentiation. As the nucleus plays an important role in regulating cell phenotype, this has led scientists to explore how nuclear mechanical properties may differ among various cell types. Several properties, such as nuclear stiffness, deformability and nuclear protein expression, have been identified as parameters that can be used to classify cells. It has been reported that nuclear shape is a quantifiable discriminant of mechanical properties in various stem cell types, such as human mesenchymal stem cells (MSCs) derived from bone marrow or adipose tissues. Both cell types were found to be more elastic and anisotropic and displayed more elongated nuclear profiles in comparison to human induced pluripotent stem cells (iPSCs), which have a higher differentiation potential [122]. Upon measuring nuclear stiffness using micromanipulation methods, Pajerowski et al. demonstrated that human embryonic stem cells (ESCs) possess a highly deformable nuclei while human adult hematopoietic stem cells (HSCs) contain nuclei of intermediate stiffness that deform irreversibly [113]. Interestingly, when lamin A/C was knocked down in human epithelial cells, which are fully differentiated cells that have relatively stiff nuclei, the resulting nuclei were softer and their deformability capacity was altered. Further examination of the nuclear lamina in stem cells has revealed that undifferentiated mouse and human ESCs express lamins B1and B2 but not lamin A/C [123]. Additionally, the finding that ESCs lack lamin A/C was further supported by the observation that lamin A/C is not expressed during the early embryonic stages (i.e. before day 9) of mouse development [124– 126]. Altogether, these results demonstrate that the absence of lamin A can potentially serve as a marker for undifferentiated ESCs. During cell differentiation and activation under physiological or pathological conditions, the mechanical property of the nucleus changes due to alterations in the expression of proteins involved in nuclear morphology and deformability. During ESC differentiation, there is an activation of lamin A/C expression, and the nucleus stiffens up to 6-fold, displaying a nuclear stiffness similar to that of differentiated cells [113,123]. Moreover, it is well known that the biophysical cues, such as matrix stiffness, can direct stem cell lineage specification [127]. It has been proposed that the nucleus acts as a mechanostat to modulate cellular mechanosensation and mechanotransduction signaling during cell differentiation [128]. Heo et al. showed that during MSC differentiation, there is a reorganization of lamin A/C and an increase in heterochromatin content that leads to stiffer nuclei that no longer deform when the cell is mechanically stretched [128]. These changes in nuclear stiffness resulted in the sensitization of stem cells to mechanicalloading-induced calcium signaling and the expression of differentiated cell markers. This sensitization was reversed when the differentiated nucleus was softened, and was enhanced when the undifferentiated nucleus was stiffened. Interestingly, in the absence of soluble differentiation factors, dynamic loading was able to stiffen and condense the nucleus of undifferentiated MSCs, demonstrating how biophysical cues could potentially replace soluble factors and alter nuclear mechanics to influence cell differentiation [128]. In response to topographical cues, human ESCs and MSCs exhibited distinct temporal changes in nuclear morphology and lamin A/C expression during neuronal differentiation [129]. Interestingly, the expression of lamin A/C in ESCs was weakly detected during the first 7 days of differentiation whereas it was highly expressed in human MSCs within 24 hours after culturing the cells on the substrate [129]. Several studies have also shown that magnitude and frequency of dynamic mechanical loading can have an effect on cell phenotype and functions, such as proliferation and differentiation [130,131]. For example, gentle cyclic loading (maximum elongation 10%, 0.2 Hz), mimicking gentle breathing, prevented myofibroblast differentiation by altering the cytoskeleton, paracrine signaling and gene expression [132,133]. On the other hand, low8

magnitude high-frequency mechanical loads (i.e. vibrations where the frequency applied ranged from 30-100 Hz) induced changes in the cytoskeleton such as increased actin content and cytoskeletal realignment that resulted in increased cell stiffness, potentially enabling cells to become more sensitive to the mechanical signals [134,135]. In addition, there is evidence that high frequency vibrations can regulate nuclear structure and LINC complex elements [136,137]. For instance, low intensity vibrations promoted MSC proliferation and the restoration of nuclear proteins Lamin A/C and Sun-2 after exposure to simulated microgravity in a LINC-complex dependent manner [136]. These findings suggest the nucleus is involved in the mechanocoupling of dynamic loading and furthermore, may even oscillate within the cytoplasm, thereby inducing fast, high amplitude loading on both the cytoskeleton and nucleus [138–140]. Moreover, recent work has revealed that lamin A expression scales with tissue stiffness and can regulate matrix elasticity-induced differentiation [48]. For example, low lamin A levels in stem cells on soft matrices can promote adipogenic differentiation whereas high levels of lamin A in stem cells on a stiff matrix improves differentiation into bone. These findings were attributed to increases in lamin A levels by tissue stiffness and stress that enabled nuclear stabilization and contributed to lineage specification [48]. A recent study provided further support for this concept as it was demonstrated that matrix stiffness can induce an increase in MSC contractility that tenses the nucleus to favor lamin A/C accumulation, resulting in a suppression of differentiation into an adipocyte lineage [141]. Together, these studies highlight the potential role of nuclear lamins and stiffness in stem cell maintenance and differentiation. Immune cells and immune responses. In a study that investigated the viscoelastic properties of human myeloid antigen presenting cells (APCs), it was demonstrated that different populations of APCs (e.g. dendritic cells (DCs), monocytes, and macrophages) exhibited distinct mechanical properties in their resting state, which were modified under inflammatory conditions and dependent on the actin content and myosin IIA activity [114]. In comparison to monocytes who had an elastic modulus of 580 + 30 Pa, macrophages were found to be stiffer (i.e. 930 + 40 Pa) whereas DCs were slightly less rigid (510 + 20 Pa). The application of different inflammatory cytokines, which induce myeloid APC phenotypic maturation, yielded opposite effects on the mechanical properties of myeloid APCs. For example, a cocktail of tumor necrosis factor-α and prostaglandin E2 induced a decrease in the rigidity of macrophages and DCs (650 + 30 Pa and 240 + 30 Pa, respectively) while interferon-γ increased cell stiffness in both cell types (i.e. 1460 + 80 Pa for macrophages and 830 + 40 Pa for DCs) [114]. On the other hand, exposure to lipopolysaccharide increased the viscoelastic properties of DCs but had no significant effect on macrophages, thus correlating with the higher sensitivity of DCs to LPS [114]. In addition, the effects of inflammation on the mechanical properties and the expression of maturation markers were found to be independent of one another. In the same study, it was also shown that human T lymphocytes were considerably less rigid than APCs (i.e. Young modulus of 85 + 5 Pa) [114]. This observation that T lymphocytes are softer can potentially be attributed to the expression level of nuclear proteins as it has been shown that resting T lymphocytes display negligible amounts of lamin A [142,143]. However, lamin A is important for T cell function as it is highly expressed during T cell receptor activation, and T cell response in vivo is impaired in mice lacking lamin A [143,144]. Another study utilized mass spectrometry-calibrated intracellular flow cytometry to quantify lamin-A:B ratios and revealed that lamin expression partitions hematopoiesis and thus, can be used as a parameter to distinguish blood cell types from human marrow and peripheral blood [115]. The lamin-A:B ratio, which regulates nuclear viscoelasticity and cell migration 9

potential, was found to be a good predictor of nuclear stiffness in hematopoietic phenotypes [115]. For example, erythroblast nuclei were stiffer due to a high lamin A:B ratio, causing cells to favor marrow retention and further promoting erythropoiesis. Similarly, human blood stem/progenitor cells that typically reside in the marrow display moderate lamin levels whereas cell types that travel into blood (e.g. white blood cells) have lower lamin levels [115]. These studies demonstrate that a wide variety of cell types can be characterized based on nuclear mechanical properties, suggesting a crucial role for these physical properties in cell function. When injury occurs, activation of inflammatory signaling recruits monocytes to enter tissue, usually near the site of injury, whereupon they differentiate into macrophages [145]. During this process, the nucleus generally becomes more rounded, and chromatin domains within monocytes are aggregated into clusters [146]. Once monocytes have fully differentiated into macrophages, the cell nucleus can undergo extensive deformation in response to environmental conditions [147]. During macrophage polarization from M1 (pro-inflammatory) to M2 (pro-healing) phenotype, macrophages undergo dramatic changes in cell shape (e.g. M2 cells exhibit a more elongated morphology) that are correlated with changes in actin organization [148,149]. More specifically, M1 macrophages have clustered F-actin structures that are typically found around the nuclei, whereas M2 macrophages display more diffused actin that is located towards the cell periphery [148]. Interestingly, controlling cell shape using biophysical cues altered macrophage phenotype from M1 to M2, in the absence of exogenous cytokines, and it was determined that the cytoskeleton plays a role in this shape-induced polarization [149]. As cytoskeletal changes are involved in macrophage polarization, actin dynamics are currently being targeted as a potential therapy to alter the phenotype of tumor-associated macrophages [150]. Furthermore, lamin A/C expression has also been shown to regulate macrophage and T cell responses through a coupling of the plasma membrane to the nucleus via the LINC complex and actin cytoskeleton, suggesting that it may be a key modulator of immune response [144,151]. Additionally, it is worth mentioning that throughout tissue damage to reconstruction, the nuclear lamina can behave as a mechanosensor and mechanotransducer to modulate inflammation and tissue specification. During inflammation, the nucleus is able to transduce cell swelling and lysis to initiate signaling that promotes an immune response. More specifically, F-actin and the nuclear lamina can regulate nuclear swelling-induced cytosolic phospholipase A2 activation, which generates pro-inflammatory eicosanoids that attract leukocytes to the sites of tissue damage [152]. Cell migration. The ability of cells to overcome the steric hindrance of the ECM is essential for migration during wound healing, tumor metastasis and foreign-body reaction. Accumulating evidence suggests that the nucleus is the main internal cause of hindrance in cell invasion and migration [153]. In addition, the nucleus can prevent cell migration when the crosssectional area of pores in the ECM is below a critical threshold, which corresponds to ~10% of the nuclear cross-sectional area [154]. Thus, modulating nuclear stiffness can potentially affect cell migration through the narrow pores. For example, increasing nuclear stiffness in cells by overexpressing lamin A results in impeded cell migration [155]. On the contrary, when nuclear stiffness is decreased by inhibiting lamin A/C expression, leukocytes can rapidly and efficiently migrate through narrow gaps [59]. Indeed, elucidating the role of nuclear stiffness in cell migration and invasion is a highly valuable for regenerative medicine. Recent evidence suggests that nuclear biophysical properties can serve as “biomarkers” for tissue diagnosis in pathology and potential therapeutic agents. Ermis et al. demonstrated that nuclear stiffness can be used as a physical parameter to evaluate 10

cancer cells based on their lineage and compared it with non-cancerous cells originating from the same tissue type [116]. Ali et al. trapped gold nanoparticles at the NE to increase nuclear stiffness and slowed cancer cell migration and invasion [156]. However, Zhang et al. found that mechano growth factor (MGF) promoted tenocyte migration by increasing nuclear stiffness [157]. In their study, MGF did not affect lamin A/C expression, but it clearly promoted chromatin condensation. As both lamin A/C and chromatin can contribute to the nuclear stiffness [47,52,53], this implies that an increase in nuclear stiffness by overexpression of lamin A/C or altering chromatin reorganization using small molecules could result in different behaviors during cell migration and invasion [158,159]. Although cells can secrete matrix metalloproteinases to cleave ECM fibers and create migration paths, it appears that nuclear stiffness and deformability are more important in determining whether a cell is able to pass through a “small pore” [155]. Many cancer cells and immune cells express lower levels of lamin A/C [114,115,160], which results in a more deformable nucleus that allows them to display a high degree of migration and invasion potential in order to pass through narrower pores and invade tissues. This phenomenon is correlated with tumor metastasis and immune response [159,161]. Additionally, when cancer cells overcome the ultimate steric hindrance of the ECM matrix and the nucleus undergoes a large degree of deformation, there is a transient opening in the NE [162,163]. During the envelope rupture, nucleo-cytoplasmic mixing can cause DNA damage that may further lead to aneuploidy and genomic instability, thereby promoting cancer progression. At the same time, the fast resealing of the nucleo-cytoplasmic barrier by endosomal sorting complexes further reduces apoptotic events and allows the cancer to progress [162–164]. It is becoming increasingly evident that mutations in LINC complex-associated proteins are correlated with various diseases, especially those resulting from lamin A mutations, which can induce diseases that are either highly tissue-specific, such as muscular dystrophy, or systemic (e.g. Hutchinson–Gilford progeria syndrome) [24]. In such cases, it has been shown that nuclear stiffness can be altered in these diseases. For example, the nucleus is stiff and fragile in Hutchinson–Gilford progeria syndrome, but unusually soft in Emery–Dreifuss muscular dystrophy [165–167]. In summary, nuclear mechanics regulates a variety of cell functions during physiological and pathological processes. Mechanobiology of the nucleus in cell engineering It is well-recognized that cells can sense and respond to mechanical and biochemical cues in the extracellular microenvironment, and these signals are transduced intracellularly to the nucleus either physically through the cytoskeleton and LINC complex and/or biochemically through the translocation of transcription factors [5,6]. Emerging evidence indicates that external mechanical forces can induce alterations in nuclear mechanics, chromatin organization, and gene expression [5,6,168], suggesting that these cues can ultimately regulate cellular behavior. Forces that elicit a mechano-response include those that are externally applied (e.g. mechanical compression or strain) or generated by biomaterials. Biomaterials provide biochemical and biophysical signals that can influence cell fate and function [1,169–171]. Biomaterial-mediated physical cues mainly consist of topography, stiffness/elasticity, ligand density/patterning and dimensionality. Herein, we will mainly focus on mechanical forces that are externally applied and generated by biomaterial-mediated physical cues, henceforth be referred to as biophysical factors/cues, and discuss their effect on the mechanobiology of the nucleus. Although it has been 11

well established that biophysical cues can regulate a variety of cellular processes, such as morphology [172], proliferation [173], migration [12,174] and differentiation [127,175–177], the exact mechanism by which these mechanical signals are transduced to the nucleus and regulate gene expression remains unknown. Moreover, whether these biophysical cues act directly on the nucleus or influence nuclear changes through downstream cytoplasmic mechanotransduction pathways has yet to be fully elucidated. More recent evidence suggests the nucleus can behave as a mechanosensor and there are currently several proposed mechanisms for nuclear mechanotransduction [5,6,30]. Mechanotransduction to the nucleus through transcription factors Biophysical factors can activate various mechanotransduction signaling pathways, including Ras homologous/Rho-associated protein kinase (Rho/ROCK) [178,179], myocardinrelated transcription factor-A (MRTF-A) [180,181], mitogen-activated protein kinase (MAPK) [182], wingless-INT [183], and Yes-associated protein/transcriptional coactivator with PDZbinding domain (YAP/TAZ) [184–186], to regulate cell function. Outside-in signals generated from biophysical cues are thought to travel from the cell surface through transmembrane proteins (e.g. integrins) that sense and transduce these signals to initiate signaling pathways that activate the small GTPase RhoA and its downstream effector, ROCK [4,14,187]. In turn, Rho-mediated signaling results in actin cytoskeletal remodeling and an increase in intracellular tension [14,187]. These alterations in actin assembly subsequently promote the translocation of transcription factors, such as YAP/TAZ and MRTF-A, from the cytoplasm into the nucleus through nuclear pore complexes to modulate gene expression and dictate cell behavior [85,188– 191]. For example, it has been reported that mechanical forces and matrix stiffness induces actin assembly to promote nuclear translocation of MRTF-A that consequently, activates the smooth muscle α-actin promoter, resulting in the activation of the myofibroblast differentiation program [192,193]. More information on the mechanoregulation of transcription factors can be found in several excellent reviews [194–197]. Interestingly, a recent study demonstrated that local stresses can be sensed by integrins, propagated through the actin cytoskeleton and transmitted across the nuclear lamina to stretch chromatin and upregulate transcription [95], demonstrating the effect of direct cytoskeleton-lamin-chromatin coupling on gene regulation in an open chromatin structure. Biophysical regulation of epigenetic state and cell functions The epigenetic state is the “memory” of cell identity that controls the on/off state of phenotypic genes [198,199]. It can be regulated by various mechanisms, including histone modifications, such as acetylation/deacetylation and methylation/demethylation of histone tails, DNA cytosine methylation and small non-coding RNAs, each of which modulates gene expression without altering DNA sequence [199,200]. Considering the physical link that exists between the cytoskeleton and the nucleus and the association of the nuclear lamina to chromatin, it may be possible that biophysical cues impart deformations onto cells that lead to changes in chromatin structure, organization or location. As a result, chromatin segments can reorganize into transcriptionally active or repressive regions, thereby modulating gene expression (e.g. activating or repressing genes) [201]. This chromatin remodeling process, which is highly dependent on histone modifications and chromatin condensation changes, is essential for many cellular processes, such as proliferation and differentiation [202–204]. As illustrated in Figure 3, 12

many studies have demonstrated that biophysical cues can regulate the epigenetic state to influence cell behavior and disease progression. Effects of cell adhesion substrates. Numerous studies have shown that biophysical cues, such as ligand micropatterning [205–207], micro- and nano-topography [208–211], matrix stiffness [212,213] and mechanical stretch [56,140], can induce changes in cell shape that lead to a subsequent change in nuclear shape. In turn, these nuclear deformations have been shown to alter the epigenetic state to regulate cell function. For example, biophysically-induced nuclear deformation can cause changes in chromatin condensation that influences cell proliferation. Utilizing micropatterned adhesive islands to independently control cell spreading and elongation, Roch-Sachs et al. determined that increases in cell spreading led to a larger nuclear volume, chromatin decondensation, increased DNA synthesis and lower proliferation rates [214]. On the other hand, Versaevel et al. showed that cell elongation decreased nuclear volume, resulting in drastic condensation of the chromatin and a reduction in cell proliferation [206]. Likewise, it has been reported that cell elongation directed by micro- or nanotopography can lead to a decrease in DNA synthesis and lower proliferation rates [210,215]. More recent evidence indicates that biophysical cues can also induce changes in chromatin organization [216–218]. Using high-resolution fluorescence anisotropy imaging, it has been found that the application of mechanical forces results in alterations to chromatin assembly, followed by nuclear deformation [217]. Interestingly, these events occurred before the translocation of MRTF-A into the nucleus was evident, suggesting that mechanical cues can temporally activate different events within cells. Recent work has also revealed that histone modifications are indirectly influenced by biophysical factors, which promote the activation of enzymes that interact with and modify histones. It has been shown that changes in cell shape and nuclear stiffness, investigated using fibronectin coated micropatterns, can alter gene expression by influencing the nuclear translocation rates of histone modifying enzymes (e.g. histone deacetylases (HDACs)) and transcription cofactors (i.e. MRTF-A) [207,218]. Interestingly, spatial confinement of macrophages using micropatterns to control for cell spreading induced changes in chromatin compaction and epigenetic modifications (i.e. HDAC3 levels and H3K36-dimethylation), which resulted in the suppression of M1 phenotype function [189]. It was determined the cell confinement downregulated lipopolysaccharide-induced actin polymerization, thereby reducing nuclear translocation of MRTF-A and MRTF-A-dependent transcription [189]. These findings suggest that macrophage confinement may play an important role in regulating the function of pro-inflammatory macrophages. In addition, microgrooves have also been shown to decrease HDAC activity in MSCs, which led to an increase in histone acetylation [208]. Further reduction in HDAC activity was observed when these substrates were stretched perpendicular to the orientation of the grooves. It was elucidated that lamin A/C is an important transducer in this process as partial knockdown of the protein inhibited epigenetic changes due to mechanotransduction of the loading conditions. The observation that mechanical stimulation can lead to cell phenotypic changes through epigenetic alterations has also been reported in studies where the effects of tensile and compressive forces have been investigated [139,140,219–221]. It has been demonstrated that cyclic strain can modulate various HDACs, resulting in inhibition of smooth muscle cell (SMC) migration [219]. Moreover, dynamic tensile loading can induce increases in chromatin condensation in MSCs [139,140]. These changes in chromatin condensation were mediated by acto-myosin based contractility, the activity of histone methyl-transferase EZH2, and ATP13

dependent purinergic signaling. It was further elucidated that the intensity and duration of the mechanical stimulus regulated the extent of chromatin condensation, which furthermore, resulted in altered nuclear mechanics (i.e. nuclear stiffening) and the establishment of a mechanical memory in MSCs [139]. Similarly, the application of compressive forces on mouse fibroblasts cultured on micropatterns induced changes in actomyosin contractility that led to chromatin condensation and alterations in the transcriptional response, which were found to be cell geometry-dependent [221]. It was determined that HDAC3 promoted the increase in heterochromatin content, and the observed changes in condensation were reversible upon removal of the stimulus. In order for mechanical forces to elicit changes in cell phenotype, there must be transmittance of such forces through mechanotransductive pathways. There is evidence that components of the LINC complex play an important role in force transmittance to the nucleus [136,220,222–224]. For example, LINC complexes are crucial for MSCs to sense low-magnitude mechanical signals as disruption of SUN or nesprin proteins using targeted short-interfering RNAs or overexpression of a dominant negative KASH domain prevents nuclear-actin cytoskeleton mechanocoupling and as a result, inhibits the transduction of these signals to repress adipogenic differentiation [224]. Similarly, it has been reported that knockdown of nesprin-1 in oligodendrocyte progenitors leads to impaired heterochromatin formation and prevents mechanical compression-induced differentiation, suggesting that LINC components are vital for the transduction of mechanical signals that stimulate oligodendrocyte progenitor differentiation into the myelinating phenotype [220], yet the specific isoform of nesprin-1 involved in mechanotransduction needs further elucidation. Moreover, upon sensing extracellular forces, cells generate tractional forces to tune their internal stiffness to match that of their substrate and adapt their morphology accordingly [9,12,225,226], thereby proposing a correlation between cell shape and stiffness. Indeed, several studies have reported that changes in cell shape, in some cases induced by biophysical factors, can modulate cell stiffness and mechanosensitivity [172,227,228]. The stiffness of cells has been shown to be a function of both the rigidity of the microenvironment and cell shape [227–230]. For instance, upon analyzing cellular rheology in adherent (i.e. flat morphology) versus cells in a suspended state, which are rounded, it was determined that cellular stiffness decreases when cells are in suspension (i.e. elastic constant ranged from 35-430 Pa for suspended cells versus 2.6-4.3 kPa for adherent cells) [227]. However, this may be cell type-specific as the opposite has also been observed, whereby the compliance of cells decreased in suspension [231,232]. Interestingly, inactivation of myosin II in adherent cells brought into suspension resulted in cell stiffening, suggesting that myosin activity softens cells in suspension and the mechanical property of the cells may not only be due stress-fiber generated pre-stress, but might also be related to other cytoskeleton components such microtubule assembly [233]. In addition, the mechanical responsiveness of cells can also differ depending on the cell morphology and rheological properties [172,227,232,234,235]. For example, osteocytes in suspension were significantly more responsive to force, as demonstrated through nitro oxide release, compared to adherent cells [227]. These findings suggest that cell morphology plays an important role in mechanosensing and may provide insights into the relationship between cell shape and mechanosensitivity in vivo. Further investigation into the involvement of nuclear mechanics and epigenetic changes in the rheology and mechanosensitivity of adherent versus non-adherent cells remains to be explored.

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Effects of fluid flow. In addition to the application of forces through the deformation of a substrate, extracellular forces that are derived by fluid flow, such as fluid shear stress, can also impose a biophysical cue on cells, which can be transduced to influence cell shape, the epigenetic state and thus, cell behavior [1,236,237]. The endothelium has been extensively used as a model to explore how fluid shear stress regulates the epigenetic state and cell function as mechanical forces are critical in the development and homeostasis of the cardiovascular system [238–240]. As alterations in physiological fluid flow patterns are correlated with the development and progression of vascular disorders, such as atherosclerosis, a number of studies have shown that changes in shear stress can alter endothelial cell (EC) phenotype through modifications of the epigenome [241,242]. For example, upon exposing ECs to varying fluid flow patterns, laminar fluid shear stress can promote a more accessible genome through enhanced acetylation whereas oscillatory fluid shear stress increases the expression of HDACs and DNA methyltransferase 1, which in turn, causes DNA hypermethylation and endothelial dysfunction [243–245]. Recent work has also demonstrated a role for histone methyltransferase EZH2 in regulating EC phenotype in response to fluid-mediated shear stresses [246,247]. It has been reported that laminar flow, in conjunction with the mechanosensitive miR-101, can reduce a repressive epigenetic mark (i.e. trimethylation of histone 3 lysine 27 –H3K27me3) and EZH2 to promote anti-inflammatory genes in ECs [246]. In the same study, IGFBP5 (insulin-like growth factor-binding protein 5) was identified as a novel mechanosensitive gene that can be modulated by H3K27me3. Interestingly, these epigenetic changes are relevant to therapeutic applications as atherosclerotic plaques from human patients exhibit upregulated EZH2 and downregulated IGFBP5. These important findings indicate that the EZH2/H3K27me3/IGFBP5 pathway may serve as a novel target for the treatment of inflammatory disorders such as atherosclerosis. Moreover, it has also been shown that shear stress can influence various epigenetic regulators to downregulate the expression of tissue metalloproteinase inhibitor in SMCs, resulting in matrix remodeling, altered integrin signaling, and cofilin-dependent actin cytoskeleton reorganization [248]. These findings provide some insights into how SMCs may respond to aberrant shear stress upon disruption of the protective endothelial layer, which can occur during medical procedures such as angioplasty [249]. Altogether, these studies provide a connection between biophysical cues and cell phenotypic changes through modulations of the epigenetic state. In most of these studies, the actin cytoskeleton, including nuclear and perinuclear actin, and the LINC complex played a crucial role in regulating the observed changes in histone modifications, chromatin condensation and organization. However, the exact mechanisms through which gene expression is controlled by biophysical factor-induced chromatin alterations await further investigation. Effects on cell reprogramming. Cell reprogramming, the reverse process of differentiation, enables the conversion of somatic cells into induced pluripotency or distinct cell types through various mechanisms including somatic cell nuclear transfer, cell fusion, ectopic expression of lineage transcription factors, and small molecule compounds [250–252]. Since the discovery that iPSCs can generated from fibroblasts using four transcription factors (OCT4, SOX2, C-MYC and KLF-4), it has been revealed that somatic cells undergo dynamic gene expression and epigenetic alterations during reprogramming [253–255]. Additionally, during iPSC conversion, fibroblasts, which have a mesenchymal phenotype, display morphological changes into epithelial-like cells, indicative of a mesenchymal-to-epithelial transition (MET), which has been determined to be critical for nuclear reprogramming [255–258]. As cell 15

reprogramming offers a promising approach for regenerative medicine, disease modeling, and drug discovery [259,260], many groups have sought out to investigate the role of biophysical factors in this process [170]. Interestingly, several studies have shown that biophysical cues can regulate iPSC reprogramming through epigenetic modulation. For example, it has been elucidated that topographical cues, in the form of parallel microgrooves and aligned nanofibers, can significantly enhance the reprogramming efficiency through mechanomodulation of the epigenetic state of fibroblasts [261]. More specifically, these cues were found to promote MET and iPSC reprogramming by eliciting changes in cell morphology that provoked an increase in histone acetylation and H3K4methylation, suggesting that topographical cues may result in a more permissive chromatin state for reprogramming. These findings further support a linkage of biophysical cues to the activity of specific histone modification enzymes. Moreover, the physical confinement of cells can promote cell shape-induced epigenetic changes that have an impact on cell reprogramming. It has been reported that mouse and human iPSC reprogramming efficiency could be improved by more than 2-fold in a 3D PEG-based hydrogel system. It was determined that the physical confinement of the 3D microenvironment exerted cell morphological changes that accelerated MET and increased acetylation and methylation of Histone 3 [262], which is in accordance with previous findings [261]. More recently, it has been revealed that micropatterned substrates can laterally confine the growth of fibroblasts leading to changes in the epigenetic state that promotes cell reprogramming into iPSC-like cells [263]. Intriguingly, this physically-induced cell conversion occurred in the absence of any exogenous reprogramming factors. Additionally, electromagnetic fields (EMFs) can have a significant effect on various cellular processes, including cell reprogramming [264– 268]. More specifically, extremely low-frequency EMFs regulate dynamic epigenetic changes through the activation of the histone lysine methyltransferase MII2 to promote efficient somatic cell reprogramming into pluripotency [267]. However, the mechanotransductive mechanisms that may be involved during EMF stimulation are not clear. Emerging evidence indicates that topographical cues can also regulate the epigenome to enhance direct reprogramming, a process by which cells do not proceed through a pluripotent state and thus, differs from iPSC reprogramming [269,270]. It has been demonstrated that microgrooves activated the translocation of transcription factor MRTF-A and increased histone acetylation to improve the yield of functional cardiomyocytes from mouse fibroblasts [271]. Similarly, nanogrooved substrates were shown to enhance the efficiency of converting mouse embryonic fibroblasts into induced dopaminergic neurons through an upregulation of MET gene expression and specific histone modifications (i.e. H3K4me3) [272]. In these direct conversion studies, micro- and nano-topographical cues were shown to direct and promote the derivation of mature cell phenotypes, which is highly valuable and more appropriate for disease modeling and drug screening as these cells may be more representative of the in vivo targets [270]. In several of the aforementioned studies [261,271], it was also discovered that biomaterial topography could replace small molecule epigenetic modifiers and significantly enhance the reprogramming efficiency, providing a safer and a more clinically relevant alternative for therapeutic purposes. In addition to topography, EMFs were recently shown to facilitate the direct conversion of somatic fibroblasts cultured on gold nanoparticles into induced dopaminergic neurons both in vitro and in vivo [268]. More importantly, it was shown that EMF exposure in the presence of gold nanoparticles and reprogramming factors was able to not only improve the efficiency of in vivo direct lineage reprogramming, but also restored dopaminergic function in various mouse Parkinson’s disease models. The mechanism that enabled the enhancement in the reprogramming 16

efficiency was due to an induction of histone acetyltransferase Brd2 that further led to H3K27 acetylation and neuronal gene activation [268]. As nanoparticles and EMFs are increasingly being utilized for the treatment of various disorders [273–276], it would be interesting to see whether this EMF/nanoparticle platform can be applied to other cell reprogramming paradigms and potentially serve as a novel therapeutic strategy. Effects on stem cell differentiation. Mounting evidence has revealed that during stem cell lineage specification, there is extensive reorganization of long-range chromatin contacts, in particular rearrangement of active and inactive chromatin compartments, that enable cells to undergo dynamic changes in gene expression, which are regulated by various epigenetic mechanisms [277,278]. Considerable progress has shown that biophysical cues can influence cell differentiation [1,169,279,280]. However, the epigenetic mechanisms that enable this biophysical regulation to occur remains limited. Several studies have reported that topography both on the micro and nano scale can modulate the epigenetic state of stem cells to regulate cell fate determination [129,210,281–283]. For example, parallel microgrooves can promote histone acetylation and myocardin sumoylation in order to enhance the directed differentiation of cardiac progenitors into cardiomyocytes [281]. It has also been demonstrated that titanium dioxide nanotubes promote osteogenic differentiation by inhibiting histone demethylase retinoblastoma protein 2 and increasing H3K4 methylation in localized promoter regions [282]. Apart from topography, mechanical forces (i.e. mechanical strain and compression) can also induce epigenetic changes that affect lineage commitment [220,284,285]. Compressive forces can promote oligodendrocyte progenitor differentiation through a redistribution of actin that is coupled with heterochromatin formation and dependent on Nesprin-1 [220]. In contrast, when cyclic mechanical strain was applied to epidermal stem cells, it induced changes in emerin localization, non-muscle myosin IIA activity and the actin cytoskeleton, resulting in transcriptional repression and chromatin compaction that influenced stem cell differentiation [284]. Fluid flow shear stress is another biophysical cue that has been implicated in cell lineage specification through epigenetic regulation. Laminar fluid shear stress can alter the epigenetic state to enhance lineage-specific markers and direct mouse ESCs to differentiate into cardiovascular cells [286]. On the other hand, oscillatory fluid flow, a physical cue that naturally occurs in the bone microenvironment and is regulated by bone loading, can induce osteogenic differentiation from MSCs through a reduction of DNA methylation on the promoter of osteogenic genes that consequently increases their expression [287]. These studies highlight how mechanical forces representative of those found in the in vivo microenvironment can influence cell differentiation into appropriate lineages. Micro-RNAs (miRNAs) [288,289] and long non-coding RNAs [290], which contain highly conserved noncoding RNAs, play an important role in regulating various cellular processes, such as proliferation [291], differentiation [291–293] and reprogramming [294,295], via pre- and post-transcriptional regulation of gene expression. For instance, large intergenic non-coding RNAs were demonstrated to have a critical function in modulating iPSC reprogramming [294]. Though the role of non-coding RNAs in cell-substrate interactions is not well understood, few studies have provided some insights into their function as they can be differentially expressed in response to biophysical cues, and in turn, regulate gene expression and cell behavior [248,296–298]. As an example, it has been shown that microtopography can modulate miRNA expression to guide cell proliferation and differentiation, respectively [297,298]. As the mechanical and physical inputs from the tissue microenvironment dictate cell 17

behavior through mechanotransduction mechanisms to maintain normal physiology, disruption of these cues can also lead to the initiation and development of a variety of diseases [2,3,299,300]. In addition to regulating tissue development and homeostasis in a mechanicallydependent manner, miRNA expression can also become dysregulated leading to diseases, such as cancer [301–304]. It has been reported that increased matrix stiffness can induce a mechanicallyregulated miRNA circuit that drives tumor progression and thus, can potentially be used as a prognosis for patients with luminal breast cancers [304]. A recent study has shed light on the influence of matrix stiffness on the epigenetic state of cancer stem cells, which play an important role in the initiation and progression of cancer [305]. It was determined that soft 3D fibrin matrices induce cell softening, H3K9 demethylation and Sox2 expression to promote the selfrenewal of melanoma tumor-initiating cells [305].

Future Directions We have sought to highlight the most recent developments of nuclear biomechanics and mechanobiology with a particular focus on how the mechanical properties of the nucleus and biophysical factor-induced epigenetic changes modulate cell behavior. It is well known that cell function is dependent on the stiffness of the matrix, yet how various cells define their sensitive range of stiffness is not clear. A previous study showed that when cells interact with substrates (e.g. matrices of varying stiffness), there is a sensible interaction window in which cells are able to sense and respond to changes in substrate modulus [306]. How matrix stiffness inside or outside this sensible interaction window will induce different mechanotransduction pathways to transduce the mechanical signal to the nucleus and elicit changes in the epigenetic state remains to be explored. To date, most mechanotransduction studies have utilized substrates (e.g. hydrogels) that are elastic and generally static [307]. However, the ECM is a dynamic environment that displays viscoelastic behavior (i.e. time-dependent response to deformation or mechanical loading), enabling cellular traction forces to remodel the ECM [308,309]. As such, this has motivated the development of novel dynamic, reversible hydrogels that exhibit elastic or viscoelastic properties, providing tunable platforms to study mechanotransduction in an elastic or viscoelastic context [308,309]. As recent work has begun to recognize the importance of viscoelasticity in cellular fate and behavior [310–314], further exploration into the influence of stress relaxation and creep on nuclear mechanics and mechanobiology will provide new insights into cellular mechanosensing. Additionally, cells undergo major epigenetic changes during cell differentiation and reprogramming, which might be due to or result in changes in the mechanical properties of the nucleus. Thus, it still remains to be investigated how nuclear stiffness is involved in cell lineage specification. Although there are abundant studies demonstrating that biophysical cues can induce phenotypic changes to alter cell function, the influence of these cues on many other epigenetic pathways such as non-coding RNAs and DNA methylation is not well known. Future research can include to determine whether these epigenetic regulatory mechanisms can be modulated by biophysical cues and whether they a play any role in the biophysical regulation of cell behavior. Additionally, various biomaterial properties have been shown to play a role in cell reprogramming and differentiation. However, the mechanistic details of how these biochemical and biophysical cues regulate the epigenetic state during these processes has not been fully elucidated. Future in-depth studies exploring this topic will contribute to our knowledge of how 18

microenvironmental cues influence mechanotransductive pathways, the epigenetic state and the consequent cell behavior. Significant advancements in next-generation sequencing approaches in recent years has enabled the development of novel genomic, transcriptomic, epigenomic, and proteomic technologies to perform single-cell analysis [315–317]. The integration of these technologies with high throughput methods has provided a high-resolution approach for understanding cell specification without the noise associated with a mixed cell population, and dissecting sample heterogeneity, in particular in tumors. Thus, findings from future studies investigating the biophysical regulation of the epigenetic state at the single-cell level will have important implications for basic biological research and clinical applications. Finally, advances in gene editing and epigenome editing tools such as the clustered regulatory interspaced short palindromic repeat (CRISPR)-Cas system enables site-specific epigenetic modifications. How the global or regional epigenetic effects of biophysical factors can be combined with site-specific molecular engineering approach to study nuclear mechanobiology remains to be explored in future studies. Furthermore, our current understanding of mechanotransduction mechanisms has coincided with the creation of innovative technologies to study this process [6,318]. For example, fluorescence resonance energy transfer (FRET)-based reporters have been engineered to monitor histone modification and chromatin condensation in living cells [319–322]. Recent studies have also used fluorescence lifetime imaging to quantify and spatially resolve the condensation of fluorescently-labeled chromatin based on chromatin viscosity [322,323]. Thus, further development of novel tools to study nuclear mechanotransduction will provide new insights into how mechanical signals are relayed to the nucleus and regulate gene expression. By gaining a deeper understanding of nuclear biomechanics and how biophysical cues can modify the epigenetic state to regulate cell fate and function, we can uncover important regulatory mechanisms for the development and prevention of disease. Author Contributions Statement YS, JS, BC and SL: wrote the manuscript. YS, JS, LY and SL: revised and edited the manuscript. Conflict of Interest Statement The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Acknowledgements The authors are supported in part by a grant from the National Institute of Health (HL121450, to S.L.), the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the NIH under the Ruth L. Kirschstein National Research Service Award (T32AR059033, to J.S.), UCLA Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research Innovation Award, and the National Nature Science Foundation of China (11532004, to Y.S.). The content is solely 19

the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health and other funding agencies.

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Figure Legends Figure 1. Elements of the cytoskeleton and nucleus involved in mechanotransduction. Mechanical forces from the extracellular environment are sensed and transduced by receptors (e.g. integrins) and transmitted across the cytoskeleton network (e.g. actin filaments, microtubules, and intermediate filaments) into the nucleus via the LINC (linker of the nucleoskeleton and cytoskeleton) complex, thereby impacting chromatin organization, gene expression and cell function. Nesprins at the outer nuclear membrane (ONM) interact with cytoskeletal elements whereas Sad1/UNC-84 (SUN) proteins at the inner nuclear membrane (INM) interact with the nuclear lamina and chromatin, enabling force transmission between the cytoskeleton and nucleus. ECM, extracellular matrix; NPC, nuclear pore complex; BAF, barrierto-autointegration factor; LAP2, lamina-associated polypeptide 2. Figure is not drawn to scale. Figure 2. Cell and nuclear mechanical measurement techniques. Various approaches can be utilized to measure cellular and nuclear mechanical properties, including micropipette aspiration, atomic force microscopy, optical tweezers and microfluidic platforms. Micropipette aspiration was the first technique developed to measure the elastic and viscoelastic properties of cells and nuclei. This method involves aspirating a nucleus into a micro-sized pipette and measuring the length of the nuclei extruded into it to determine the mechanical property. Atomic force microscopy, the most widely used method, can be used for mechanical testing and topological scanning using various cantilevers (with or without a tip). It enables precise nanoindentation measurements from which nuclear mechanical properties can be derived. In an optimal tweezer platform, two microbeads are attached to a nucleus where one is trapped by a highly-focused laser and other is adhered to the surface of a glass slide. In order to determine the mechanical properties, the nuclei is stretched by moving the glass slide. In contrast to these methods where one nucleus can be measured at a time, microfluidic-based platforms provide a high-throughput method for mechanical measurements. Microfluidic chips are advantageous in that they can be customized depending on the need. The mathematical models corresponding to each technique are depicted in the figure. Figure is not drawn to scale. Figure 3. Influence of biophysical cues on the epigenetic state and cell function. Biophysical factors in the microenvironment regulate the epigenetic state through mechanotransduction pathways to consequently, alter cell fate and behavior. Mitogen-activated protein kinase, MAPK; HMT, histone methyltransferase; HDMT, histone demethyltransferase; HAT, histone acetyltransferase; HDAC, histone deacetylase; DNMT, DNA methyltransferase; long non-coding RNAs, lncRNAs. Figure is not drawn to scale.

43

ECM

Focal adhesion Focal adhesion F-Actin F-Actin Intermediate Intermediate Filaments Filaments

Microtubules Microtubules Nesprin 1/2 Plectin Plectin Nesprin Nesprin Nesprin 33

Cytoplasm Cytoplasm

Nesprin Nesprin 44 NPC NPC

ONM Heterochromatin Heterochromatin

ONM ONM

Euchromatin Euchromatin INM INM

Integrins

Myosin

SUN

Dynein

Kinesin

Emerin

BAF

LAP2

Lamin A/C Lamin B1/2

Histone

Micropipette Aspiration

Atomic Force Microscope Photodiode

Micropipette

Laser Nucleus

Standard linear solid model

Nucleus Hertz model

• Original measurement method • Low throughput nuclear analysis

Optical Tweezers

Laser

• Precise indentation measurements • Low throughput nuclear analysis

Microfluidics Chip Shear stress

Microbead Nucleus Nucleus

Stretching

Maxwell model

• No physical contact with sample • Low throughput nuclear analysis

• Customizable chip design • High throughput nuclear analysis

Biophysical Cues Mechanical Forces

Topography Substrate Stiffness

Ligand density/patterning Micro/Nanostructures

Stretch Fluid shear stress

3D microenvironment

Geometry/Shape

F

F Signaling pathways (e.g. MAPK)

Focal adhesions

Myosin II

Nesprin Signaling pathways (e.g. MAPK)

RhoA

Sun F

Actin filament

F

LINC complex

MRTF-A

Nuclear pore complex

ROCK

Nuclear lamina

YAP/TAZ

Nuclear Mechanics

Epigenetic Remodeling

MRTF-A

YAP/TAZ

Histone modification

DNA methylation DNMT Nesprin

HMT, HDMT HAT, HDAC

Actin monomer

Sun Focal adhesions

Chromatin Myosin II remodeling Actin filament

Nuclear pore Non-coding complex RNAs LINC complex Nuclear lamina lncRNA

Gene Expression

miRNA Protein Expression

Nesprin Sun LINC complex

Focal adhesions

Cell Functions Nuclear pore complex

Myosin II

Migration Actin filament

Differentiation Proliferation

Reprogramming

Nuclear lamina

AUTHOR DECLARATION We the undersigned declare that this manuscript is original, has not been published before and is not currently being considered for publication elsewhere. We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). He/she is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from [email protected]. Signed by all authors as follows: [LIST AUTHORS AND DATED SIGNATURES ALONGSIDE]

Yang Song

Jennifer Soto

Date: 7/1/2019

____

_______________

Date: 7/1/2019

Binru Chen

Date: 7/1/2019

Li Yang

Date: 7/1/2019

Song Li

Date: 7/1/2019

Declaration of interests    ☒ The authors declare that they have no known competing financial interests or personal relationships  that could have appeared to influence the work reported in this paper.    ☐The authors declare the following financial interests/personal relationships which may be considered  as potential competing interests: