Anchorage of Vinculin to Lipid Membranes Influences Cell Mechanical Properties

The focal adhesion protein vinculin (1066 residues) can be separated into a 95-kDa head and a 30-kDa tail domain. Vinculin's lipid binding sites localized on the tail, helix 3 (residues 944-978) and the unstructured C-terminal arm (residues 1052-1066, the so-called lipid anchor), influence focal adhesion turnover and are important for cell migration and adhesion. Using magnetic tweezers, we characterized the cell mechanical behavior in mouse embryonic fibroblast (MEF)-vin(−/−) cells transfected with EGFP-linked-vinculin deficient of the lipid anchor (vinΔC, residues 1-1051). MEF-vinΔC cells incubated with fibronectin-coated paramagnetic beads were less stiff, and more beads detached during these experiments compared to MEF-rescue cells. Cells expressing vinΔC formed fewer focal contacts as determined by confocal microscopy. Two-dimensional traction measurements showed that MEF-vinΔC cells generate less force compared to rescue cells. Attenuated traction forces were also found in cells that expressed vinculin with point mutations (R1060 and K1061 to Q) of the lipid anchor that impaired lipid binding. However, traction generation was not diminished in cells that expressed vinculin with impaired lipid binding caused by point mutations on helix 3. Mutating the src-phosphorylation site (Y1065 to F) resulted in reduced traction generation. These observations show that both the lipid binding and the src-phosphorylation of vinculin's C-terminus are important for cell mechanical behavior.     

Preparation,Mechanical and Biological Properties of Inkjet Printed Alginate/Gelatin Hydrogel

3D printing has made remarkable progress in soft tissue reconstruction enabling the custom design of complex material implants with patient specific geometry.Th...     

Growth Factor Stimulation Improves the Structure and Properties of Scaffold-Free Engineered Auricular Cartilage Constructs

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.     

Characterization of Engineered Channelrhodopsin Variants with Improved Properties and Kinetics

Channelrhodopsin 2 (ChR2), a light-activated nonselective cationic channel from Chlamydomonas reinhardtii, has become a useful tool to excite neurons into which it is transfected. The other ChR from Chlamydomonas, ChR1, has attracted less attention because of its proton-selective permeability. By making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis, we developed a ChR variant, named ChEF, that exhibits significantly less inactivation during persistent light stimulation. ChEF undergoes only 33% inactivation, compared with 77% for ChR2. Point mutation of Ile¹⁷⁰ of ChEF to Val (yielding “ChIEF”) accelerates the rate of channel closure while retaining reduced inactivation, leading to more consistent responses when stimulated above 25 Hz in both HEK293 cells and cultured hippocampal neurons. In addition, these variants have altered spectral responses, light sensitivity, and channel selectivity. ChEF and ChIEF allow more precise temporal control of depolarization, and can induce action potential trains that more closely resemble natural spiking patterns.     

Novel Tag-and-Exchange (RMCE) Strategies Generate Master Cell Clones with Predictable and Stable Transgene Expression Properties

Site-specific recombinases have revolutionized the systematic generation of transgenic cell lines and embryonic stem cells/animals and will ultimately also reveal their potential in the genetic modification of induced pluripotent stem cells. Introduced in 1994, our Flp recombinase-mediated cassette exchange strategy permits the exchange of a target cassette for a cassette with the gene of interest, introduced as a part of an exchange vector. The process is “clean” in the sense that it does not co-introduce prokaryotic vector parts; neither does it leave behind a selection marker. Stringent selection principles provide master cell lines permitting subsequent recombinase-mediated cassette exchange cycles in the absence of a drug selection and with a considerable efficiency (∼ 10%). Exemplified by Chinese hamster ovary cells, the strategy proves to be successful even for cell lines with an unstable genotype.     

Microwave-Assisted Growth of Silver Nanoparticle Films with Tunable Plasmon Properties and Asymmetrical Particle Geometry for Applications as Radiation Sensors

We report a simple and fast microwave-assisted method to grow silver nanoparticle films with tunable plasmon resonance band. Microwaving time controls nucleation and growth as well as particle agglomeration, cluster formation, particle morphology, and the plasmonic properties. Films produced with times shorter than 30 s presented a single well-defined plasmon resonance band (~ 400 nm), whereas films produced with times longer than 40 s presented higher wavelength resonances modes (> 500 nm). Plasmon band position and intensity can be easily tuned by controlling microwaving time and power. SEM and AFM images suggested the growth of asymmetrical silver nanoparticles. Simulated extinction spectra considering particles as spheres, hemispheres, and spherical caps were performed. The films were employed to enhance the sensitivity of ionizing radiation detectors assessed by optically stimulated luminescence (OSL) via plasmon-enhanced luminescence. By tuning the plasmon resonance band to overlap with the OSL stimulation (530 nm), luminescence enhancements of greater than 100-fold were obtained, demonstrating the importance of tuning the plasmon resonance band to maximize the OSL intensity and detector sensitivity. This versatile method to produce silver nanoparticle films with tunable plasmonic properties is a promising platform for developing small-sized radiation detectors and advanced sensing technologies.

Graphical Abstract

     

Auxin- and Acid-Induced Changes in the Mechanical Properties of the Cell Wall

Auxin- and acid-induced changes in the mechanical propertiesof the cell wall were analyzed by measuring the creep of thecell wall using pumpkin (Cucurbita moschata Duch cv. Shirakikuza)hypocotyl segments. Hypocotyl segments were treated with orwithout IAA and stored in 50% glycerol at –15°C formore than 2 weeks before measurements. Creep rate increasedwith the increase in the load. The increase was first very slowup to the phase shift point (yield threshold, y), and afterthat, it was steep. The rate of the creep rate increase (creepcoefficient, Cm) was larger and y was smaller at pH 4.5 thanpH 6.8. This indicates cell wall loosening was facilitated underacidic conditions. IAA-pretreatment of the segments resultedin the lowering of y at pH 6.8 only. Around pH 5 and 45°C,Cm was highest and y was lowest. Boiling in distilled wateralmost lost the differential effect of Cm and y on pH and IAA.The differential effect of pH on Cm and y were recovered bythe addition of a crude extract of cell wall-bound proteins.It is implied that some enzymatic processes are involved inthe control of acid-induced cell wall extension. ¹Present address: Nihon Shokuhin Kako Co., Ltd. 30 Tajima, Fuji-City,Shizuoka, 417-8530 Japan. ²Present address: Graduate School of Environmental Earth Science,Hokkaido University, Sapporo, 060 Japan.     

CNS Cell Distribution and Axon Orientation Determine Local Spinal Cord Mechanical Properties

Mechanical signaling plays an important role in cell physiology and pathology. Many cell types, including neurons and glial cells, respond to the mechanical properties of their environment. Yet, for spinal cord tissue, data on tissue stiffness are sparse. To investigate the regional and direction-dependent mechanical properties of spinal cord tissue at a spatial resolution relevant to individual cells, we conducted atomic force microscopy (AFM) indentation and tensile measurements on acutely isolated mouse spinal cord tissue sectioned along the three major anatomical planes, and correlated local mechanical properties with the underlying cellular structures. Stiffness maps revealed that gray matter is significantly stiffer than white matter irrespective of directionality (transverse, coronal, and sagittal planes) and force direction (compression or tension) (K_g= ∼130 Pa vs. K_w= ∼70 Pa); both matters stiffened with increasing strain. When all data were pooled for each plane, gray matter behaved like an isotropic material under compression; however, subregions of the gray matter were rather heterogeneous and anisotropic. For example, in sagittal sections the dorsal horn was significantly stiffer than the ventral horn. In contrast, white matter behaved transversely isotropic, with the elastic stiffness along the craniocaudal (i.e., longitudinal) axis being lower than perpendicular to it. The stiffness distributions we found under compression strongly correlated with the orientation of axons, the areas of cell nuclei, and cellular in plane proximity. Based on these morphological parameters, we developed a phenomenological model to estimate local mechanical properties of central nervous system (CNS) tissue. Our study may thus ultimately help predicting local tissue stiffness, and hence cell behavior in response to mechanical signaling under physiological and pathological conditions, purely based on histological data.     

A Novel Multifunctional Crosslinking PVA/CMCS Hydrogel Containing Cyclic Peptide Actinomycin X2 and PA@Fe with Excellent Antibacterial and Commendable Mechanical Properties

Bacterial infected environments and resulting bacterial infections have been threatening the human health globally. Due to increased bacterial resistance caused by improper and excessive use of antibiotics, antibacterial biomaterials are being developed as alternatives to antibiotics in some cases. Herein, an advanced multifunctional hydrogel with excellent antibacterial properties, enhanced mechanical properties, biocompatibility and self-healing performance, was designed through freezing-thawing method. This hydrogel network is composed of polyvinyl alcohol (PVA), carboxymethyl chitosan (CMCS), protocatechualdehyde (PA), ferric iron (Fe) and an antimicrobial cyclic peptide actinomycin X2 (Ac.X2). The double dynamic bonds among protocatechualdehyde (PA), ferric iron (Fe) and carboxymethyl chitosan containing coordinate bond (catechol-Fe) as well as dynamic Schiff base bonds and hydrogen bonds endowed the hydrogel with enhanced mechanical properties. Successful formation of hydrogel was confirmed through ATR-IR and XRD, and structural evaluation through SEM analysis, whereas mechanical properties were tested with electromechanical universal testing machine. The resulting PVA/CMCS/Ac.X2/PA@Fe (PCXPA) hydrogel has favorable biocompatibility and excellent broad-spectrum antimicrobial activity against both S. aureus (95.3 %) and E. coli (90.2 %) compared with free-soluble Ac.X2, which exhibited subpar performance against E. coli reported in our previous studies. This work provides a new insight on preparing multifunctional hydrogels containing antimicrobial peptides as antibacterial material.     

Effect of Bioglass on Growth and Biomineralization of SaOS-2 Cells in Hydrogel after 3D Cell Bioprinting

We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related SaOS-2 cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP•Ca²⁺-complex, or silica, or as biosilica that had been enzymatically prepared from ortho-silicate by silicatein. These hydrogels, together with SaOS-2 cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO₂∶CaO∶P₂O₅ of 55∶40∶5, did not affect the growth of the encapsulated cells. If silica, biosilica, or polyP•Ca²⁺-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped SaOS-2 cells to mineralize. If compared with the extent of the cells to form mineral deposits in the absence of bioglass, the cells exposed to bioglass together with 100 µmoles/L polyP•Ca²⁺-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP•Ca²⁺-complex and 50 µmoles/L biosilica) from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by SaOS-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted SaOS-2 cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass, provide a suitable strategy for the fabrication of morphogenetically active and biodegradable implants.     

Development of Polydimethylsiloxane Substrates with Tunable Elastic Modulus to Study Cell Mechanobiology in Muscle and Nerve

Mechanics is an important component in the regulation of cell shape, proliferation, migration and differentiation during normal homeostasis and disease states. Biomaterials that match the elastic modulus of soft tissues have been effective for studying this cell mechanobiology, but improvements are needed in order to investigate a wider range of physicochemical properties in a controlled manner. We hypothesized that polydimethylsiloxane (PDMS) blends could be used as the basis of a tunable system where the elastic modulus could be adjusted to match most types of soft tissue. To test this we formulated blends of two commercially available PDMS types, Sylgard 527 and Sylgard 184, which enabled us to fabricate substrates with an elastic modulus anywhere from 5 kPa up to 1.72 MPa. This is a three order-of-magnitude range of tunability, exceeding what is possible with other hydrogel and PDMS systems. Uniquely, the elastic modulus can be controlled independently of other materials properties including surface roughness, surface energy and the ability to functionalize the surface by protein adsorption and microcontact printing. For biological validation, PC12 (neuronal inducible-pheochromocytoma cell line) and C2C12 (muscle cell line) were used to demonstrate that these PDMS formulations support cell attachment and growth and that these substrates can be used to probe the mechanosensitivity of various cellular processes including neurite extension and muscle differentiation.     

单细胞机械性能检测方法与应用研究进展

细胞机械性能与细胞的生理状态与功能存在密切联系。早期对于细胞机械性能的研究受制于技术条件,只能获得细胞群的弹性或剪切模量,使得少量异质细胞的机械表型被淹没。近年来,单细胞机械性能检测技术得到了蓬勃发展。原子力显微镜、微吸管技术、光镊与光学拉伸、磁扭转流变仪与磁镊等单细胞机械性能检测技术展现出非常高的检测精度,但检测通量相对较低。新型微流控高通量检测方法的出现使检测通量呈几何式增长,有望解决大样本快速检测的需求。本文首先综述原子力显微镜、微吸管、光镊与光学拉伸和磁扭转流变仪与磁镊等单细胞机械性能检测技术。在此基础上,重点介绍细胞过孔、剪切诱导细胞变形和拉伸诱导细胞变形3种新兴微流控高通量检测技术的工作原理及最新研究进展,探讨各类方法的优缺点。最后,本文展望单细胞机械性能检测技术的未来发展方向。     

Chronic Exposure to Combined Carcinogens Enhances Breast Cell Carcinogenesis with Mesenchymal and Stem-Like Cell Properties

Breast cancer is the most common type of cancer affecting women in North America and Europe. More than 85% of breast cancers are sporadic and attributable to long-term exposure to small quantities of multiple carcinogens. To understand how multiple carcinogens act together to induce cellular carcinogenesis, we studied the activity of environmental carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (B[a]P), and dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) using our breast cell carcinogenesis model. Our study revealed, for the first time, that combined NNK and B[a]P enhanced breast cell carcinogenesis chronically induced by PhIP in both non-cancerous and cancerous breast cells. Co-exposure was more potent than sequential exposure to combined NNK and B[a]P followed by PhIP in inducing carcinogenesis. Initiation of carcinogenesis was measured by transient endpoints induced in a single exposure, while progression of carcinogenesis was measured by acquisition of constitutive endpoints in cumulative exposures. Transient endpoints included DNA damage, Ras-Erk-Nox pathway activation, reactive oxygen species elevation, and increased cellular proliferation. Constitutive endpoints included various cancer-associated properties and signaling modulators, as well as enrichment of cancer stem-like cell population and activation of the epithelial-to-mesenchymal transition program. Using transient and constitutive endpoints as targets, we detected that a combination of the green tea catechins ECG and EGCG, at non-cytotoxic levels, was more effective than individual agents in intervention of cellular carcinogenesis induced by combined NNK, B[a]P, and PhIP. Thus, use of combined ECG and EGCG should be seriously considered for early intervention of breast cell carcinogenesis associated with long-term exposure to environmental and dietary carcinogens.     

3D Fluid-Structure Interaction Canine Heart Model with Patch to Quantify Mechanical Conditions for Optimal Myocardium Stem Cell Growth and Tissue Regeneration

Right ventricular (RV) dysfunction is a common cause of heart failure in patients with congenital heart defects and often leads to impaired functional capacity and premature death. Myocardial tissue regeneration techniques are being developed for the potential that viable myocardium may be regenerated to replace scar tissues in the heart or used as patch material in heart surgery. 3D computational RV/LV/Patch models with fluid-structure interactions (FSI) were constructed based on data from a healthy dog heart to obtain local fluid dynamics and structural stress/strain information and identify optimal conditions under which tissue regeneration techniques could achieve best outcome. RV/LV/Patch geometry and blood pressure data were obtained from a dog following established procedures. Four FSI models were used to quantify the influence of different patch materials (Dacron scaffold, treated pericardium) on local environment around the patch area, especially focusing on the thickness and stiffness of the patch. Our results indicated that changes in patch stiffness had little impact on the ejection fraction of the right ventricle because the total patch area was small. However, patch stiffness had huge impact on local RV maximum principal stress (Stress-P1) and strain (Strain-P1) around the patch area. Compared to the no-patch model, patch models had increased Stress-P1 and decreased Strain-P1 values in the patch area. Softer patches were associated with greater stress/strain variations. Thinner patch led to complex local flow environment which may have impact on myocytes seeding and RV remodeling. Our multi-physics RV/LV/Patch FSI model can serve as a useful tool to investigate cellular biology and tissue regeneration under localized flow and structural stress environment.     

Cell Growth and the Structure and Mechanical Properties of the Wall in Internodal Cells of Nitella opaca: I. WALL STRUCTURE AND GROWTH

This is the first of two papers dealing with the relationshipbetween growth and the mechanical properties of the wall ininternodal cells of Nitella opaca L. The submicroscopic structure of the cell wall of this alga,as determined by chemical analysis, X-ray crystallography, polarizingmicroscopy, electron microscopy, swelling measurements, andinfra-red spectrography, is described in detail and the changesduring growth are recorded. It has been found that the wallcontains cellulose in the form of cellulose I (type B). Theconstituent microfibrils are preferentially oriented, usuallyin slow helices with considerable angular dispersion about thecommon direction. They are arranged in discrete layers withpectic substances providing an amorphous matrix between microfibrillar-reinforcedlaminations. It is shown that, as the cell elongates, both thestreaming direction in the cell and the mean microfibrillarorientation in the wall change in such a way as to allow thepossibility of a causal connexion between streaming and microfibrillarorientation in a new wall lamella. The orientation in such alamella is undoubtedly modified by subsequent passive extensionmuch as implied in the multi-net growth hypothesis of Roelofsen.     

Structural and Functional Properties of Platelet-Derived Growth Factor and Stem Cell Factor Receptors

The receptors for platelet-derived growth factor (PDGF) and stem cell factor (SCF) are members of the type III class of PTK receptors, which are characterized by five Ig-like domains extracellularly and a split kinase domain intracellularly. The receptors are activated by ligand-induced dimerization, leading to autophosphorylation on specific tyrosine residues. Thereby the kinase activities of the receptors are activated and docking sites for downstream SH2 domain signal transduction molecules are created; activation of these pathways promotes cell growth, survival, and migration. These receptors mediate important signals during the embryonal development, and control tissue homeostasis in the adult. Their overactivity is seen in malignancies and other diseases involving excessive cell proliferation, such as atherosclerosis and fibrotic diseases. In cancer, mutations of PDGF and SCF receptors—including gene fusions, point mutations, and amplifications—drive subpopulations of certain malignancies, such as gastrointestinal stromal tumors, chronic myelomonocytic leukemia, hypereosinophilic syndrome, glioblastoma, acute myeloid leukemia, mastocytosis, and melanoma.The type III tyrosine kinase receptor family consists of platelet-derived growth factor (PDGF) receptor α and β, stem cell factor (SCF) receptor (Kit), colony-stimulating factor-1 (CSF-1) receptor, and Flt-3 (Blume-Jensen and Hunter 2001). Members of this receptor family are characterized by five Ig-like domains in their extracellular part, a single transmembrane domain, and an intracellular part consisting of a rather well-conserved juxtamembrane domain, a tyrosine kinase domain with a characteristic inserted sequence without homology with kinases, and a less well-conserved carboxy-terminal tail. The ligands for these receptors are all dimeric molecules, and on binding they induce receptor dimerization. Although the overall mechanisms for the activation of the type III tyrosine kinase receptors and the signaling pathways they induce are similar, the receptors are expressed on different cell types and thus have different functions in vivo.Here we will describe the structural and functional properties of the PDGF receptors and Kit.     

Cell Walls of Seed Hairs fromLygeum spartum: Ultrastructure, Composition and Mechanical Properties

The silky hairs covering the whole surface ofLygeum spartumdispersalunits are unicellular, unlignified epidermal structures whoselength varies from 5 to 17 mm. The cell walls present a stratifiedtexture after extraction of matrix material. Their compositiondepends on the geographical origin of the grasses. Polysaccharidesextracted with boiling water represent approx. 18% of the cellwall dry matter in samples harvested in Algerian arid high-plateausand only 3% in samples harvested in semi-arid coastal zones.In the latter case, hemicellulose and cellulose contents amountedto approx. 50 and 40%, respectively. Slightly lower values werefound in high-plateau samples. Cinnamic acids were detectedonly in coastal region specimens which exhibited a higher mechanicalresistance and a lower extensibility. These results supportthe existence of two distinct populations colonizing arid orsemi-arid areas respectively.Copyright 1998 Annals of BotanyCompany Cell wall; extensibility; polysaccharides;Lygeum spartum; seed hairs.     

Differential effect of Calcium and Magnesium on Mechanical Properties of Pea Stem Cell Walls

Replacement of calcium ion with magnesium ion in the cell wallof the pea epicotyl makes the wall more extensible. A possiblerole of this differential effect of Ca²⁺ and Mg^2– in regulatingcell elongation in pea epicotyl is discussed. The ratio of the content of calcium ion to that of magnesiumion (Ca²⁺/Mg²⁺) in the walls decreased markedly in the orderof the first > the second > the third internodes of thepea epicotyl. The capacity of the walls for cation exchangeincreased in the same order, whereas the calcium-magnesium ionexchange selectivity of the walls was virtually constant. Ourresults indicate that the changes in the Ca²⁺/Mg²⁺ ratio amongthe internodes is not attributable to the ion exchange propertiesof the walk per se, but is due to other physiological conditionswhich regulate the activities of free calcium and magnesiumions in the environment with which the walls are in equilibrium. ¹ Present address: Wakayama Research Laboratories, Kao SoapCo., Ltd., Wakayama 640-91, Japan ² Present address: Foold Development Laboratories, Meiji SeikaKaisha Ltd., Kawasaki 210, Japan (Received May 1, 1981; Accepted August 26, 1981)     

Cell Growth and the Structure and Mechanical Properties of the Wall in Internodal Cells of Nitella opaca: II. MECHANICAL PROPERTIES OF THE WALLS

The internodal cells of Nitella opaca L. have been used in anattempt to assess the part which mechanical properties of thewall may play in the control of cell growth. It is shown thatthe wall is mechanically anisotropic in both its plastic andelastic properties, and evidence is presented which indicatesthat this arises from its anisotropy of structure. The degreeof anisotropy is greater in cells with a high growth-rate thanin those with a low growth-rate. Evidence is presented thatthis variation in properties with growth-rate is due wholly,or in part, to changes in the orientation of the crystallinecomponent, in the relative proportion of wall constituents,and in the condition of active groups of the wall components.The findings are in harmony with the theory that extension growthof the cell wall is due to ‘creep’, i.e. disturbancesof the molecular forces within the wall leading to a slow plasticyielding to turgor pressure.     

Biophysical Assays to Probe the Mechanical Properties of the Interphase Cell Nucleus: Substrate Strain Application and Microneedle Manipulation

In most eukaryotic cells, the nucleus is the largest organelle and is typically 2 to 10 times stiffer than the surrounding cytoskeleton; consequently, the physical properties of the nucleus contribute significantly to the overall biomechanical behavior of cells under physiological and pathological conditions. For example, in migrating neutrophils and invading cancer cells, nuclear stiffness can pose a major obstacle during extravasation or passage through narrow spaces within tissues.¹ On the other hand, the nucleus of cells in mechanically active tissue such as muscle requires sufficient structural support to withstand repetitive mechanical stress. Importantly, the nucleus is tightly integrated into the cellular architecture; it is physically connected to the surrounding cytoskeleton, which is a critical requirement for the intracellular movement and positioning of the nucleus, for example, in polarized cells, synaptic nuclei at neuromuscular junctions, or in migrating cells.² Not surprisingly, mutations in nuclear envelope proteins such as lamins and nesprins, which play a critical role in determining nuclear stiffness and nucleo-cytoskeletal coupling, have been shown recently to result in a number of human diseases, including Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy, and dilated cardiomyopathy.³ To investigate the biophysical function of diverse nuclear envelope proteins and the effect of specific mutations, we have developed experimental methods to study the physical properties of the nucleus in single, living cells subjected to global or localized mechanical perturbation. Measuring induced nuclear deformations in response to precisely applied substrate strain application yields important information on the deformability of the nucleus and allows quantitative comparison between different mutations or cell lines deficient for specific nuclear envelope proteins. Localized cytoskeletal strain application with a microneedle is used to complement this assay and can yield additional information on intracellular force transmission between the nucleus and the cytoskeleton. Studying nuclear mechanics in intact living cells preserves the normal intracellular architecture and avoids potential artifacts that can arise when working with isolated nuclei. Furthermore, substrate strain application presents a good model for the physiological stress experienced by cells in muscle or other tissues (e.g., vascular smooth muscle cells exposed to vessel strain). Lastly, while these tools have been developed primarily to study nuclear mechanics, they can also be applied to investigate the function of cytoskeletal proteins and mechanotransduction signaling. Download video file.^{(105M, mov)}