scholarly journals The effect of remodelling and contractility of the actin cytoskeleton on the shear resistance of single cells: a computational and experimental investigation

2012 ◽  
Vol 9 (77) ◽  
pp. 3469-3479 ◽  
Author(s):  
Enda P. Dowling ◽  
William Ronan ◽  
Gidon Ofek ◽  
Vikram S. Deshpande ◽  
Robert M. McMeeking ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Single Cells ◽  
Three Dimensional ◽  
Mechanical Stimuli ◽  
Indentation Curve ◽  
Modelling Framework ◽  
Two Factors ◽  
Hyperelastic Model ◽  
Linear Force

The biomechanisms that govern the response of chondrocytes to mechanical stimuli are poorly understood. In this study, a series of in vitro tests are performed, in which single chondrocytes are subjected to shear deformation by a horizontally moving probe. Dramatically different probe force–indentation curves are obtained for untreated cells and for cells in which the actin cytoskeleton has been disrupted. Untreated cells exhibit a rapid increase in force upon probe contact followed by yielding behaviour. Cells in which the contractile actin cytoskeleton was removed exhibit a linear force–indentation response. In order to investigate the mechanisms underlying this behaviour, a three-dimensional active modelling framework incorporating stress fibre (SF) remodelling and contractility is used to simulate the in vitro tests. Simulations reveal that the characteristic force–indentation curve observed for untreated chondrocytes occurs as a result of two factors: (i) yielding of SFs due to stretching of the cytoplasm near the probe and (ii) dissociation of SFs due to reduced cytoplasm tension at the front of the cell. In contrast, a passive hyperelastic model predicts a linear force–indentation curve similar to that observed for cells in which the actin cytoskeleton has been disrupted. This combined modelling–experimental study offers a novel insight into the role of the active contractility and remodelling of the actin cytoskeleton in the response of chondrocytes to mechanical loading.

Three-dimensional organization of actin cytoskeleton and podosomal contact structures in neoplastic cells in vitro

1998 ◽  
pp. 219-243
Author(s):  
Pavel Vesely ◽  
Luboslava Pavlikova ◽  
Jiri Plachy ◽  
Katerina Trejbalova ◽  
Jiri Hejnar ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Three Dimensional ◽  
Contact Structures ◽  
Neoplastic Cells

scholarly journals Mechanisms of endothelial cell coverage by pericytes: computational modelling of cell wrapping and in vitro experiments

2020 ◽  
Vol 17 (162) ◽  
pp. 20190739
Author(s):  
Kei Sugihara ◽  
Saori Sasaki ◽  
Akiyoshi Uemura ◽  
Satoru Kidoaki ◽  
Takashi Miura
Keyword(s):  
Cell Adhesion ◽  
Three Dimensional ◽  
Computational Modelling ◽  
Cellular Level ◽  
Cellular Potts Model ◽  
Contributing Factors ◽  
Modelling Framework ◽  
Cell Cell

Pericytes (PCs) wrap around endothelial cells (ECs) and perform diverse functions in physiological and pathological processes. Although molecular interactions between ECs and PCs have been extensively studied, the morphological processes at the cellular level and their underlying mechanisms have remained elusive. In this study, using a simple cellular Potts model, we explored the mechanisms for EC wrapping by PCs. Based on the observed in vitro cell wrapping in three-dimensional PC–EC coculture, the model identified four putative contributing factors: preferential adhesion of PCs to the extracellular matrix (ECM), strong cell–cell adhesion, PC surface softness and larger PC size. While cell–cell adhesion can contribute to the prevention of cell segregation and the degree of cell wrapping, it cannot determine the orientation of cell wrapping alone. While atomic force microscopy revealed that PCs have a larger Young’s modulus than ECs, the experimental analyses supported preferential ECM adhesion and size asymmetry. We also formulated the corresponding energy minimization problem and numerically solved this problem for specific cases. These results give biological insights into the role of PC–ECM adhesion in PC coverage. The modelling framework presented here should also be applicable to other cell wrapping phenomena observed in vivo .

scholarly journals Identification of Novel Recognition Motifs and Regulatory Targets for the Yeast Actin-regulating Kinase Prk1p

2003 ◽  
Vol 14 (12) ◽  
pp. 4871-4884 ◽  
Author(s):  
Bo Huang ◽  
Guisheng Zeng ◽  
Alvin Y.J. Ng ◽  
Mingjie Cai
Keyword(s):  
Actin Cytoskeleton ◽  
Phosphorylation Site ◽  
Three Dimensional ◽  
Kinase Domain ◽  
Yeast Genome ◽  
Three Dimensional Model ◽  
Recognition Sequence ◽  
Yeast Saccharomyces Cerevisiae

Prk1p is a serine/threonine kinase involved in the regulation of the actin cytoskeleton organization in the yeast Saccharomyces cerevisiae. Previously, we have identified LxxQxTG as the phosphorylation site of Prk1p. In this report, the recognition sequence for Prk1p is investigated more thoroughly. It is found that the presence of a hydrophobic residue at the position of P-5 is necessary for Prk1p phosphorylation and L, I, V, and M are all able to confer the phosphorylation at various efficiencies. The residue flexibility at P-2 has also been identified to include Q, N, T, and S. A homology-based three-dimensional model of the kinase domain of Prk1p provided some structural interpretations for these substrate specificities. The characterization of the [L/I/V/M]xx[Q/N/T/S]xTG motif led to the identification of a spectrum of potential targets for Prk1p from yeast genome. One of them, Scd5p, which contains three LxxTxTG motifs and is previously known to be important for endocytosis and actin organization, has been chosen to demonstrate its relationship with Prk1p. Phosphorylation of Scd5p by Prk1p at the three LxxTxTG motifs could be detected in vitro and in vivo, and deletion of PRK1 suppressed the defects in actin cytoskeleton and endocytosis in one of the scd5 mutants. These results allowed us to conclude that Scd5p is likely another regulatory target of Prk1p.

scholarly journals Optical Cellular Micromotion: A New Paradigm to Measure Tumour Cells Invasion in 3D Tumour Environments

2021 ◽  
Author(s):  
Zhaobin Guo ◽  
Chih-Tsung Yang ◽  
Chia-Chi Chien ◽  
Luke Selth ◽  
Pierre Bagnaninchi ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Invasion ◽  
Tumour Cell ◽  
Single Cells ◽  
Three Dimensional ◽  
Tumour Cells ◽  
Digital Holographic Microscopy ◽  
New Paradigm ◽  
Cell Invasiveness

Measuring tumour cell invasiveness through three-dimensional (3D) tissues, particularly at the single cell level, can provide important mechanistic understanding and assist in identifying therapeutic targets of tumour invasion. However, current experimental approaches, including standard in vitro invasion assays, have limited physiological relevance and offer insufficient insight about the vast heterogeneity in tumour cell migration through tissues. To address these issues, here we report on the concept of optical cellular micromotion, where digital holographic microscopy (DHM) is used to map the optical thickness fluctuations at sub-micron scale within single cells. These fluctuations are driven by the dynamic movement of subcellular structures including the cytoskeleton and inherently associated with the biological processes involved in cell invasion within tissues. We experimentally demonstrate that the optical cellular micromotion correlates with tumour cells motility and invasiveness both at the population and single cell levels. In addition, the optical cellular micromotion significantly reduced upon treatment with migrastatic drugs that inhibit tumour cell invasion. These results demonstrate that micromotion measurements can rapidly and non-invasively determine the invasive behaviour of single tumour cells within tissues, yielding a new and powerful tool to assess the efficacy of approaches targeting tumour cell invasiveness.

scholarly journals In Vitro Tissue Microarrays for Quick and Efficient Spheroid Characterization

2017 ◽  
Vol 23 (2) ◽  
pp. 211-217 ◽  
Author(s):  
D. P. Ivanov ◽  
A. M. Grabowska
Keyword(s):  
Single Cells ◽  
Three Dimensional ◽  
Tissue Microarrays ◽  
3D Models ◽  
Morphological Examination ◽  
Protein Targets ◽  
Cell Monolayers ◽  
Her 2 ◽  
Models Of Disease

Three-dimensional (3D) in vitro microphysiological cultures, such as spheroids and organoids, promise increased patient relevance and therapeutic predictivity compared with reductionist cell monolayers. However, high-throughput characterization techniques for 3D models are currently limited to simplistic live/dead assays. By sectioning and staining in vitro microtissues, researchers can examine their structure; detect DNA, RNA, and protein targets; and visualize them at the level of single cells. The morphological examination and immunochemistry staining for in vitro cultures has historically been done in a laborious manner involving testing one set of cultures at a time. We have developed a technology to rapidly screen spheroid phenotype and protein expression by arranging 66 spheroids in a gel array for paraffin embedding, sectioning, and immunohistochemsitry. The process is quick, mostly automatable, and uses 11 times less reagents than conventional techniques. Here we showcase the capabilities of the technique in an array made up of 11 different cell lines stained in conventional hematoxylin and eosin (H&E) staining, as well as immunohistochemistry staining for estrogen (ER), progesterone (PR), and human epidermal growth factor (Her-2) receptors, and TP53. This new methodology can be used in optimizing stem cell–based models of disease and development, for tissue engineering, safety screening, and efficacy screens in cancer research.

Tubulogenesis from isolated single cells of adult mammalian kidney: clonal analysis with a recombinant retrovirus

1996 ◽  
Vol 271 (1) ◽  
pp. F42-F49 ◽  
Author(s):  
H. D. Humes ◽  
J. C. Krauss ◽  
D. A. Cieslinski ◽  
A. J. Funke
Keyword(s):  
Single Cells ◽  
Three Dimensional ◽  
High Capacity ◽  
Cell Lineage ◽  
Growth Conditions ◽  
Rabbit Kidney ◽  
Adult Rabbit ◽  
Collagen Gels ◽  
Mammalian Kidney

The adult mammalian kidney tubule epithelium exists in a relatively dormant, slowly replicative state but has a large potential for regenerative morphogenesis following severe ischemic or toxic injury. Under selective serum-free growth conditions, which included epidermal growth factor and retinoic acid, a subpopulation of renal proximal tubule cells isolated from adult rabbit kidney were grown in cell culture. These cells possessed two important characteristics: 1) an ability to differentiate morphogenically into tubule structures when grown in three-dimensional collagen gels and 2) a high capacity for self-renewal, since cell lineage analysis with a recombinant retrovirus demonstrated that in vitro tubulogenesis arose from clonal expansion of a single cell. Thus individual cells in the adult kidney have retained the ability for kidney tubulogenesis in vitro.

Remodeling and Contractility of the Actin Cytoskeleton During the Shear Deformation of Single Chondrocytes

2011 ◽  
Author(s):  
Enda P. Dowling ◽  
William Ronan ◽  
Vikram S. Deshpande ◽  
Robert M. McMeeking ◽  
Kyriacos A. Athanasiou ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Actin Polymerization ◽  
Type Ii Collagen ◽  
Mechanical Stimuli ◽  
Type Ii ◽  
Cell Models ◽  
Predictive Capability ◽  
Computational Cell ◽  
External Loads

Numerous in vitro studies have demonstrated that chondrocytes react to mechanical stimuli. Compression of chondrocytes in an agarose gel induces disruption of the actin cytoskeleton [1]. In addition, chondrogenic gene expression decreases for chondrocytes cultured in monolayer, while inhibition of actin polymerization causes an increase in type II collagen and GAG production [2]. Despite such extensive in vitro investigations, the mechanisms by which chondrocytes actively respond to mechanical loading are not well understood. Simple hyperelastic and viscoelastic computational cell models have previously been used to model chondrocytes. However, such passive models ignore the key biomechanisms by which cells sense and react to external loads, and hence offer limited insight or predictive capability [3].

scholarly journals Bioprinting on 3D Printed Titanium Scaffolds for Periodontal Ligament Regeneration

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1337
Author(s):  
Ui-Lyong Lee ◽  
Seokhwan Yun ◽  
Hua-Lian Cao ◽  
Geunseon Ahn ◽  
Jin-Hyung Shim ◽  
...  
Keyword(s):  
Dental Implants ◽  
Periodontal Ligament ◽  
Defense Mechanisms ◽  
Animal Experiments ◽  
Three Dimensional ◽  
Artificial Organs ◽  
Artificial Organ ◽  
Mechanical Stimuli ◽  
3D Printed

The three-dimensional (3D) cell-printing technique has been identified as a new biofabrication platform because of its ability to locate living cells in pre-defined spatial locations with scaffolds and various growth factors. Osseointegrated dental implants have been regarded as very reliable and have long-term reliability. However, host defense mechanisms against infections and micro-movements have been known to be impaired around a dental implant because of the lack of a periodontal ligament. In this study, we fabricated a hybrid artificial organ with a periodontal ligament on the surface of titanium using 3D printing technology. CEMP-1, a known cementogenic factor, was enhanced in vitro. In animal experiments, when the hybrid artificial organ was transplanted to the calvarial defect model, it was observed that the amount of connective tissue increased. 3D-printed hybrid artificial organs can be used with dental implants, establishing physiological tooth functions, including the ability to react to mechanical stimuli and the ability to resist infections.

scholarly journals Limbal Niche Cells in Three-Dimensional Matrigel Induced Dedifferentiation of Mature Corneal Epithelial Cells toward a Progenitor State

2020 ◽  
Author(s):  
Hui Zhu ◽  
Wei Wang ◽  
Lingjuan Xu ◽  
Menglin Jiang ◽  
Yongyao Tan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Epithelial Cells ◽  
Single Cells ◽  
Three Dimensional ◽  
Stem Cell Marker ◽  
Epithelial Stem Cells ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial ◽  
Epithelial Marker

ABSTRACTPurposeTo investigate the possibility and the key factors of stably committed mature corneal epithelial cells dedifferentiate into corneal epithelial stem cells in vitro.MethodsMature cornea epithelia cell (MCEC) sheets or limbal epithelial progenitor cell (LEPC) sheets were isolated from central corneas or limbal segments by Dispase II and further digested with 0.25% trypsin/1 mM EDTA (T/E) to yield single cells. Limbal niche cells (LNC) were isolated from the limbal stroma by collagenase A and expanded on 5% Matrigel coated plastic. Single MCECs were seeded on 50% Matrigel with or without LNC culturing for 10 days, regarding as three-dimensional MCEC (3D-MCEC) group or three-dimensional MCEC+LNC (3D-MCEC+LNC) group. Expression of CK12, p63α, PCK, Vimentin were analyzed with immunofluorescence staining.ResultsThe expression of mature cornea epithelial marker (CK12) in MCEC was higher than that in LEPC (P=0.020) but epithelial stem cell marker (p63α) was lower than that in LEPC (P=0.000). When seeded in 3D Matrigel, single MCEC cells could form spheres within 72 hours, and the expression of CK12 reduced (P=0.005) and the expression of p63α also reduced to zero (P=0.000) compared to MCEC. Serial passages of LNC which were expanded in coated Matrigel could form spheres in 3D Matrigel. After mixing MCECs with LNC, rounder spheres emerged within 24 hours which consisted of both epithelia cells (PCK+/Vim-) and LNC (PCK-/Vim+). Moreover, epithelia cells in 3D-MCEC+LNC group expressed less CK12 and more p63α than those in MCEC group (P=0.043, 0.000). Besides, the diameter of spheres in 3D-MCEC+LNC group were larger than that in 3D-MCEC group (P=0.000).ConclusionHuman LNC and three-dimensional Matrigel could induce the dedifferentiation of mature corneal epithelial cells into corneal epithelial stem cells.

scholarly journals Quantification of nematic cell polarity in three-dimensional tissues

2020 ◽  
Vol 16 (12) ◽  
pp. e1008412
Author(s):  
André Scholich ◽  
Simon Syga ◽  
Hernán Morales-Navarrete ◽  
Fabián Segovia-Miranda ◽  
Hidenori Nonaka ◽  
...  
Keyword(s):  
Cell Polarity ◽  
Liver Tissue ◽  
Cell Biology ◽  
Single Cells ◽  
Orientational Order ◽  
Three Dimensional ◽  
Order Parameters ◽  
Imaging Data ◽  
Unique Type

How epithelial cells coordinate their polarity to form functional tissues is an open question in cell biology. Here, we characterize a unique type of polarity found in liver tissue, nematic cell polarity, which is different from vectorial cell polarity in simple, sheet-like epithelia. We propose a conceptual and algorithmic framework to characterize complex patterns of polarity proteins on the surface of a cell in terms of a multipole expansion. To rigorously quantify previously observed tissue-level patterns of nematic cell polarity (Morales-Navarrete et al., eLife 2019), we introduce the concept of co-orientational order parameters, which generalize the known biaxial order parameters of the theory of liquid crystals. Applying these concepts to three-dimensional reconstructions of single cells from high-resolution imaging data of mouse liver tissue, we show that the axes of nematic cell polarity of hepatocytes exhibit local coordination and are aligned with the biaxially anisotropic sinusoidal network for blood transport. Our study characterizes liver tissue as a biological example of a biaxial liquid crystal. The general methodology developed here could be applied to other tissues and in-vitro organoids.

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