Probing Cytoskeletal Mechanics Using Biochemical Inhibitors

Quantifying the mechanics of the cytoskeletons of living cells is important for understanding several physiologic and pathologic cellular functions, such as wound healing and cellular migration in cancer. Our laboratory develops three-dimensional tissue constructs for assaying cytoskeletal mechanics in controlled conditions. These tissue constructs consist of defined components such as chick embryo fibroblasts and reconstituted rat tail collagen; fibroblasts remodel the collagen extracellular matrix (ECM) and develop a structural environment representative of that which would exist in a natural tissue. Our protocol for quantifying the microscale mechanics of the proteins that comprise the cytoskeleton involves mechanical testing of a tissue construct first in a bath that contains nutrition medium to support the active physiologic functioning of the cells, and next in the presence of inhibitors that selectively eliminate specific cytoskeletal structures. By solving an inverse homogenization problem, the mechanical functioning of these proteins at the cellular level can be estimated. Here, we present a combination of mechanical testing and imaging results to quantify the effects of specific inhibitors on cytoskeletal and extracellular matrix form and function.

Download Full-text

Quantifying Vascular Density in Tissue Engineered Constructs Using Machine Learning

Frontiers in Physiology ◽

10.3389/fphys.2021.650714 ◽

2021 ◽

Vol 12 ◽

Author(s):

Hannah A. Strobel ◽

Alex Schultz ◽

Sarah M. Moss ◽

Rob Eli ◽

James B. Hoying

Keyword(s):

Machine Learning ◽

Three Dimensional ◽

Vascular Density ◽

Form And Function ◽

Tissue Constructs ◽

Automated Method ◽

Frequent Monitoring ◽

Ideal Tool ◽

Engineered Tissue ◽

And Function

Given the considerable research efforts in understanding and manipulating the vasculature in tissue health and function, making effective measurements of vascular density is critical for a variety of biomedical applications. However, because the vasculature is a heterogeneous collection of vessel segments, arranged in a complex three-dimensional architecture, which is dynamic in form and function, it is difficult to effectively measure. Here, we developed a semi-automated method that leverages machine learning to identify and quantify vascular metrics in an angiogenesis model imaged with different modalities. This software, BioSegment, is designed to make high throughput vascular density measurements of fluorescent or phase contrast images. Furthermore, the rapidity of assessments makes it an ideal tool for incorporation in tissue manufacturing workflows, where engineered tissue constructs may require frequent monitoring, to ensure that vascular growth benchmarks are met.

Download Full-text

Weighing trees with lasers: advances, challenges and opportunities

Interface Focus ◽

10.1098/rsfs.2017.0048 ◽

2018 ◽

Vol 8 (2) ◽

pp. 20170048 ◽

Cited By ~ 44

Author(s):

M. I. Disney ◽

M. Boni Vicari ◽

A. Burt ◽

K. Calders ◽

S. L. Lewis ◽

...

Keyword(s):

Laser Scanning ◽

Large Scale ◽

Three Dimensional ◽

Point Clouds ◽

3D Models ◽

Tree Form ◽

Form And Function ◽

Challenges And Opportunities ◽

And Function ◽

Non Destructive

Terrestrial laser scanning (TLS) is providing exciting new ways to quantify tree and forest structure, particularly above-ground biomass (AGB). We show how TLS can address some of the key uncertainties and limitations of current approaches to estimating AGB based on empirical allometric scaling equations (ASEs) that underpin all large-scale estimates of AGB. TLS provides extremely detailed non-destructive measurements of tree form independent of tree size and shape. We show examples of three-dimensional (3D) TLS measurements from various tropical and temperate forests and describe how the resulting TLS point clouds can be used to produce quantitative 3D models of branch and trunk size, shape and distribution. These models can drastically improve estimates of AGB, provide new, improved large-scale ASEs, and deliver insights into a range of fundamental tree properties related to structure. Large quantities of detailed measurements of individual 3D tree structure also have the potential to open new and exciting avenues of research in areas where difficulties of measurement have until now prevented statistical approaches to detecting and understanding underlying patterns of scaling, form and function. We discuss these opportunities and some of the challenges that remain to be overcome to enable wider adoption of TLS methods.

Download Full-text

Quantitative analysis of subcellular distributions with an open-source, object-based tool

Biology Open ◽

10.1242/bio.055228 ◽

2020 ◽

Vol 9 (10) ◽

pp. bio055228 ◽

Cited By ~ 2

Author(s):

Pearl V. Ryder ◽

Dorothy A. Lerit

Keyword(s):

Image Analysis ◽

Open Source ◽

Three Dimensional ◽

Analysis Data ◽

Form And Function ◽

Object Based Image Analysis ◽

Object Based ◽

And Function ◽

Microscopy Images ◽

Complete Process

ABSTRACTThe subcellular localization of objects, such as organelles, proteins, or other molecules, instructs cellular form and function. Understanding the underlying spatial relationships between objects through colocalization analysis of microscopy images is a fundamental approach used to inform biological mechanisms. We generated an automated and customizable computational tool, the SubcellularDistribution pipeline, to facilitate object-based image analysis from three-dimensional (3D) fluorescence microcopy images. To test the utility of the SubcellularDistribution pipeline, we examined the subcellular distribution of mRNA relative to centrosomes within syncytial Drosophila embryos. Centrosomes are microtubule-organizing centers, and RNA enrichments at centrosomes are of emerging importance. Our open-source and freely available software detected RNA distributions comparably to commercially available image analysis software. The SubcellularDistribution pipeline is designed to guide the user through the complete process of preparing image analysis data for publication, from image segmentation and data processing to visualization.This article has an associated First Person interview with the first author of the paper.

Download Full-text

Virtual paleontology: computer-aided analysis of fossil form and function

Journal of Paleontology ◽

10.1666/13-001i ◽

2014 ◽

Vol 88 (4) ◽

pp. 633-635 ◽

Cited By ~ 8

Author(s):

Imran A. Rahman ◽

Selena Y. Smith

Keyword(s):

Imaging Techniques ◽

Three Dimensional ◽

Fossil Plants ◽

Element Analysis ◽

X Ray ◽

Form And Function ◽

Wide Range ◽

History Of ◽

Micro Tomography ◽

And Function

‘Virtual paleontology’ entails the use of computational methods to assist in the three-dimensional (3-D) visualization and analysis of fossils, and has emerged as a powerful approach for research on the history of life. Three-dimensional imaging techniques allow poorly understood or previously unknown anatomies of fossil plants, invertebrates, and vertebrates, as well as microfossils and trace fossils, to be described in much greater detail than formerly possible, and are applicable to a wide range of preservation types and specimen sizes (Table 1). These methods include non-destructive high-resolution scanning technologies such as conventional X-ray micro-tomography and synchrotron-based X-ray tomography. In addition, form and function can be rigorously investigated through quantitative analysis of computer models, for example finite-element analysis.

Download Full-text

A three-dimensional muscle model: A quantified relation between form and function of skeletal muscles

Journal of Morphology ◽

10.1002/jmor.1051820107 ◽

1984 ◽

Vol 182 (1) ◽

pp. 95-113 ◽

Cited By ~ 163

Author(s):

R. D. Woittiez ◽

P. A. Huijing ◽

H. B. K. Boom ◽

R. H. Rozendal

Keyword(s):

Skeletal Muscles ◽

Three Dimensional ◽

Muscle Model ◽

Form And Function ◽

And Function

Download Full-text

Cell-to-cell contact and extracellular matrix: Integration of form and function: the central role of adhesion molecules

Current Opinion in Cell Biology ◽

10.1016/s0955-0674(99)00029-0 ◽

1999 ◽

Vol 11 (5) ◽

pp. 537-539 ◽

Cited By ~ 13

Author(s):

Mina J Bissell ◽

W James Nelson

Keyword(s):

Extracellular Matrix ◽

Adhesion Molecules ◽

Cell Contact ◽

Form And Function ◽

And Function

Download Full-text

Human endothelial cell morphology and autacoid expression

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1995.268.4.h1613 ◽

1995 ◽

Vol 268 (4) ◽

pp. H1613-H1620

Author(s):

C. J. de Groot ◽

V. A. Chao ◽

J. M. Roberts ◽

R. N. Taylor

Keyword(s):

Gene Expression ◽

Extracellular Matrix ◽

Morphological Changes ◽

Umbilical Vein ◽

Three Dimensional ◽

Endothelin 1 ◽

And Function ◽

Cellular Fibronectin ◽

Cells Cultured

Human umbilical vein endothelial (HUVE) cells plated on plastic or gelatin-coated dishes grow as a “cobblestone” monolayer. By contrast, endothelial cells cultured on a complex matrix (e.g., Matrigel) form three-dimensional, capillary-like structures. In the current study, we verified the capillary phenotype of the latter structures and asked whether the morphological changes induced by extracellular matrix also affect human endothelial gene expression and function in vitro. Concentrations of cellular fibronectin, prostacyclin, and endothelin-1 were measured in the conditioned media by enzyme-linked immunosorbent and radioimmunoassays. Steady-state concentrations of HUVE mRNA were estimated by reverse transcription-polymerase chain reaction and quantified by Northern analyses to assess fibronectin and endothelin-1 gene expression. We found that the subjacent extracellular matrix affects the morphology, proliferation, and differentiation of HUVE cells in vitro. Cells cultured on gelatin were more mitotically active, expressed significantly less cellular fibronectin, made similar amounts of prostacyclin, and secreted significantly more endothelin-1 compared with the same cells grown on a Matrigel substrate.

Download Full-text

Hyaluronan: Metabolism and Function

Biomolecules ◽

10.3390/biom10111525 ◽

2020 ◽

Vol 10 (11) ◽

pp. 1525 ◽

Cited By ~ 1

Author(s):

Takashi Kobayashi ◽

Theerawut Chanmee ◽

Naoki Itano

Keyword(s):

Extracellular Matrix ◽

Tissue Concentration ◽

Molecular Size ◽

Cellular Functions ◽

Altered Expression ◽

Surface Receptors ◽

Hyaluronan Synthases ◽

Wide Range ◽

And Migration ◽

And Function

As a major polysaccharide component of the extracellular matrix, hyaluronan plays essential roles in the organization of tissue architecture and the regulation of cellular functions, such as cell proliferation and migration, through interactions with cell-surface receptors and binding molecules. Metabolic pathways for biosynthesis and degradation tightly control the turnover rate, concentration, and molecular size of hyaluronan in tissues. Despite the relatively simple chemical composition of this polysaccharide, its wide range of molecular weights mediate diverse functions that depend on molecular size and tissue concentration. Genetic engineering and pharmacological approaches have demonstrated close associations between hyaluronan metabolism and functions in many physiological and pathological events, including morphogenesis, wound healing, and inflammation. Moreover, emerging evidence has suggested that the accumulation of hyaluronan extracellular matrix and fragments due to the altered expression of hyaluronan synthases and hyaluronidases potentiates cancer development and progression by remodeling the tumor microenvironment. In addition to the well-known functions exerted by extracellular hyaluronan, recent metabolomic approaches have also revealed that its synthesis can regulate cellular functions via the reprogramming of cellular metabolism. This review highlights the current advances in knowledge on the biosynthesis and catabolism of hyaluronan and describes the diverse functions associated with hyaluronan metabolism.

Download Full-text

RGD-modified injectable hydrogel maintains islet beta-cell survival and function

Journal of Applied Biomaterials & Functional Materials ◽

10.1177/2280800020963473 ◽

2020 ◽

Vol 18 ◽

pp. 228080002096347

Author(s):

Tianshu Lan ◽

Jingyi Guo ◽

Xiaoming Bai ◽

Zengjiong Huang ◽

Zhimin Wei ◽

...

Keyword(s):

Extracellular Matrix ◽

Islet Transplantation ◽

Pancreatic Beta Cells ◽

Three Dimensional ◽

High Concentration ◽

Β Cells ◽

Glucose Levels ◽

And Function

Objective: A potential solution for islet transplantation and drug discovery vis-à-vis treating diabetes is the production of functional islets in a three-dimensional extracellular matrix. Although several scaffold materials have been reported as viable candidates, a clinically applicable one that is injectable and can maintain long-term functionality and survival of islet pancreatic beta-cells (β-cells) is far from being established. Results: In the current study, we evaluated a ready-to-use and injectable hydrogel’s impact on β-cells’ function and viability, both in vitro and in vivo. We found that β-cells in high concentration with hydrogels functionalized via Arg-Gly-Asp (RGD) demonstrated better viability and insulin secretory capacity in vitro. Moreover, it is a biocompatible hydrogel that can maintain β-cell proliferation and vascularization without stimulating inflammation after subcutaneous injection. Meanwhile, modifying the hydrogel with RGD can maintain β-cells’ secretion of insulin, regulating the blood glucose levels of mice with streptozotocin-induced diabetes. Conclusions: Thus, these preliminary results indicate that this RGD-modified hydrogel is a potential extracellular matrix for islet transplantation at extrahepatic sites, and they also provide a reference for future tissue engineering study.

Download Full-text