Biology subjects

Knothe Tate, M. L.

Publications and source records attributed to Knothe Tate, M. L..

3 recordsLinked to original sources

bioRxiv2025

The Distribution of Elastin and Collagen Underpinning the Smart Properties of the Interosseous Membrane

The interosseous membrane (IOM), a ligament-like structure spanning the radius and ulna, reduces strain in the ulna and structurally stiffens the radio-ulnar complex of the forearm. Using two-photon and second-harmonic-imaging we measured collagen and elastin signal intensity to test the hypothesis that their spatial distributions correspond to predominant loading patterns in the IOM. Distinct spatial gradients in collagen and elastin, as well as cruciate ligament-like architectures, were observed at the submicron and the micron to mesoscopic length scales. Quantitative analysis revealed anisotropies in the elastin-collagen composite comprising the IOM, with elastin 4-6 times higher than collagen concentrations at radius/ulna - IOM interfaces, and organized in the tensile loading direction, i.e. along the major Centroidal Axis, of the IOM. Hence, the IOM exhibits a composite structure comprising elastin and collagen, with spatial distribution of elastin higher than collagen at bone-IOM interfaces and decreasing from the interface with the ulna to that of the radius. These increased concentrations of elastin at interfaces are expected to confer elasticity (spring function). In contrast, peaks in collagen concentrations represent collagens organization into fibers, parallel to the length of the IOM, bridging the radius and ulna, and conferring toughness and damping function to the IOM and forearm construct. Mapping the cross-scale elastin and collagen composition of the IOM gives unprecedented insight into its emergent properties and associated mechanical function, an understanding of which may guide future surgical treatments, implant and medical textile design and manufacture, as well as physical therapy protocols to promote healing.

physiology↗

bioRxiv2024

Stem cell mechanoadaptation - Part A - Effect of microtubule stabilization and volume changing stresses on cytoskeletal remodeling

Here we report on the first part of a two-part experimental series to elucidate spatiotemporal cytoskeletal remodeling, which underpins the evolution of stem cell shape and fate, and the emergence of tissue structure and function. In Part A of these studies, we first develop protocols to stabilize microtubules exogenously using paclitaxel (PAX) in a standardized model murine embryonic stem cell line (C3H/10T1/2) to maximize comparability with previous published studies. We then probe native and microtubule stabilized stem cells capacity to adapt to volume changing stresses effected by seeding at increasing cell densities, which emulates local compression and tissue template formation during development. Within the concentration range 1 - 100 nM, microtubule stabilized stem cells maintain viability and reduce proliferation. PAX-stabilization of microtubules is associated with increased cell volume as well as flattening of the cell and nucleus. Compared to control cells, microtubule stabilized cells exhibit thick, bundled microtubules and highly aligned, thicker and longer F-actin fibers, corresponding to an increase in the Youngs Modulus of the cell. Both F-actin and microtubule concentration increase with increasing PAX concentration, whereby the increase in F-actin is more prominent in the basal region of the cell. The corresponding increase in microtubule is observed more globally across the apical and basal region of the cell. Seeding at increasing target densities induces local compression on cells. This increase in local compression modulates cell volume and concomitant increases in F-actin and microtubule concentration to a greater degree than microtubule stabilization via PAX. Cells seeded at high density (HD) exhibit higher bulk modulus than corresponding cells seeded at low density (LD). These data demonstrate the capacity of stem cells to adapt to an interplay of mechanical and chemical cues, i.e. respective compression and exogenous microtubule stabilization; the resulting cytoskeletal remodeling manifests as evolution of mechanical properties relevant to development of multicellular tissue constructs. Significance statementElucidation of mechanisms by which stem cells adapt across length and time scales may prove enabling for the development of regenerative medicine therapies and devices that emulate natural processes. Dynamic cytoskeletal remodeling underpins the emergence of structure-function relationships at the tissue length scale. Here we stabilized the tubulin cytoskeleton exogenously using paclitaxel (PAX), a microtubule depolymerization inhibitor. We probed stem cell mechanoadaptation by seeding at increasing density to introduce local compression to cells. Changes in cytoskeletal architecture and concentration of F-actin and tubulin per cell occurred in a PAX concentration-dependent manner. Compression from increasing seeding density modulated this PAX-induced cytoskeletal remodeling and mechanical properties of the multicellular constructs. Hence, mechanical cues counterbalance concentration-dependent effects of exogenous chemical microtubule stabilization.

cell biology↗

bioRxiv2024

Stem cell mechanoadaptation Part B - Microtubule stabilization and substrate compliance effects on cytoskeletal remodeling

Stem cells adapt to their local mechanical environment by rearranging their cytoskeleton, which underpins the evolution of their shape and fate, as well as the emergence of tissue structure and function. Here we report on the second part of a two-part experimental series to elucidate spatiotemporal cytoskeletal remodeling and resulting changes in morphology and mechanical properties of cells, their nuclei, akin to mechanical testing of the most basic living and adapting unit of life, in situ in model tissue templates. We probed the native and PAX-exposed (inhibiting cytoskeleton tubulin depolymerization) stem cells cytoskeletal adaptation capacity on substrates of different compliance (exerting local tension on cells) and in combination with exposure to local compression effected with increased target seeding densities (5000 cells/cm2 - Low Density, LD; 15,000 cells/cm2, High Density, HD). On 10 and 100 kPa gels, cells seeded at both LD and cells proliferated to HD exhibited bulk moduli that nearly matched those of their respective substrates, hence exhibiting a greater increase in Youngs Modulus after microtubule stabilization than cells cultured on glass. Culture on compliant substrates also reduced the PAX-mediated F-actin and microtubule concentration increase. On gels, F-actin alignment decreased as more randomly oriented, short actin crosslinks were observed, representing emergent adaptation to the compliant substrate, mediated through myosin II contractility. We conclude that stem cell adaptation to compliant substrates facilitates the accommodation of larger loads from the PAX-stabilized polymerizing microtubule, which in turn exerts a larger effect in determining cells capacity to stiffen and remodel the cytoskeleton. Taken as a whole, these studies establish correlations between cytoskeleton and physical and mechanical parameters of stem cells that progress our understanding of the dynamic cytoskeleton, as well as shape changes in cells and their nuclei, culminating in emergent tissue development and healing. Significance StatementStem cells adapt to their dynamic environment by means of cytoskeleton rearrangements - underpinning the emergence of tissue structure-function relationship; this represents a current gap in knowledge that needs to be addressed, to better target tissue neogenesis and healing in context of regenerative medicine. We introduced compression via increasing seeding density and tension via compliant substrates to create tissue templates, while stabilizing microtubules. We found that mechanical and biophysical cues exert a greater effect in modulating cytoskeletal adaptation than exogenous chemical agents targeting the cytoskeleton, thus counterbalancing the concentration-dependent effect on cell physical and mechanical properties. We further found that stem cells with stabilized microtubules are sensitive to a range of substrate stiffness and seeding density that allowed cells or multicellular constructs to broaden their capacity to adapt their mechanical properties.

cell biology↗