Biology subjects

Crandall, C.

Publications and source records attributed to Crandall, C..

3 recordsLinked to original sources

bioRxiv2025

Small Accelerations of the cell generate sufficient nuclear motion to modulate transcriptional activity, driving cellular response independent of matrix strain

The cells mechanical environment is a fundamental determinant of its activity. Ostensibly, the cellular response is dependent on interactions between extracellular matrix deformations and the cell adhesome. Low-intensity vibration (LIV) induces sinusoidal mechanical accelerations that stimulate mesenchymal stem cell (MSC) anabolism despite generating minimal matrix strain. In this study, we tested the hypothesis that accelerations of less than 1g cause nuclear motions relative to the cell membrane in adherent cells, resulting in elevated stresses in the cytoskeleton that promote transcriptional activity. Coupling a piezoelectric vibration platform with real-time microscopy, we applied a 0.7g, 90Hz LIV signal that oscillates the cell with displacements of up to {+/-}11 {micro}m. Live-cell tracking revealed that the sinusoidal vibrations caused the nucleus to move {+/-}1.27 {micro}m (17% of total displacement) out of phase with the cell membrane. Disruption of the LINC complex, which mechanically couples the nucleoskeleton to the cytoskeleton, doubled the magnitude of this relative motion, indicating that the nucleo-cytoskeletal configuration plays a major role in regulating nuclear motion. Consistent with a previously reported increase in nuclear stiffness caused by LIV, machine-learning-based image segmentation of confocal micrographs showed that LIV increased both apical and basal F-actin fiber numbers, generating a denser, more branched actin network near the nucleus. Following six 20 min bouts of LIV applied to MSC, RNA sequencing identified 372 differentially expressed genes. Upregulated gene sets were linked to F-actin assembly and focal adhesion pathways. Finite element simulations showed that nuclear stresses increased by LIV up to 18% were associated with nuclei flattening and a 30-50% increase in actin-generated forces. These findings demonstrate that low-intensity accelerations, independent of matrix strain, can directly activate a response of the nucleus, leading to cytoskeletal reorganization and heightened nuclear stresses. Thus, even very small oscillatory mechanical signals can markedly influence cell outcomes, establishing a mechanosensing pathway independent of extracellular strains.

bioengineering↗

bioRxiv2024

Low-intensity vibration does not induce changes in microtubule dynamics in vitro

Microtubules (MTs) are cytoskeletal filaments responsible for many vital cellular processes including intracellular organelle organization and enabling the movement of intracellular components. While MTs were shown to respond to low frequency and large mechanical signals like substrate strain, how MTs may respond to a high frequency mechanical signal like low-intensity vibrations (LIV) is unknown. Here we quantified the polymerization dynamics of MTs under an acute 1-day LIV protocol applied at 90 Hz and 0.7 xg, a signal we have shown to be effective for altering F-actin dynamics and nuclear stiffness. LIV treatments were compared against Taxol, a potent regulator of MT acetylation. Using mouse mesenchymal stem cells (MSCs) in vitro, we quantified tubulin polymerization via centrifugal fractionation and western blots as well as alpha-tubulin acetylation via immunostaining. Finally, MT growth dynamics were quantified using machine learning-assisted analysis of live cell fluorescence microscopy of MT plus end binding protein EB1. Our results were not able to detect differences between LIV and control groups while Taxol treatment was effective in all measured outcomes. Our findings indicate that LIV applied at 90 Hz and 0.7 xg does not affect MT dynamics in MSCs, suggesting a higher mechanical threshold of MTs when compared to F-actin cytoskeleton.

bioengineering↗

bioRxiv2023

Data driven and cell specific determination of nuclei-associated actin structure

Quantitative and volumetric assessment of filamentous actin fibers (F-actin) remains challenging due to their interconnected nature, leading researchers to utilize threshold based or qualitative measurement methods with poor reproducibility. Here we introduce a novel machine learning based methodology for accurate quantification and reconstruction of nuclei-associated F-actin. Utilizing a Convolutional Neural Network (CNN), we segment actin filaments and nuclei from 3D confocal microscopy images and then reconstruct each fiber by connecting intersecting contours on cross-sectional slices. This allowed measurement of the total number of actin filaments and individual actin filament length and volume in a reproducible fashion. Focusing on the role of F-actin in supporting nucleocytoskeletal connectivity, we quantified apical F-actin, basal F-actin, and nuclear architecture in mesenchymal stem cells (MSCs) following the disruption of the Linker of Nucleoskeleton and Cytoskeleton (LINC) Complexes. Disabling LINC in mesenchymal stem cells (MSCs) generated F-actin disorganization at the nuclear envelope characterized by shorter length and volume of actin fibers contributing a less elongated nuclear shape. Our findings not only present a new tool for mechanobiology but introduce a novel pipeline for developing realistic computational models based on quantitative measures of F- actin.

bioengineering↗