Biology subjects

Howard, S. M.

Publications and source records attributed to Howard, S. M..

4 recordsLinked to original sources

bioRxiv2026

Chronological Aging Stiffens Cells, Alters Mitochondria, and Impairs Mitochondrial Mechanical Responsiveness in Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and adipocytes, play a critical role for maintaining bone homeostasis. Although, aging impairs MSC function and contributes to several complications, the effects of aging on subcellular structure and related gene expression need further investigation. Here we established an in vitro system to study MSCs isolated from bones of 5, 12, and 24 months old mice which showed significantly decreased bone volume and exercise performance with age. RNA sequencing revealed downregulation of genes related to cell-matrix interactions, cell metabolism, and division. Functionally MSCs extracted from 24mo showed significantly increased adipogenesis and reduced osteogenesis compared to 5mo, which was accompanied by reduced levels of cell proliferative marker Ki67 and increased expression of senescence protein p16. Data-driven segmentation of F-actin architecture revealed no cell-wide or nuclei-associated alteration, while 24mo MSCs showed decreased nuclear volume and increased spreading and higher nuclear stiffness compared to 5mo. Mitochondria from 12mo and 24mo showed increased length and volume of fibers when compared to 5mo, which was accompanied by gene expression changes associated with mitochondrial inflammation and oxidative phosphorylation. To test the functional consequences, MSCs were subjected to mechanical stress for 72 hours using 90Hz, 07g low-intensity vibration (LIV). While 5mo MSCs showed a robust fusing of individual mitochondrial fibers, LIV response of 12mo and 24mo MSCs were progressively less, indicating an already stressed mitochondria compromised to mechanical challenge. In summary, this study has established an in vitro assay system, revealing age-associated impairments in differentiation, mitochondrial function, and mechanotransduction capacity.

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↗

bioRxiv2019

CtIP promotes the motor activity of DNA2 to accelerate long-range DNA end resection

BLM or WRN helicases function with the DNA2 helicase-nuclease to resect DNA doublestrand breaks and initiate homologous recombination. Upon DNA unwinding by BLM/WRN, RPA directs the DNA2 nuclease to degrade the 5-strand, revealing the 3 overhang needed for recombination. RPA bound to ssDNA also represents a barrier, explaining the need for the motor activity of DNA2 to displace RPA prior to resection. Using ensemble and single molecule biochemistry, we show that phosphorylated CtIP dramatically stimulates the ATP hydrolysis driven motor activity of DNA2. This activation in turn strongly promotes the degradation of RPA-coated ssDNA by DNA2. The domains of CtIP required to stimulate DNA2 are separable from those that regulate the MRN complex. These results establish that CtIP couples both MRE11-dependent short and DNA2-dependent long-range resection, and show how the motor activity of DNA2 promotes resection. Our data explain the less severe resection defects of MRE11 nuclease-deficient cells compared to those lacking CtIP.\n\nHighlightsO_LIPhosphorylated CtIP stimulates the motor activity of DNA2\nC_LIO_LIThe activated DNA2 translocase facilitates degradation of RPA-coated ssDNA\nC_LIO_LICtIP promotes both MRN and DNA2 nucleases coupling short and long-range resection\nC_LIO_LIThe CtIP domains required to promote DNA2 and MRN are distinct and fully separable\nC_LI

biochemistry↗

bioRxiv2018

A role for RNA and DNA:RNA hybrids in the modulation of DNA repair by homologous recombination

DNA double-strand breaks (DSBs) are toxic DNA lesions which, if not properly repaired, may lead to genomic instability, cell death and senescence. Damage-induced long non-coding RNAs (dilncRNAs) are transcribed from broken DNA ends and contribute to DNA damage response (DDR) signaling. Here we show that dilncRNAs play a role in DSB repair by homologous recombination (HR) by contributing to the recruitment of the HR proteins BRCA1, BRCA2, and RAD51, without affecting DNA-end resection. In S/G2-phase cells, dilncRNAs pair to the resected DNA ends and form DNA:RNA hybrids, which are recognized by BRCA1 and promote its recruitment to DSBs. We also show that RNase H2 is in a complex with the HR proteins BRCA1, PALB2, BRCA2, and RAD51, and that it localizes to DSBs in the S/G2 cell-cycle phase. BRCA2 controls DNA:RNA hybrid levels at DSBs by mediating RNase H2 recruitment and, therefore, hybrids degradation. These results demonstrate that regulated DNA:RNA hybrid levels at DSBs contribute to HR-mediated repair.

cell biology↗