A Role for the Actin Cytoskeleton and Plastin-3 in Osteoblast Mineralization and Mechanosensation

ORCID

https://orcid.org0000-0003-1158-2694

Date of Award

3-6-2025

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

The great diversity in actin network architectures and dynamics is exploited by cells to drive fundamental biological processes including cell migration, intracellular trafficking, and cell division. One such system that relies on the actin cytoskeleton to fulfill its physiological function are osteoblasts, which are mesenchymal lineage bone cells that promote bone formation through depositing a collagen scaffold and subsequently promoting mineralization of this matrix. A number of factors are known to drive osteogenic differentiation of pre-osteoblast cells including extracellular matrix cues such as substrate stiffness, which are primarily detected via mechanosensitive machinery linked to the actin cytoskeleton. Disruptions to β-actin, actin cytoskeletal dynamics, and actin-associated proteins have all been shown to disrupt both osteoblast physiology at the cellular level as well as the integrity of bone structure in model systems. This emphasizes that careful tuning of actin filament dynamics is required for osteoblast differentiation and function, which thus raises the question of how actin-binding proteins contribute to these processes. In this work, we take two approaches to explore the significance of regulatory mechanisms governing actin cytoskeleton dynamics. In the first part of this work, we directly compare how processing and modification of the N terminus of β-actin affects intrinsic polymerization dynamics and its remodeling by actin-binding proteins that are essential for cell migration. In the second part of this work, we investigate the role of plastin-3 (PLS3) in osteoblast physiology. PLS3 is a calcium-sensitive actin-bundling protein that has recently been linked to the development of childhood-onset osteoporosis, however, the underlying mechanism remains elusive. To investigate the role of PLS3 in osteoblasts, we generated MC3T3-E1 pre- osteoblast cells that are stably depleted of PLS3. Comparison of osteogenic differentiation in control and PLS3 knockdown cells reveals that depletion of PLS3 does not alter the first stage of osteoblast differentiation in which a collagen matrix is deposited, but severely affects the subsequent mineralization of that matrix. Osteogenic differentiation heavily relies on mechanosensitive pathways including those linked to focal adhesions to drive mineral deposition. We observed PLS3 prominently localizes to focal adhesions (FAs) and that depletion of PLS3 rendered osteoblasts unresponsive to changes in ECM stiffness. More specifically, our results reveal pre-osteoblasts depleted of PLS3 exhibit similar cell sizes, FA lengths, and number of FAs when plated on soft (6 kPa) versus stiff (100 kPa) substrates in contrast to control cells, which showed an increased in each of these parameters when plated on 100 kPa substrates. Defective cell spreading of PLS3 KD cells on stiff substrates could be rescued by expression of wildtype PLS3, but not by expression of three PLS3 mutants that were identified in patients with early-onset osteoporosis and that have aberrant actin-bundling activity. Altogether, our results show that actin-bundling by PLS3 is part of the mechanosensitive mechanism that promotes osteoblast mineralization and thus begins to elucidate how PLS3 contributes to the development of bone defects such as osteoporosis.

Language

English (en)

Chair and Committee

Silvia Jansen

Committee Members

Martha Bagnall ; Roberto Civitelli ; John Cooper ; David Piston ; Deborah Veis

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