Abstract
Mutations in actin-bundling protein plastin 3 (PLS3) emerged as a cause of congenital osteoporosis, but neither the role of PLS3 in bone development nor the mechanisms underlying PLS3-dependent osteoporosis are understood. Of the over 20 identified osteoporosis-linked PLS3 mutations, we investigated all five that are expected to produce full-length protein. One of the mutations distorted an actin-binding loop in the second actin-binding domain of PLS3 and abolished F-actin bundling as revealed by cryo-EM reconstruction and protein interaction assays. Surprisingly, the remaining four mutants fully retained F-actin bundling ability. However, they displayed defects in Ca2+ sensitivity: two of the mutants lost the ability to be inhibited by Ca2+, while the other two became hypersensitive to Ca2+. Each group of the mutants with similar biochemical properties showed highly characteristic cellular behavior. Wild-type PLS3 was distributed between lamellipodia and focal adhesions. In striking contrast, the Ca2+-hyposensitive mutants were not found at the leading edge but localized exclusively at focal adhesions/stress fibers, which displayed reinforced morphology. Consistently, the Ca2+-hypersensitive PLS3 mutants were restricted to lamellipodia, while chelation of Ca2+ caused their redistribution to focal adhesions. Finally, the bundling-deficient mutant failed to co-localize with any F-actin structures in cells despite a preserved F-actin binding through a non-mutation-bearing actin-binding domain. Our findings revealed that severe osteoporosis can be caused by a mutational disruption of the Ca2+-controlled PLS3’s cycling between adhesion complexes and the leading edge. Integration of the structural, biochemical, and cell biology insights enabled us to propose a molecular mechanism of plastin activity regulation by Ca2+.
Highlights
Osteoporosis is a disease defined by low bone density and disruption of the bone architecture resulting in fragility and fractures.[1]
Protein instability is a likely reason of osteogenesis imperfecta (OI) phenotype in case of the frameshift but not the insertion and missense plastin 3 (PLS3) mutations To check whether the OI phenotype can be a consequence of destabilized/denatured protein, seven recombinant OI PLS3 mutants and wild-type (WT) PLS3 (Fig. 1a, b) were expressed and purified from Escherichia coli
Tm melting temperature determined by differential scanning fluorimetry (DSF), Kd equilibrium dissociation constant of PLS3 binding to F-actin, [PLS3]50% bundling efficiency of PLS3, pCa50% Ca2+ sensitivity of PLS3 (-log[Ca2+] at 50% reduction in light scattering of actin bundles), SE standard error of the mean, standard deviations (SD) standard deviation of the mean attempts to purify the truncation constructs from the minor soluble fraction failed due to their proteolytic degradation (Supplementary Fig. S1d)
Summary
Osteoporosis is a disease defined by low bone density and disruption of the bone architecture resulting in fragility and fractures.[1]. Among three vertebrate tissue-specific plastin isoforms,[18] PLS3. ( known as T-plastin) is ubiquitously expressed in solid tissues[19] and involved in cell migration,[20] endocytosis,[21] DNA repair,[22] and membrane trafficking.[23] In agreement with the essential role of PLS3 in bone and connective tissue development in vertebrates, a pls[3] knockdown in zebrafish results in craniofacial dysplasia and malformations of body axis and tail,[13] whereas PLS3 knockout mouse models showed impaired cortical bone acquisition with decreased osteoblast mineralization capacity[24] and defects in the development of the epidermal basal membrane.[25]
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