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Osteogenesis imperfecta mutations in plastin 3 lead to impaired calcium regulation of actin bundling.

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⁺.

SUBMITTER: Schwebach CL

PROVIDER: S-EPMC7244493 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Osteogenesis imperfecta mutations in plastin 3 lead to impaired calcium regulation of actin bundling.

Schwebach Christopher L CL Kudryashova Elena E Zheng Weili W Orchard Matthew M Smith Harper H Runyan Lucas A LA Egelman Edward H EH Kudryashov Dmitri S DS

Bone research 20200522

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 b ...[more]

PMID: 32509377

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Project description:Regulation of the cytoskeleton is essential for cell migration in health and disease. Lymphocyte cytosolic protein 1 (lcp1, also called L-plastin) is a hematopoietic-specific actin-bundling protein that is highly conserved in zebrafish, mice and humans. In addition, L-plastin expression is documented as both a genetic marker and a cellular mechanism contributing to the invasiveness of tumors and transformed cell lines. Despite L-plastin's role in both immunity and cancer, in zebrafish there are no direct studies of its function, and no mutant, knockout or reporter lines available. Using CRISPR-Cas9 genome editing, we generated null alleles of zebrafish lcp1 and examined the phenotypes of these fish throughout the life cycle. Our editing strategy used gRNA to target the second exon of lcp1, producing F0 mosaic fish that were outcrossed to wild types to confirm germline transmission. F1 heterozygotes were then sequenced to identify three unique null alleles, here called 'Charlie', 'Foxtrot' and 'Lima'. In silico, each allele truncates the endogenous protein to less than 5% normal size and removes both essential actin-binding domains (ABD1 and ABD2). Although none of the null lines express detectable LCP1 protein, homozygous mutant zebrafish (-/-) can develop and reproduce normally, a finding consistent with that of the L-plastin null mouse (LPL -/-). However, such mice do have a profound immune defect when challenged by lung bacteria. Interestingly, we observed reduced long-term survival of zebrafish lcp1 -/- homozygotes (~30% below the expected numbers) in all three of our knockout lines, with greatest mortality corresponding to the period (4-6 weeks post-fertilization) when the innate immune system is functional, but the adaptive immune system is not yet mature. This suggests that null zebrafish may have reduced capacity to combat opportunistic infections, which are more easily transmissible in the aquatic environment. Overall, our novel mutant lines establish a sound genetic model and an enhanced platform for further studies of L-plastin gene function in hematopoiesis and cancer.

Dataset Information

Osteogenesis imperfecta mutations in plastin 3 lead to impaired calcium regulation of actin bundling.

Publications

Osteogenesis imperfecta mutations in plastin 3 lead to impaired calcium regulation of actin bundling.

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