Talk Abstracts

Thursday 09:35-09:55am: Just enough: the right level of plastin’s sensitivity to calcium for strong bones.

Dmitri Kudryashov (The Ohio State University), Elena Kudryashova, Christropher Schwebach (The Ohio State University), Weili Zheng, Edward Egelman (University of Virginia), Matthew Orchard (The Ohio State University), Harper Smitrh (The Ohio State University), Lucas Runyan (The Ohio State University)

Abstract:
Research in Kudryashov’s lab is focused on two broadly defined areas: host-pathogen interactions and the actin cytoskeleton organization and function in health and disease.
Osteogenesis imperfecta (OI) is the congenital form of osteoporosis that develops largely due to defects in collagen biogenesis. However, mutations in actin-bundling protein plastin-3 (PLS3) have recently emerged as an alternative cause of this “fragile bone disease”. Plastins belong to a large family of tandem calponin homology (tCH) actin-binding proteins. Plastins contain four CH domains, organized in two actin-binding domains (ABDs), and the N-terminal calcium-binding regulatory domain. Binding of Ca²⁺ to plastins reduces their actin-bundling ability.
Of nearly two dozen recognized osteoporosis-linked mutations in the PLS3 gene, only five are predicted to form full-length protein products. Of these, only the PLS3 mutant with L478P substitution in the second actin binding domain (ABD2) lost the ability to bundle actin due to major disorganization of an actin contacting loop. All the remaining mutants bundled actin well but showed either increased or reduced sensitivity to Ca²⁺, despite that the Ca²⁺-binding domain is not directly affected by the mutations. While wt PLS3 that is distributed between lamellipodia and focal adhesions, the OI-linked calcium-hypersensitive mutants were restricted to lamellipodia, while calcium-hyposensitive mutants were enriched only in focal adhesions/stress fibers. We propose a model of PLS3 regulation by Ca²⁺ and demonstrate that the finely-tuned Ca²⁺ regulation of PLS3 controls its cycling between the leading edge and adhesion complexes, contributing to bone formation via yet unknown mechanisms.

Keywords: congenital osteoporosis, actin cytoskeleton, regulation by calcium

Thursday 09:55-10:15am: Complementarity of Native MS and CryoEM

Vicki Wysocki (Department of Chemistry and Biochemistry)

Abstract:
Characterization of the overall topology and inter-subunit contacts of protein complexes, and their assembly/disassembly and unfolding pathways, is critical because protein complexes regulate key biological processes, including processes important in understanding and controlling disease. Tools to address structural biology problems continue to improve. Native mass spectrometry (nMS) and associated technologies such as ion mobility are becoming an increasingly important component of the structural biology toolbox. When the mass spectrometry approach is used early or mid-course in a structural characterization project, it can provide answers quickly using small sample amounts and samples that are not fully purified. Integration of sample preparation/purification with effective dissociation methods (e.g., surface-induced dissociation), ion mobility, and computational approaches provide a MS workflow that can be enabling in biochemical, synthetic biology, and systems biology approaches. Native MS can determine whether the complex of interest exists in a single or in multiple oligomeric states and can provide characterization of topology/intersubunit connectivity, and other structural features. Beyond its strengths as a stand-alone tool, nMS can also guide and/or be integrated with other structural biology approaches such as SEC, SAXS, AUC, NMR, X-ray crystallography, and cryoEM Examples will be presented to illustrate complementary data provided by native MS/SID and cryoEM.

Keywords:

Thursday 10:15-10:30am: Towards Solving the Structure of Inner Ear and Synaptic Proteins, Cadherin-23 and Calsyntenins 1-3

Marissa Boyer (The Ohio State University Department of Chemistry and Biochemistry), Marcos Sotomayor (The Ohio State University Department of Chemistry and Biochemistry)