The Centrosomal Swiss Army Knife: A combined in silico and in vivo approach to the structure-function annotation of SPD-2 provides mechanistic insight into its functional diversity

Abstract

AbstractCentrosomes are organelles that function as hubs of microtubule nucleation and organization, with key roles in organelle positioning, asymmetric cell division, and ciliogenesis. Aberrant centrosome structure or function is linked to neurodegenerative diseases, developmental abnormalities, ciliopathies, and tumor development. A major regulator of centrosome biogenesis and function in C. elegans is the highly conserved protein Spindle-defective protein 2 (SPD-2), a homolog of the human CEP-192 protein. CeSPD-2 is required for centrosome maturation, centriole duplication, spindle assembly and cell polarity establishment. Despite its importance, the specific molecular mechanism of CeSPD-2 function is poorly understood. To address this gap in knowledge, we combined computational analysis with cell biology approaches to uncover structure-function relationships of CeSPD-2 that may shed mechanistic light on its function. Domain prediction analysis corroborated and refined previously identified coiled-coils and ASH (Aspm-SPD-2 Hydin) domains and identified new domains and motifs: an additional coiled-coil, a GEF domain, an Ig-like domain, and a PDZ-like domain. Our findings suggest that ASH domain belongs to the same superfold as PapD chaperone domains and Major Sperm Protein (MSP) domains within the larger Immunoglobulin superfamily. We have identified a large novel basic region in the CeSPD-2 ASH domain that harbors most of the predicted protein and nucleic acid contact residues in the ASH domain. In vivo, ASH::GFP localized to centrosomes and centrosome associated microtubules, and forms aggregates in the cytosol when overexpressed. This study lays the groundwork for designing rational hypothesis-based experiments for future analyses to further elaborate the mechanisms of CeSPD-2 function in vivo.