Affiliation: Kasturba Medical College, Manipal, India
Keywords: v. cell biology of disease; e. intracellular transport
Primary cilia (PC) are tubular antenna like structures present on the mammalian cell surface transducing extracellular cues like hormones, growth factors and mechanical stimuli. Also, playing an important role in cellular development, differentiation and homeostasis. These microtubular structures are maintained by intraflagellar transport (IFT) machinery involved in bidirectional transport of ciliary proteins. IFT52 is a component of the IFT complex and associated with a novel skeletal ciliopathy, short-rib thoracic dysplasia 16 with or without polydactyly (MIM# 617102). Cellular mechanism of IFT52 function in modulating primary cilia biogenesis, signalling and its dependent regulation of osteogenesis and chondrogenesis is not known.
In our study we have characterized PC during osteogenic (OS) and chrondrogenic (CH) differentiation in vitro and elucidated the molecular mechanism of IFT52 function in a PC dependent manner duringosteogenesis and chondrogenesis in C3H10T1/2 and ATDC5 cell lines and Drosophila systems. OS and CH differentiation was induced in both cell linesand ascertained by assaying for known differentiation markers, calcium and glycosaminoglycan deposition in the extracellular matrix. Ift52 transcript levels were studied using qRT-PCR. Additionally, we characterized the IFT52 ortholog, Osm6 in Drosophila by IFT52 knockdown (KD) in embryonic neuroblasts by RNA interference using pan neuronal Gal4 drivers.
OS differentiation was evident at 7 days following induction via alizarin red (AR) staining to reveal extracellular matrix (ECM) mineralization. CH differentiation was first detected at 14 days post induction by sulfated proteoglycan deposition and ECM mineralization using alcian blue and AR staining. We found Ift52 transcript levels were upregulated at 14 and 21 days of OS differentiation, while in CH differentiation they were initially upregulated at 7 days and subsequently depressed. Ift52 KD resulted in reduced protein expression and significant reduction of PC prevalence, length and OS differentiation. IFT52 KD in embryonic neuroblasts of Drosophila melanogaster resulted in significant loss of chordotonal function like locomotion and hearing in adults and larva. Also, IFT52 KD embryos showed structural ciliary defects like profound depletion of the Ich5 cilia in embryos. Our findings are anticipated to remit a comprehensive molecular characterization of the Ift52 gene and enhance our understanding of its allied human anomalies.