MBI PhD Oral Defense

Time: 3pm
Date: 6 March 2019, Wednesday
Venue: NUS, E3-06-04

Supervisor(s): Dr Low Boon Chuan (main sup), Dr Cynthia He (co-sup)

T. brucei Spef1/CLAMP is required for anchorage of cytoskeletal organelles to the basal bodies

by DONG Xiaoduo,  Low Boon Chuan Group

Sperm flagellar protein 1, also known as Spef1/CLAMP, was first discovered in mouse sperm flagellum and has been implicated in spermatogenesis and central pair assembly. It binds and bundles microtubules (MTs), and facilitates embryonic intercalation with its MT functions in Xenopus. Additionally, it has been shown to play a role in planar cell polarity, which underlies global polarity at tissue level and is essential for a large variety of developmental processes and collective cell behaviors.

Trypanosoma brucei (T. brucei), the causative agent of trypanosomiasis, is an excellent model for organelle biology studies for its simple cellular organization with a series of single-copied organelles, which replicate and divide following a strict temporal and spatial order.Previously, T. brucei  Spef1 ortholog (TbSpef1) has been shown to be associated with a specialized microtubule quartet (MTQ), which originates from a region between the pro- and mature basal bodies. Knockdown of TbSpef1 by RNA interference (RNAi) results in defects in MTQ assembly and duplication/segregation of several single-copied cytoskeletal organelles, suggesting a role of TbSpef1 in co-ordinated organelle biogenesis/division during the T. bruceicell cycle.

In this study, the microtubule binding and bundling effects of TbSpef1 were demonstrated by in vitro assays. Using BioID, a protein-protein interaction screening method, 37 putative interacting partners of TbSpef1 have been identified with 8 further characterized. All 8 proteins were in close association to TbSpef1, validating the proximity-based method.One candidate was found to be localized between the pro- and mature basal bodies. RNAi of this protein did not affect duplication of the basal bodies nor any other organelles, but severed the connection between the MTQ and the basal bodies. This led to changes in the positioning of several cytoskeletal organelles relative to the basal bodies, and morphological changes in membrane-bound organelles including the flagellar pocket (FP). This study provided the first molecular identification of the connection between the MTQ and the basal bodies in T. brucei. The results also revealed a conserved cellular function of Spef1 in polarized cell development mediated by MT anchorage, possibly via its MT binding and bundling activities.

**Please note the examination following the seminar is closed-door**