MSc Exit Seminar-Reuben Philip

Characterizing the Microtubule Organizing Centres in Osteoclasts

The skeleton is a metabolically active organ that undergoes continuous remodeling in order to uphold structural integrity and to repair bone following injury. Osteoclasts are highly specialized, multinucleated cells responsible for the selective resorption of bone matrix components, however, they are also responsible for the pathological bone destruction found in microgravity environments, periodontitis, and osteoporosis. Our study investigates the origin of the microtubule cytoskeleton during differentiation and bone resorption. Microtubule nucleation is generally restricted to specific subcellular sites called microtubule organizing centres (MTOCs) and is primarily fulfilled by the centrosome in mitotic animal cells. While mononuclear osteoclast precursor cells contain centrosomal MTOCs, previous research has suggested that functional centrosomal MTOCs do not exist in osteoclasts. To revisit and characterize the MTOCs in osteoclasts, both cell line and primary cell-derived murine osteoclasts were subjected to high-resolution imaging to track centrosome behaviour and their ability to organize microtubules. Through live-cell imaging, fixed immunofluorescence, and ultrastructural analyses, we observed that most, if not all centrosomes donated from precursor cells clustered early in osteoclastogenesis and persisted post-differentiation in non-resorbing and resorbing osteoclasts. Drug-induced microtubule regrowth assays revealed that centrosomes remained individually functional post-differentiation but clustered in a microtubule-dependent manner in order to organize microtubules. Quantification of microtubules emanating from centrosome clusters showed that they were capable of nucleating more microtubules compared to lone centrosomes. Finally, by visualizing Golgi reorganization and the nucleation of Golgi-derived microtubules, we identified the Golgi as a possible non-centrosomal MTOC that potentially facilitates the production of polarized microtubule arrays in osteoclasts. Together these findings show that multinucleated osteoclasts employ unique centrosomal and non-centrosomal MTOCs to organize microtubules.

Supervisor: Prof. Rene Harrison

MSc Exit Seminar- Aditi Aggarwal

Effect of thermal stress on the intracellular localization of constitutively expressed heat shock protein HSPA8 (Hsc70) in differentiated and undifferentiated cultured human neuronal cells

Heat shock protein HSPA8 (Hsc70) is a constitutively expressed member of the Hsp70 multigene family that is abundantly expressed in neuronal cells. Previous studies in our laboratory suggest that HSPA8 may play an important role in neuronal pre-protection against cellular stress. This thesis highlights the importance of HSPA8 and its role as a fast responder to cellular stress in differentiated human SH-SY5Y neuronal cells. The effect of thermal stress on the intracellular localization of HSPA8 is compared in differentiated and undifferentiated neuronal cells. HSPA8 rapidly translocated into the nuclei of differentiated neuronal cells after heat shock and co-localized at transcription sites with DNAJB1 (Hsp40) and HSPH1 (Hsp105α) components of the protein disaggregation/ refolding machine. The rapid targeting and assembly of an HSPA8 based disaggregation/ refolding machine acts as a nuclear protective mechanism in differentiated neurons without the time lag needed to induce stress-inducible Hsp70.

Supervisor:  Prof. Ian Brown

MSc Exit Seminar- Stefan Schuetz

Characterizing the basis of strigolactone perception by HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE 2

Abstract:

The HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE 2 (HTL/KAI2) α/β hydrolases likely play a critical role in the life cycle of parasitic plants of the genus Striga. Several of these hydrolases serve as receptors for strigolactones (SLs), a class of compounds exuded by the roots of some plants, including many economically significant crops. Upon perception of minute levels of SLs, Striga seeds germinate and parasitize the nearby host, often leading to massive losses in crop yields in affected regions. We have produced a series of mutant variants of the Arabidopsis thaliana homolog of HTL/KAI2, which is only weakly responsive to SL. By substituting certain key amino acids in the protein’s active site, we have created a receptor conferring increased Arabidopsis germination under inhibitory conditions in the presence of SL. Additional study of this mutant receptor may offer insight into the biochemical basis of Striga’s SL sensitivity.

Supervisor: Prof. Shelley Lumba