Abstract
[Truncated] The small GTPases of the Ras family are present at distinct trafficking pathways during vesicle transport. Bone resorption in osteoclasts requires the formation of the ruffled border domain by accelerated fusion of intracellular vesicles to the plasma membrane, an event comparable with vesicle trafficking in other mammalian cells. The Rab3 subfamily consists of four isoforms, Rab3A, Rab3B, Rab3C and Rab3D, which are involved in the mediation of exocytic vesicle transport. This thesis sought to clarify the mechanism of vesicle trafficking mediated by Rab3D in the osteoclast precursor cellline, RAW264.7.
Using RT-PCR analysis it was demonstrated that RAW264.7 cells expressed both Rab3C and Rab3D. Rab3D was considered the dominant isoform in RAW264.7 cells and was shown to be upregulated during osteoclastogenesis. To further examine the subcellular localisation and role of Rab3D in intracellular trafficking in RAW264.7 cells confocal microscopy and GFP protein techniques were used. Rab3D was shown to localise to pleomorphic vesicular compartments in the cytoplasm of the RAW264.7 cells that associate with microtubules as well as the trans-Golgi network. The expression of three Rab3D mutants, Q81L, N135I and CXC , in RAW264.7 cells showed the importance of prenylation and GTP/GDP cycling in Rab proteins.
Using RT-PCR analysis it was demonstrated that RAW264.7 cells expressed both Rab3C and Rab3D. Rab3D was considered the dominant isoform in RAW264.7 cells and was shown to be upregulated during osteoclastogenesis. To further examine the subcellular localisation and role of Rab3D in intracellular trafficking in RAW264.7 cells confocal microscopy and GFP protein techniques were used. Rab3D was shown to localise to pleomorphic vesicular compartments in the cytoplasm of the RAW264.7 cells that associate with microtubules as well as the trans-Golgi network. The expression of three Rab3D mutants, Q81L, N135I and CXC , in RAW264.7 cells showed the importance of prenylation and GTP/GDP cycling in Rab proteins.
| Original language | English |
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| Qualification | Masters |
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| Publication status | Unpublished - 2002 |
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