1. Investigating the Role of Inner-arm Dynein Knockdowns in Trypanosoma brucei Kathryn Kinzel

2. Introduction Trypanosoma brucei –Unicellular eukaryote –Causative agent of African Sleeping Sickness –Model system for flagellar defects

3. Structure and Motility Flagellar structure dictates movement Microtubule movement leads to flagellar bending –Bending results in wave formation Movement driven by dynein “motors” Hill, 2003 Alberts, 2002

4. Why this is important in T. brucei Motility Cell division Kinetoplast attachment to basal body Exocytosis/endocytosis/signaling

5. The Flagellum T. brucei flagellum has several components HIGHLY conserved 9+2 axoneme Structure present in motile flagella/cilia Alberts, 2002Hill, 2003

6. Dyneins “Molecular motors" Complicated structures Different repeating lengths Flagellar beat versus wave Wirschell, 2007 Alberts, 2002

7. This study: DNAH10 - 1  -HC –Sequence shares homology to flagellar heavy chain –Chlamydomonas mutants swim slowly IC95 - IC138 –Shows similarity to IC138 –Marked phenotype seen in Chlamydomonas PURPOSE: To characterize these mutants and elucidate function. Wirschell, 2007

8. Strain Preparation Engineered strains created by Noël Rosenthal ‘07 –Allows for stable RNA interference (RNAi) Single-cell dilution –Creating clonal cell lines from one cell RNAi to induce mutations

9. dsRNARNAi Effective knockdown of gene product Inducible RNAi

10. Analytical Methods Growth curves Sedimentation assays Time-lapse photo microscopy Fluorescence microscopy Electron microscopy

11. Growth curves Cell titers were measured every 24 hours for 120 hours after induction

12. Sedimentation A measure of optical density (OD) of the sample over time Low OD indicates motility defect  High OD  Low OD

13. Sedimentation

14. Time-lapse photo microscopy Cell movement tracked for 30-second intervals, classified based on quality of movement –Runners, Tumblers, Immotile

15. Time-lapse photo microscopy

16. Immotility develops over time New flagella are affected - increase in immotility mirrors cell division Proteins in old flagella may be exchanged

17. Ongoing work Fluorescence microscopy –Using DAPI to determine cell cycle stage

18. Ongoing work Scanning electron microscopy –Observation of cell division defects –Clumping cells

19. Ongoing work Transmission electron microscopy –Observation of dynein arms –Presence/absence of dyneins or other proteins in axoneme

20. Conclusions Knockdowns exhibit motility and growth defects –DNAH10 more severe Inner-arm dyneins critical for proper movement Gradual change result of new flagella and/or protein replacement –Exact dynein change to be determined

21. Acknowledgements Amy Springer Springer Lab –Tenaya Vallery Michele Klingbeil Anthi Vandoros Marian Rice Biology Department Dreyfus Foundation