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

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

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

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

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

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

This study: DNAH  -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

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

dsRNARNAi Effective knockdown of gene product Inducible RNAi

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

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

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

Sedimentation

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

Time-lapse photo microscopy

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

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

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

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

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

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