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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

Hill, 2003 Alberts, 2002


Dyneins
• “Molecular motors"
• Complicated structures
• Different repeating lengths
• Flagellar beat versus wave

Alberts, 2002

Wirschell, 2007
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 Wirschell, 2007

these mutants and elucidate


function.
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
Inducible RNAi

dsRNA RNAi

Effective knockdown
of gene product
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

DNAH10 Growth Curve IC95 Growth Curve

2.50E+07 2.50E+07

2.00E+07 2.00E+07

Cell Titer (cells/mL)


Cell Titer (cells/mL)

1.50E+07
DNAH10 1.50E+07 IC95
uninduced uninduced

1.00E+07
DNAH10 1.00E+07 IC95
induced induced

5.00E+06 5.00E+06

0.00E+00 0.00E+00
0 24 48 72 96 120 0 24 48 72 96 120
Hours post-induction Hours post-induction
Sedimentation
• A measure of optical density (OD) of the
sample over time
• Low OD indicates motility defect

 High OD

 Low OD
Sedimentation
Sedimentation Assay, ²OD/time

-0.05

-0.1

-0.15
DNAH10 uninduced
²OD600

DNAH10 induced
-0.2
IC95 uninduced
IC95 induced
-0.25

-0.3

-0.35

-0.4
0 1 2 3 4 5 6 7 8 9
Hours
Time-lapse photo microscopy
• Cell movement tracked for 30-second
intervals, classified based on quality of
movement
– Runners, Tumblers, Immotile

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Time-lapse photo microscopy
DNA H10 Uninduced DNAH10 Induced

6% 13%

20%
Runners 19% Runners
Tumblers Tumblers
Immotile Immotile
68%
74%

IC95 Uninduced IC95 Induced

17% 19%

Runners 42% Runners


46%
Tumblers Tumblers
Immotile Immotile

37%
39%
Immotility develops over time
Immotile DNAH10 cells over time

80.00%
70.00%
60.00%
% immotile

50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
Uninduced 12 14 16 24 48
Hours post-induction

•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