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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, 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
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
2.50E+07 2.50E+07
2.00E+07 2.00E+07
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
6% 13%
20%
Runners 19% Runners
Tumblers Tumblers
Immotile Immotile
68%
74%
17% 19%
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