Transcript Trypanosoma brucei

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
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
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
IC95 Growth Curve
2.50E+07
2.50E+07
2.00E+07
2.00E+07
1.50E+07
DNAH10
uninduced
1.00E+07
DNAH10
induced
5.00E+06
0.00E+00
Cell Titer (cells/mL)
Cell Titer (cells/mL)
DNAH10 Growth Curve
1.50E+07
IC95
uninduced
1.00E+07
IC95
induced
5.00E+06
0.00E+00
0
24
48
72
96
Hours post-induction
120
0
24
48
72
96
Hours post-induction
120
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
-0.05
-0.1
²OD600
-0.15
DNAH10 uninduced
DNAH10 induced
-0.2
IC95 uninduced
IC95 induced
-0.25
-0.3
-0.35
-0.4
0
1
2
3
4
Hours
5
6
7
8
9
Time-lapse photo microscopy
• Cell movement tracked for 30-second
intervals, classified based on quality of
movement
– Runners, Tumblers, Immotile
QuickTime™ and a
H.264 decompressor
are needed to see this picture.
QuickTime™ and a
H.264 decompressor
are needed to see this picture.
Time-lapse photo microscopy
DNA H10 Uninduced
DNAH10 Induced
6%
13%
20%
Runners
19%
Tumblers
Immotile
Tumblers
Immotile
68%
74%
IC95 Uninduced
IC95 Induced
17%
19%
46%
37%
Runners
Runners
Runners
42%
Tumblers
Tumblers
Immotile
Immotile
39%
Immotility develops over time
Immotile DNAH10 cells over time
80.00%
% immotile
70.00%
60.00%
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