intraflagellar transport in trypanosomes filippo rusconi muséum national d’histoire naturelle...
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INTRAFLAGELLAR TRANSPORT IN TRYPANOSOMES
Filippo RUSCONI
Muséum National d’Histoire NaturelleINSERM U565 & CNRS UMR 5153 & MNHN 0503
IntraFlagellar Transport (IFT)
Radial spoke
Anterograde IFT: FLA10 Kinesin II (towards flagellum tip, + end MT)
Retrograde IFT: Cytoplasmic Dynein DHC1b (towards cell body)
IFT particle
Raft
FLA10 KinesinII
Cytoplasmic Dynein
Cole et al., J Cell Biol. 141, 993-1008, 1998
Phase Anti-flagellum
+ DAPI
nucleus
Trypanosoma brucei (protozoan)
• Flagellated organism easy to grow in vitro
• Genome sequenced• Straight forward
molecular biology • Reverse genetics (RNAi,
inducible expression)• Responsible for
sleeping sickness
• Flagellum attached to the cell body
IFT in trypanosomes
PFRA
T7 tet-inducible promoters
T7 transcription terminators
drug resistance
Wang et al., JBC (2000) 275, 40174
Durand-Dubief, Kohl & Bastin, MBP (2003) 129, 11-21
intermolecular
Gene Characteristics IFT in T. b.Motors
DHC1b dynein heavy chain +DHC1bis dynein heavy chain +LC8 dynein light chain -KIF3A kinesin motor -KIF3B kinesin motor NDKAP cargo binding ND
Raft components
IFT20 coiled-coil +IFT27 G protein ?IFT52 coiled-coil +IFT55 coiled-coil +IFT74 coiled-coil NDIFT88 TPR +IFT122 WD40 +IFT172 WD40 +
Regulators
LF4 kinase +/-
Vector for RNAi silencing
The trypanosome cell cycle
Green: Flagellum, Red lines: FAZ, Red dots: basal body, Blue: DAPI
Generation of non-flagellated cells
non-induced
Green: Flagellum, Red lines: FAZ, Red dots: basal body, Blue: DAPI
induced
Defects: No new flagellum, but FAZ is still there but ...
FAZ is too short
Shorter flagella, shorter cells ?
Green: Flagellum, Red lines: FAZ, Red dots: basal body, Blue: DAPI
DIC (TbDHC1b cells, RNAi silencing induced for 48 hrs)
30201000
10
20
30
40
Flagellum length (µm)
cell
siz
e (µ
m)
Flagellum and cell size are linked
0
10
20
DHC1b
IFT88
Induced cells don’t have a flagellum
Characterisation of IFT RNAi mutants
% F
lag
ella
ted
cel
ls
FAZ FORMATION AND CYTOKINESIS
no new flagellum
new FAZ
no new flagellum
no new FAZ
no new flagellum
no new FAZ
No cytokinesis
Nuclear division OK
Blue: DAPIRed lines: FAZRed spots: basal bodies
Non-flagellated
sibling
Flagellated sibling
Multinucleated cells
Summary
1) IFT controls flagellum assembly2) IFT controls flagellum length3) Flagellum length dictates FAZ elongation
and defines cytokinesis initiation4) Flagellum and FAZ are required for cell
morphogenesis (shape, polarity)
Kohl, Robinson & Bastin, EMBO J. (2003) 22,5336-5346
See also comments inHurtley S. (2003) A dead-end short cut. Science 302, 535 Jones S. (2003) Trypanosome flagellum-motility and more. Nature Rev. Microbiol. 1, 172-173.
Non-flagellated cells show dispersion of their endocytic network
Red: anti-clathrin
Normal
Non-flagellatedBlue: DAPI
Dispersion of :-rab5 (early endosomes)-rab11 (recycling pathway)-p67 (lysosomes)
Group Philippe BASTIN(Muséum National d’Histoire Naturelle, Paris, Dir. C. GIOVANNANGELI & J.S. SUN)
Linda KOHL Filippo RUSCONI
Sabrina BENGHANEMGéraldine TOUTIRAIS
Mickaël DURAND-DUBIEF Carole BRANCHE
Gwénola DORE
Derrick ROBINSON (Université Bordeaux II)
Mark FIELD (Imperial College, UK)
Thanks toK. GULL, P. ENGLUND, C. CLAYTON, G. CROSS