INTRAFLAGELLAR TRANSPORT IN TRYPANOSOMES

Filippo RUSCONI

Muséum National d’Histoire NaturelleINSERM U565 & CNRS UMR 5153 & MNHN 0503

rusconi@mnhn.fr

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