Understanding basal body and ciliary structure and their relationship to human disease

1
Understanding basal body and ciliary structure
and their relationship to human disease
Susan K. Dutcher Washington University School of
Medicine and 3-Dimensional Fine Structure of
Cells and Tissue
2
3. Cilia and basal bodies are biochemically
complex (800 polypeptides) 4. Cilia are
conserved from basal eukaryotes to humans with
loss in several phylogenetic lineages 5. Cilia
are found on most cell types in humans and play
important roles
1. Cilia (or flagella) are ancient organelles
Membrane bound projections Generally have
doublet microtubules2. Basal bodies are
organizing centers for cilia Generally have
triplet microtubules
3
Cilia in mammals are widespread
Diseases of Sensory Cilia Polycystic kidney
Central obesity Polydactly Retinal
degeneration Bile duct dysplasia Anosmia LR-
reversal Life span (C. elegans)
Diseases of Motile Cilia Infertility
Bronchitis LR-reversal
Heart defects
4

• Primary cilia have no known function in
  mammalian cells and are likely to be vestigial
  organelles
• Molecular Biology of the Cell
• Alberts et al., 2002

5
Signaling via the sensory cilia in mammals

• Polycystins- PKD
• Progesterone receptor
• Somatostatin-3 receptor
• Platlet-derived growth factor receptor
• Olfactory receptors
• Smoothened/Hedgehog

6
Intraflagellar transport particle movement
IFT moves ciliary components out to tip for
assembly IFT moves components back to the cell
body for signaling
Carlo Iomini
7
Loss of IFT in mice suggests an essential role
for cilia

• Embryonic lethality
• Neural tube closure defects

Loss of IFT in just the eye results in retinal
degeneration

• Retinitis pigmentosum

8
Centrioles and basal bodies

• Centrioles and basal bodies both have cylinders
  of nine triplet microtubule blades
• Centrioles play roles in spindle assembly and
  progression through the cell cycle
• Basal bodies are templates for cilia and docking
  sites for IFT

9
Centrosome Ultrastructure
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Breast cancer cells often have centrosome
amplification

• Too many centrioles results in multipolar
  spindles and genome instability
• Centrioles are often morphologically abnormal in
  breast cancer cells

11
Using Chlamydomonas to study cilia and basal body
structure

• Cryo EM tomography
• Epsilon tubulin
• STP2
• BBS5

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Cryofixation of Chlamydomonas
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Aligned Tilt Series of a Proximal Basal Body
Region

• 250 nm -400 nm thick section
• 15 nm gold used as
• alignment markers
• 1.5o increments
• /- 60 degrees
• Dual axis

Eileen OToole
14
Wild-type basal body structure
Basal body Transition zone
Eileen OToole
15
Tomography of basal bodies
16
Using Chlamydomonas to study cilia and basal body
structure

• Cryo EM tomography
• BLD2 Epsilon tubulin
• STP2
• BBS5

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The tubulin superfamily
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Mutants in ?-tubulin
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?????-tubulin encircles the basal bodies. and
acts as a scaffold to stabilize the microtubule
cylinder
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A missense mutation in epsilon tubulin

• Maintainence of the integrity of the amorphous
  material and microtubule blades

21
Using Chlamydomonas to study cilia and basal body
structure

• Cryo EM tomography
• BLD2 Epsilon tubulin
• STP2
• BBS5

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STP2 Cilia asssembly and MT assembly
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New mechanisms for taxol sensitivity

• Aberrant basal bodies/centrioles
• STP2

Mitra and Sept, 2006
24
Using Chlamydomonas to study cilia and basal body
structure

• Cryo EM tomography
• Epsilon tubulin
• STP2
• BBS5

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Obesity
Increasing incidence of obesity in Missouri 12
in 1991 22.5 in 2001 Causes? Increased
consumption Cilia defects?
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Comparative genomics
Chlamydomonas Human
4000
Jin Billy Li
BLASTP
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Comparative genomics
688/322
700/ 322
Chlamydomonas Human
Arabidopsis
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Vertebrate homologs
RNAi Phenotypes in Chlamydomonas

• PARCG Spermatogenesis
• Dynein DNAH5 PCD
• Hydin Infertility, hydrocephaly in mice
• Spag16 Infertility, hydrocephaly in mice
• Seahorse Cystic kidney in zebrafish
• Qilin Cystic kidney in zebrafish
• Polaris Polycystic kidney disease (IFT88)
• Wimple Neural tube closure (IFT172)
• Nephrocystin 4 Juvenile renal defects, retinal
  degeneration
• HRG4 (unc119) retinal degeneration
• Pkhd/Fibrocystin Renal defects
• PC2 Polycystic kidney disease
• Meckels syndrome bile duct dysplasia
• TULP Retinal degeneration

Slowly swimming
Slowly swimming
Altered movement
Paralyzed flagella
Mulitflagellated
Aflagellate
Aflagellate
Aflagellate

• Phototaxis

Aflagellate
Short, slowly beating
Phototaxis
Phototaxis
Altered movement
Linya Li
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Bardet Biedl Syndrome is a cilia defect

• Rare autosomal recessive human disease
• central obesity, diabetes, renal cysts, anosmia,
  retinal degeneration, learning disabilities,
  polydactyl
• Bardet Biedl Syndrome 5 of the 6 genes (BBS1,
  BBS2, BBS4, BBS7, BBS8) were in the computational
  dataset

BBS5
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Multiple bbs5 patients have mutations in
DKFZp762194
NFB9 Splice junction change in exon 6
KK63 L142X in exon 6
PB108 263-271 indel
PB127 W59X
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Bbs5 localizes to basal bodies in mice
Li et al., 2004
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Bbs5 protein localizes to basal bodies
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Two transition zone defect in two RNAibbs5
Chlamydomonas strains
Cap could be an intermediate in the assembly
process Bbs5p may help to remove the cap
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Cilia and human disease

• Cilia play essential roles in humans and defects
• Provide sensing or monitoring of environment
• Alterations in basal bodies and cilia result in
  increased sensitivity to taxol
• Role of bbs5 in preparing basal body to assemble
  cilia

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• Basal body mutant phenotypes
• Andrea Preble (CU)
• John Stanga
• Naomi Morrissette
• Jessica Esparza
• Andrew Lippa
• Jen Heelly
• Jacob Till
• Leslie Meyer

• Cryo-Electron tomography
• Dick McIntosh
• Eileen OToole
• Tom Giddings
• Andreas Hochstrasser
• Comparative Genomics
• Jin Billy Li
• Gary Stormo
• RNA interference/two hybrid
• Linya Li
• Jenny Keller
• Human BBS genetics
• Nicholas Katsanis (Johns Hopkins)
• Richard Lewis (Baylor)
• Brad Yoder (Alabama)
• Lisa Guay-Woodford (Alabama)
• Willy Davidson (Simon Fraser)
• Phil Beales (University College, London)

• Membrane proteins
• Carlo Iomini
• Gianni Piperno (Mt. Sinai)
• Katelyn McGary

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MUDPit Proteomics are complementary
400
85
14/
175/
Keller et al., 2005
Pazour et al., 2005

• We are missing tubulins, kinesins, phosphatases,
  kinases, EF hand, glycolytic enzymes, small G
  proteins, some coiled coil
• Proteomics is missing BBS proteins, DTI1, DTI2,
  Tubby
• Our set must have other conserved, but not
  ciliary genes, but we do not know which ones yet

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Basal bodies and flagella are complex
But they were lost multiple times during
evolution
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Human Chlamy - Yeast
Cr Tusp
Cr TBY2
Yeast 6,100
Cr TBY1
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RNAi and Localization of Bardet Biedl Syndrome
homologs in Chlamydomonas

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Bbs proteins localize to transition zone and to
basal bodies
BBS1, 5, 8 BBS2, 4
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Tubby and tubby family members
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Longer cylinder vs. cap?
uni3-1 uni1-1
RNAibbs5
Other mutants have longer transition zone regions
and assemble flagella, but the cap is unique to
bbs5 Cap could be an intermediate in the
assembly process and Bbs5p may help to remove the
cap
43
Loss of axoneme structures
Chlamydomonas axoneme Mouse oviduct axoneme
44
Two transition zone defect in two RNAibbs5
strains
Ratio of Distal to Proximal Region wild-type
bbs5 N15 N12
H
1.24 1.4
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Longer cylinder vs. cap?
uni3-1 uni1-1
RNAibbs5
Other mutants have longer transition zone regions
and assemble flagella, but the cap is unique to
bbs5 Cap could be an intermediate in the
assembly process and Bbs5p may help to remove the
cap