Cilium formation and function
Esben Lorentzen
Department of Structural Cell Biology
Max Planck Institute of Biochemistry
Martinsried, Germany
The eukaryotic cell is highly organized into
different organelles with specific functions
The eukaryotic cell is highly organized into
different organelles with specific functions:
which brings about the requirement for intracellular transport
(DNA)
Cellular
Power plant
ATP generation
Some organelles in the eukaryotic cell
originate from bacteria (chloroplasts and mitochondria)
A photosynthetic bacterium is engulfed by an
early eukaryotic cell to form a chloroplast
The cilium protrudes from the surface of most
eukaryotic cells (with the exceptions of fungi and higher plants)
EM micrograph of a cilium
Bacterial and eukaryotic flagella
are evolutionarily unrelated
Bacterial flagellum
Hollow tube of the flagellin protein
Not membrane-surrounded
Latin: flagellum= "whip"
Eukaryotic flagellum
Microtubule-based
Membrane surrounded
All cilia share a common and well-conserved
microtubule-based architecture
ciliary membrane membrane-­‐bound structures emana/ng from the cell surface conserved between single-­‐celled algae (Chlamydomonas) and higher eukaryotes such as mammals /p axoneme MT-­‐based ciliary membrane is con/nuous with plasma membrane but contains dis/nct lipids and proteins microtubule-­‐based axoneme is built onto a ‘basal body’ at the base of the cilium (modified centriole) 9 + 2 cilium highly ordered arrangement of microtubules in both the basal body (9 triplets) and the axomeme (either ‘9+2’ or ‘9+0’) basal body is aBached to the membrane by ‘transi/on fibers’ à restric/on of free diffusion of macromolecules à ‘ciliary pore’ plasma membrane environment base cell body basal body 9 + 0 transi/on fibers Cilia and flagella come in different shapes and
sizes and can be motile or non-motile cilia
•  Motile cilia typically have nine outer MT-doublets surrounding a
central pair of MT (9+2)
•  Non-motile cilia typically have only the nine outer MT (9+0)
Silverman & Leroux, Trends in Cell Biology, 2009 A short introduction to microtubules (MT)
Most MTs of the cell are highly dynamic
However, MTs of the cilium are relatively stable
Structural organization of flagella
MT of cilia are found as triplets, doublets and singlets
EM tomographic structure of basal bodies
(from which MT triplets grow)
EM tomographic structure
of basal bodies
Li et al., EMBO J 2012
Molecular basis for the 9-fold symmetry of cilia
Axoneme EM structure
EM tomogram of the axoneme from
sea urchin sperm flagellum
Nicastro et al., PNAS 2005
The Sas6 self-organizes into a ring with 9-fold
symmetry dictating the assembly of basal bodies
Figure from Mizuno et al., JMB 2012
Motile cilia in singled cell organisms
•  Motile cilia protrude from eukaryotic cells, often in
multiple copies, and beat in coordinated waves to move the
cell towards a chemical substance (chemotaxis) or light
(phototaxis)
Tetrahymena
(a ciliated protozoan)
(chemotaxis)
Chlamydomonas
(green alga)
(phototaxis)
Two flagella
Hundreds
of flagella
Phototaxis of green algae
Chlamydomonas
(green alga)
(phototaxis)
Light
Wild type
Flagella
mutant
Motile cilia in mammals
•  Motile cilia serve to remove mucus and dust from the lungs
•  Important in reproduction (sperm cells and fallopian tubes)
Sperm cell
(chemotaxis)
Motile cilia move the egg from
the ovaries to the uterus
Motile cilia remove particles from the lungs
Scanning EM picture of
a human fallopian tube
Lyons R et al. Hum. Reprod. 2006
Motile cilia in mammalian development
Mouse embryonic node during development Nonaka et al., Cell, 1998
Filegaut et al., Nat Rev, 2007
Motile cilia in the brain (spinal cord of zebrafish)
Motile cilia (green) found on cells of the
spinal cord of zebrafish embryos are required
for proper fluid flow and formation of organs
Structural organization of flagella
MT of cilia are found as triplets, doublets and singlets
Motile cilia are powered by dynein motors
Immotile cilia (primary cilia)
Primary cilia on epithelial cells •  Most animal cells have a cilium present in one copy per
cell
•  The immotile cilium was long thought to be an evolutionary
remnant much like the appendix
•  Recent research show that the primary cilium is involved in
sensory reception including photoreceptor cells of the eye and
olfactory neurons (sight, smell and hearing)
•  Clustering of receptors in the ciliary membrane makes it
important in signal transduction pathways (Sonic Hedgehog,
epidermal growth factor, 5HT6 serotonin, Wnt)
Primary cilia on mammalian cells were
discovered more than 100 years ago
1898
- Non-motile
- Sensory reception function suggested
Primary cilia on mammalian cells were
discovered more than 100 years ago:
and then rediscovered and named rudimentary cilium after WWII
1962
Primary cilia: important organelles for signaling
Primary cilium:
•  recently shown to play an important role in the sonic
hedgehog signaling pathway. PDGF and noncanonical Wnt signaling have also been suggested
to require the cilium for proper function.
•  Antennae of the cell
•  Important for signaling because of ciliary localization
of trans-membrane receptors and intracellular
signaling molecules
•  explains why complete lack of cilia in knock-out
mice results in early embryonic lethality
à important for developmental signaling
•  Many disease mutations map to ciliary genes
Ciliary localiza/on of ‘Smoothened’ (transmembrane protein in Shh-­‐pathway) from Corbit et al, Nature, 2005 Cilia in sensory reception:
photoreceptor cells (Sight)
Retinal pigmented epithelium
Outer segment
2000 opsin molecules per minute are
transported from the inner segment
to the outer segment via the cilium
Connecting cilium
Inner segment
Electronmicroscopy
picture
Nucleus
Photoreceptor cell
Ramamurthy and Cayouette, Clinical Gen., 2009
Cilia in sensory reception:
olfactory sensory cells (smell)
Olfactory sensory cell
Cilia packed with
olfactory receptors
that bind odor molecules
Ramamurthy and Cayouette, Clinical Gen., 2009
The cilium as a flow sensor in kidney cells
Fliegaut et al., Nat Rev, 2007
Defects in ciliary functions result in diseases (ciliopathies)
Motile cilia
Non-motile cilia
Defects in ciliary functions result in diseases (ciliopathies)
Motile cilia
Non-motile cilia
Primary and motile cilia are
built by intraflagellar transport (IFT)
•  About 600 proteins localize
to the cilium
ciliary membrane •  Cilia do not contain protein
synthesis machinery (no
ribosomes)
•  Transition zone fibers connect
the basal body to the
membrane creating a diffusion
barrier for macromolecules
3. anterograde IFT (kinesin) assembly site 3p axoneme 5. Retrograde IFT (dynein) cilium 4. cargo unloading, complex remodeling transi3on zone fibers ‘ciliary pore’ environment 2. import and IFT complex assembly 6. IFT complex disassembly plasma membrane base cell body 1. targe3ng protein synthesis Basal body IFT proteins Intraflagellar Transport (IFT)
IFT in Chlamydomonas
From Kozminski and
Rosenbaum, 1998
•  IFT was first discovered microscopically in 1993 (Kozminski et al., 1993)
•  The motors for movement are heterotrimeric kinesin-II (anterograde transport) and
cytoplasmic dynein-1b (retrograde transport)
•  Necessary for building and maintaining functional cilia (axoneme grows from the tip,
not from the base)
•  Anterograde IFT: Building blocks (e.g. tubulin) and ciliary membrane protein (e.g.
signaling receptors)
•  Retrograde IFT: Ciliary turnover products and signal transduction proteins
IFT particles form long arrays in the cilium (so-called trains)
•  Electron dense material span the distance between the
ciliary membrane and the axoneme microtubules
EM micrograph of a Chlamydomonas cilium
EM tomographic reconstruction of IFT-particles
?IFT complex + cargo?
Pigino et al., JCB, 2009
Kozminski et al., JCB, 1995
How is a protein recognized as a cilium protein?
Sorting signals for different compartments
Examples of Intracellular sorting sequences (ZIP codes):
Into the nucleus (NLS) :
Out of the nucleus (NES):
into mitochondria
into peroxisomes:
into ER:
PPKKKRKV
LALKLAGLDI
Nterm-MLSLRQSIRFFKPATRTLCSSRYLL
SKL-Cterm
Nterm-MMSFVSLLLVGILFWATEAEQLTKCEVFQ
Suggested motifs for ciliary localization:
Rhodopsin:
Smoothened:
Fibrocystein:
Opsin/Rhodopsin:
Polycystin-2:
Serotonin receptor 6:
MLNKQFRNCMLTTL
ATLLIWRRTWCRLT
LVCCWFKKSKTRKIKP
SSSQVSPA
NSSRVQPQQPGDA
LTTGTATAGQALETLQV
Corbit et al., Nature 2005
Follit et al., JCB 2010
Tam et al., JCB 2000
Geng et al., JCS 2006
Berbari et al., MBC 2008
Nucleocytoplasmic transport:
a well understood system
Sorting transport signal peptides are
recognized by transport receptors
The IFT complex: biochemical purification
IFT-B
70
46
88
52
Cole et al., JCB 1998 22
80
172
IFT-B ‘core’
74/72
81
81
74/72
27
25
IFT-A
43
122A
•  IFT complexes were first purified from isolated Chlamydomonas flagella (Cole et al., 1998)
•  IFT proteins forming this complex are named after their approximate size (on SDS-PAGE)
•  The IFT complex consists of 2 distinct sub-complexes (IFT-A and IFT-B), which separate already at a low
salt concentration (around 50 mM NaCl)
- IFT-A: 6 proteins, mutant phenotypes similar to dynein-1b à involved in retrograde transport
- IFT-B: 14 proteins, mutant phenotype similar to kinesin-II
à involved in anterograde transport
•  IFT-B can further be reduced to an IFT-B ‘core’ complex (at 300 mM NaCl)
•  Only limited knowledge about overall architecture of the complex, no structures available
139
144
57 54
20
122B
140
IFT complex proteins:
prediction of domain composition from sequence
WD (β-propeller)
IFT121
IFT122
IFT140
IFT144
IFT172
WD (β-propeller)
IFT80
α-solenoid
Nup214
Sec13/31
(nuclear pore complex)
(vesicular trafficking)
TPR, α-helical repeats
IFT70
IFT88
IFT139
Importin α
(nucleo-cytoplasmic transport)
IFT74
IFT81
IFT20
IFT57
IFT54
Coiled-coil
Sec2
(vesicle trafficking)
IFT25
IFT22
IFT27
Small GTPase
(regulatory switch molecule)
Jelly-roll fold
Calcium-binding protein
IFT46 ???
IFT52 ?sugar-binding domain?
IFT43 ???
IFT proteins are essential for cilium formation
WT
IFT88 mutant
•  Mouse knockouts of genes encoding IFT core proteins are lethal
Structural studies of intraflagellar transport
Research aims
•  Reconstitute IFT complexes for structural and
functional characterization
•  Determine the architecture of the IFT complex
using a combination of X-ray crystallography,
electron-microscopy and biochemical methods
•  Molecular basis for intraflagellar transport:
1) How does the IFT complex recognize proteins
to be transported to the cilium?
2) How does the IFT complex associate with
dynein and kinesin motors?
Project 1: Purification of IFT proteins/complexes
IFT-B
80
70
46
88
52
IFT-B ‘core’
74/72
81
81
74/72
Cr46/52/ Cr46/52/70
70/88
Cr46/52
22
172
27
25
Cr43
IFT-A
43
122A
122B
139
144
140
57 54
20
Cr52 ∆C
Cr25/27
Cr46
Hs22
Hs: Homo sapiens
Cr: Chlamydomonas reinhardtii
Mm: Mus musculus
Cr57∆C
Mm20 Cr20
Hs74/81 Hs25
Assembly of the C.reinhardtii IFT-B core: two stable sub-complexes
Size exclusion chromatography
SDS gel of peak fractions
E.coli expression
Insect cell expression
Reconstitution of larger complexes: IFT-B core octamer
Size exclusion chromatography
The IFT-­‐B core octamer appears stoichiometric and is stable in >2M NaCl SDS gel of peak fractions
IFT-­‐B core octamer Reconstitution of larger complexes: IFT-B core nonamer
Size exclusion
chromatography
Superose
6
116
66
80
45
MW (kDa)
Absorbance A280 (mAU)
100
60
40
20
0
SDS gel of peak fractions
35
25
*
*
*
*
IFT88
IFT70
IFT81∆N
IFT74∆N
IFT52
IFT46
His-IFT27
His-IFT22
18
8
10
12
14
IFT25∆C
elution volume (ml)
IFT-­‐B core nonamer The current interaction map of the IFT-B core complex
Using biochemical methods it is possible to map
the direct protein protein interactions within the complex
Negative stain EM of IFT complexes
Class averages of 5000 particles
MW = 500 kDa
Superose 6
116
80
45
60
40
20
0
IFT88
66
MW (kDa)
Absorbance A280 (mAU)
100
35
25
*
*
*
*
IFT70
IFT81∆N
IFT74∆N
IFT52
IFT46
His-IFT27
His-IFT22
18
8
10
12
elution volume (ml)
14
IFT25∆C
Crystals of a ‘minimal’ IFT-B core hexamer
1200
IFT81ΔN
IFT74ΔN
45
800
400
0
10
SDS gel of peak fractions
116
66
1600
MW (kDa)
Absorbance A280 (mAU)
Size exclusion chromatography
15
elution volume (ml)
35
His-IFT27
25
18
IFT46ΔN
IFT25ΔC
14
IFT52ΔN.
‘Minimal’ IFT-­‐B hexamer Crystals 50 µm
Project 2: How is tubulin transported into the cilium?
4. cargo unloading
+
5. tubulin
incorporation
3p Large amounts of tubulin
is needed in the cilium
cilium Calculations show that each IFT complex
carries 1-2 tubulin dimers
3. transport to
ciliary tip (IFT)
environment plasma membrane 2. IFT complex assembly/
tubulin-binding
base 1. ciliary targeting/import
cell body α/β tubulin dimers Basal body IFT proteins Intraflagellar transport proteins 54, 74 and 81
contain N-terminal domains of unknown function
++++
N-terminal
domain
N-terminal
domain
Predicted coiled-coil
Middle
domain
Predicted
coiled-coil
he N-terminal region of IFT81 is conserved
HsIFT81N
CfIFT81N
DrIFT81N
CeIFT81N
CrIFT81N
TbIFT81
---------MSDQIK-FIMDSLNKEPFRKNYNLITFDSLEPMQLLQVLSDVLAEIDPK-Q
---------MSDQIK-FIVDNLNKEPFRKNYNLITFDSLEPMQLLQVLNDVLAEIDPK-Q
---------MSEQLK-FIVEQLNKEPFKKNFNLITFDSLEPMQLLQTLSDVLAEIDPK-Q
---------MSNDIQGFILHFLNEEPFNLNLSSLQFDQLPPQQLLQILSNVLSWVSDT-D
----------MGDVS-YIVDSLGLPPFSYQMSLLSFTEKGPQELLQLLSDVFSTISPKHQ
MRQNPPKEPSEEEVLQYIVDNVN-KLLSRHYSLVEFDAIQGTDLLQILADIFGTLSPA-Q
:: :*:. :. : : . : *
:*** * :::. :. :
49
49
49
50
49
58
HsIFT81N
CfIFT81N
DrIFT81N
CeIFT81N
CrIFT81N
TbIFT81
LVDIREEMPEQTAKRMLSLLG-ILKYKPSGNATDMSTFRQGLVIGSKPVIYPVLHWLLQR
VVDIREEMPEQTAKRMLSLLG-ILKYKPPGNATDMSTFRQGLVIGSKPVIYPVLHWLLQR
AIDIREELPEQTAKRMFTLLG-MLKYKPSGGMSEVSSFRQGLVSGSKPVVHPILHWLLQR
RIDIKREAAEETAIRILNMLR-ILRYRPPQDQDEQEEWRAGIVEGRKTSLYPLLVFLFEN
KVDVAKEVPDQTADRLIGFLK-IIKYRP--NVQDPLLFRQLVAAGDRETLYQILRWVVPQ
QIDMGVAPTDEAAASMLEFLTKTLGYRVPPMLADS--FPTSFSRAEPTVIYPTLYWVLSN
:*:
.:::* :: :* : *:
: : . .
:: * ::. .
108
108
108
109
106
116
HsIFT81N
CfIFT81N
DrIFT81N
CeIFT81N
CrIFT81N
TbIFT81
TNELKKRAYLARF--TSELKKRAYLARF--IPELKKRAYLARF--SEGLKERAYLSKY--AQLLEKRAFVGYYLSF
MQQNEKRVYLARFLQR
::*.::. :
121
121
121
122
122
132
IFT54 and IFT81 contain an N-terminal CH domain
CH: Calponin homology:
~120 residue alpha-helical domain implicated in MT binding
Cr IFT54N
Purification
Crystals
15 kDa 1.6Å, Rfree= 24.4% Cr: Chlamydomonas reinhardtii
Mm: Mus musculus
Cr IFT81N
Mm IFT54N
Purification
15 kDa 1.6Å, Rfree= 21.6% Crystals
Purification
Crystals
15 kDa 2.3Å, Rfree= 25.0% IFT81N is structurally similar to
microtubule (MT)-binding CH-domains
Of all the structures in the protein data bank, IFT81N is most similar to NDC80, a kinetochore-­‐MT bridging protein NDC80 CH domain (Kinetochore-­‐microtubule bridging protein) Ciferri et al., Cell 2008"
IFT81N CH domain MT-binding residues are conserved in IFT81N
Tubulin dimer Posi/vely charged patch that binds microtubules Alushin et al., Nature 2010 NDC80 CH domain IFT81N CH domain Posi/vely charged Nega/vely charged Electrosta/c surface poten/al of IFT81N Predicted MT binding residues in IFT81N
HsIFT81N
CfIFT81N
DrIFT81N
CeIFT81N
CrIFT81N
TbIFT81
---------MSDQIK-FIMDSLNKEPFRKNYNLITFDSLEPMQLLQVLSDVLAEIDPK-Q
---------MSDQIK-FIVDNLNKEPFRKNYNLITFDSLEPMQLLQVLNDVLAEIDPK-Q
---------MSEQLK-FIVEQLNKEPFKKNFNLITFDSLEPMQLLQTLSDVLAEIDPK-Q
---------MSNDIQGFILHFLNEEPFNLNLSSLQFDQLPPQQLLQILSNVLSWVSDT-D
----------MGDVS-YIVDSLGLPPFSYQMSLLSFTEKGPQELLQLLSDVFSTISPKHQ
MRQNPPKEPSEEEVLQYIVDNVN-KLLSRHYSLVEFDAIQGTDLLQILADIFGTLSPA-Q
:: :*:. :. : : . : *
:*** * :::. :. :
49
49
49
50
49
58
HsIFT81N
CfIFT81N
DrIFT81N
CeIFT81N
CrIFT81N
TbIFT81
LVDIREEMPEQTAKRMLSLLG-ILKYKPSGNATDMSTFRQGLVIGSKPVIYPVLHWLLQR
VVDIREEMPEQTAKRMLSLLG-ILKYKPPGNATDMSTFRQGLVIGSKPVIYPVLHWLLQR
AIDIREELPEQTAKRMFTLLG-MLKYKPSGGMSEVSSFRQGLVSGSKPVVHPILHWLLQR
RIDIKREAAEETAIRILNMLR-ILRYRPPQDQDEQEEWRAGIVEGRKTSLYPLLVFLFEN
KVDVAKEVPDQTADRLIGFLK-IIKYRP--NVQDPLLFRQLVAAGDRETLYQILRWVVPQ
QIDMGVAPTDEAAASMLEFLTKTLGYRVPPMLADS--FPTSFSRAEPTVIYPTLYWVLSN
:*:
.:::* :: :* : *:
: : . .
:: * ::. .
108
108
108
109
106
116
HsIFT81N
CfIFT81N
DrIFT81N
CeIFT81N
CrIFT81N
TbIFT81
TNELKKRAYLARF--TSELKKRAYLARF--IPELKKRAYLARF--SEGLKERAYLSKY--AQLLEKRAFVGYYLSF
MQQNEKRVYLARFLQR
::*.::. :
121
121
121
122
122
132
C.reinhardtii IFT81N binds MT in vitro (weakly)
main
116
+
+
+
-
66
45
MW (kDa)
B35
116
25
66
MW (kDa)
45
50 µM GST-IFT81N
6 µM microtubules
tubulin
+
+
+
-
N N GST-IFT81N
50 µM GST-IFT81N
6 µM microtubules
C
C
+
+
IFT81 IFT74 25/27 ain
B
+
+
+
-
+
-
MT
IFT81N
(10 µM)
tubulin
GST-IFT81N
35
18
IFT25ΔC 25
14
18
Microtubule sedimentation assay
Coomassie stained protein gel
14
nega/ve stain electron micrographs M
I
(
IFT74 N-terminus is highly basic
§  N-terminus of NDC80 is highly basic in nature and is proposed to
bind to the acidic C-terminal tail of tubulin subunits
§  Deletion of the basic part of the NDC80 dramatically reduces the
affinity of this protein towards MT
§  IFT74 contains a highly basic N-terminal region
NDC80 ++++ CH Coiled coil CH IFT81 & 74 ++++ IFT81
IFT74
Coiled coil MASNHKSSAARPVSRGGVGLTGRPPSGIRPLSGNIRVATAMPPGTARPGSRGCPIGTGGV
LSSQIKVAHRPVTQQGLTGMKTGTKGPQRQ
Hs74N pI = 12.55
Purification of a truncated human IFT74/81 complex
CH IFT81 & 74 ++++ IFT81
IFT74
Coiled coil 676
600
insoluble
peak fractions from
gel filtration
CH IFT81
IFT74
++++ truncated
IFT74/81
complex
45 kDa IFT74N_CC
35 kDa IFT81N_CC
Human IFT74N/IFT81N binds MT:
The acidic C-terminal part of tubulin is required for binding
-
+
Tubulin
+
+
+
+
MT
Hs74/81 complex (2 µM)
Sub/lisin treated, to cut off the C-­‐terminal tail of (β)-­‐tubulin IFT74N_CC
IFT81N_CC
Microtubule sedimentation assay
Coomassie stained protein gel
Human IFT74N/IFT81N complex binds αβ-tubulin dimers
GST pull down
Human IFT74N/IFT81N complex binds αβ-tubulin dimers
Binding constants for αβ-tubulin:
1600
1100
1400
Kd=0.9±0.2µM
1000
1200
900
Kd=15.8±2.0µM
1000
800
0
50000
100000
Hs74/81 concentration (nM)
150000
600
0
Kd=186.9±36.6µM
1200
1000
800
700
1400
Thermophoresis with
no jump
Thermophoresis with
no jump
Thermophoresis with
no jump
1200
IFT81N alone: 16 µM
IFT81N/IFT74N: 1 µM
100000
200000
Hs81N concentration (nM)
300000
800
0
200000
400000
600000
Hs81N(K73E_K75E) concentration (nM)
IFT81N mutant: 187 µM
Is the MT/tubulin binding motif required for localization of
IFT-complexes to the cilium or for tubulin transport into the cilium?
Both Full length IFT81 and N-terminal deletion mutant (dN)
localize along the ciliary axoneme/tip of the cilium in RPE1 cells
IFT81-Flag (dN)
IFT81-Flag (full length)
Flag / Acetylated
tubulin
Flag / Acetylated
tubulin
Flag
Flag
Ac.tubulin
Ac.tubulin
IFT81 dN overexpression impairs cilia formation
and/or maintenance RPE1 cells
Flag
B- cilia induced by 0.5uM cytochalasin D
IFT81-dN-Flag
IFT81-Flag
A- cilia induced by serum starvation
Arl13b
Flag/Arl13b/
CAP350
DAPI
CAP 350
Flag
Flag/Arl13b/CAP350
DAPI
CAP 350
Arl13b