Centrosomes and Cilia
SMC6052/BIM6027/516-604D
William Y. Tsang
Research Unit Director, Cell Division and Centrosome Biology, IRCM
Chercheur Adjoint, Faculté de medécine, Université de Montréal
Adjunct Professor, Division of Experimental Medicine, McGill University
Outline
-A Brief History
-Centrosome function
-Centrosome structure
-Microtubule structure
-Microtubule assembly, stability and dynamics
-The Cell Cycle
-The Centrosome cycle
-Cilium function
-Cilium structure
-Cilium assembly
-Human diseases
Early Studies on Centrosomes
©2004 WILEY-VCH
1877 Edouard Van Beneden’s drawings of
mitosis during the first cleavage division
of the mesozoan Dicyemella. He drew
small dots/circles at the spindle poles
which he called the polar corpuscle.
1888 Theodore Boveri’s observations of mitosis
during the first cleavage division of the
nematode
Ascaris
megalocephala.
(A)
Metaphase; (B) End of mitosis. He introduced
the terms centriole and centrosome.
Centrosome: A Tiny Organelle
Mammalian cell: ~2000-4000 µm3 in volume
~20 µm in diameter
~700 µm3 in volume
~5-10 µm in diameter
Nucleus
Centrosome
~ 1 µm3 in volume
Centriole: 0.2 X 0.5µm
-is non-membrane bound
-does not contain DNA
PCM
Centrosome Function
-Cell Division
-Cell Shape
-Cell Cycle Progression
-Cell Polarity
-Intracellular transport
-Cell Migration
-Cilia Formation
J. Cell Sci (2012)
Centrosome: The Main Microtubule Organizing Center
-A pair of centrioles (mother and daughter centriole) and an amorphous pericentriolar matrix (PCM)
-9 sets of microtubule triplets
-Microtubule polarity
-Sub-distal and distal appendages at the + end
-Connecting fibers at the - end
-Microtubule (MT) nucleation (γ-TuRC complex)
-Microtubule anchoring (Ninein)
-Cytoplasmic versus centriolar microtubules
Cytoplasmic Versus Centriolar Microtubules
Within a Cell
In Different Cell Types
Centriolar
Microtubules
A Cartwheel Protein, SAS-6 Dictates The Universal 9-Fold Symmetry of Centrioles
Pierre Gonczy
SAS-6 Dimer
C
B
A
Central hub
Microtubules
Spoke
Pinhead
SAS-6 organizes the cartwheel central hub and spokes. (A) SAS-6 monomer. (B) Two SAS-6
proteins homodimerize. (C) Pairs of SAS-6 homodimers interact. (D) Assembly of a cartwheel
(central hub and spokes) with nine homodimers. (E) The cartwheel connects with the pinhead
which in turn connects with microtubules.
Cep135 Constitutes The Pinhead That Stabilizes The Cartwheel
Procentriole
Spoke
EMBO J. (2013)
Cep135 organizes the pinhead and connects SAS-6 with microtubules and CPAP.
CPAP binds αβ dimer and γ-TuRC and promotes centriolar microtubule assembly/elongation.
The Tubulin Superfamily
©2004 WILEY-VCH
α and β Tubulins are Building Blocks of Microtubules
-α and β tubulins form αβ heterodimers
-End-to-end joining of αβ tubulin heterodimers
followed by hydrolysis of GTP bound to β-tubulin
-13 protofilaments surrounding a hollow core
-Elongation occurs at the + end
Singlet, Doublet and Triplet Microtubules
© 2000 by W. H. Freeman and Company
Cytoplasmic microtubules
Caenorhabditis elegans centrioles
Cilia
Flagella
Drosophila centrioles
Centrioles of most eukaryotes
-A tubule: 13 protofilaments
-B and C tubules: ~10 protofilaments fused to the wall of another tubule
Microtubule Assembly
©2004 WILEY-VCH
α +β + γ tubulins
Microtubule nucleation:
-is thermodynamically unfavorable
-occurs spontaneously in vitro
-requires γ-tubulin in vivo
α + β tubulins
The γ-tubulin Ring Complex Mediates Microtubule Nucleation
©2004 WILEY-VCH
γ-tubulin
GCP2
GCP3
GCP4, GCP5, GCP6
and other
accessory proteins
GCP = γ-Tubulin complex protein
γ-tubulin ring complex (γ-TuRC) = 12-14 γ-tubulin molecules + several GCP proteins
γ-tubulin binds between protofilaments at the - end
Ninein Mediates Microtubule Anchoring
N
Apical-basal microtubule arrays (blue in (a) and green
in (b)) and centrosomes (orange) in polarized cochlear
inner pillar epithelial cells. (a) N denotes the nucleus.
(b) Centrosomes were stained with γ-tubulin.
Nucleating complex (γ-TuRC) vs. anchoring Complex (Ninein)
γ-tubulin and ninein are highly enriched at the centrosome.
Possible fates:
1. Microtubule release following nucleation
2. Microtubule - end capping by a anchoring complex
3. Microtubule release from the centrosome or firm
anchorage within the centrosome
4. Release of anchoring complexes from the centrosome
and their transport along a microtubule
Ninein
(yellow)
is
concentrated at the
centrosome and at the
apical sites of inner
pillar
cells.
Ninein
speckles are also found
within the apical half of
the cytoplasm.
©2004 WILEY-VCH
Microtubule Dynamic Instability and Treadmilling
The Plant Cell (2004)
+ end is dynamic.
- end never grows, but can be stabilized or depolymerized.
Microtubule-Associated Proteins Regulate Microtubule Stability
Ninein complex anchors
at the minus end
(Plus end tracking proteins)
Ex: EB1, EB2, EB3
CLASP1, CLASP2
Image: MBoC Panel 16-3
Post-translational Modifications of Microtubules
Glycylation, Glutamylation, Acetylation and Detryosination
Post-translational Modifications Regulate Microtubule Stability
Nature Reviews Molecular Cell Biology (2011)
Centriolar and axonemal microtubules are
extremely stable.
Microtubule Motor Proteins
-Two major classes: kinesin and dynein
Kinesin: 2 heavy chains (head domain) and 2 light chains
Dynein: 2 heavy chains (head domain) and multiple light and intermediate chains
A kinesin or dynein contains:
-a microtubule binding site
-a ATP binding site
-a cargo binding site
Microtubule Motors Mediate Cargo Transport
Cargoes:
Membrane vesicles
Protein complexes
Chromosomes
mRNAs
Organelles
Microtubules
Science (1998)
Structural and Functional Diversity of Microtubules
Within a Cell
In Different Cell Types
Centriolar
Microtubules
Centrosome Function
-Cell Division
-Cell Shape
-Cell Cycle Progression
-Cell Polarity
-Intracellular transport
-Cell Migration
-Cilia Formation
J. Cell Sci (2012)
The Cell Cycle
Centrosome
Centrosome
Centrosome
Centrosome
Centrosome
Centrosome
The Centrosome Cycle
1)
1) Centriole Disengagement
-licenses centrioles for
duplication in the next cell
cycle
2) Centriole Duplication
-procentriole assembly
4)
2)
3)
3) Centrosome Maturation
-procentrioles elongate to
become daughters
-the daughter centriole
acquires appendages and
becomes the mother
centriole
-PCM enlargement
4) Centrosome Separation
-the two centrosomes
migrate to opposite poles of
the cell
Coordination Between The Centrosome Cycle and The Cell Cycle
Major cell cycle regulators: Cyclin/Cyclin-dependent kinase (CDK)
Polo-like kinase (PLK)
Never in mitosis A-related kinase (NEK)
Anaphase-promoting complex (APC)
Phosphorylation of:
CP110: prevents premature
centrosome separation
Nucleophosmin: dissociates
from centrosomes and
triggers duplication
Mps1: accumulates at
centrosomes and promotes
duplication
Asp: promotes microtubule
nucleation in concert with γtubulin
C-Nap1: dissociates from
centrosomes and induces
centrosome splitting
Eg-5: binds to microtubules,
slides microtubules apart and
induces centrosome
separation
APC
Kendrin
Separase
Destruction of
Kendrin: induces centriole
disengagement and license
centrosomes for the next
round of duplication
Canonical Centriole Duplication Versus De novo Centriole Assembly
Canonical Pathway
De novo Pathway
Operates when:
1) there are no centrosomes
2) extra centrioles are needed to
template cilia
Deuterosome
Mother/
Daughter
Centriole
Nat Cell Biol. (2013)
Procentriole
-generates 1 procentriole per existing centriole
Procentriole
Deuterosome
-resembles a ring/sphere
-is devoid of microtubules
-is smaller in diameter (0.15µm)
compared to a centriole (0.2µm)
-supports simultaneous generation of
multiple procentrioles
Centrosome Amplification Frequently Occurs in Human Cancers
Staining of pericentrin in white/red
and DAPI in blue.
(C) Non-tumor cell line
(D, F) Breast tumor cell line
(E, G) Prostate tumor cell line
(H) Cell dissociated from a human
breast tumor
Cancer Res. (1998)
The Origin of Centrosome Amplification
Nat. Reviews (2002)
Consequences of Centrosome Amplification
Lagging chromosome as a result of a chromatid
bounded to more than one spindle pole
Centrosome clustering
J. Cell Sci.(2012)
The Relationship Between Centrosomes and Cilia
Cilium
(1-10 µm in length)
Proliferating
-Centrosome is located
near the cell center
Non-proliferating/Non-mitotic
-Centrosome is located near the cell
surface
-Only the mother centriole/basal body
can template a cilium.
Conservation of Centrosomes and Cilia in Eukaryotes
BMC Biology 2013
Organisms possessing (1) centrosomes (mother and daughter centrioles); (2) mother
centrioles/basal bodies; and (3) cilia.
Cilium Diversity
Neurons
Epithelial Cells
-motile versus non-motile (primary) cilia
-cilia number per cell
-cell/tissue types
Genetics in Medicine (2009)
Cilium Function
-Motility
Sperm movement
Mucus clearance
Cerebrospinal fluid circulation
Ovum transport
Left-right asymmetry
-Sensory
Mechanosensation
Chemosensation
Light sensation
Thermosensation
Motility Function
Mucus clearance
Sperm movement
Sensory Function
Mechanosensation
The polycystin-1–polycystin-2 complex (PC1–PC2), which is sensitive to shear stress, is localized within the ciliary membrane
(left panel). Fluid-induced ciliary bending activates this Ca2+ channel. The Ca2+ influx (right panel) causes Ca2+ release from
ryanodine-sensitive intracellular stores and subsequent downstream responses such as activating protein-1 (AP1)dependent gene transcription by the Ca2+-dependent kinase PKC. Mutations in PC1 or PC2 might disable cilia-mediated
mechanosensation, which is normally required for tissue morphogenesis, and thus can cause polycystic kidney disease.
Sensory Function
Chemosensation
The transcriptional factor glioma (GLI) and regulator suppressor of fused (SUFU) are transported to the
ciliary tip. GLI is processed into a transcriptional repressor, which is transported back to the cell body. Upon
Hedgehog (Hh) binding to its receptor patched-1 (PTCH1), smoothened (SMO) is released and transported to
the ciliary tip, where it turns off GLI processing by interacting with SUFU. The activator form of GLI is
transported to the cell body and enters the nucleus where it induces the expression of genes , such as those
involved in tissue patterning, cell proliferation and differentiation. Defective Hh signaling can lead to
polydactyly, cerebellar hypoplasia and craniofacial development. Hyperactive Hh signaling, on the other
hand, can lead to basal cell carcinoma and medulloblastoma.
Nature Reviews Molecular Cell Biology (2007)
Cilium Structure
Daughter centriole
Transition zone
Mother centriole/
Basal body
Transitional fibers/distal
appendages
-A cilium is made up of:
-an axoneme surrounded by a ciliary membrane
-9 sets of microtubule doublets with or without a central pair
-Distal appendages dock the mother centriole to the membrane
-Transition zone provides a diffusion barrier for selective transport
Nature (2007)
Cilium Assembly
Dev. Dyn. (2007)
Intraflagellar Transport (IFT)
-IFT was first reported in 1993
-IFT is a microtubule-dependent
bidirectional motility along ciliary
axonemes
-IFT is essential for cilia formation
and maintenance, and cargo
transport
transition
zone
Mother centriole/
Basal body
-IFT particles contain at least 20
subunits and can be divided into
two sub-complexes, IFT-A (required
for retrograde transport) and IFT-B
(required for anterograde transport)
/distal appendages
Daughter
centriole
-IFT
requires
kinesin-2
for
anterograde transport and dynein1b for retrograde transport. Motors
move along the outer doublet
microtubules of ciliary axonemes.
-Cargos are attached to IFT particles
Frontiers in Bioscience (2008)
Intraflagellar Transport (IFT)-Continued
Ciliary tip compartment
Frontiers in Bioscience (2008)
IFT is proposed to consist of 6 phases: (1) IFT particles accumulate within the basal body region, with the transitional fibers
possibly serving as a docking point. (2) Active kinesin-2 transports IFT particles and inactive dynein-1b to the ciliary tip.
Kinesin-2 associates with IFT-A (A), and dynein-1b (HC) associates with IFT-B (B). (3) Release of anterograde IFT assemblies
into the ciliary tip compartment (CTC), followed by dissociation of assemblies. Kinesin-2 does not enter the CTC. (4) Active
dynein-1b first associates with IFT-A, which then associates with IFT-B. Retrograde IFT assemblies exit the CTC, where
inactive kinesin-2 now associates with dynein-1b. (5) Dynein-1b returns IFT particles to the ciliary base. (6) Retrograde
assemblies are reorganised in preparation for another IFT cycle. Active IFT motors are coloured green and inactive IFT
motors are coloured red.
Centrosomes and Cilia in Human Disease
Primary Microcephaly (small brain)
Dwarfism (short stature)
Non-motile Ciliopathies
Polycystic Kidney Disease (kidney cysts)
Centrosome
Aberrations
Genomic
Instability;
aneuploidy
Cancer
Leber Congenital Amaurosis (loss of
vision)
Bardet-Biedl Syndrome (retinal
degeneration, obesity, diabetes, male
infertility, polydactyly, cognitive
impairment)
Cilia
Aberrations
Joubert Syndrome (cerebellar
malformation, ataxia, hypotonia)
Motile Ciliopathies
Primary Ciliary Dyskinesia
(infertility,
bronchiectasis,
chronic
sinusitis, situs inversus)
CP110 (Centrosomal Protein of 110 kDa), A Novel Cyclin/CDK –Binding
Protein
CP110 is phosphorylated by Cyclin/CDKs
In vitro kinase assay: purified GST-CP110 + cyclin/cdk + radiolabeled ATP
Dev. Cell (2002)
CP110 is a Centriolar Protein
Centrin
γ-tubulin
CP110
Centrin
Merge
G1
M
Dev. Cell (2002)
CP110 is a Protein Localized to The Distal End of Centrioles
Dev. Cell (2007)
Schematic of CP110 Motifs/Domains
Motifs/Domains:
SP/TP - CDK phosphorylation
RXL/KXL - cyclin binding
D box - destruction box
KEN box
Coiled-coil
Cyclin/CDK
APC
Protein-protein interaction
Dev. Cell (2002)
CP110 Expression is Regulated During The Cell Cycle
Northern Blot
Western Blot
Dev. Cell (2002)
CP110-Interacting Partners
Immunoprecipitation and mass spectrometry
Putative CP110-interacting proteins:
Calmodulin
Cep290
Cep97
Cep76
Kif24
Cell (2007); Dev. Cell (2008); Dev. Cell (2009); Cell (2011)
Phenotype of CP110 Loss
Control
CP110KD
Control
CP110KD
Polaris
Ac. tub
Non-ciliated Cells
Elongated centrioles
Ciliated Cells
Premature cilia formation
Cell (2007); Curr. Biol. (2008)
Phenotype of CP110 Over-Expression
Flag
Merge
Control
Control
CP110
100%
80%
60%
40%
20%
0%
CP110
Ac. tub
% of
Ciliated Cells
DAPI
Loss of cilia in quiescent cells
Cell (2007); Dev. Cell (2008)
CP110 is Absent From The mother Centriole in Quiescent Cells
Glu. tubulin
CP110
Merge
Cell (2007)
CP110 Knockout in Drosophila
Projections of an EM tomogram of a centriole in a WT (A and C) and a CP110Δ (B and D) larval
wing disc cell (A and B) or brain cell (C and D). The centriolar MTs extend dramatically (arrows)
beyond the centriole (brackets) in CP110Δ cells.
J. Cell Biol. (2013)
Up-regulation of CP110 in Patients with Chronic Sinusitis
J. Allergy Clin. Immunol. (2011)
Nature (2014)
Sinonasal mucosa from patients with CS is poorly ciliated and over-expresses CP110. A, Control mucosa
evinces near-complete coverage by cilia, whereas mucosa from patients with CS has significantly less ciliated
coverage. B, CP110 mRNA copy number is increased in mucosa from patients with CS. C, CP110 protein
expression is increased in mucosa from patients with CS.
Suggested Readings
Bornens. Science (2012) 335: 422
Nigg and Raff. Cell (2009) 139: 663
Nigg and Stearns. Nat. Cell Biol. (2011) 13: 1154
Fu et al. Cold Spring Harb. Perspect. Biol. (2015) 7: a015800
Tsang and Dynlacht. Cilia (2013) 2: 9