Centrosomes and Cilia
SMC6052/BIM6028/EXMD604
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 structure
-Microtubule structure
-Centrosome function
-Cilia structure
-Cilia function
-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.
The Centrosome is 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
-stable
-non-membrane bound
-no DNA
PCM
The Centrosome is the Major Microtubule Organizing Center
©2004 WILEY-VCH
-A pair of centrioles (mother and daughter centriole) and a pericentriolar material (PCM)
-centrioles are made up of 9 sets of microtubule triplets
-Microtubules (MTs) are polar
-Sub-distal and distal appendages at the + end of the mother centriole
-Connecting fibers at the - end of centrioles
-Cytoplasmic versus centriolar microtubules
-Microtubule nucleation (γ-TuRC complex)
-Microtubule anchoring (Ninein)
α and β Tubulins are Building Blocks of Microtubules
-α and β tubulins bound to GTP form αβ heterodimers
-Head-to-tail joining of αβ tubulin heterodimers followed by hydrolysis
of GTP bound to β-tubulin leads to a protofilament
-Protofilament elongation occurs primarily at the + end
-a microtubule contains 13 protofilaments surrounded by a hollow core
Singlet, Doublet and Triplet Microtubules
© 2000 by W. H. Freeman and Company
Cytoplasmic microtubules
Caenorhabditis elegans centrioles
Cilia
Eukaryotic flagella
Drosophila centrioles
Centrioles of most eukaryotes
-A tubule: 13 protofilaments
-B and C tubules: ~10 protofilaments fused to the wall of another tubule
A Cartwheel Protein SAS-6 Dictates The Universal 9-Fold Symmetry of Centrioles
SAS-6 Undergoes Dimerization and Oligomerization to Form The Cartwheel
Pierre Gonczy
C
B
A
Central hub
Microtubules
Spoke
Pinhead
SAS-6 organizes the cartwheel structure. (A) A SAS-6 monomer. (B) Two SAS-6 molecules
dimerize. (C) Two SAS-6 homodimers interact. (D) A cartwheel (central hub and spokes) with
nine homodimers. (E) Each spoke of a cartwheel connects with a pinhead which in turn connects
with microtubules.
Cep135 is a “Pinhead” Protein
Spoke
EMBO J. (2013)
Cep135 constitutes the pinhead and links the cartwheel to microtubules.
CPAP binds to αβ tubulin and γ-tubulin ring complex.
The Tubulin Superfamily
©2004 WILEY-VCH
γ-tubulin Mediates Microtubule Nucleation
©2004 WILEY-VCH
α +β + γ tubulins
α + β tubulins
The γ-tubulin Ring Complex Mediates Microtubule Nucleation
+
©2004 WILEY-VCH
γ-tubulin
GCP2
Other accessory
proteins
GCP3
-
GCP = γ-tubulin complex protein
γ-tubulin ring complex (γ-TuRC) contains 12-14 γ-tubulin molecules + 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
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
Microtubules are Highly Dynamic Structures
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 Stabilize Microtubules
Nature Reviews Molecular Cell Biology (2011)
Centriolar (centrosomes) and axonemal microtubules
(cilia) are extremely stable.
Centrosome Function
Cell division
Cell shape
Intracellular transport
Cell migration
Cell polarity
Cilia assembly
MBoC 6th edition
Tang and Marshall, JCS 2012
Cell Division: Functional Diversity of Microtubules Organized by The
Centrosome
The Cell Cycle
Centrosome
Centrosome
Centrosome
Centrosome
Centrosome
Centrosome
The Centrosome Cycle
1) Centriole Disengagement
(late M)
-licenses
centrioles
for
duplication in the next cell
cycle
1)
2) Centriole Duplication (S)
-synthesis of two
daughter centrioles
3) Centrosome
(S/G2)
new
Maturation
-daughter centrioles elongate
4)
2)
-the
original
daughter
centriole acquires appendages
and becomes the mother
centriole
-PCM enlarges
3)
4) Centrosome
(G2/M)
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)
Phosphorylation of:
CP110: prevents premature
centrosome separation
Nucleophosmin: dissociates
from centrosomes to induce
duplication
Mps1: accumulates at
centrosomes and promotes
duplication
Asp: promotes microtubule
nucleation in concert with γtubulin
C-Nap1: dissociates from
centrosomes and induces
centrosome separation
Eg-5: binds to microtubules,
slides microtubules apart and
induces centrosome
separation
Centrosome Amplification Frequently Occurs in Human Cancers
Staining of centrosomes n white/red
and DNA 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)
How do Cells Cope With Centrosome Amplification?
Centrosome
clustering
Lagging chromosome occurs when a kinetochore is
attached to microtubules emanating from both
spindle poles!
J. Cell Sci.(2012)
The Consequences of Centrosome Amplification
Nat. Rev. Mol. Cell Biol. (2009)
Intracellular Transport: Microtubule Motor Proteins
Two major classes of microtubule motor proteins: kinesin (2 heavy chains and 4 light
chains) and dynein (2 heavy chains and variable light/intermediate chains)
A heavy chain is composed of:
a head domain: ATP and microtubule binding
a tail domain: cargo binding
Direction of movement differs!
Microtubule Motor Proteins Mediate Cargo Transport
The Relationship Between Centrosomes and Cilia
Cilium
(1-10 µm in length)
0.5 µm in length
Proliferating cell
Non-proliferating cell
-The centrosome is
located near the cell
center
-The centrosome is located near the cell
surface
-Only the mother centriole/basal body
templates a cilium.
Cilia Types
-motile versus non-motile (primary) cilia
-cilia number per cell
Genetics in Medicine (2009)
Cilia are Present in Most Human Cells
Brain development
Smell
Cardiogenesis
Mucus
clearance
Vision
Reproduction
Kidney
development
Left-right asymmetry
Reproduction
Brown and Witman, Bioscience 2014
Cilia Assembly
Dev. Dyn. (2007)
Cilia 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)
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)
Cilia 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
Mechanosensation
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.
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)
Light Sensation
Cilia
Transition
zone
Centrosomes and Cilia in Human Disease
Primary Microcephaly (small brain)
Dwarfism (short stature)
Centrosome
Aberrations
Genomic
Instability;
aneuploidy
?
Cancer
Non-motile Ciliopathies
Polycystic Kidney Disease (kidney cysts)
Leber Congenital Amaurosis (retinal
degeneration)
Cilia
Aberrations
Bardet-Biedl Syndrome (retinal
degeneration, obesity, diabetes, male
infertility, polydactyly, cognitive
impairment)
Joubert Syndrome (cerebellar
malformation, ataxia, hypotonia)
Motile Ciliopathies
Primary Ciliary Dyskinesia
(infertility,
bronchiectasis,
chronic
sinusitis, situs inversus)
Summary
-Centrosomes and cilia are microtubule-based structures that are
extremely stable.
-Centrosomes and cilia are intimately related.
-Centrosome duplication and cilia assembly are tightly regulated in a
cell cycle-dependent manner.
-Centrosomes and cilia perform different cellular functions.
-Centrosome and cilia dysfunction can lead to different human
diseases.