centriole duplication,cell cycle and copy number control, length control,procentriole, deregulation role in cancer, use in therapeautics

1. Once and only once:
Mechanisms of
Centriole duplication
and their deregulation
in disease
Erich a. Nigg and Andrew j. Holland
Nature reviews | Molecular cell
Biology Volume 19 | May 2018

2. Contents
What are
Centrosomes ??
What are
Centrioles??
Why are Centrioles
Important??
How are
Centrioles made ??
How is Centriole
length controlled
??
How is Pericentriolar
Material
Assembled??
2
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Experimental Proof for
PCM assembly through
Phase Separation??
How Centriole
Numbers are
Controlled??
How Does
Centriole
Duplicate??
How cells Sense
centriole
Number??
Centrosome
Anomalies and there
Effects
Centrosome as
therapeutic Targets

4. Centrosomes are composed of
two Centrioles arranged at right-
angles to each other, and assembles
an amorphous mass of protein termed
the pericentriolar material (PCM).
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What are
Centrosomes?
?
• Cytoskeleton is framework of intermediate filaments,
microtubules, and actin filaments.
• Microtubules are hollow cylinders made of the protein tubulin.
They are long, straight and have one end attached to
microtubule-organizing centre (MTOC) called centrosome.
(Nucleation site).

5. Centrioles are cylindrical structures
characterized by an evolutionarily
conserved radial nine-fold symmetry.
• Protein components of centrioles are α and β-tubulin.
• First observed by Edouard van Beneden in 1883.
• Centrioles are present in most Animal cells, but are rarely
find in Plant Cells especially Angiosperms.
• Pro Centrioles are newly constructed centrioles that are
unable to duplicate.
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What are
Centrioles??

6. Dysfunction can lead to ciliopathies and
Cancer
Acts as MTOC (Microtubule organizing centres)
Regulate cell shape, polarity and motility
Spindle formation
Chromosome segregation
Cytokinesis
Pericentriolar material (PCM) harbors proteins which
are regulators of the cell cycle and its checkpoints.
Centrioles function as basal bodies for the formation of cilia
and flagella.
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Why are
Centriole
Required??

7. Centriole
Structure and
How are they
Made ??
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• Walls of centrioles are nine triplet microtubule blades that are
arranged circumferentially.
• Subdistal appendages for anchoring cytoskeletal microtubules
• Distal appendages for membrane docking during ciliogenesis
• Proximal part of the Procentriole has Transient scaffolding
known as the cartwheel onto which microtubules are added
Cartwheel Scaffolds Microtubules to
form Centriole.

8. SAS6 is required for 9 fold symmetry of
centriole
• Centre of the cartwheel is a ring-shaped hub, from which
nine spoke connect to the ‘A tubule’ of triplet.
• A typical microtubule triplet is composed of A, B and C
tubules, with the innermost ‘A tubule’.
• Cartwheel ring is composed of nine homodimers of
spindle assembly abnormal protein 6 homologue (SAS6)
proteins.
• SAS6 imparts the typical nine-fold symmetry to
centrioles.
• The deposition of microtubules onto the cartwheel
(wall of centriole) clearly requires centrosomal
P4.1-associated protein (CPAP).
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9. How Centriole
length is
controlled??
• Centrioles have constant Dimensions in most cells of
any given organism ex: Human centrioles 450–500 nm.
• For centriole length, the polymerization and
depolymerization of centriolar microtubules is critical.
• Kinesin-like protein (Klp10A) acts as a microtubule
depolymerase to control centriole length.
• CP110 protein, caps the distal tips of centrioles and its
depletion results in overly long centriolar microtubules.
• CPAP controls the speed of microtubule growth.
• Polyglutamylation for long-term stability of Centriolar
Microtubules.
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Centriole Length is controlled by
polymerization and De-polymerization of
Microtubules, Due to various proteins
(Klp10A, CP110, CPAP) and
Modifications

10. How is
pericentriolar
Material
assembled??
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• Centrosomes has ~200–300 proteins
• Centrosome composition rapidly changes through
trafficking on microtubules anchored within the
centrosome or through dynamic cytoplasmic granules
termed as centriolar satellites.
• Centriolar satellites Deliver centriolar/centrosomal
components from the cytoplasm. These particles
constantly move around the centrosome.
• Centrosomes are not surrounded by membranes.
• PCM forms by an initial phase separation that
concentrates components, which subsequently harden
into a gel-like or solid structure with ordered protein–
protein interactions.
Phase Separation and ordered Protein
interactions

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Regulation of centriolar satellite integrity and its physiology by Akiko Hori and Takashi Toda

12. The Centrosome Is
a Selective
Condensate that
Nucleates
Microtubules by
Concentrating
Tubulin
Jeffrey B. Woodruff , Beatriz
Ferreira Gomes, Per O. Widlund ,
Julia Mahamid , Alf Honigmann ,
and Anthony A. Hyman
Cell | June 1, 2017

13. Condensate are viscous liquids that
rapidly into a more viscous material like
a gel or Glass.
• SPD-5(Spindle defective protein 5) is a PCM
scaffold protein that can self organize themselves
into condensates in crowded environment in-vitro.
• PCM rapidly expands in preparation for mitosis,
then stops during the metaphase-anaphase transition.
• SPD-5 incorporates throughout the PCM while it
grows, indicating that PCM expands isotopically.
• Pericentriolar material that surrounds centrioles is a
phase separated compartment that nucleates
microtubule arrays through localized concentration
of tubulin.
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15. SPD-5 protein
assembles into
spherical
compartments in a
crowded
environment in
vitro
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16. How
Centriole
Numbers are
controlled??
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• Centrosome duplication is tightly regulated to
ensure Cell cycle control and copy number
control
• Duplication and segregation of centrosomes is
co-regulated with the chromosome duplication
and segregation cycle.
By cell cycle and copy number control
through Co-regulation with cell Division
cycle

17. How does
Centriole
Duplicate??
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• For understanding centrosome duplication cycle has
been divided into three main stages.
a) Licensing centrioles for a new round of duplication
b) The birth of a new centriole
c) Maturation of centrioles and centrosomes
Stage A
• Competence for Duplication is acquired after mitosis.
• Licensing depends on two main processes
1. Centriole disengagement, which permits the
reduplication of the parent centriole. (Occurs in late
M/early G1)
2. Centriole-to-centrosome conversion, required for the
procentriole to acquire competence for duplication.

18. • The parent centriole forms a tight and orthogonal
connection with pro-centriole, blocks its
reduplication.
• Polo-like kinase 1 (PLK1) and protease Separase
help in disengagement.
• Separase acts on PCM component pericentrin
(PCNT) and Centriole associated cohesin.
• Removal of the cartwheel from the procentriole is
mediated by cyclin-dependent kinase 1 (CDK1).
• Procentriole then acquire PCM, by centriole-to-
centrosome conversion governed by CDK1 and
PLK1.
• These activities provide procentrioles the
competence to duplicate and also provides Cell Cycle
Control.
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Stage B (Birth of New Centriole)
• Protein PLK4 (Polo-like kinase 4) is linchpin for copy
number control.
• At the G1/S transition one new procentriole begins to
assemble 900 to the parent centriole, remains closely
linked to it and elongates throughout G2.
• Trans-autophosphorylation of PLK4 trigger
recruitment of SAS6 and cartwheel formation.
But how is the construction site for a new procentriole
is chosen on circumference of the parent centriole ??
• Scaffolding proteins form rings around parent
centrioles and then PLK4, SAS6 coalesce to a precise
region on parent centriole that marks the site of
procentriole assembly(Symmetry Breaking)

20. But what ensures copy number control ??
• There is intrinsic choice of a building site and
suppression other potential sites. But its
Regulation in time and space is still Unknown.
• Acc. To Mould model lumen of the parent
centriole acts as a mould for the assembly of a
cartwheel that is released and used to form of a
procentriole.
 But how cells limit the use of the mould to once
per centriole and cell cycle ??
How the cartwheel is transferred from the lumen
to the wall of the parent centriole?? Are still not
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• Parent centrioles connected by tether containing rootletin protein.
• At the G2/M transition, PCM expands for mitotic spindle formation called centrosome maturation.
Stage C
(Maturation)

22. Summary
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• Centrosomes are composed of two Centrioles and
an amorphous mass of protein termed the pericentriolar
material (PCM).
• Centrioles are cylindrical structures made of tubulin protein
with 9 fold symmetry.
• Cartwheel Scaffolds Microtubules to form Centriole.
• SAS6 is required for 9 fold symmetry of centriole
• Centriole Length is controlled by polymerization and De-
polymerization of Microtubules
• PCM is assembled through Phase Separation and ordered
Protein interactions.
• Centriole Number is controlled by cell cycle and copy
number control through Co-regulation with cell Division
cycle
• Centrosome duplication occurs through Licensing, Birth and
Maturation.

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How cells Sense
centriole
Number??
Centrosome
Anomalies and there
Effects
Centrosome as
therapeutic Targets
Contents

24. Centrosome
depletion and
cell
proliferation
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25. • Centrosomes are generally required for the sustained proliferation of mammalian
cells.
• Mouse embryos lacking centrioles undergo widespread p53 dependant apoptosis at
an earlier developmental stage than mutants that lack cilia.
• In cultured mammalian cells, centrosome loss led to a robust cell cycle arrest within
a few divisions.
• This could be reversed by the removal of p53.
Centrosome depletion and p53?
Genome wide knockout screens done
Led to the identification of the Mitotic
Surveillance Pathway 25
Centrosome depletion can activate p53 dependent pathways

26. • USP28-53BP1-p53-p21 signalling axis
USP28: Ubiquitin carboxy-terminal hydrolase 28
53BP1: TP53 binding protein 1
p21: CDKN1A [Cyclin Dependant Kinase Inhibitor 1A]
LOSS
NORMAL CENTRIOLE NUMBER
USP2853BP1
p53 STABILISATION
+
DEATHARREST
DELAYED
MITOSIS
26
The mitotic surveillance pathway

27. Centrosome
amplification
and cell
proliferation
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28. • Like centrosome loss, centrosome amplification,(an increase in centrosome number)can also suppress the proliferation of cells.
• Initial insight came when tetraploid cells were shown to stabilise p53 through Hippo Pathway serine/threonine-protein kinase LATS2
• Inducing extra chromosome led to LATS2 dependant stabilisation of p53.
LATS2 plays a role in preventing the proliferation of cells with extra chromosomes.
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Centrosome amplification can activate p53 dependant pathways

29. Centrosome amplification can activate p53 dependant pathways
• Recently, another pathway controlled by the PIDDosome was found to be important in preventing the proliferation of cells with extra
centrosomes.
• PIDDosome controls the proximity induced activation of caspase 2 which is required for the stabilisation of p53.
• Some PIDDosome components localise to the older parent centriole, suggesting that its activation may be controlled by the presence
of additional mature centrioles.
PIDDosome plays a role in preventing proliferation of cells with extra centrosomes
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30. Centrosome amplification can activate p53 dependant pathways
• Hippo pathway: a signalling pathway in animals that controls organ size in animals by restraining cell
proliferation and promoting apoptosis.
• LATS2: Large Tumour Suppressor Kinase2, responsible for stabilising p53.
• PIDDosome: Protein complex composed of a death domain containing protein CRADD and a p53-induced
death domain (PIDD1), that is implicated in activation of caspase 2.
• MDM2: Mouse Double Minute 2 homolog, negative regulator of p53.
Amplification
NORMAL
CENTRIOLE
NUMBER
Caspase2
LATS2
p53 STABILISATION
DEATHARREST
MDM2
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31. Sensing centriole number : The cellular sixth sense
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32. Centrosome
anomalies and
their
consequences
32

33. CENTROSOME ANOMALIES
Numerical
Alterations
Structural
Alterations
Due to alterations in
the levels or activity of
centrosome proteins
Due to acquisition of
an excessive number
of centrioles
Both these types of anomalies often coexist in
tumours! 33

34. Centrosome anomalies and cancer
Theodor Boveri
1862-1915
Postulated that centrosome aberrations could contribute to human cancers.
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35. Centrosome amplification led to tumorigenesis in model organisms
• Pioneering work done in flies, showed that centrosome amplification doesn’t
promote development of spontaneous tumours.
• Neuroblasts and epithelial cells with extra centrosomes can initiate tumorigenesis
when transplanted into host flies.
• In mouse models, centrosome abnormalities were not able to initiate spontaneous
tumorigenesis or enhance the development of carcinogen-induced skin tumours.
• But, centrosome amplification did lead to accelerated tumorigenesis in p53-deficient
epidermis
Supernumerary centrosomes do play a role in tumorigenesis, yet the mechanisms of tumour promotion remain to be clarified
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36. The origin of centrosome defects in tumour cells
• Cancer cell lines show wide variation in penetrance and extent of centrosome
amplification.
• Genes encoding centrosome proteins are rarely mutated in human cancers.
• Increased or decreased expression of these centrosome proteins is more common.
Gene symbol Function Links to disease
PLK4 Centriole duplication Over expressed in breast cancer
NLP Microtubule nucleation Over expressed in many cancers
Some centriole genes linked to tumorigenesis:
36

37. Centrosome defects lead to genome instability
• Cells with supernumerary centrosomes form multipolar mitotic spindles.
• Multipolar divisions lead to the production of highly aneuploid daughter cells that are
typically non-viable.
• In addition to creating whole chromosome aneuploidy, mitotic errors caused by extra
centrosomes can promote the acquisition of DNA double-strand breaks that result in
chromosomal rearrangements.
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38. Joining the
dots
38
Linking centrosome
defects with
chromosomal instability

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40. Introduction
• Chromosome instability is a hallmark of many tumours and correlates with the
presence of extra chromosomes.
• However, there was no direct mechanistic link between extra chromosomes and
chromosome instability (CIN).
• It had been proposed that extra centrosomes generate CIN by promoting multipolar
anaphase.
• Ganem et al. through the use of long-term live cell imaging demonstrated that cells
with multiple centrosomes rarely undergo multipolar divisions and the progeny of
these divisions are typically inviable.
40

41. • Chromosome instability(CIN)- A feature of cancer cells to gain or lose whole
chromosomes at an elevated rate.
• Cells with CIN missegregate chromosomes with 10-100 times more frequency
than non-transformed or chromosomally stable diploid cancer cells.
• Thus, CIN is a major source of aneuploidy, which enables cancer cells to
expand clonally with proliferative advantages, metastatic potential or
chemoresistance.
• The mechanism leading to CIN in most cancers was not defined.
Chromosomal instability leads to aneuploidy in tumour cells
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42. Materials and methods
• A variety of cancer cell lines were generated from different tissues of
origin.
• These cells were stably expressing the chromosome marker H2B-
GFP.
• Long-term live cell imaging was performed to directly visualise the
relationship between multipolar cell division and cell viability
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43. Results
43

44. Multipolar cell divisions are rare and progeny typically inviable
fraction of cells undergoing multipolar cell division, defined by the segregation of chromosomes to 3 or more poles during
anaphase, was always markedly less than the fraction of cells possessing extra centrosomes.
44

45. Multipolar cell divisions are rare and progeny typically inviable
• vast majority of progeny (P1) of multipolar cells died or arrested, regardless of tissue of origin or whether the cells were
mono- or poly-nucleated
• when rare P1 progeny from multipolar divisions completed a second round of mitosis to generate P2 progeny, even fewer
of the resulting daughter cells were capable of further division
45

46. Multipolar cell divisions are rare and progeny typically inviable
Cell cycle
arrest
Successful
division
Death in
interphase
• representative SCC114 cell undergoing several rounds of bipolar cell
division (top row);
• or a single multipolar cell division (bottom row).
• Coloured arrows track the fate of the three progeny from the multipolar cell
division. 46

47. Inference of the study
• Cells with multiple centrosomes rarely undergo multipolar cell divisions
• The progeny of these divisions are typically inviable
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49. Mutations in genes encoding centrosome localised proteins cause microcephaly
• Microcephaly, a severe developmental disorder is caused by reduced neuronal
proliferation during embryonic development.
• It is characterised by small brain size and mental retardation.
• Major genetic causes of microcephaly are mutations in widely expressed genes coding
for proteins working at the centrosome.
Mutations in 12 genes encoding centrosome localised proteins have been shown to cause MCPH, out of which 8 are involved in centrosome
duplication.
Suggests that anomalies in centriole biogenesis are an underlying cause of neurogenesis defects in
microcephaly!
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50. Centrosomes
as therapeutic
targets
50

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•PLK4 inhibitors could offer therapeutic value in suppressing functions of
PLK4 that promote invasion and metastasis.
•Forcing cancer cells with extra centrosomes into lethal multipolar divisions.
•Pathologically activating the pathways to reverse the adverse effects of
centrosomal anomalies.
Centrosomes offer a new strategy of cancer therapy

52. Summary
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• Centrosome depletion and its effects on
cell proliferation
• Centrosome amplification and its
effects on cell proliferation
• Centrosome anomalies and their
consequences.
• Centrosome defects and chromosomal
instability.
• Centrosomes offer a new strategy of
cancer therapy

53. Questions ??
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Presented by
Ayush Jain (MP18004)
Syed Azeez Tahseen (MP 18027)