Centrosomes and Cilia

Transcription

1 Centrosomes and Cilia SMC6052/BIM6027/ D 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

2 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

3 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 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.

4 Centrosome: A Tiny Organelle Mammalian cell: ~ µm 3 in volume ~20 µm in diameter ~700 µm 3 in volume ~5-10 µm in diameter Nucleus Centrosome ~ 1 µm 3 in volume Centriole: 0.2 X 0.5µm PCM -is non-membrane bound -does not contain DNA

5 Centrosome Function -Cell Division -Cell Shape -Cell Cycle Progression -Cell Polarity -Intracellular transport -Cell Migration -Cilia Formation J. Cell Sci (2012)

6 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

7 Cytoplasmic Versus Centriolar Microtubules In Different Cell Types Within a Cell Centriolar Microtubules

8 A Cartwheel Protein, SAS-6 Dictates The Universal 9-Fold Symmetry of Centrioles Pierre Gonczy SAS-6 Dimer Microtubules B C A Central hub 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.

9 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.

10 The Tubulin Superfamily 2004 WILEY-VCH

11 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

12 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

13 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

14 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) = γ-tubulin molecules + several GCP proteins γ-tubulin binds between protofilaments at the - end

15 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 WILEY-VCH

16 Microtubule Dynamic Instability and Treadmilling The Plant Cell (2004) + end is dynamic. - end never grows, but can be stabilized or depolymerized.

17 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

18 Post-translational Modifications of Microtubules Glycylation, Glutamylation, Acetylation and Detryosination

19 Post-translational Modifications Regulate Microtubule Stability Nature Reviews Molecular Cell Biology (2011) Centriolar and axonemal microtubules are extremely stable.

20 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

21 Microtubule Motors Mediate Cargo Transport Cargoes: Membrane vesicles Protein complexes Chromosomes mrnas Organelles Microtubules Science (1998)

22 Structural and Functional Diversity of Microtubules In Different Cell Types Within a Cell Centriolar Microtubules

23 Centrosome Function -Cell Division -Cell Shape -Cell Cycle Progression -Cell Polarity -Intracellular transport -Cell Migration -Cilia Formation J. Cell Sci (2012)

24 The Cell Cycle Centrosome Centrosome Centrosome Centrosome Centrosome Centrosome

25 The Centrosome Cycle 1) 1) Centriole Disengagement -licenses centrioles for duplication in the next cell cycle 4) 2) 2) Centriole Duplication -procentriole assembly 3) Centrosome Maturation -procentrioles elongate to become daughters -the daughter centriole acquires appendages and becomes the mother centriole -PCM enlargement 3) 4) Centrosome Separation -the two centrosomes migrate to opposite poles of the cell

26 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 γ- tubulin C-Nap1: dissociates from centrosomes and induces centrosome splitting Kendrin Separase APC Eg-5: binds to microtubules, slides microtubules apart and induces centrosome separation Destruction of Kendrin: induces centriole disengagement and license centrosomes for the next round of duplication

27 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 Procentriole -generates 1 procentriole per existing centriole 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

28 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)

29 The Origin of Centrosome Amplification Nat. Reviews (2002)

30 Consequences of Centrosome Amplification Centrosome clustering Lagging chromosome as a result of a chromatid bounded to more than one spindle pole J. Cell Sci.(2012)

31 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.

32 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.

33 Cilium Diversity -motile versus non-motile (primary) cilia -cilia number per cell -cell/tissue types Neurons Epithelial Cells Genetics in Medicine (2009)

34 Cilium Function -Motility Sperm movement Mucus clearance Cerebrospinal fluid circulation Ovum transport Left-right asymmetry -Sensory Mechanosensation Chemosensation Light sensation Thermosensation

35 Motility Function Mucus clearance Sperm movement

36 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 Ca 2+ channel. The Ca 2+ influx (right panel) causes Ca 2+ release from ryanodine-sensitive intracellular stores and subsequent downstream responses such as activating protein-1 (AP1)- dependent gene transcription by the Ca 2+ -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.

37 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)

38 Cilium Structure Transition zone Daughter centriole 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 Nature (2007) -Distal appendages dock the mother centriole to the membrane -Transition zone provides a diffusion barrier for selective transport

39 Cilium Assembly Dev. Dyn. (2007)

40 Intraflagellar Transport (IFT) -IFT was first reported in 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 Frontiers in Bioscience (2008) Daughter centriole /distal appendages -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) -IFT requires kinesin-2 for anterograde transport and dynein- 1b for retrograde transport. Motors move along the outer doublet microtubules of ciliary axonemes. -Cargos are attached to IFT particles

41 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.

42 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 (loss of vision) 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)

43 CP110 (Centrosomal Protein of 110 kda), A Novel Cyclin/CDK Binding Protein

44 CP110 is phosphorylated by Cyclin/CDKs In vitro kinase assay: purified GST-CP110 + cyclin/cdk + radiolabeled ATP Dev. Cell (2002)

45 CP110 is a Centriolar Protein Centrin γ-tubulin CP110 Centrin Merge G1 M Dev. Cell (2002)

46 CP110 is a Protein Localized to The Distal End of Centrioles Dev. Cell (2007)

47 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)

48 CP110 Expression is Regulated During The Cell Cycle Northern Blot Western Blot Dev. Cell (2002)

49 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)

50 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)

51 Phenotype of CP110 Over-Expression % of Ciliated Cells Control CP110 DAPI Ac. tub Flag Merge Control CP % 80% 60% 40% 20% 0% Loss of cilia in quiescent cells Cell (2007); Dev. Cell (2008)

52 CP110 is Absent From The mother Centriole in Quiescent Cells Glu. tubulin CP110 Merge Cell (2007)

53 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)

54 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.

55 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: a Tsang and Dynlacht. Cilia (2013) 2: 9