Centrosomes and Cilia

Transcription

1 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

2 Outline -A Brief History -Microtubules -Centrosome structure -Centrosome function -Cilia structure -Cilia function -Human diseases -Work on centrosomes and cilia

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 The Centrosome is 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 -stable -dynamic -non-membrane bound -no DNA

5 The Centrosome is the Major Microtubule Organizing Center 2004 WILEY-VCH -A pair of centrioles (mother and daughter centriole) surrounded by pericentriolar material (PCM) -Centrioles are made up of 9 sets of microtubule triplets -Microtubules are polar -Sub-distal and distal appendages at the + end of the mother centriole -Interconnecting fibers at the - end of centrioles -Cytoplasmic versus centriolar microtubules

6 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

7 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

8 The Tubulin Superfamily 2004 WILEY-VCH

9 γ-tubulin Mediates Microtubule Nucleation (Seed Formation) 2004 WILEY-VCH α +β + γ tubulins α + β tubulins

10 The γ-tubulin Ring Complex Mediates Microtubule Nucleation + γ-tubulin GCP2 GCP3 - Other accessory proteins 2004 WILEY-VCH GCP = γ-tubulin complex protein γ-tubulin ring complex (γ-turc) contains γ-tubulin molecules + GCP proteins γ-tubulin binds between protofilaments at the - end

11 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 WILEY-VCH

12 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

13 Post-translational Modifications Stabilize Microtubules Nature Reviews Molecular Cell Biology (2011) Centriolar (centrosomes) and axonemal microtubules (cilia) are extremely stable.

14 Centrosome Function Intracellular transport Cell cycle Cell division Cell polarity Cell shape Cell migration Cilia assembly MBoC 6 th edition Tang and Marshall, JCS 2012

15 Cell Division: Functional Diversity of Microtubules Organized by The Centrosome

16 The Cell Cycle Centrosome Centrosome Centrosome Centrosome Centrosome Centrosome

17 The Centrosome Cycle 1) 1) Centriole Disengagement (late M) -licenses centrioles for duplication in the next cell cycle 4) 2) 2) Centriole Duplication (S) -synthesis of two new daughter centrioles 3) Centrosome Maturation (S/G2) -daughter centrioles elongate -the original daughter centriole acquires appendages and becomes the mother centriole -PCM enlarges 3) 4) Centrosome Separation (G2/M) -the two centrosomes migrate to opposite poles of the cell

18 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 gene 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 γ- tubulin C-Nap1: dissociates from centrosomes and induces centrosome separation Eg-5: binds to microtubules, slides microtubules apart and induces centrosome separation

19 Centrosome Amplification is Frequently Detected 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)

20 Origin of Centrosome Amplification Nat. Reviews (2002)

21 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)

22 Consequences of Centrosome Amplification Nat. Rev. Mol. Cell Biol. (2009)

23 Early Studies on Cilia Anton van Leeuwanhoek s early microscopic observations of sperm cells in mid 1670s.

24 Cilia are Present in Most Human Cells Brain development Smell Cardiogenesis Mucus clearance Vision Reproduction Left-right asymmetry Kidney development Reproduction Brown and Witman, Bioscience 2014

25 Cilia Types Choksi et al, Development, 2014

26 Cilia Types -motile versus non-motile (primary) cilia -cilia number per cell Genetics in Medicine (2009)

27 Relationship Between Centrosomes and Cilia Cilium (1-10 µm in length) 0.5 µm in length Proliferating cell -The centrosome is located near the cell center Non-proliferating cell -The centrosome is located near the cell surface -Only the mother centriole/basal body templates a cilium.

28 Cilia 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

29 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

30 Non-motile (Primary) Cilia Assembly Dev. Dyn. (2007)

31 Motile Cilia Assembly Deuterosome Nat Cell Biol. (2013) Science (2014)

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

33 Motility Mucus clearance Sperm movement

34 Mechanosensation Mechanosensation Nature Reviews Molecular Cell Biology (2007) The polycystin-1 polycystin-2 complex (PC1 PC2) is a Ca 2+ channel localized to the ciliary membrane of epithelial cells (left panel). Fluid-induced ciliary bending activates this channel. Ca 2+ influx (right panel) causes Ca 2+ release from ryanodinesensitive intracellular stores and subsequent downstream responses such as activating protein 1 (c-jun/c-fos) -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 can cause polycystic kidney disease.

35 Chemosensation The role of primary cilia in Shh signalling. (a) In the absence of Shh, its receptor Ptch1 localizes to primary cilia and prevents Smo from entering primary cilia. Gli3 is constitutively proteolytically cleaved to a Gli3 repressor (Gli3R) to repress Shh target genes. When Shh binds to Ptch1, Ptch1 disappears from the cilia allowing Smo to enter primary cilia, where it activates Gli2 to become transcriptional activator (Gli2A) and inhibits Gli3R formation, leading to Shh target gene expression. (b) In the absence of cilia, neither Gli2A nor Gli3R form. Shh target genes are involved in tissue patterning, cell proliferation and differentiation. Defective Shh signaling can lead to polydactyly, cerebellar hypoplasia and craniofacial development. Hyperactive Shh signaling, on the other hand, can lead to basal cell carcinoma and medulloblastoma. Curr Opin Neurobiol (2010)

36 Cilia Lost is Detected in Some Human Cancers Melanocytes (HEMn-LP) Primary melanoma cells (WM115 and WM278) Metastatic melanoma cells (WM266-4 and WM1617) J Invest Dermatol (2015)

37 Cilia Can Promote or Suppress Shh Pathway-dependent Tumorigenesis Constitutively active Smo + Cilia = Cancer Constitutively active Smo + No cilia = No cancer Constitutively active Gli2 + Cilia = No cancer Constitutively active Gli2 + No cilia = Cancer Curr Opin Neurobiol (2010)

38 Centrosomes and Cilia in Human Disease Genomic Instability; aneuploidy Primary Microcephaly (small brain) Dwarfism (short stature) Cancer Centrosome Aberrations Non-motile Ciliopathies Polycystic Kidney Disease (kidney cysts) Leber Congenital Amaurosis (retinal degeneration) Cilia Aberrations Senior Løken syndrome (kidney cysts, retinal degeneration) Bardet-Biedl Syndrome (retinal degeneration, obesity, male infertility, polydactyly, cognitive impairment) Motile Ciliopathies Primary Ciliary Dyskinesia (infertility, bronchiectasis, chronic sinusitis, situs inversus)

39 Loss of Cep76 Induces Centriole Amplification Control sirna Cep76 sirna Tsang et al, Dev Cell 2009

40 Ectopic Expression of Cep76 Suppresses Centriole Amplification Tsang et al, Dev Cell 2009

41 Cep76 Protein Levels Peak in S phase Tsang et al, Dev Cell 2009

42 A working Model G1 Cep76 M S G2 Fully grown centriole Growing centriole PCM Cilium

43 Ciliopathy Proteins Cep290 and NPHP5 Interact IN NPHP5 Cep290 Control Cep290 is mutated in: Leber congenital amaurosis Senior-Løken syndrome Bardet-Biedl syndrome Joubert syndrome Meckel syndrome IP Cep290 NPHP5 is mutated in: NPHP5 Leber congenital amaurosis Senior-Løken syndrome Barbelanne et al, HMG (2013)

44 Cep290 and NPHP5 are Required for Cilia Assembly sirna NS sirna Cep290 sirna NPHP5 9 μm Tsang et al, Dev Cell (2008) Barbelanne et al, HMG (2013)

45 NPHP5 Centrin/NPHP5 Cep290 is Required for NPHP5 Localization to the Centrosome Cep 290 Centrin/Cep290 sirna Control sirna NPHP5 sirna Cep290 sirna Control sirna NPHP5 sirna Cep290 9 μm 9 μm 9 μm 9 μm 9 μm 9 μm Barbelanne et al, HMG (2013)

46 NPHP5 Disease Mutants Do Not Interact With Cep290 And Are Mis- Localized Control 1-598(FL) F142fsX146 R332X R461X R502X H506fsX518 IP: Flag NPHP5 Amino Acids Cep290- binding Centrosomal Localization 1-598(FL) F142fsX146 R332X R461X R502X H506fsX Cep290 Flag-NPHP5 IN: Cep (FL) F142fsX146 R332X R461X R502X H506fsX518 Flag Centrin Merge Hum. Mol. Genet μm 9 μm 9 μm 9 μm 9 μm 9 μm Barbelanne et al, HMG (2013)

47 A working Model NPHP5 Cep290 G0 G1 Cep76 M S G2 Fully grown centriole Growing centriole PCM Cilium

48 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 have different cellular functions. -Centrosome and cilia dysfunction give rise to different human diseases.