Jay Gopalakrishnan - assoc. RG

Our aim was to apply our interdisciplinary expertise of genetics, biochemistry and structural biology to underpin how functional centrosomes are built and how do they control cell proliferation, differentiatuion and organ development. To accomplish the overall goal, we solved the crystal structure of CPAP-tubulin protein complex. To dissect the significance of this interaction, we generated transgenic Drosophilaand identified that this molecular interaction is critically required for centriole length determination (Zheng et al 2016 Nature Communications).We then extended our study and identified a chemical probe that prevents CPAP-tubulin interaction and stimulate centrosomes to recruit enhanced PCM (Mariappan et al 2019, The EMBO Journal).Finally, to understand the spatiotemporal regulation of PCM during cell cycle, we have identified that Sas-4 (in human it is CPAP) is being phosphorylated by Polo kinase, which is required to assemble PCM at the onset of mitosis (Ramani et al 2018 Cell).In this progress project report, we summarize our results. Collectively, our interdisciplinary collaborative studies have provided new and fundamental mechanistic insights into the assembly of a functional centrosome and centrosomal mechanisms those can be targeted in cancer cells.

Introduction 

Centrosomes are conserved organelles in eukaryotic cells consisting of centrioles surrounded by a protein network of peri-centriolar-material (PCM). Centrosomes are essential for fundamental cellular functions such as microtubule nucleation, accurate cell division and cilium formation. Within a centrosome, the centriole is the template for cilia formation during interphase, while the PCM assembles a spindle pole during mitosis to nucleate astral and spindle microtubules. In order for centrosomes to be functional organelle as a microtubule-organizing center (MTOC) of a cell, it must first successfully recruit PCM around centrioles. The complexity of PCM assembly, recruitment to centrosomes and their regulation remains a mystery. 

Molecular basis of CPAP-Tubulin interaction in regulating centriolar and ciliary length

By solving the co-crystal structure of PN2-3 domain of CPAP bound to tubulin dimer,we identified an unexpected role of CPAP PN2-3 in controlling centriolar and ciliary length. To uncover how centriolar and ciliary length is regulated, we undertook combinatorial approachthat 

included biochemistry, structural biology and genetics. From the CPAP-tubulin crystal structure, we identified certain CPAP residues (CPAP^EE343) to regulate the delivery of its bound cytoplasmic tubulin at the site of centriole and ciliary microtubule formation (Figure 1).

By generating human RPE1 cell linesand Drosophila expressing CPAP^EE343RRand Sas-4^EE112RR(fly counter part of CPAP) respectively, we identified that CPAP regulates delivery of its bound-tubulin to define the size of microtubule-based cellular structures of basal bodies and cilia (Zheng et al., 2016 Nature Commun).

Activation of centrosomes by a CPAP-tubulin inhibitor: 

Our studies in Drosophila have shown that cytoplasmic free tubulin negatively regulates the microtubule-nucleating activity of centrosomes through its direct interaction with Sas-4 (CPAP in humans) 18. Mutated Sas-4, which cannot interact with tubulin, prematurely activates interphase centrosomes to nucleate robust microtubules by recruiting increasing amounts of PCM proteins 18. To test this in cells, we established AlphaScreen, a proximity-based protein-protein interaction assay that identified CCB02, a selective inhibitor of CPAP-tubulin interaction. To do this, we exploited our recently solved CPAP-tubulin structure (Fig. 1). Further structural studies have indeed identified that CCB02 as a tubulin binder that competes for the CPAP binding site of β-tubulin, a previously uncharacterized site that has not been occupied by known conventional tubulin binders. 

We use CBB02 not only to substantiate that tubulin negatively regulates CPAP-dependent peri-centriolar material recruitment but also to prematurely activate extra centrosomes of cancer cells. Here, we briefly explain our second year work, which is under review in Nature Chemical Biology.

Centrosome amplification is a hallmark of human cancers that can trigger cancer cell invasion.

To survive, cancer cells cluster extra centrosomes to achieve pseudo-bipolar division. Here, we set out to prevent clustering of extra centrosomes by prematurely activating centrosomes to induce microtubule nucleation before mitotic onset. 

Tubulin, by interacting with the centrosomal protein CPAP, negatively regulates CPAP-dependent peri-centriolar material recruitment and concurrently microtubule nucleation. Screening for compounds that perturb CPAP-tubulin interaction lead to the identification of CCB02, which selectively binds at the CPAP binding site of tubulin. 

CCB02 prematurely activates extra centrosomes to nucleate microtubules causing cancer cells to undergo centrosome declustering, multipolar mitosis, prolonged mitotic arrest and cell death (Fig. 2).

3D-organotypic invasive assays reveal CCB02 to have broad anti-invasive activity against various cancers including tyrosine-kinase inhibitor (TKI)-resistant EGFR- and KRAS-mutant non-small cell lung cancers. Thus, we identify a vulnerability of cancer cells to extra centrosomal activation, which may serve as a global approach to target various cancers including drug-resistant cancers.

Selected publications  

1)  Anand Ramani Mariappan A, Gottardo M, Mandad S, Urlaub H, Avidor-Reiss T, Riparbelli M, Callaini G, Debec A, Feederle R,et al., Gopalakrishnan J*.Plk1/Polo phosphorylates Sas-4 at the onset of mitosis for an efficient recruitment of pericentriolar material to centrosomes. Cell Reports. Dec 26;25(13):3618-3630.e6. doi: 10.1016/j.celrep.2018.11.102. (2018)

2)  Arul Mariappan Soni K, Schorpp K, Zhao F, Minakar A, Zheng X, Mandad S, Macheleidt I, Ramani A, Kubelka T, Dawidowski M, Golfmann K, Wason A, Yang C, Simons J, Schmalz HG, Hyman AA, Aneja R, Ullrich R, Urlaub H, Odenthal M, Büttner R, Li H, Sattler M, Hadian K,Gopalakrishnan J*.Inhibition of CPAP-Tubulin interaction prevents the proliferation of centrosome amplified cancer cells.EMBO Journal Dec 10. pii: e99876. doi: 10.15252/embj.201899876(2018) (Highlighted in EMBO J as Magic Bullet).

3)  Goranci-Buzhala G, Gabriel E, Mariappan A, Gopalakrishnan J*Losers of Primary Cilia Gain the Benefit of survival.Cancer Discovery 12, 1374-1375 (2017).

4)  Zheng X, Ramani A, Soni K, Gottardo M, Zheng S, Ming Gooi L, Li W, Feng S, Mariappan A, Wason A, Widlund P, Pozniakovsky A, Poser I, Deng H, Ou G, Riparbelli M, Giuliano C, Hyman AA, Sattler M, Gopalakrishnan J*, Li H*. Molecular basis for CPAP-tubulin interaction in controlling centriolar and ciliary length.Nat Commun7, 11874 (2016). 

5)   Gabriel E, Ramani A, Karow U, Gottardo M, Natarajan K, Gooi LM, Goranci-Buzhala G, Krut O, Peters F, Nikolic M, Kuivanen S, Korhonen E, Smura T, Vapalahti O, Papantonis A, Schmidt-Chanasit J, Riparbelli M, Callaini G, Krönke M, Utermöhlen O, Gopalakrishnan J*.Recent Zika virus isolates induce premature differentiation of neural progenitors in human brain organoids.Cell Stem Cell 20, 1-10 (2017).