Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function

Gulluni, Federico; Martini, Miriam; Santis, Maria Chiara De; Campa, Carlo Cosimo; Ghigo, Alessandra; Margaria, Jean Piero; Ciraolo, Elisa; Franco, Irene; Ala, Ugo; Annaratone, Laura; Disalvatore, Davide; Bertalot, Giovanni; Viale, Giuseppe; Noatynska, Anna; Compagno, Mara; Sigismund, Sara; Montemurro, Filippo; Thelen, Marcus; Fan, Fan; Meraldi, Patrick; Marchiò, Caterina; Pece, Salvatore; Sapino, Anna; Chiarle, Roberto; Fiore, Pier Paolo Di; Hirsch, Emilio

doi:10.1016/j.ccell.2017.09.002

Cited by 70 publications

(105 citation statements)

References 61 publications

(94 reference statements)

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“…Therefore, many of the phenotypes attributed to complex disruption (i.e., depletion of CHC or TACC3) may partly result from delocalization of GTSE1 from spindles and loss of MCAK inhibition. Interestingly, while CHC and TACC3 homologues are conserved among eukaryotes (Peset and Vernos, 2008;Gergely, 2002), published reports of CHC and TACC3 interacting on the mitotic spindle have been limited to vertebrate model systems (human, mouse, chicken, and Xenopus), and it has been proposed that this function of clathrin may have arisen later in evolution (Gulluni et al, 2017;Royle, 2013;Hubner et al, 2010;Lin et al, 2010;Fu et al, 2010;Burgess et al, 2015). Our finding that homologues of human GTSE1 containing the important C-terminal domain exist in only vertebrates raises the question of whether the appearance of GTSE1 (and its interaction with CHC) may be evolutionarily linked to clathrin localization to the spindle and its MT-stabilizing function during mitosis.…”

Section: Discussionmentioning

confidence: 99%

Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

Rondelet

Lin

Singh

et al. 2020

Journal of Cell Biology

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Clathrin ensures mitotic spindle stability and efficient chromosome alignment, independently of its vesicle trafficking function. Although clathrin localizes to the mitotic spindle and kinetochore fiber microtubule bundles, the mechanisms by which clathrin stabilizes microtubules are unclear. We show that clathrin adaptor interaction sites on clathrin heavy chain (CHC) are repurposed during mitosis to directly recruit the microtubule-stabilizing protein GTSE1 to the spindle. Structural analyses reveal that these sites interact directly with clathrin-box motifs on GTSE1. Disruption of this interaction releases GTSE1 from spindles, causing defects in chromosome alignment. Surprisingly, this disruption destabilizes astral microtubules, but not kinetochore-microtubule attachments, and chromosome alignment defects are due to a failure of chromosome congression independent of kinetochore–microtubule attachment stability. GTSE1 recruited to the spindle by clathrin stabilizes microtubules by inhibiting the microtubule depolymerase MCAK. This work uncovers a novel role of clathrin adaptor-type interactions to stabilize nonkinetochore fiber microtubules to support chromosome congression, defining for the first time a repurposing of this endocytic interaction mechanism during mitosis.

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Section: Discussionmentioning

confidence: 99%

Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

Rondelet

Lin

Singh

et al. 2020

Journal of Cell Biology

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“…Interestingly, while CHC and TACC3 homologs are conserved among eukaryotes (Peset and Vernos, 2008;Gergely, 2002), published reports of CHC and TACC3 interacting on the mitotic spindle have been limited to vertebrate model systems (human, mouse, chicken, and Xenopus), and it has been proposed that this function of clathrin may have arisen later in 14 evolution (Gulluni et al, 2017;Royle, 2013;Hubner et al, 2010;Lin et al, 2010;Fu et al, 2010;Burgess et al, 2015). Our finding that homologs of human GTSE1 containing the important Cterminal domain only exist in vertebrates raises the question of whether the appearance of GTSE1 (and its interaction with CHC) may be evolutionarily linked to clathrin localization to the spindle and its MT stabilizing function during mitosis.…”

Section: Discussionmentioning

confidence: 99%

“…Importantly, these mitotic roles of clathrin are independent of its role in endocytosis and membrane trafficking (Royle et al, 2005;Smith and Chircop, 2012;Cheeseman et al, 2013;Royle, 2013). During mitosis, clathrin forms a complex with the proteins TACC3, PI3K-C2 , and the microtubule polymerase ch-Tog (Hubner et al, 2010;Lin et al, 2010;Fu et al, 2010;Booth et al, 2011;Gulluni et al, 2017). Formation of this complex (hereafter referred to as the CHC/TACC3 complex) and its recruitment to spindles depends on the direct interaction between clathrin heavy chain (CHC) and TACC3 phosphorylated on serine S558 by the Aurora A kinase, thereby restricting the function of this clathrin complex on microtubules to mitosis.…”

Section: Introductionmentioning

confidence: 99%

Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

Rondelet

Lin

Singh

et al. 2019

Preprint

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Clathrin plays an important role to ensure mitotic spindle stability and efficient chromosome alignment, independently of its well-characterized functions in vesicle trafficking. While clathrin clearly localizes to the mitotic spindle and kinetochore-fiber microtubule bundles, the mechanisms by which clathrin stabilizes microtubules have remained elusive. Here we show that clathrin adaptor interaction sites on clathrin heavy chain (CHC) are repurposed during mitosis to directly recruit the microtubule-stabilizing protein GTSE1 to the mitotic spindle.Structural analyses reveal that multiple clathrin-box motifs on GTSE1 interact directly with different clathrin adaptor interaction sites on CHC, in a manner structurally analogous to that which occurs between adaptor proteins and CHC near membranes. Specific disruption of this interaction in cells releases GTSE1 from spindles and causes defects in chromosome alignment. Surprisingly, this disruption causes destabilization of astral microtubules, but not kinetochore-microtubule attachments, and the resulting chromosome alignment defect is due to a failure of chromosome congression independent of kinetochore-microtubule attachment stability. Finally, we show that GTSE1 recruited to the spindle by clathrin stabilizes 2 microtubules and promotes chromosome congression by inhibiting the activity of the microtubule depolymerase MCAK. This work thus uncovers a novel role of clathrin to stabilize non-kinetochore-fiber microtubules to support chromosome congression. This role is carried out via clathrin adaptor-type interactions of CHC with GTSE1, defining for the first time an important repurposing of this endocytic interaction mechanism during mitosis. Rizk et al., 2009; Walczak et al., 1996;Wordeman and Mitchison, 1995) (Moore et al., 2005). It was proposed that a function of TACC3 is to antagonize MCAK's potent activity at centrosomes to stabilize astral MTs, presumably by promoting activity of the microtubule depolymerase XMAP215/ch-Tog (Kinoshita et al., 2005). However, whether clathrin (or the CHC/TACC3 complex) is required for astral microtubule stabilization has not been addressed.Another potential functional member of the CHC/TACC3 complex is GTSE1 (pronounced "jitsee-one" (Monte et al., 2000)), an intrinsically disordered protein that has been shown by proteomic analysis to interact with the CHC/TACC3 complex in cells (Hubner et al., 2010). Two recent studies showed that GTSE1 localizes to the spindle during mitosis, and like TACC3, is necessary for astral microtubule stabilization and efficient chromosome alignment (Bendre et al., 2016;Tipton et al., 2017). We showed that GTSE1 stabilizes MTs in mitosis by inhibiting MCAK activity (Bendre et al., 2016). In that study, cancer cells either depleted or knocked-out of GTSE1 showed destabilization of both astral MTs and kinetochore-MT attachments.Another study reported that a specific "slow turnover" population of MTs become stabilized upon depletion of GTSE1(Tipton et al., 2017). The reason for these seemingly contr...

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“…One potential explanation for this discrepancy is functional differences between human PIK3C2A and the mouse ortholog. However, the involvement of both human and mouse PIK3C2A in cilia formation, PI metabolism, and cellular proliferation suggests a high degree of functional conservation at the cellular level (Franco et al, 2014;Gulluni et al, 2017). An alternate possibility is that the species viability differences associated with PIK3C2A deficiency result from altered compensation from other PI metabolizing enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…However, they are generally thought to catalyze the phosphorylation of PI and/or PI 4-phosphate [PI(4)P] to generate PI(3)P and PI(3,4)P2, respectively (Jean and Kiger, 2014). PIK3C2A has been attributed a wide-range of biological functions including glucose transport, angiogenesis, Akt activation, endosomal trafficking, phagosome maturation, mitotic spindle organization, exocytosis, and autophagy (Behrends et al, 2010;Campa et al, 2015;Devereaux et al, 2013;Falasca and Maffucci, 2012;Franco et al, 2014;Gulluni et al, 2017;Krag et al, 2010;Leibiger et al, 2010;Posor et al, 2013;Yoshioka et al, 2012). In addition, PIK3C2A is critical for the formation and function of primary cilia (Falasca 5 and Maffucci, 2012;Franco et al, 2014).…”

Section: Introductionmentioning

confidence: 99%

Mutations In PIK3C2A Cause Syndromic Short Stature, Skeletal Abnormalities, and Cataracts Associated With Ciliary Dysfunction

Tiosano

Feldman

Chen

et al. 2018

Preprint

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PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2. We identified homozygous loss-of-function mutations in PIK3C2A in children from three independent consanguineous families with short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations, among other findings. Cellular studies of patient-derived fibroblasts found that they lacked PIK3C2A protein, had impaired cilia formation and function, and demonstrated reduced proliferative capacity.Collectively, the genetic and molecular data implicate mutations in PIK3C2A in a new Mendelian disorder of PI metabolism, thereby shedding light on the critical role of a class II PI3K in growth, vision, skeletal formation and neurological development. This discovery expands what is known about disorders of PI metabolism and helps unravel the role of PIK3C2A and class II PI3Ks in health and disease.

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Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function

Cited by 70 publications

References 61 publications

Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

Clathrin’s adaptor interaction sites are repurposed to stabilize microtubules during mitosis

Mutations In PIK3C2A Cause Syndromic Short Stature, Skeletal Abnormalities, and Cataracts Associated With Ciliary Dysfunction

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