A farnesyl-dependent structural role for CENP-E in expansion of the fibrous corona

Correct chromosome segregation during cell division depends on proper connections between spindle microtubules and kinetochores. During prometaphase, kinetochores are temporarily covered with a dense protein meshwork known as the fibrous corona. Formed by oligomerization of ROD/ZW10/ZWILCH-SPINDLY (RZZ-S) complexes, the fibrous corona promotes spindle assembly, chromosome orientation, and spindle checkpoint signaling. The molecular requirements for formation of the fibrous corona are not fully understood. Here, we show that the fibrous corona depends on the mitotic kinesin CENP-E and that poorly expanded fibrous coronas after CENP-E depletion are functionally compromised. This previously unrecognized role for CENP-E does not require its motor activity but instead is driven by farnesyl modification of its C-terminal kinetochore- and microtubule-binding domain. We show that in cells, CENP-E binds Spindly and recruits RZZ-S complexes to ectopic locations in a farnesyl-dependent manner. CENP-E is recruited to kinetochores following RZZ-S, and-while not required for RZZ-S oligomerization per se-promotes subsequent fibrous corona expansion. Our comparative genomics analyses suggest that the farnesylation motif in CENP-E orthologs emerged alongside the full RZZ-S module in an ancestral lineage close to the fungi-animal split (Obazoa), revealing potential conservation of the mechanisms for fibrous corona formation. Our results show that proper spindle assembly has a potentially conserved non-motor contribution from the kinesin CENP-E through stabilization of the fibrous corona meshwork during its formation.</p

A farnesyl-dependent structural role for CENP-E in expansion of the fibrous corona

Correct chromosome segregation during cell division depends on proper connections between spindle microtubules and kinetochores. During prometaphase, kinetochores are temporarily covered with a dense protein meshwork known as the fibrous corona. Formed by oligomerization of ROD/ZW10/ZWILCH-SPINDLY (RZZ-S) complexes, the fibrous corona promotes spindle assembly, chromosome orientation, and spindle checkpoint signaling. The molecular requirements for formation of the fibrous corona are not fully understood. Here, we show that the fibrous corona depends on the mitotic kinesin CENP-E and that poorly expanded fibrous coronas after CENP-E depletion are functionally compromised. This previously unrecognized role for CENP-E does not require its motor activity but instead is driven by farnesyl modification of its C-terminal kinetochore- and microtubule-binding domain. We show that in cells, CENP-E binds Spindly and recruits RZZ-S complexes to ectopic locations in a farnesyl-dependent manner. CENP-E is recruited to kinetochores following RZZ-S, and-while not required for RZZ-S oligomerization per se-promotes subsequent fibrous corona expansion. Our comparative genomics analyses suggest that the farnesylation motif in CENP-E orthologs emerged alongside the full RZZ-S module in an ancestral lineage close to the fungi-animal split (Obazoa), revealing potential conservation of the mechanisms for fibrous corona formation. Our results show that proper spindle assembly has a potentially conserved non-motor contribution from the kinesin CENP-E through stabilization of the fibrous corona meshwork during its formation.</p

A farnesyl-dependent structural role for CENP-E in expansion of the fibrous corona

Correct chromosome segregation during cell division depends on proper connections between spindle microtubules and kinetochores. During prometaphase, kinetochores are temporarily covered with a dense protein meshwork known as the fibrous corona. Formed by oligomerization of ROD/ZW10/ZWILCH-SPINDLY (RZZ-S) complexes, the fibrous corona promotes spindle assembly, chromosome orientation, and spindle checkpoint signaling. The molecular requirements for formation of the fibrous corona are not fully understood. Here, we show that the fibrous corona depends on the mitotic kinesin CENP-E and that poorly expanded fibrous coronas after CENP-E depletion are functionally compromised. This previously unrecognized role for CENP-E does not require its motor activity but instead is driven by farnesyl modification of its C-terminal kinetochore- and microtubule-binding domain. We show that in cells, CENP-E binds Spindly and recruits RZZ-S complexes to ectopic locations in a farnesyl-dependent manner. CENP-E is recruited to kinetochores following RZZ-S, and-while not required for RZZ-S oligomerization per se-promotes subsequent fibrous corona expansion. Our comparative genomics analyses suggest that the farnesylation motif in CENP-E orthologs emerged alongside the full RZZ-S module in an ancestral lineage close to the fungi-animal split (Obazoa), revealing potential conservation of the mechanisms for fibrous corona formation. Our results show that proper spindle assembly has a potentially conserved non-motor contribution from the kinesin CENP-E through stabilization of the fibrous corona meshwork during its formation.</p

A farnesyl-dependent structural role for CENP-E in expansion of the fibrous corona

Correct chromosome segregation during cell division depends on proper connections between spindle microtubules and kinetochores. During prometaphase, kinetochores are temporarily covered with a dense protein meshwork known as the fibrous corona. Formed by oligomerization of ROD/ZW10/ZWILCH-SPINDLY (RZZ-S) complexes, the fibrous corona promotes spindle assembly, chromosome orientation, and spindle checkpoint signaling. The molecular requirements for formation of the fibrous corona are not fully understood. Here, we show that the fibrous corona depends on the mitotic kinesin CENP-E and that poorly expanded fibrous coronas after CENP-E depletion are functionally compromised. This previously unrecognized role for CENP-E does not require its motor activity but instead is driven by farnesyl modification of its C-terminal kinetochore- and microtubule-binding domain. We show that in cells, CENP-E binds Spindly and recruits RZZ-S complexes to ectopic locations in a farnesyl-dependent manner. CENP-E is recruited to kinetochores following RZZ-S, and-while not required for RZZ-S oligomerization per se-promotes subsequent fibrous corona expansion. Our comparative genomics analyses suggest that the farnesylation motif in CENP-E orthologs emerged alongside the full RZZ-S module in an ancestral lineage close to the fungi-animal split (Obazoa), revealing potential conservation of the mechanisms for fibrous corona formation. Our results show that proper spindle assembly has a potentially conserved non-motor contribution from the kinesin CENP-E through stabilization of the fibrous corona meshwork during its formation.</p

A farnesyl-dependent structural role for CENP-E in expansion of the fibrous corona

Correct chromosome segregation during cell division depends on proper connections between spindle microtubules and kinetochores. During prometaphase, kinetochores are temporarily covered with a dense protein meshwork known as the fibrous corona. Formed by oligomerization of ROD/ZW10/ZWILCH-SPINDLY (RZZ-S) complexes, the fibrous corona promotes spindle assembly, chromosome orientation, and spindle checkpoint signaling. The molecular requirements for formation of the fibrous corona are not fully understood. Here, we show that the fibrous corona depends on the mitotic kinesin CENP-E and that poorly expanded fibrous coronas after CENP-E depletion are functionally compromised. This previously unrecognized role for CENP-E does not require its motor activity but instead is driven by farnesyl modification of its C-terminal kinetochore- and microtubule-binding domain. We show that in cells, CENP-E binds Spindly and recruits RZZ-S complexes to ectopic locations in a farnesyl-dependent manner. CENP-E is recruited to kinetochores following RZZ-S, and-while not required for RZZ-S oligomerization per se-promotes subsequent fibrous corona expansion. Our comparative genomics analyses suggest that the farnesylation motif in CENP-E orthologs emerged alongside the full RZZ-S module in an ancestral lineage close to the fungi-animal split (Obazoa), revealing potential conservation of the mechanisms for fibrous corona formation. Our results show that proper spindle assembly has a potentially conserved non-motor contribution from the kinesin CENP-E through stabilization of the fibrous corona meshwork during its formation.</p

A farnesyl-dependent structural role for CENP-E in expansion of the fibrous corona

Correct chromosome segregation during cell division depends on proper connections between spindle microtubules and kinetochores. During prometaphase, kinetochores are temporarily covered with a dense protein meshwork known as the fibrous corona. Formed by oligomerization of ROD/ZW10/ZWILCH-SPINDLY (RZZ-S) complexes, the fibrous corona promotes spindle assembly, chromosome orientation, and spindle checkpoint signaling. The molecular requirements for formation of the fibrous corona are not fully understood. Here, we show that the fibrous corona depends on the mitotic kinesin CENP-E and that poorly expanded fibrous coronas after CENP-E depletion are functionally compromised. This previously unrecognized role for CENP-E does not require its motor activity but instead is driven by farnesyl modification of its C-terminal kinetochore- and microtubule-binding domain. We show that in cells, CENP-E binds Spindly and recruits RZZ-S complexes to ectopic locations in a farnesyl-dependent manner. CENP-E is recruited to kinetochores following RZZ-S, and-while not required for RZZ-S oligomerization per se-promotes subsequent fibrous corona expansion. Our comparative genomics analyses suggest that the farnesylation motif in CENP-E orthologs emerged alongside the full RZZ-S module in an ancestral lineage close to the fungi-animal split (Obazoa), revealing potential conservation of the mechanisms for fibrous corona formation. Our results show that proper spindle assembly has a potentially conserved non-motor contribution from the kinesin CENP-E through stabilization of the fibrous corona meshwork during its formation.</p

Galectin-3 and prohibitin 1 are autoantigens in IgG4-related cholangitis without clear-cut protective effects against toxic bile acids

Background and aimsIgG4-related cholangitis (IRC) is the hepatobiliary manifestation of IgG4-related disease, a systemic B cell-driven fibro-inflammatory disorder. Four autoantigens have recently been described in IgG4-RD: annexin A11, galectin-3, laminin 511-E8, and prohibitin 1. We have previously reported a protective role of annexin A11 and laminin 511-E8 in human cholangiocytes against toxic bile acids. Here, we explored the potentially protective role of the carbohydrate-binding lectin galectin-3 and the scaffold proteins prohibitins 1 and 2.MethodsAnti-galectin-3, anti-prohibitin 1 and 2 autoantibody positivity in IRC and healthy and disease (primary sclerosing cholangitis (PSC)) control sera was assessed by ELISA/liquid chromatography–tandem mass spectrometry (LC-MS/MS). Human H69 cholangiocytes were subjected to short hairpin RNA (shRNA) knockdown targeting galectin-3 (LGALS3), prohibitin 1 (PHB1), and prohibitin 2 (PHB2). H69 cholangiocytes were also exposed to recombinant galectin-3, the inhibitor GB1107, recombinant prohibitin 1, and the pan-prohibitin inhibitor rocaglamide. Protection against bile acid toxicity was assessed by intracellular pH (pHi) measurements using BCECF-AM, 22,23-3H-glycochenodeoxycholic acid (3H-GCDC) influx, and GCDC-induced apoptosis using Caspase-3/7 assays.ResultsAnti-galectin-3 autoantibodies were detected in 13.5% of individuals with IRC but not in PSC. Knockdown of LGALS3 and galectin-3 inhibition with GB1107 did not affect pHi, whereas recombinant galectin-3 incubation lowered pHi. LGALS3 knockdown increased GCDC-influx but not GCDC-induced apoptosis. GB1107 reduced GCDC-influx and GCDC-induced apoptosis. Recombinant galectin-3 tended to decrease GCDC-influx and GCDC-induced apoptosis. Anti-prohibitin 1 autoantibodies were detected in 61.5% and 35.7% of individuals with IRC and PSC, respectively. Knockdown of PHB1, combined PHB1/2 KD, treatment with rocaglamide, and recombinant prohibitin 1 all lowered pHi. Knockdown of PHB1, PHB2, or combined PHB1/2 did not alter GCDC-influx, yet knockdown of PHB1 increased GCDC-induced apoptosis. Conversely, rocaglamide reduced GCDC-influx but did not attenuate GCDC-induced apoptosis. Recombinant prohibitin 1 did not affect GCDC-influx or GCDC-induced apoptosis. Finally, anti-galectin-3 and anti-prohibitin 1 autoantibody pretreatment did not lead to increased GCDC-influx.ConclusionsA subset of individuals with IRC have autoantibodies against galectin-3 and prohibitin 1. Gene-specific knockdown, pharmacological inhibition, and recombinant protein substitution did not clearly disclose a protective role of these autoantigens in human cholangiocytes against toxic bile acids. The involvement of these autoantibodies in processes surpassing epithelial secretion remains to be elucidated

Chk1 and 14-3-3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest

The atypical E2Fs, E2F7 and E2F8, act as potent transcriptional repressors of DNA replication genes providing them with the ability to induce a permanent S-phase arrest and suppress tumorigenesis. Surprisingly in human cancer, transcript levels of atypical E2Fs are frequently elevated in proliferating cancer cells, suggesting that the tumor suppressor functions of atypical E2Fs might be inhibited through unknown post-translational mechanisms. Here, we show that atypical E2Fs can be directly phosphorylated by checkpoint kinase 1 (Chk1) to prevent a permanent cell cycle arrest. We found that 14-3-3 protein isoforms interact with both E2Fs in a Chk1-dependent manner. Strikingly, Chk1 phosphorylation and 14-3-3-binding did not relocate or degrade atypical E2Fs, but instead, 14-3-3 is recruited to E2F7/8 target gene promoters to possibly interfere with transcription. We observed that high levels of 14-3-3 strongly correlate with upregulated transcription of atypical E2F target genes in human cancer. Thus, we reveal that Chk1 and 14-3-3 proteins cooperate to inactivate the transcriptional repressor functions of atypical E2Fs. This mechanism might be of particular importance to cancer cells, since they are exposed frequently to DNA-damaging therapeutic reagents

Chaperonin CCT Checkpoint Function in Basal Transcription Factor TFIID Assembly

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Cardiomyocytes stimulate angiogenesis after ischemic injury in a ZEB2-dependent manner

The disruption in blood supply due to myocardial infarction is a critical determinant for infarct size and subsequent deterioration in function. The identification of factors that enhance cardiac repair by the restoration of the vascular network is, therefore, of great significance. Here, we show that the transcription factor Zinc finger E-box-binding homeobox 2 (ZEB2) is increased in stressed cardiomyocytes and induces a cardioprotective cross-talk between cardiomyocytes and endothelial cells to enhance angiogenesis after ischemia. Single-cell sequencing indicates ZEB2 to be enriched in injured cardiomyocytes. Cardiomyocyte-specific deletion of ZEB2 results in impaired cardiac contractility and infarct healing post-myocardial infarction (post-MI), while cardiomyocyte-specific ZEB2 overexpression improves cardiomyocyte survival and cardiac function. We identified Thymosin β4 (TMSB4) and Prothymosin α (PTMA) as main paracrine factors released from cardiomyocytes to stimulate angiogenesis by enhancing endothelial cell migration, and whose regulation is validated in our in vivo models. Therapeutic delivery of ZEB2 to cardiomyocytes in the infarcted heart induces the expression of TMSB4 and PTMA, which enhances angiogenesis and prevents cardiac dysfunction. These findings reveal ZEB2 as a beneficial factor during ischemic injury, which may hold promise for the identification of new therapies