Biology subjects

Gruneberg, U.

Publications and source records attributed to Gruneberg, U..

10 recordsLinked to original sources

bioRxiv2026

Spatial separation provides the force-coupling mechanism for the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) prevents anaphase until chromosomes have formed correct bipolar attachments to the mitotic spindle. Spatial separation of pericentromeric Aurora B from outer-kinetochore substrates explains how tension-dependent biorientation stabilises microtubule-kinetochore attachments. How this geometry controls the SAC and is ultimately sensed by the key checkpoint kinase MPS1 has remained unclear. Here, we show that MPS1 recruitment is regulated by the spatial relationship between pericentromeric Aurora B-INCENP and the outer kinetochore. Shortening INCENP reduces MPS1 recruitment, whereas extending INCENP delays MPS1 removal and SAC silencing. Partial NDC80 depletion prevents normal Aurora B-kinetochore separation while retaining MPS1-dependent checkpoint signalling. Conversely, acute recruitment of Aurora B to microtubule-attached outer kinetochores under tension bypasses spatial separation and rapidly restores MPS1 and downstream SAC signalling. We propose that error correction and MPS1-dependent SAC signalling share an upstream spatial-sensing mechanism in which force-dependent separation from Aurora B favours attachment stabilisation while limiting MPS1 recruitment.

cell biology↗

bioRxiv2023

Spindle checkpoint signalling in anaphase is prevented by KNL1 release from kinetochores

CDK1-cyclin B1 kinase is the main driver of mitosis and initiates the morphological changes that characterise mitosis, including mitotic spindle assembly and formation of the outer kinetochore. CDK1-cyclin B1 activity is also critically required for spindle assembly checkpoint (SAC) signalling during mitosis. In particular, CDK1-cyclin B1 promotes the targeting of the principal spindle checkpoint kinase MPS1 to kinetochores, leading to the recruitment of SAC proteins to the outer kinetochore scaffold protein KNL1 and initiation of checkpoint signalling. However, cells expressing kinetochore-tethered MPS1 still require CDK1 activity for SAC signalling, suggesting that CDK1 plays both MPS1-dependent and -independent roles in regulating the SAC. Here we show that the latter is due to CDK1-mediated kinetochore recruitment of KNL1, which is reversed by the PP1 phosphatase at the metaphase-to-anaphase transition. Our findings explain the abrupt and irreversible termination of spindle checkpoint signalling in anaphase, since the drop of CDK1 activity means both MPS1 and the spindle checkpoint scaffold KNL1 are lost from kinetochores. SummaryLim-Manley and Gruneberg investigate MPS1-independent roles of CDK1 in spindle checkpoint signalling. They reveal how PP1 activity following CDK1 inactivation results in the rapid removal of KNL1 from kinetochores at anaphase onset, contributing to prompt spindle checkpoint silencing.

cell biology↗

bioRxiv2022

Regulated Degradation of the Inner Nuclear Membrane Protein SUN2 Maintains Nuclear Envelope Architecture and Function

Nuclear architecture and functions depend on dynamic interactions between nuclear components (such as chromatin) and inner nuclear membrane (INM) proteins. Mutations in INM proteins interfering with these interactions result in disease. However, mechanisms controlling the levels and turnover of INM proteins remain unknown. Here, we describe a mechanism of regulated degradation of the INM SUN domain-containing protein 2 (SUN2). We show that Casein Kinase II and the C-terminal domain Nuclear Envelope Phosphatase 1 (CTDNEP1) have opposing effects on SUN2 levels by regulating SUN2 binding to the ubiquitin ligase Skp/Cullin1/F-Box{beta}TrCP (SCF{beta}TrCP). Upon binding to phosphorylated SUN2, SCF{beta}TrCP promotes its ubiquitination. Ubiquitinated SUN2 is membrane extracted by the AAA ATPase p97 and delivered to the proteasome for degradation. Importantly, accumulation of non-degradable SUN2 results in aberrant nuclear architecture, vulnerability to DNA damage and increased lagging chromosomes in mitosis. These findings uncover a central role of proteolysis in INM protein homeostasis.

cell biology↗

bioRxiv2022

PP6-regulated Aurora A activity towards NDC80 limits mitotic spindle size to prevent micronucleation

Amplification of the mitotic kinase Aurora A or loss of its regulator protein phosphatase 6 (PP6) have emerged as drivers of genome instability. Cells lacking PPP6C, the catalytic subunit of PP6, have amplified Aurora A activity and as we show here, enlarged mitotic spindles which fail to hold chromosomes tightly together in anaphase, causing defective nuclear structure. Using functional genomics to shed light on the processes underpinning these changes, we discover synthetic lethality between PPP6C and the kinetochore protein NDC80. We find that NDC80 is phosphorylated on multiple N-terminal sites during spindle formation by Aurora A-TPX2, exclusively at checkpoint-silenced, microtubule-attached kinetochores. NDC80 phosphorylation persists until spindle disassembly in telophase, is increased in PPP6C-knockout cells and, and is Aurora B-independent. An Aurora-phosphorylation-deficient NDC80-9A mutant reduces spindle size and suppresses defective nuclear structure in PPP6C-knockout cells. By regulating NDC80 phosphorylation by Aurora A-TPX2, PP6 plays an important role in mitotic spindle formation and size control, and thus the fidelity of cell division.

cell biology↗

bioRxiv2022

MPS1 localizes to microtubule-attached kinetochores and actively promotesmicrotubule release

SummaryIn eukaryotes, the spindle assembly checkpoint protects genome stability in mitosis by preventing anaphase onset until incorrect microtubule-kinetochore attachment geometries have been eliminated and chromosome biorientation has been completed. These error correction and checkpoint processes are linked by two conserved serine/threonine kinases, Aurora B and MPS1 1, 2. In the prevailing model for spindle checkpoint signaling, MPS1 detects microtubule-free kinetochores generated by the Aurora B-dependent error correction pathway, and initiates spindle checkpoint signaling. However, we find that MPS1 initially localizes to microtubule-attached kinetochores in a manner dependent on the relative activities of Aurora B and its counteracting phosphatase PP2A-B56, and then actively promotes microtubule release. Thus, MPS1 is not a passive sensor for the microtubule binding state of kinetochores but actively generates microtubule-free checkpoint signaling kinetochores. MPS1 is thus instrumental for both the initial resolution of incorrect microtubule-kinetochore attachments and the downstream propagation of spindle checkpoint signaling.

cell biology↗

bioRxiv2020

The association of Plk1 with the Astrin-Kinastrin complex promotes formation and maintenance of a metaphase plate

Errors in mitotic chromosome segregation can lead to DNA damage and aneuploidy, both hallmarks of cancer. To achieve synchronous error-free segregation, mitotic chromosomes must align at the metaphase plate with stable amphitelic attachments to microtubules emanating from opposing spindle poles. The Astrin-Kinastrin/SKAP complex, also containing DYNLL1 and MYCBP, is a spindle and kinetochore protein complex with important roles in bipolar spindle formation, chromosome alignment and microtubule-kinetochore attachment. However, the molecular mechanisms by which Astrin-Kinastrin fulfils these diverse roles are not fully understood. Here we characterise a direct interaction between Astrin and the mitotic kinase Plk1. We identify the Plk1-binding site on Astrin as well as four Plk1 phosphorylation sites on Astrin. Regulation of Astrin-Kinastrin by Plk1 is dispensable for bipolar spindle formation and bulk chromosome congression but promotes stable microtubule-kinetochore attachments and metaphase plate maintenance. It is known that Plk1 activity is required for effective microtubule-kinetochore attachment formation, and we suggest that Astrin phosphorylation by Plk1 contributes to this process.Summary We demonstrate that Plk1 binds to and phosphorylates the N-terminus of Astrin. This interaction promotes recruitment of the Astrin-complex to kinetochores and stabilises microtubule-kinetochore-attachments in situations when mitosis is delayed.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2020

PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20

Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20 form the main ubiquitin E3 ligase for these proteins. APC/CCDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylations. Here we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilises cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1-phosphorylation defective CDC206A mutants. These CDC206A cells show a normal spindle checkpoint response, but once all chromosomes have aligned rapidly degrade cyclin B and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase by promoting APC/CCDC20-dependent destruction of cyclin B in human cells.

cell biology↗

bioRxiv2020

The C-terminal helix of BubR1 is essential for CENP-E-dependent chromosome alignment

During cell division, misaligned chromosomes are captured and aligned by motors before their segregation. The CENP-E motor is recruited to polar unattached kinetochores, to facilitate chromosome alignment. The spindle checkpoint protein BubR1 has been reported as a CENP-E interacting partner, but to what extent, if at all, BubR1 contributes to CENP-E localization at kinetochores, has remained controversial. Here we define the molecular determinants that specify the interaction between BubR1 and CENP-E. The basic C-terminal helix of BubR1 is necessary but not sufficient for CENP-E interaction, while a minimal key acidic patch on the kinetochore-targeting domain of CENP-E, is also essential. We then demonstrate that BubR1 is required for the recruitment of CENP-E to kinetochores to facilitate chromosome alignment. This BubR1-CENP-E axis is critical to align chromosomes that have failed to congress through other pathways and recapitulates the major known function of CENP-E. Overall, our studies define the molecular basis and the function for CENP-E recruitment to BubR1 at kinetochores during mammalian mitosis.

cell biology↗

bioRxiv2019

Aurora A promotes chromosome congression by activating the condensin-dependent pool of KIF4A

Aurora kinases create phosphorylation gradients within the spindle during prometaphase and anaphase. These locally regulate factors that promote spindle organisation, chromosome condensation and movement, and cytokinesis. We show that one such factor is the kinesin KIF4A, which is present along the chromosome axes throughout mitosis and the central spindle in anaphase. These two pools of KIF4A depend on condensin I and PRC1, respectively. Previous work has shown KIF4A is activated by Aurora B at the anaphase central spindle. However, whether or not chromosome-associated KIF4A bound to condensin I is regulated by Aurora kinases remain unclear. To determine the roles of the two different pools of KIF4A, we generated specific point mutants that are unable to interact with either condensin I or PRC1, or are deficient for Aurora kinase regulation. By analysing these mutants, we show that Aurora kinases phosphorylate the condensin I dependent pool of KIF4A and thus actively promote chromosome congression from the spindle poles to the metaphase plate.

cell biology↗

bioRxiv2019

Checkpoint signaling and error correction require regulation of the MPS1 T-loop by PP2A-B56

During mitosis, the formation of microtubule-kinetochore attachments is monitored by the serine/threonine kinase Mono-Polar Spindle 1 (MPS1). MPS1 is recruited to unattached kinetochores where it phosphorylates KNL1, BUB1 and MAD1 to initiate the spindle checkpoint. This arrests the cell cycle until all kinetochores have been stably captured by microtubules. MPS1 also contributes to the error correction process rectifying incorrect kinetochore attachments. MPS1 activity at kinetochores requires auto-phosphorylation at multiple sites including T676 in the activation segment or \"T-loop\". We now demonstrate that a BUBR1-bound pool of PP2A-B56 regulates MPS1 T-loop autophosphorylation and hence activation status in mammalian cells. Overriding this regulation using phospho-mimetic mutations in the MPS1 T-loop to generate a constitutively active kinase results in a prolonged mitotic arrest with continuous turn-over of microtubule-kinetochore attachments. Dynamic regulation of MPS1 catalytic activity by kinetochore-localized PP2A-B56 is thus critical for controlled MPS1 activity and timely cell cycle progression.

cell biology↗