Anaphase-promoting Complex Research Articles

Principles and dynamics of spindle assembly checkpoint signalling.

The transmission of a complete set of chromosomes to daughter cells during cell division is vital for development and tissue homeostasis. The spindle assembly checkpoint (SAC) ensures correct segregation by informing the cell cycle machinery of potential errors in the interactions of chromosomes with spindle microtubules prior to anaphase. To do so, the SAC monitors microtubule engagement by specialized structures known as kinetochores and integrates local mechanical and chemical cues such that it can signal in a sensitive, responsive and robust manner. In this Review, we discuss how SAC proteins interact to allow production of the mitotic checkpoint complex (MCC) that halts anaphase progression by inhibiting the anaphase-promoting complex/cyclosome (APC/C). We highlight recent advances aimed at understanding the dynamic signalling properties of the SAC and how it interprets various naturally occurring intermediate attachment states. Further, we discuss SAC signalling in the context of the mammalian multisite kinetochore and address the impact of the fibrous corona. We also identify current challenges in understanding how the SAC ensures high-fidelity chromosome segregation.

DNA replication initiation factor RECQ4 possesses a role in antagonizing DNA replication initiation

Deletion of the conserved C-terminus of the Rothmund-Thomson syndrome helicase RECQ4 is highly tumorigenic. However, while the RECQ4 N-terminus is known to facilitate DNA replication initiation, the function of its C-terminus remains unclear. Using an unbiased proteomic approach, we identify an interaction between the RECQ4 N-terminus and the anaphase-promoting complex/cyclosome (APC/C) on human chromatin. We further show that this interaction stabilizes APC/C co-activator CDH1 and enhances APC/C-dependent degradation of the replication inhibitor Geminin, allowing replication factors to accumulate on chromatin. In contrast, the function is blocked by the RECQ4 C-terminus, which binds to protein inhibitors of APC/C. A cancer-prone, C-terminal-deleted RECQ4 mutation increases origin firing frequency, accelerates G1/S transition, and supports abnormally high DNA content. Our study reveals a role of the human RECQ4 C-terminus in antagonizing its N-terminus, thereby suppressing replication initiation, and this suppression is impaired by oncogenic mutations.

Cross-talk between energy and redox metabolism in astrocyte-neuron functional cooperation

Astrocytes show unique anatomical, morphological, and metabolic features to take up substrates from the blood and metabolize them for local delivery to active synapses to sustain neuron function. In the present review, we specifically focus on key molecular aspects of energy and redox metabolism that facilitate this astrocyte-neuronal coupling in a controlled manner. Basal glycolysis is co-ordinated by the anaphase-promoting complex/cyclosome (APC/C)-Cdh1, a ubiquitin ligase that targets the proglycolytic enzyme 6-phosphofructokinase-2,6-bisphosphastate-3 (PFKFB3) for degradation. APC/C-Cdh1 activity is more robust in neurons than in astrocytes, which determine that PFKFB3 abundance and glycolytic rate are weaker in neurons. The low PFKFB3 activity in neurons facilitates glucose-6-phosphate oxidation via the pentose-phosphate pathway, which promotes antioxidant protection. Conversely, the high PFKFB3 activity in astrocytes allows the production and release of glycolytic lactate, which is taken up by neurons that use it as an oxidizable substrate. Importantly, the mitochondrial respiratory chain is tighter assembled in neurons than in astrocytes, thus the bioenergetic efficiency of mitochondria is higher in neurons. Because of this, the production of reactive oxygen species (mROS) by mitochondrial complex I is very low in neurons and very high in astrocytes. Such a naturally occurring high abundance of mROS in astrocytes physiologically determines a specific transcriptional fingerprint that contributes to sustaining cognitive performance. We conclude that the energy and redox metabolism of astrocytes must complementarily match that of neurons to regulate brain function and animal welfare.

The APC/C Activator Cdh1p Plays a Role in Mitochondrial Metabolic Remodelling in Yeast

Cdh1p is one of the two substrate adaptor proteins of the anaphase promoting complex/cyclosome (APC/C), a ubiquitin ligase that regulates proteolysis during cell cycle. In this work, using a proteomic approach, we found 135 mitochondrial proteins whose abundance was significantly altered in the cdh1Δ mutant, with 43 up-regulated proteins and 92 down-regulated proteins. The group of significantly up-regulated proteins included subunits of the mitochondrial respiratory chain, enzymes from the tricarboxylic acid cycle and regulators of mitochondrial organization, suggesting a metabolic remodelling towards an increase in mitochondrial respiration. In accordance, mitochondrial oxygen consumption and Cytochrome c oxidase activity increased in Cdh1p-deficient cells. These effects seem to be mediated by the transcriptional activator Yap1p, a major regulator of the yeast oxidative stress response. YAP1 deletion suppressed the increased Cyc1p levels and mitochondrial respiration in cdh1Δ cells. In agreement, Yap1p is transcriptionally more active in cdh1Δ cells and responsible for the higher oxidative stress tolerance of cdh1Δ mutant cells. Overall, our results unveil a new role for APC/C-Cdh1p in the regulation of the mitochondrial metabolic remodelling through Yap1p activity.

Structural investigation of CDCA3-Cdh1 protein-protein interactions using in vitro studies and molecular dynamics simulation.

The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase and its cofactor, Cdh1, regulate the expression of several cell-cycle proteins and their functions during mitosis. Levels of the protein cell division cycle-associated protein 3 (CDCA3), which is functionally required for mitotic entry, are regulated by APC/CCdh1 . CDCA3 is an intrinsically disordered protein and contains both C-terminal KEN box and D-box recognition motifs, enabling binding to Cdh1. Our previous findings demonstrate that CDCA3 has a phosphorylation-dependent non-canonical ABBA-like motif within the linker region bridging these two recognition motifs and is required for efficient binding to Cdh1. Here, we sought to identify and further characterize additional residues that participate within this ABBA-like motif using detailed in vitro experiments and in silico modeling studies. We identified the role of H-bonds, hydrophobic and ionic interactions across the CDCA3 ABBA-like motif in the linker region between KEN and D-box motifs. This linker region adopts a well-defined structure when bound to Cdh1 in the presence of phosphorylation. Upon alanine mutation, the structure of this region is lost, leading to higher flexibility, and alteration in affinities due to binding to alternate sites on Cdh1. Our findings identify roles for the anchoring residues in the non-canonical ABBA-like motif to promote binding to the APC/CCdh1 and regulation of CDCA3 protein levels.

Single-cell analyses identify anaphase-promoting complex subunit 11 as a switch controlling neuronal differentiation of glioblastoma cells

Single-cell analyses identify anaphase-promoting complex subunit 11 as a switch controlling neuronal differentiation of glioblastoma cells

Targeting the anaphase-promoting complex/cyclosome (APC/C) enhanced antiproliferative and apoptotic response in bladder cancer

Improving the chemotherapy sensitivity of bladder cancer is a current clinical challenge. It is critical to seek out effective combination therapies that include low doses of cisplatin due to its dose-limiting toxicity. This study aims to investigate the cytotoxic effects of the combination therapy including proTAME, a small molecule inhibitor, targeting Cdc-20 and to determine the expression levels of several APC/C pathway-related genes that may play a role in the chemotherapy response of RT-4 (bladder cancer) and ARPE-19 (normal epithelial) cells. The IC20 and IC50 values were determined by MTS assay. The expression levels of apoptosis-associated (Bax and Bcl-2) and APC/C-associated (Cdc-20, Cyclin-B1, Securin, and Cdh-1) genes were assessed by qRT-PCR. Cell colonization ability and apoptosis were examined by clonogenic survival experiment and Annexin V/PI staining, respectively. Low-dose combination therapy showed a superior inhibition effect on RT-4 cells by increasing cell death and inhibiting colony formation. Triple-agent combination therapy further increased the percentage of late apoptotic and necrotic cells compared to the doublet-therapy with gemcitabine and cisplatin. ProTAME-containing combination therapies resulted in an elevation in Bax/Bcl-2 ratio in RT-4 cells, while a significant decrease was observed in proTAME-treated ARPE-19 cells. Cdc-20 expression in proTAME combined treatment groups were found to be decreased compared to their control groups. Low-dose triple-agent combination induced cytotoxicity and apoptosis in RT-4 cells effectively. It is essential to evaluate the role of APC/C pathway-associated potential biomarkers as therapeutic targets and define new combination therapy regimens to achieve improved tolerability in bladder cancer patients in the future.

Inactivation of Mad2B Enhances Apoptosis in Human Cervical Cancer Cell Line upon Cisplatin-Induced DNA Damage.

Mad2B (Mad2L2), the human homolog of the yeast Rev7 protein, is a regulatory subunit of DNA polymerase ζ that shares sequence similarity with the mitotic checkpoint protein Mad2A. Previous studies on Mad2B have concluded that it is a mitotic checkpoint protein that functions by inhibiting the anaphase-promoting complex/cyclosome (APC/C). Here, we demonstrate that Mad2B is activated in response to cisplatin-induced DNA damage. Mad2B co-localizes at nuclear foci with DNA damage markers, such as proliferating cell nuclear antigen and gamma histone H2AX (γ-H2AX), following cisplatin-induced DNA damage. However, unlike Mad2A, the binding of Mad2B to Cdc20 does not inhibit the activity of APC/C in vitro. In contrast to Mad2A, Mad2B does not localize to kinetochores or binds to Cdc20 in spindle assembly checkpoint-activated cells. Loss of the Mad2B protein leads to damaged nuclei following cisplatin-induced DNA damage. Mad2B/Rev7 depletion causes the accumulation of damaged nuclei, thereby accelerating apoptosis in human cancer cells in response to cisplatin-induced DNA damage. Therefore, our results suggest that Mad2B may be a critical modulator of DNA damage response.

Abstract B006: Does TFPI1 drive development of multiple drug resistant cancer and can the anaphase promoting complex prevent it

Abstract Chemotherapy is often rendered ineffective by the development of multiple drug resistance (MDR). Given time MDR occurs almost universally and once developed applies in a non-specific manner. The lack of MDR specificity means many drugs, including those a patient has no pervious exposure to, are affected which limits treatment options. The goal of this research is to show nuclear localized tissue factor pathway inhibitor 1 (TFPI1) plays a driving role in development of MDR, and that the Anaphase Promoting Complex (APC) is capable of inhibiting MDR by targeting TFPI1 for degradation. A coagulation regulator, TFPI1 normally inhibits the tissue factor pathway to prevent blood clot formation. However, TFPI1 is highly elevated in many types of MDR cancer where we believe it mediates resistance development. Previous work in our lab has shown plasmid overexpression of TFPI1 induces MDR in MCF7 breast cancer cells, in MDR cells TFPI1 does not localize to the cell surface but instead to the nucleus, and a putative TFPI1 nuclear localization sequence (NLS) was identified. To confirm the putative NLS is required for TFPI1’s localization to nucleus, and subsequent MDR development, NLS mutant or wild type TFPI1-GFP were overexpressed in MCF7 cells. Localization of TFPI1-GFP was observed using widefield and confocal microscopy. Wild type TFPI1-GFP localized to the nucleus while the NLS mutant TFPI1 did not. These results help to confirm the putative NLS and its importance to TFPI1s role in MDR development. The APC is a nuclear E3 ubiquitin ligase and highly conserved regulator of cell cycle progression. Defects in APC function are associated with reduced longevity and cancer. Other work in our lab focuses on the APC’s role in longevity using a yeast model. However, we believe the APC also plays a role in TFPI1’s promotion of MDR. Our observations suggest APC dysfunction exists within MDR cancer cells where APC substrates accumulate, as does TFPI1. We hypothesize that nuclear TFPI1 is an APC substrate, and that APC activation will result in TFPI1 degradation slowing MDR development. Our lab has isolated several small peptides we believe bind and activate the APC, and have used these peptides in conjugation with known APC activators such as Mad2 Inhibitor-1 (M2I-1) to show that APC activation is capable of reducing TFPI1 abundance in MCF7 cells treated with the chemotherapeutic Doxorubicin (Dox). These results suggest TFPI1 is an APC substrate. To summarize, we believe TFPI1 drives development of MDR cancer, for which TFPI1’s accumulation within the nucleus is important. We have shown TFPI1’s NLS is required for localization of the protein to the nucleus, and activation of the APC reduces nuclear accumulation of TFPI1. Moving forward our goal is to demonstrate the effect TFPI1 NLS knockout and APC activation have on the development of MDR in MCF7 cells undergoing drug selection with Dox. By exploring TFPI1’s role in MDR we may elucidate means to slow resistance development and extend chemotherapy effectiveness. Citation Format: Mathew Lubachowski, Troy Harkness. Does TFPI1 drive development of multiple drug resistant cancer and can the anaphase promoting complex prevent it [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B006.

Identification and sequencing of temperature sensitive alleles of the Anaphase Promoting Complex component mat-3 in C. elegans.

The Anaphase Promoting Complex (APC) regulates the transition from metaphase to anaphase during cell division and is important to prevent defects in chromosome segregation. In a recent temperature sensitive genetic screen looking for further genes involved in fertilization, we isolated a new temperature sensitive allele of mat-3 (as49) . We also sequenced a previously identified mat-3 ( or344 ) allele that did not previously have an annotated sequence. We determined that the as49 allele was an Alanine to Threonine (A451T) mutation in the sixth exon and the or344 mutation was a Leucine to Phenylalanine (L474F) mutation in the seventh exon. These locations of the mutant alleles are consistent with other previously annotated alleles that displayed the same metaphase to anaphase transition defect phenotype and further reinforce the importance of the tetratricopeptide repeats to mediate protein interactions.

The anaphase promoting complex/cyclosome ubiquitylates histone H2B on the promoter during UbcH10 transactivation.

Among various post-translational modifications of histones, ubiquitylation plays a crucial role in transcription regulation. Histone mono-ubiquitylation by RING finger motif-containing ubiquitin ligases is documented in this respect. The RING finger ligases primarily regulate the cell cycle, where the anaphase-promoting complex/cyclosome (APC/C) takes charge as mitotic ubiquitin machinery. Reportedly, APC/C participates in transcriptional activation of the ubiquitin carrier protein UbcH10. However, the ubiquitylation activity of APC/C on the UBCH10 promoter remains elusive. This study shows that APC/C, with its adapter protein Cdc20, catalyses mono-ubiquitylation of Lysine-120 in histone 2B on the UBCH10 promoter. This study also identified a cell-cycle-specific pattern of this modification. Finally, APC/C-driven crosstalk of acetylation and ubiquitylation was found operational on UBCH10 trans-regulation. Together, these findings suggest a role for APC/C catalysed promoter ubiquitylation in managing transcription of cell cycle regulatory genes.

APC/C-Cdh1-targeted substrates as potential therapies for Alzheimer's disease.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder and the main cause of dementia in the elderly. The disease has a high impact on individuals and their families and represents a growing public health and socio-economic burden. Despite this, there is no effective treatment options to cure or modify the disease progression, highlighting the need to identify new therapeutic targets. Synapse dysfunction and loss are early pathological features of Alzheimer's disease, correlate with cognitive decline and proceed with neuronal death. In the last years, the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C) has emerged as a key regulator of synaptic plasticity and neuronal survival. To this end, the ligase binds Cdh1, its main activator in the brain. However, inactivation of the anaphase promoting complex/cyclosome-Cdh1 complex triggers dendrite disruption, synapse loss and neurodegeneration, leading to memory and learning impairment. Interestingly, oligomerized amyloid-β (Aβ) peptide, which is involved in Alzheimer's disease onset and progression, induces Cdh1 phosphorylation leading to anaphase promoting complex/cyclosome-Cdh1 complex disassembly and inactivation. This causes the aberrant accumulation of several anaphase promoting complex/cyclosome-Cdh1 targets in the damaged areas of Alzheimer's disease brains, including Rock2 and Cyclin B1. Here we review the function of anaphase promoting complex/cyclosome-Cdh1 dysregulation in the pathogenesis of Alzheimer's disease, paying particular attention in the neurotoxicity induced by its molecular targets. Understanding the role of anaphase promoting complex/cyclosome-Cdh1-targeted substrates in Alzheimer's disease may be useful in the development of new effective disease-modifying treatments for this neurological disorder.

The Secret Life of the Inhibitor of Virus Replication.

The inhibitor of virus replication (IVR) is an inducible protein that is not virus-target-specific and can be induced by several viruses. The GenBank was interrogated for sequences closely related to the tobacco IVR. Various RNA fragments from tobacco, tomato, and potato and their genomic DNA contained IVR-like sequences. However, IVRs were part of larger proteins encoded by these genomic DNA sequences, which were identified in Arabidopsis as being related to the cyclosome protein designated anaphase-promoting complex 7 (APC7). Sequence analysis of the putative APC7s of nine plant species showed proteins of 558-561 amino acids highly conserved in sequence containing at least six protein-binding elements of 34 amino acids called tetratricopeptide repeats (TPRs), which form helix-turn-helix structures. The structures of Arabidopsis APC7 and the tobacco IVR proteins were modeled using the AlphaFold program and superimposed, showing that IVR had the same structure as the C-terminal 34% of APC7, indicating that IVR was a product of the APC7 gene. Based on the presence of various transcription factor binding sites in the APC7 sequences upstream of the IVR coding sequences, we propose that IVR could be expressed by these APC7 gene sequences involving the transcription factor SHE1.

CENP-F-dependent DRP1 function regulates APC/C activity during oocyte meiosis I

Chromosome segregation is initiated by cohesin degradation, which is driven by anaphase-promoting complex/cyclosome (APC/C). Chromosome cohesin is removed by activated separase, with the degradation of securin and cyclinB1. Dynamin-related protein 1 (DRP1), a component of the mitochondrial fission machinery, is related to cyclin dynamics in mitosis progression. Here, we show that DRP1 is recruited to the kinetochore by centromeric Centromere protein F (CENP-F) after nuclear envelope breakdown in mouse oocytes. Loss of DRP1 during prometaphase leads to premature cohesin degradation and chromosome segregation. Importantly, acute DRP1 depletion activates separase by initiating cyclinB1 and securin degradation during the metaphase-to-anaphase transition. Finally, we demonstrate that DRP1 is bound to APC2 to restrain the E3 ligase activity of APC/C. In conclusion, DRP1 is a CENP-F-dependent atypical spindle assembly checkpoint (SAC) protein that modulates metaphase-to-anaphase transition by controlling APC/C activity during meiosis I in oocytes.

APC/CCDC20 targets SCFFBL17 to activate replication stress responses in Arabidopsis.

DNA replication stress threatens genome stability and affects plant growth and development. How plants resolve replication stress is poorly understood. The protein kinase WEE1-mediated cell cycle arrest is required for replication stress responses. The E3 ubiquitin ligases anaphase-promoting complex/cyclosome (APC/C) and Skp1/Cullin 1/F-box (SCF) are essential regulators of the cell cycle. Here, we show that APC/CCDC20 mediates the degradation of SCFFBL17 during replication stress responses in Arabidopsis thaliana. Biochemically, WEE1 interacts with and phosphorylates the APC/C co-activator APC10, which enhances the interaction between F-BOX-LIKE17 (FBL17) and CELL DIVISION CYCLE 20 (CDC20), an activator of APC/C. Both APC10 and CDC20 are required for the polyubiquitination and degradation of FBL17. Genetically, silencing CDC20 or APC10 confers plant hypersensitivity to replication stress, which is suppressed by loss of FBL17. Collectively, our study suggests that WEE1 activates APC/C to inhibit FBL17, providing insight into replication stress responses in plants.

The Anaphase-Promoting Complex/Cyclosome Is a Cellular Ageing Regulator

The anaphase-promoting complex/cyclosome (APC/C) is a complicated cellular component that plays significant roles in regulating the cell cycle process of eukaryotic organisms. The spatiotemporal regulation mechanisms of APC/C in distinct cell cycle transitions are no longer mysterious, and the components of this protein complex are gradually identified and characterized. Given the close relationship between the cell cycle and lifespan, it is urgent to understand the roles of APC/C in lifespan regulation, but this field still seems to have not been systematically summarized. Furthermore, although several reviews have reported the roles of APC/C in cancer, there are still gaps in the summary of its roles in other age-related diseases. In this review, we propose that the APC/C is a novel cellular ageing regulator based on its indispensable role in the regulation of lifespan and its involvement in age-associated diseases. This work provides an extensive review of aspects related to the underlying mechanisms of APC/C in lifespan regulation and how it participates in age-associated diseases. More comprehensive recognition and understanding of the relationship between APC/C and ageing and age-related diseases will increase the development of targeted strategies for human health.

Full and D-Box-Deficient PTTG1 Isoforms: Effects on Cell Proliferation

Human securin (PTTG1) is a protooncogene whose expression is elevated in many types of malignant cells. We previously discovered a minor short isoform of securin lacking exons 3 and 4. The missing exons encode the main recognition site (D-box) of the anaphase-promoting complex (APC/C). We show that these two PTTG1 isoforms have different effects on transcription. Here, we have studied the effects of overexpression and selective knockdown of the short and complete securin isoforms on cell proliferation using the xCELLigence system. Notably, selective knockdown of the short isoform mRNA led to a dramatic decrease in cell growth, while overexpression of both isoforms accelerated cell growth. To search for genes with alternative isoforms similar to securin, we analyzed the GENCODE database and found that 54 of 128 genes with a PTTG1-like set of APC/C recognition sites have known isoforms without the D-box. Overall, the data obtained indicate the existence of a new class of alternative isoforms and reinstates the importance of minor isoforms.

Revisiting degron motifs in human AURKA required for its targeting by APC/CFZR1.

Mitotic kinase Aurora A (AURKA) diverges from other kinases in its multiple active conformations that may explain its interphase roles and the limited efficacy of drugs targeting the kinase pocket. Regulation of AURKA activity by the cell is critically dependent on destruction mediated by the anaphase-promoting complex (APC/CFZR1) during mitotic exit and G1 phase and requires an atypical N-terminal degron in AURKA called the "A-box" in addition to a reported canonical D-box degron in the C-terminus. Here, we find that the reported C-terminal D-box of AURKA does not act as a degron and instead mediates essential structural features of the protein. In living cells, the N-terminal intrinsically disordered region of AURKA containing the A-box is sufficient to confer FZR1-dependent mitotic degradation. Both in silico and in cellulo assays predict the QRVL short linear interacting motif of the A-box to be a phospho-regulated D-box. We propose that degradation of full-length AURKA also depends on an intact C-terminal domain because of critical conformational parameters permissive for both activity and mitotic degradation of AURKA.

Genome-wide identification and expression analysis of anaphase promoting complex/cyclosome (APC/C) in rose

The anaphase promoting complex/cyclosome (APC/C) is a large multi-subunit complex, regulating plant development and cell cycle. In plants, the APC/C gene family has been identified in Arabidopsis, rice, and maize. The APC/Cs in rose has not yet been reported. In this study, a total of 19 APC/C genes were identified in rose. Furthermore, we also investigated phylogenetic relationships, chromosomal distribution, gene structure, motif analysis, promoter sequence analysis and expression pattern of RhAPC/C genes. Synteny analysis indicated that AtAPC/Cs and RhAPC/Cs show a high degree of conservation. RhAPC/C promoters contains numerous cis-elements involved in plant morphogenesis, hormone response and stress response. Based on the transcription of RhAPC/Cs in different tissues and developmental stages, it appears that RhAPC/Cs may play a variety of roles in rose growth and development. RhAPC/Cs have limitations in the time and space during which they respond to hormones and abiotic stress. RhAPC5, RhAPC11d, RhAPC13a and RhAPC13c may play a role in rose responding to abiotic stress. The expression of RhAPC10 was altered by infection with fungal pathogen. Our study will serve as a basis for determining the functional role of APC/C genes in roses and help future research on woody plants.

Septin 9 controls CCNB1 stabilization via APC/CCDC20 during meiotic metaphase I/anaphase I transition in mouse oocytes.

The anaphase promoting complex/cyclosome (APC/C) and its cofactors CDH1 and CDC20 regulate the accumulation/degradation of CCNB1 during mouse oocyte meiotic maturation. Generally, the CCNB1 degradation mediated by APC/CCDC20 activity is essential for the transition from metaphase to anaphase. Here, by using siRNA and mRNA microinjection, as well as time-lapse live imaging, we showed that Septin 9, which mediates the binding of septins to microtubules, is critical for oocyte meiotic cell cycle progression. The oocytes were arrested at the MI stage and the connection between chromosome kinetochores and spindle microtubules was disrupted after Septin 9 depletion. As it is well known that spindle assembly checkpoint (SAC) is an important regulator of the MI-AI transition, we thus detected the SAC activity and the expression of CDC20 and CCNB1 which were the downstream proteins of SAC during this critical period. The signals of Mad1 and BubR1 still remained on the kinetochores of chromosomes in Septin 9 siRNA oocytes at 9.5h of in vitro culture when most control oocytes entered anaphase I. The expression of CCNB1 did not decrease and the expression of CDC20 did not increase at 9.5h in Septin 9 siRNA oocytes. Microinjection of mRNA encoding Septin 9 or CDC20 could partially rescue MI arrest caused by Septin 9 siRNA. These results suggest that Septin 9 is required for meiotic MI-AI transition by regulating the kinetochore-microtubule connection and SAC protein localization on kinetochores, whose effects are transmitted to APC/CCDC20 activity and CCNB1 degradation in mouse oocytes.