Degradation of cyclin B is critical for nuclear division inTrypanosoma brucei

Abstract

Summary statementTrypanosomes do not have a spindle checkpoint but have an ability to regulate the timing of nuclear division by modulating the cyclin B protein level. AbstractKinetoplastids have a nucleus that contains the nuclear genome and a kinetoplast that contains the mitochondrial genome. These single-copy organelles must be duplicated and segregated faithfully to daughter cells at each cell division. In Trypanosoma brucei, although duplication of both organelles starts around the same time, segregation of the kinetoplast precedes that of the nucleus. Cytokinesis subsequently takes place so that daughter cells inherit a single copy of each organelle. Very little is known about the molecular mechanism that governs the timing of these events.Furthermore, it is thought that T. brucei lacks a spindle checkpoint that delays the onset of nuclear division in response to spindle defects. Here we show that a mitotic cyclin CYC6 has a dynamic localization pattern during the cell cycle, including kinetochore localization. Using CYC6 as a molecular cell cycle marker, we confirmed that T. brucei cannot delay the onset of anaphase in response to a bipolar spindle assembly defect. Interestingly, expression of a stabilized form of CYC6 caused the nucleus to arrest in a metaphase-like state without preventing cytokinesis. We propose that trypanosomes have an ability to regulate the timing of nuclear division by modulating the CYC6 protein level, without a spindle checkpoint.

The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip–coupling protein

“…In our previous analysis of endogenously YFP-tagged KKT proteins, we noticed that KKT4 localizes not only to kinetochores but also near the poles of the metaphase spindle ( Akiyoshi and Gull, 2014 ). To further examine its cellular localization pattern, we used additional markers on kinetochores (KKT2) and on spindle microtubules (MAP103; Hayashi and Akiyoshi, 2018 ; Fig. 1, A and B ).…”

A kinetochore-based ATM/ATR-independent DNA damage checkpoint maintains genomic integrity in trypanosomes

“…To understand the molecular basis of MMS-induced metaphase defect, we monitored the degradation kinetics of the mitotic B-type cyclin CYC6, since its degradation is required for metaphase-to-anaphase transition (10). Cycloheximide chase analysis showed that MMS treatment significantly slowed down CYC6 degradation, increasing its half-life from ∼4 to >8 h (Figure 1C).…”

“…Alternatively, KKIP5 may act as an upstream factor of the SAC in mediating the DDR for targeting the SAC components to kinetochores. However, whether T. brucei has a SAC is still in doubt (10,35). If there is no SAC, it is unclear how T. brucei maintains genomic stability when kinetochore-microtubule attachment errors occur.…”

“…APC/C promotes the degradation of securin (Pds1 in yeast), an inhibitor of the separase protease, thus activating separase, and the activated separase then cleaves the cohesin subunit SCC1, leading to sister chromatid separation (8). APC/C also targets the mitotic B-type cyclin for degradation to promote anaphase onset (9,10) and mitotic exit (11). The signal for SAC activation is generated from unattached kinetochores, to which the SAC components are recruited through binding to the outer kinetochore component KNL1 (12–14), an essential subunit of the KMN complex that also includes the Mis12 sub-complex and the Ndc80 sub-complex (15).…”

Structural characterisation of KKT4, an unconventional microtubule-binding kinetochore protein

“…We previously showed that KKT4 co-purifies with the APC/C subunits (Akiyoshi and Gull, 2014). Furthermore, although trypanosomes lack a canonical spindle checkpoint (Ploubidou et al, 1999), cells could control the cell cycle progression by regulating the level of cyclin B in the nucleus (Hayashi and Akiyoshi, 2018). We therefore speculate that the interaction between the BRCT domain and the microtubule-binding domain might be governed by the attachment status, which in turn controls APC/C activities and the cell cycle progression.…”