The cooperative roles of PHO80‐like cyclins in regulating the G1/S transition and posterior cytoskeletal morphogenesis in Trypanosoma brucei

Abstract

Cyclins and cyclin-dependent kinases (CDKs) represent the fundamental, crucial regulators of the cell division cycle in eukaryotes. Trypanosoma brucei expresses a large number of cyclins and Cdc2-related kinases (CRKs). However, how these cyclins and CRKs cooperate to regulate cell cycle progression remains poorly understood. Here, we carry out directional yeast two-hybrid assays to identify the interactions between the 10 cyclins and the 11 CRKs and detect a total of 26 cyclin-CRK pairs, among which 20 pairs are new. Our current efforts are focused on four PHO80-like cyclins, CYC2, CYC4, CYC5, and CYC7, and their physical and functional interactions with CRK1. Silencing of the four cyclins and CRK1 leads to the increase of G1 cells and defective DNA replication, suggesting their important roles in promoting the G1/S transition. Additionally, CYC2-, CYC7-, and CRK1-deficient cells possess an elongated posterior that is filled with newly assembled microtubules. Further, we show that the four cyclins display distinct subcellular localizations and half-lives, suggesting that they likely undergo distinct regulation. Altogether, our results demonstrate the involvement of four CRK1-associated cyclins, CYC2, CYC4, CYC5, and CYC7, in promoting the G1/S transition and the requirement of CYC2 and CYC7 in maintaining posterior cytoskeletal morphogenesis during the G1/S transition.

The G1 Cyclin-dependent Kinase CRK1 in Trypanosoma brucei Regulates Anterograde Protein Transport by Phosphorylating the COPII Subunit Sec31

“…Immunofluorescence microscopy showed that CRK1-PTP was detected in many punctate dots in the cytosol (Fig. 3C), as reported previously (27), and it co-localized with Sec31-HA at the ERES (Fig. 3C).…”

“…5, A and B), suggesting that CRK1 is required for TbGRASP localization. CRK1 is known to associate with four PHO80-like cyclins, CYC2, CYC4, CYC5, and CYC7 (27). However, only the depletion of CYC2 or CYC7 generated cells with an elongated posterior, as the depletion of CRK1 (27), suggesting that CYC2 and CYC7 play specialized functions with CRK1.…”

“…CRK1 is known to associate with four PHO80-like cyclins, CYC2, CYC4, CYC5, and CYC7 (27). However, only the depletion of CYC2 or CYC7 generated cells with an elongated posterior, as the depletion of CRK1 (27), suggesting that CYC2 and CYC7 play specialized functions with CRK1. Therefore, we examined whether depletion of CYC2 and CYC7 also affected TbGRASP localization, and the results showed that knockdown of CYC2 or CYC7 alone did not affect TbGRASP localization, but depletion of CYC2 and CYC7 simultaneously disrupted TbGRASP localization in ϳ65% of the cells after RNAi induction for 3 days (Fig.…”

“…CRK1 and CRK3 are involved in controlling the G 1 /S transition and the G 2 /M transition, respectively (24 -26). CRK1 associates with four PHO80-like cyclins, CYC2, CYC4, CYC5, and CYC7, that appear to play distinct cellular roles in addition to driving the G 1 /S transition in the procyclic form of T. brucei (27). CYC2 and CYC7 cooperate with CRK1 to regulate posterior cytoskeletal morphogenesis, whereas the CYC4-CRK1 complex appears to regulate certain unidentified nuclear processes required for the G 1 /S transition (27).…”

“…CRK1 associates with four PHO80-like cyclins, CYC2, CYC4, CYC5, and CYC7, that appear to play distinct cellular roles in addition to driving the G 1 /S transition in the procyclic form of T. brucei (27). CYC2 and CYC7 cooperate with CRK1 to regulate posterior cytoskeletal morphogenesis, whereas the CYC4-CRK1 complex appears to regulate certain unidentified nuclear processes required for the G 1 /S transition (27). Despite the essential functions of CRK1, however, its mechanistic roles remain poorly understood, mainly because its downstream substrates have not been identified.…”