The centrosome orientation checkpoint is germline stem cell specific and operates prior to the spindle assembly checkpoint in Drosophila testis

Abstract

Asymmetric cell division is utilized by a broad range of cell types to generate two daughter cells with distinct cell fates. In stem cell populations asymmetric cell division is believed to be crucial for maintaining tissue homeostasis, failure of which can lead to tissue degeneration or hyperplasia/tumorigenesis. Asymmetric cell divisions also underlie cell fate diversification during development. Accordingly, the mechanisms by which asymmetric cell division is achieved have been extensively studied, although the check points that are in place to protect against potential perturbation of the process are poorly understood. Drosophila melanogaster male germline stem cells (GSCs) possess a checkpoint, termed the centrosome orientation checkpoint (COC), that monitors correct centrosome orientation with respect to the component cells of the niche to ensure asymmetric stem cell division. To our knowledge, the COC is the only checkpoint mechanism identified to date that specializes in monitoring the orientation of cell division in multicellular organisms. Here, by establishing colcemid-induced microtubule depolymerization as a sensitive assay, we examined the characteristics of COC activity and find that it functions uniquely in GSCs but not in their differentiating progeny. We show that the COC operates in the G2 phase of the cell cycle, independently of the spindle assembly checkpoint. This study may provide a framework for identifying and understanding similar mechanisms that might be in place in other asymmetrically dividing cell types.

Cytokine receptor-Eb1 interaction couples cell polarity and fate during asymmetric cell division

“…These results validate the efficiency of RNAi-26 mediated knockdown of JAK-STAT components. We initially focused on day 4 after 27 induction of RNAi, when downregulation of STAT is clear but GSC loss is incomplete 28 (~5 GSCs/testis after 4 days of RNAi induction, compared to ~9 GSCs/testis in control 29 testis)(Figure 1-figure supplement 1), such that centrosome/spindle orientation can be 30 orientation checkpoint (COC)(Cheng et al, 2008, Venkei andYamashita, 2015). The 23…”

A kinesin Klp10A mediates cell cycle-dependent shuttling of Piwi between nucleus and nuage

“…To this end, we first examined the localization of Piwi and Vasa in control vs. klp10A RNAi GSCs/SGs. We further detected physical interaction and colocalization between Vasa and Piwi specifically when cells were blocked in metaphase with MG132 (a proteasome inhibitor that arrests cells in mitosis [52]), or with colcemid (S9 Fig) [31]. These results suggest that Piwi gains access to nuage specifically during mitosis.…”

“…GBs further undergo four rounds of mitotic divisions as spermatogonia (SGs), which then enter meiotic program as spermatocytes (SCs) ( Fig 1A). Asymmetric divisions of Drosophila male GSCs, as well as several other systems, are mediated by stereotypical positioning of the centrosomes [27][28][29][30][31][32][33][34][35][36]. Therefore, stem cell-specific centrosomal components are of significant interest in understanding the mechanisms that regulate asymmetric stem cell divisions.…”

“…To achieve this, we combined ex vivo colcemid treatment with a mad2 mutation. Colcemid treatment effectively depolymerized MTs (S9F Fig)[31], which would normally cause metaphase arrest due to the spindle assembly checkpoint (S9B and S9D Fig) [31,53]. However, when this is combined with a mad2 mutation, which inactivates the spindle assembly checkpoint, cells exit mitosis even in the absence of MTs (S13 Fig) (Li et al 2010).…”

Regulation of Stem Cells in Their Niche

“…Apart from the COC, the SAC (spindle assembly checkpoint) also exists in GSCs and monitors the tension at kinetochores generated by microtubule attachments during the metaphase-anaphase transition [23]. Together, these checkpoints ensure that correct centrosome and spindle orientation is achieved to accurately segregate chromosomes and cell fate determinants for asymmetric cell division.…”