Description

Human hTERT-RPE1 cells represent wild-type monocentric cells as they were immortalized, via telomerase expression, from normal somatic retinal pigment epithelium cells (Jiang et al. 1999). Silkworm BmN4-SID1 cells represent wild-type holocentric cells as they were spontaneously immortalized from somatic ovary cells, and modified with the SID1 gene from C. elegans for RNAi-mediated depletion (Mon et al. 2012). Interestingly, cancer such as leukemia and lymphoma often exhibits dicentric or tricentric chromosomes (Herry et al. 2007; M’Kacher et al. 2020), a condition reminiscent of holocentric cells. Further, monocentric cells induced to be dicentric showed transformed cancerous properties (Gascoigne and Cheeseman 2013). We seek to understand the differences in spindle assembly dynamics and chromosome segregation in wild-type monocentric RPE1 and holocentric BmN4 cells, to potentially reveal mechanisms of how di- or tricentric cancer cells divide.

We first established protocols for optimal live-cell imaging of RPE1 and BmN4 cells undergoing mitosis (see Methods). Using the vital dyes SPY-tubulin and SPY-DNA to label cells, and the spinning disk confocal microscope, we imaged RPE1 cells for 3 hr and BmN4 cells for 9 hr with no discernible photo damage. In comparing mitosis between RPE1 and BmN4 cells, striking differences in spindle dynamics and spindle architecture were observed. The RPE1 spindles exhibited the canonical mitotic phase transition (Fig. 1A). At prophase we observed the initial assembly of the bipolar spindle from two foci, likely the centrosome; at metaphase we observed the stable-length spindle with compacted metaphase plate-aligned chromosomes and the canonical “oval” shape spindle, with an aspect ratio of ~2 (15.1 μm length/8.0 μm width, Fig. 1C); at anaphase we observed chromosome separation concurrent with spindle elongation; at telophase we observed the compacted microtubule midzone, the future midbody (Fig. 1A). Total mitosis duration, from prophase to telophase, in RPE1 cells was ~38 min, with the onset of anaphase at ~22 min (Fig. 1B, 1C). In contrast, BmN4 spindles also exhibited clear mitotic phase transition, but with distinct differences. At prophase we mostly observed the assembly of the bipolar spindle from two foci, but in ~14% of cells spindle microtubules nucleated from more than two foci, with small microtubules present everywhere near the chromosomes, indicating potential centrosomal and acentrosomal MT nucleation (Mon et al. 2014); at metaphase we observed the stable-length spindle with compacted metaphase plate-aligned chromosomes and striking “square” shape spindle, with an aspect ratio of ~1 (16.7 μm length/18.0 μm width, Fig. 1C), and no clear foci indicative of spindle poles; at anaphase we observed chromosome separation concurrent with spindle elongation; at telophase we observed the compacted microtubule midzone (Fig. 1A). The square spindle of BmN4 cells is reminiscent of the square spindle of plant cells, which are also holocentric (de Keijzer et al. 2014; Zhang and Dawe 2011). Total mitosis duration in BmN4 cells was ~113 min, with the onset of anaphase at ~92 min (Fig. 1B, 1C). BmN4 cells took ~4X longer time to complete mitosis, with corresponding ~4X longer time spent in metaphase, compare to RPE1 cells (Fig. 1C). We also observed that mitotic failure, defined as spontaneous cell death or aneuploid cell division, occurred in ~1% (1 of 79 spindles) of RPE1 cells, compared to ~10% (22 of 207 spindles) of BmN4 cells (Fig. 1C). Given that RPE1 and BmN4 cells have similar chromosome number and round mitotic cell size (Fig. 1C), we propose that holocentricity, where multiple kinetochores are present on one chromosome, requires longer time to make proper kinetochore-to-MT attachment at metaphase as compared to monocentricity. An exception maybe at embryogenesis or early development, where mitosis occurs relatively fast, compared to adult somatic cells such as RPE1 and BmN4. The extended metaphase duration and higher frequency of failed mitosis may be a consequence of the unusual “square” spindle architecture, or of the multiple kinetochores per chromosome.

Spindle assembly dynamic is orchestrated by microtubule-associated proteins, many of which are kinesins (Mountain and Compton 2000). In human cells, kinesin-5 Kif11 is essential for bipolar spindle assembly and elongation, as its inhibition results in monopolar spindle and subsequent cell death (Mayer et al. 1999; Sawin et al. 1992); and Kinesin-14 HSET is required to focus the microtubule minus ends at the spindle poles, and its inhibition, while having little effect on the metaphase spindle in normal cells, causes catastrophic multipolar spindle defects in cancer cells with supernumerary centrosomes (Kleylein-Sohn et al. 2012; Kwon et al. 2008). To compare the function of kinesin-5 and -14 in RPE1 and BmN4 cells, we performed RNAi-mediated depletion. Consistent with previous reports, we observed monopolar spindles in kinesins-5 siRNA-treated RPE1 cells, and seemingly normal metaphase spindles in kinesin-14 siRNA-treated RPE1 cells (Fig. 1D). In contrast, while we also observed monopolar spindles in kinesins-5 dsRNA-treated BmN4 cells, we observed mal-shaped metaphase spindles and unaligned chromosomes in kinesins-14 dsRNA-treated BmN4 cells (Fig. 1D). The result indicates that BmN4 cells need kinesin-14 for proper spindle assembly and chromosome segregation, more so than RPE1 cells, and highlights the importance of differential motor function in the context of mono- and holocentricity.

In summary, the holocentric silkworm cell BmN4 is an emergent model system to investigate novel pathways of spindle assembly and centromere-kinetochore assembly (Cortes-Silva et al. 2020; Li et al. 2019; Mon et al. 2014; Mon et al. 2017; Senaratne et al. 2021). We define similarities and differences in spindle architecture and dynamics between BmN4 and monocentric human RPE1 cells (Fig. 1E). Future work should probe the impact of the centrosome, the centromere/kinetochore, the spindle assembly checkpoint, on spindle architecture and dynamics and chromosome segregation. This work sets the stage for pinpointing and investigating specific genes/proteins affecting only BmN4 cells, but not RPE1 cells, in mono- and tricentric human cancer cells.