bioRxiv preprint doi: https://doi.org/10.1101/442368; this version posted October 12, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Centrosome-nuclear envelope tethering and microtubule motor-based pulling forces collaborate in centrosome positioning during mitotic entry Vincent Boudreau1, Richard Chen1, Alan Edwards1, Muhammad Sulaimain1, Paul S. Maddox1* 1 Department of Biology, University of North Carolina at Chapel Hill * Direct all correspondence to this author at [email protected]. Centrosome positioning relative to the nucleus and cell nuclear envelope and at the cortex to ensure proper centrosome shape is highly regulated across cell types, during cell migration positioning (De Simone et al., 2016). Dynein regulators including and during spindle formation in cell division. Across most sexual- LIS-1 are also required for centrosome separation in the embryo ly reproducing animals, centrosomes are provided to the oocyte (Cockell et al., 2004), although their spatio-temporal contributions through fertilization and must be positioned properly to establish remain elusive. Despite our understanding of microtubule cyto- the zygotic mitotic spindle. How centrosomes are positioned in skeleton-based pulling forces, the contribution of key mitotic kinas- space and time through the concerted action of key mitotic en- es and phosphatases to centrosome positioning remains unclear. try biochemical regulators including Protein Phosphatase 2A The net effect of molecular regulators is a biophysical (PP2A-B55/SUR-6), biophysical regulators including Dynein mechanism required for positioning centrosomes during mitotic and the nuclear lamina is unclear. Here, we uncover a role for entry in the early embryo and the contributions of the nuclear PP2A-B55/SUR-6 in regulating centrosome positioning. Mecha- envelope and its centrosome tethering components are as yet un- nistically, PP2A-B55/SUR-6 regulates nuclear size prior to mitotic certain. Centrosomes are tethered to the nuclear lamina through entry, in turn affecting nuclear envelope-based Dynein density the linker of nucleoskeleton and cytoskeleton (LINC) complex, and motor capacity. Using computational simulations, PP2A-B55/ which is composed of SUN proteins tethered to the nuclear lamina SUR-6 regulation of nuclear size and nuclear envelope Dynein and KASH proteins tethered to the microtubule cytoskeleton (re- density were both predicted to be required for proper centrosome viewed in Chang et al., 2015). In the C. elegans embryo, both the positioning. Conversely, compromising nuclear lamina integrity SUN protein SUN-1 and the KASH protein ZYG-12 are required led to centrosome detachment from the nuclear envelope and for centrosome positioning (Malone et al., 2003). Centrosome-nu- migration defects. Removal of PP2A-B55/SUR-6 and the nuclear clear envelope tethering is thought to be achieved through lamina simultaneously further disrupted centrosome positioning, Dynein as ZYG-12 directly binds Dynein’s light chain (Malone leading to unseparated centrosome pairs dissociated from the et al., 2003) and Dynein is required for tethering (Gonczy et al., nuclear envelope. Taken together, we propose a model in which 1999). Dynein-based microtubule pulling forces and the centro- centrosomes migrate and are positioned through the concerted some-nuclear envelope tethering machinery are therefore likely action of nuclear envelope-based Dynein pulling forces and cen- to be coordinated in centrosome positioning during mitotic entry. trosome-nuclear envelope tethering. Biochemically, mitotic entry is defined by the activities of INTRODUCTION master mitotic kinases and phosphatases that ensure faithful cell Fertilization provides centrosomes to the developing cycle progression (Krasinka et al., 2011; Mochida et al, 2016). As embryo in most known systems. Associated to the sperm pro- Cyclin Dependent Kinase 1 (CDK1) activity increases at mitotic nucleus, centrosomes must grow, separate and migrate in a entry, its main counteracting phosphatase Protein Phosphatase coordinated fashion to facilitate female and male pronuclear 2A (PP2A) is also regulated. In metazoans, PP2A mainly functions meeting prior to mitosis. In doing so, centrosomes orchestrate as a heterotrimeric enzyme using its B55/SUR-6 adaptor subunit the microtubule cytoskeleton in the embryo such that the mi- for substrate recognition and is the main phosphatase respon- totic spindle is properly positioned, ensuring polarity during sible for dephosphorylating CDK1 substrates (Mayer-Jaekel et asymmetric cell division. Simultaneously, as the zygote enters al., 1994; Castilho et al., 2009; Mochida et al., 2009). To identify mitosis, collaboration between mitotic master regulators and potential converse functions of the CDK1 counteracting phos- microtubule-based biophysical regulators must be organized. phatase PP2A-B55/SUR-6, we previously used a maternal effect Mechanistically, centrosomes are positioned through the genetic screen in the fly embryo (Mehsen et al., 2018) in which concerted action of cytoplasmic flow, cytoplasmic and cortical mi- several components were identified as PP2A-B55/SUR-6 collab- crotubule pulling forces, and microtubule pushing forces against orators for proper cell cycle progression, including Lamin. The the cortex (Garzon-Coral et al., 2016; Nazockdast et al., 2017). biology of D. melanogaster early development (e.g. the zygotic di- On the molecular scale, it has been well established that microtu- vision) precluded centrosome tracking and therefore fundamental bule-based motors including Dynein (Gonczy et al., 1999; Robin- probing of the biophysical mechanisms at play, thus we examined son et al., 1999) and Eg5 (Blangy et al., 1995; Sawin et al., 1995) the contributions of the genetic interaction between PP2A-B55/ play important roles in centrosome migration at mitotic entry. Re- SUR-6 and Lamin/LMN-1 to the first developmental mitosis in cently, a combination of experimental and theoretical approaches the C. elegans embryo (the zygote). We identify a novel role for have been used to distinguish the roles of different pools of motors PP2A-B55/SUR-6 in regulating centrosome positioning in the em- (i.e. nuclear and cortical), cortical flows (De Simone et al., 2016, bryo through the regulation of nuclear envelope-tethered Dynein 2017) and cytoplasmic drag (De Simone et al., 2018) in regulating motor activity. We propose a simple model in which Dynein-based centrosome positioning at the mesoscale. In the C. elegans zy- pulling forces collaborate with centrosome-nuclear envelope teth- gote, Dynein-based microtubule pulling forces are produced at the ering to ensure proper centrosome positioning during mitotic entry. Boudreau et al. | bioRxiv | October 12th 2018 | 1 bioRxiv preprint doi: https://doi.org/10.1101/442368; this version posted October 12, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. RESULTS nuclear envelope and failed to separate from one another (Video PP2A-B55/SUR-6 and LMN-1 play critical roles in centrosome S2) (Fig. 1B and F) while depleting lmn-1 caused centrosomes positioning in the C. elegans zygote to detach from nuclear envelopes and dramatically increase To identify potential roles for the uncovered genetic inter- in distance between one another (Video S3) (Fig. 1D and G). actions in single embryonic mitoses, we depleted individual com- We hypothesized that PP2A-B55/SUR-6 may regulate ponents by RNAi in the C. elegans embryo – a system in which centrosome migration through biochemical regulation of known late meiotic and the first mitotic events can be readily observed by centrosome separation components including Dynein. Partial light microscopy (Video S1) (Fig. 1A). Depleting sur-6, the PP2A RNAi-mediated depletion of the Dynein motor heavy chain dhc-1 adapterFIGURE subunit, 1 in worms expressing H2B:GFP and g-tubulin:G- led to centrosome migration defects while centrosomes remained FP (strain TH32) led to centrosomes that were tethered to the tethered to the nuclear envelope (Video S4) (Fig. 1C and F). H2B:GFP + γ-tubulin:GFP t = 0s 100s 200s 300s 400s A CTRL B sur-6 C dhc-1 16h RNAi D lmn-1 E zyg-12 F 20 G 20 18 CTRL 18 CTRL 16 sur-6 16 lmn-1 14 14 dhc-1 zyg-12 12 16h RNAi 12 10 10 8 8 6 6 distance (µm) distance (µm) 4 4 2 2 Centrosome-centrosome Centrosome-centrosome 0 0 0 100 200 300 400 500 600 700 0 100 200 300 400 500 600 700 Time (sec.) Time (sec.) Centrosome Centrosome separation separation Figure 1 | sur-6 and lmn-1 RNAi affect centrosome separation akin to dhc-1 and zyg-12. (A)(B)(C)(D)(E) Representative maximum intensity projections of embryonic confocal stacks. All RNAi treatments were performed with L4 stage worms expressing H2B:GFP and g-tubulin:GFP for 24 hours via feeding unless otherwise noted. Circles highlight individual centrosomes and arrows denote detached centrosomes. Scale bar = 10 µm. (F)(G) Centrosome distance was measured in 2D using maximum intensity projections of confocal stacks. Error bars are SEM and are represented as shaded areas around means. n of embryos analyzed per condition > 15. Time t = 0 s corresponds to centrosome separation. Boudreau et al. | bioRxiv | October 12th 2018 | 2 bioRxiv preprint doi: https://doi.org/10.1101/442368; this version posted October 12, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under FIGURE 2 aCC-BY-NC-ND 4.0 International license. Figure 2 | Regulation of Dynein-dependent micro- A 50 G tubule growth velocity acceleration at the outer CTRL 70% nuclear envelope. (A)(B)(C)(D)(E)(F) Frequency 40 distributions of microtubule velocities for microtubules 30 F M contacting the nuclear envelope of the female pronu- cleus prior to pronuclear migration.