Condensation of pericentrin proteins in human cells illuminates phase separation in 1 centrosome assembly 2 3

At the onset of mitosis, centrosomes expand the pericentriolar material (PCM) to maximize their microtubule-organizing activity. This step, termed centrosome maturation, ensures proper spindle organization and faithful chromosome segregation. However, as the centrosome expands, how PCM proteins are recruited and held together without membrane enclosure remains elusive. We found that endogenously expressed pericentrin (PCNT), a conserved PCM scaffold protein, condenses into dynamic granules during late G2/early mitosis before incorporating into mitotic centrosomes. Furthermore, the N-terminal portion of PCNT-enriched with conserved coiled-coils (CCs) and low-complexity regions (LCRs)-phase separates into dynamic condensates that selectively recruit PCM proteins and nucleate microtubules in cells. We propose that CCs and LCRs, two prevalent sequence features in the centrosomal proteome, are preserved under evolutionary pressure in part to mediate liquid-liquid phase separation, a process that bestows upon the centrosome distinct properties critical for its assembly and functions.


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The centrosome serves as a major microtubule-organizing center (MTOC) in animal cells. It     Peng et al., 2005) 156 predicted that human PCNT is largely disordered except for the C-terminal PACT motif ( Figure  9 (Atkins et al., 2015), not all disorder predictors are in complete agreement, with each predictor 159 suggesting different degrees of disorder/order tendency [e.g., IUpred (Dosztanyi et al., 2005) 160 predicted an overall more ordered structure than PONDR did].

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Nonetheless, by comparing the conservation profile of the multi-species alignment and the 162 locations of the predicted features of human PCNT, we found that the coiled-coils and LCRs in 163 human PCNT are concentrated within the conserved orthologous regions (Figure 2A).

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Statistical analyses further demonstrated that coiled-coils and LCRs are significantly more 165 conserved than non-coiled-coils and non-LCRs, respectively, in human PCNT ( Figure 2B).

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Together, these results suggest that coiled-coils and LCRs of pericentrin orthologs are likely 167 under natural selection to preserve their molecular functions.

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To test this hypothesis, control PCNT transcription tightly, and map phase separation 178 determinants, we divided PCNT into N-and C-terminal segments, tagged each with sfGFP, and

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To further map the phase separation driver sequences, we tested GFP-tagged PCNT (2- condensates can also recruit PCM components, including structural polypeptides and "client" 232 proteins, which are also recruited to mitotic PCM (e.g., dynein and PLK1). We found that 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 The copyright holder for this preprint (which this version posted May 10, 2020. ; https://doi.org/10.1101/2020.05.08.084749 doi: bioRxiv preprint 13 assay (Jao et al., Sanders et al., 2017). In this assay, we treated cells with nocodazole to 253 depolymerize MTs, washed out the drug, and monitored renucleation of MTs by anti-α-tubulin 254 immunostaining. We found that MTs were renucleated not only from the centrosome as 255 expected, but also from the interior and surface of PCNT condensates (Figure 5A, arrows).

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The condensates also recruited endogenous PCNT as observed before. Some small PCNT 257 condensates also recruited endogenous PCNT and nucleated MTs (Figure 5B

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Our work shows that endogenously expressed PCNT, a core PCM protein important for 268 centrosome maturation, forms liquid-like granules before incorporating into mitotic centrosomes 269 in human cells. These PCNT granules are likely formed through liquid-liquid phase separation 270 because (1) they are sensitive to the acute hexanediol treatment that is known to disrupt phase-

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This process could effectively bring multiple N-terminal, phase separation-driving PCNT 288 polypeptides in close proximity. A proximity-driven phase separation can thus be envisioned 289 since phase separation is a concentration-dependent process. This also explains why these 290 . CC-BY-NC-ND 4.0 International license available under a 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 The copyright holder for this preprint (which this version posted May 10, 2020.

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. CC-BY-NC-ND 4.0 International license available under a 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 The copyright holder for this preprint (which this version posted May 10, 2020. ; https://doi.org/10.1101/2020.05.08.084749 doi: bioRxiv preprint 16 Is there a liquid-to-gel/solid-like transition in PCM assembly?

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PCNT (854-1960) condensates transition from liquid-to gel/solid-like state, resulting in a 316 scaffold with an inhomogeneous, porous appearance (Figures 3 and 4). Morphologically, this 317 pattern resembles that of salt-stripped mitotic centrosomes purified from flies and clams in early

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This region is particularly enriched with coiled-coils and LCRs (Figure 2A) and appears to 343 contain the sequence elements that drive phase separation. However, this region does not    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 The copyright holder for this preprint (which this version posted May 10, 2020.

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Importantly, coiled-coils and LCRs of human PCNT are enriched in the regions that are 379 evolutionarily conserved, suggesting that these sequence features are under natural selection to 380 preserve critical functions. We propose that PCNT is a linear multivalent protein that phase 381 separates through its conserved coiled-coils and LCRs to become spatially organized 382 condensates that scaffold PCM assembly. This process is likely initiated during its co-383 translational targeting to the centrosome when the nascent PCNT polypeptides are in close 384 proximity in the polysome (Figure 6). We propose that PCNT phase separation achieves two 385 main goals. First, it concentrates PCM components and clients as the PCNT condensates 386 selectively recruit them. This will facilitate and limit the biochemical reactions that eventually 387 19 take place at the centrosome (e.g., MT nucleation, kinase activities). Second, it enables a liquid-388 to-gel/solid-like transitioning process during centrosome assembly. This process provides the 389 PCM components a thermodynamically favored pathway to assemble into a micron-sized, 390 membraneless, and yet spatially organized PCM.

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Although coiled-coils mediate other phase separation-independent activities, it is tempting to 392 speculate that coiled-coil-mediated phase separation might be widespread among other coiled-

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Cell lines used in this study were not further authenticated after obtaining from the sources.

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All cell lines were tested negative for mycoplasma using a PCR-based testing with the Universal 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 The copyright holder for this preprint (which this version posted May 10, 2020. ; https://doi.org/10.1101/2020.05.08.084749 doi: bioRxiv preprint 26 isolate the knock-in clones, we first generated a TP53 knockout RPE-1 cell line before the 464 knock-in experiment (Figure 1-figure supplement 3). Knocking

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To generate lentiviruses expressing mScarlet-i-H2A and miRFP670-CETN2, the same 520 procedure was performed as above except for using the targeting vectors pLVX-EF1α-mScarlet-

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Deconvolution was performed using the Fusion software (Andor Technology).

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For live imaging, cells were seeded in 35-mm glass-bottom dishes 12-18 h prior to imaging 575 and were imaged in a humidified environmental chamber with 5% CO2 at 37°C.

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A FRAP photoablation module with a computer-controlled fiber optically pumped dye laser 582 was used to bleach a region-of-interest (ROI) on the condensate (~2 µm in diameter) after a few 583 pre-bleach images were acquired. After photobleaching, the same ROI continued to be imaged 584 at 2-s (young condensates) or 5-s (old condensates) intervals for 5 to 12 min. Images were 585 acquired using a Zeiss AxioObserver with a 60x objective coupled with a Yokogawa CSU-10 586 spinning disk confocal system and a Photometrics CoolSNAP HQ2 cooled CCD camera

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Imaging of different cell lines was performed with the same acquisition setting and condition (5% 596 CO2 at 37°C) using a spinning disk confocal microscope system (Dragonfly, Andor Technology).

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. CC-BY-NC-ND 4.0 International license available under a 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 The copyright holder for this preprint (which this version posted May 10, 2020.                      Data are mean ± 95% CI. n, number of condensates/scaffolds analyzed from >3 biological replicates. The percent recovery and half-life (t 1/2 ) after photobleaching were calculated after fitting the data with non-linear regression. (E) Quantification of relative protein concentrations (intensity sum per volume) in live cells expressing various GFP-tagged PCNT segments after Dox induction. Critical concentrations are mean ± 95% CI. n, number of cells analyzed from three biological replicates per segment. The p-values were determined by two-tailed Student's t-test. a.u., arbitrary unit. Scale bars, 5 µm.
. CC-BY-NC-ND 4.0 International license available under a 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 The copyright holder for this preprint (which this version posted May 10, 2020.  Figure 2A. . CC-BY-NC-ND 4.0 International license available under a 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 The copyright holder for this preprint (which this version posted May 10, 2020.   Data are mean ± CI. n, number of condensates analyzed from three biological replicates. The percent recovery and half-life (t 1/2 ) after photobleaching were calculated after fitting the data with non-linear regression. Note that the highly mobile nature of young condensates prevented us from tracking the same condensates consistently beyond 5 min in the recovery phase of the FRAP assay. Scale bars, 2 µm (A), 10 µm (B), 1 µm (C, young condensates), and 2 µm (C, old condensates).
. CC-BY-NC-ND 4.0 International license available under a 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 The copyright holder for this preprint (which this version posted May 10, 2020. . CC-BY-NC-ND 4.0 International license available under a 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 The copyright holder for this preprint (which this version posted May 10, 2020. ; https://doi.org/10.1101/2020.05.08.084749 doi: bioRxiv preprint   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 The copyright holder for this preprint (which this version posted May 10, 2020. ; https://doi.org/10.1101/2020.05.08.084749 doi: bioRxiv preprint  (A) Anti-γ-tubulin immunofluorescence of GFP-PCNT (854-1960) and GFP-HOTag3/6 condensates. Right: The line plots of the selected regions (cyan lines). Asterisks denote the centrosomes. (B and C) Fold enrichment of fluorescence signals in the PCNT or HOTag condensates relative to those in the cytoplasm was quantified. Data are mean ± 95% CI. p-values were determined by two-tailed Student's t-test. n.s., not significant. n, number of cells analyzed from two, one, and two biological replicates for γ-tubulin, PCNT, and CEP192, respectively.
. CC-BY-NC-ND 4.0 International license available under a 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 The copyright holder for this preprint (which this version posted May 10, 2020. Note that MTs were renucleated within and on the surface of the condensate (arrows). A dot in the merged channels denotes the centrosome, where MT renucleation was robust. (B) MT renucleation also occurred in small PCNT condensates (asterisks), which also recruited endogenous PCNT. (C) Quantification of MT density in GFP-PCNT (854-1960) condensates (Con) and in the surrounding cytoplasm (Cyt) during MT renucleation. Data are mean ± 95% CI. Fold enrichment mean value is noted. n, number of condensates analyzed from three biological replicates. The p-value was determined by two-tailed Student's t-test. a.u., arbitrary unit. Scale bars, 10 µm and 2 µm (inset).
. CC-BY-NC-ND 4.0 International license available under a 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 The copyright holder for this preprint (which this version posted May 10, 2020. ; https://doi.org/10.1101/2020.05.08.084749 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license available under a 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 The copyright holder for this preprint (which this version posted May 10, 2020.  Figure 6. Model for PCNT phase separation in centrosome assembly. PCNT is a linear multivalent protein that phase separates through its conserved coiled-coils and LCRs during its co-translational targeting to the centrosome when the nascent PCNT polypeptides are in close proximity in the polysome. The resulting PCNT granules/condensates promote PCM assembly by (1) selectively concentrating PCM components and clients; this will facilitate and limit the biochemical reactions that eventually take place at the centrosome (e.g., MT nucleation, kinase activities) and (2) enabling a liquid-to-gel/solid-like transitioning process during centrosome assembly; this process provides the PCM components a thermodynamically favored pathway to assemble into a micron-sized, membraneless, and yet spatially organized PCM.
. CC-BY-NC-ND 4.0 International license available under a 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 The copyright holder for this preprint (which this version posted May 10, 2020. ; https://doi.org/10.1101/2020.05.08.084749 doi: bioRxiv preprint