Par complex cluster formation mediated by phase separation

Liu, Ziheng; Yang, Ying; Gu, Aihong; Xu, Jiawen; Mao, Ying; Lu, Haojie; Hu, Weiguo; Lei, Qun-Ying; Li, Zhouhua; Zhang, Mingjie; Cai, Yu; Wen, Wenyu

doi:10.1038/s41467-020-16135-6

Download PDF

Article
Open access
Published: 08 May 2020

Par complex cluster formation mediated by phase separation

Nature Communications volume 11, Article number: 2266 (2020) Cite this article

12k Accesses
164 Citations
13 Altmetric
Metrics details

Subjects

Abstract

The evolutionarily conserved Par3/Par6/aPKC complex regulates the polarity establishment of diverse cell types and distinct polarity-driven functions. However, how the Par complex is concentrated beneath the membrane to initiate cell polarization remains unclear. Here we show that the Par complex exhibits cell cycle-dependent condensation in Drosophila neuroblasts, driven by liquid–liquid phase separation. The open conformation of Par3 undergoes autonomous phase separation likely due to its NTD-mediated oligomerization. Par6, via C-terminal tail binding to Par3 PDZ3, can be enriched to Par3 condensates and in return dramatically promote Par3 phase separation. aPKC can also be concentrated to the Par3N/Par6 condensates as a client. Interestingly, activated aPKC can disperse the Par3/Par6 condensates via phosphorylation of Par3. Perturbations of Par3/Par6 phase separation impair the establishment of apical–basal polarity during neuroblast asymmetric divisions and lead to defective lineage development. We propose that phase separation may be a common mechanism for localized cortical condensation of cell polarity complexes.

Transient loss of Polycomb components induces an epigenetic cancer fate

Article Open access 24 April 2024

Giant organelle vesicles to uncover intracellular membrane mechanics and plasticity

Article Open access 04 May 2024

Extensive protein pyrophosphorylation revealed in human cell lines

Article Open access 25 April 2024

Introduction

Cell polarity, the morphological and molecular asymmetries of cells, is essential for diverse processes in metazoan cells^1,2. A hallmark of cell polarization is local concentration of specific protein complexes at restrictive membrane regions^3,4. Among them, the Par (partitioning defective) complex, the first set of genes identified in polarization of Caenorhabditis elegans embryos during development, are evolutionarily conserved master polarity determinants from worms to mammals^5,6. The Par complex plays indispensable roles in diverse polarity-related contexts, such as asymmetric cell division (ACD)^7,8,9, establishment of apical–basal polarity in epithelial cells³, oriented cell migration¹⁰, and neuronal polarization¹¹. Dysfunction of the Par complex leads to developmental defects, tumorigenesis, and even lethality of animals¹².

The Par complex proteins, including Par3 (Bazooka, Baz in Drosophila), Par6, and atypical protein kinase (aPKC), are multi-domain proteins capable of binding to each other and a diverse range of other cell polarity-regulating proteins¹³. Par3 has three PDZ (PSD-95, DLG, and ZO-1) domains that mediate protein–protein interactions¹⁴. How Par3 interacts with Par6 remains controversial. Mammalian Par3 was reported to interact with Par6 through its PDZ domains^15,16, though it is unclear through which PDZ domain Par3 binds to Par6. A recent study showed that both PDZ1 and PDZ3 of Baz weakly bind to a PDZ-binding motif (PBM) of Drosophila Par6 (with a dissociation constant > 50 µM)¹⁷. aPKC, which forms a stable subcomplex with Par6 through their PB1 domains¹⁸, binds to Par3 conserved region 3 (CR3) through its kinase domain, and this inhibitory interaction keeps aPKC in a stable Par complex for the establishment of cell polarity¹⁹. Activation of aPKC through other regulators (e.g., Aurora-A and Cdc42) leads to the phosphorylation of Par3 CR3 and its subsequent dissociation from Par6/aPKC (ref. ²⁰). These specific interactions ensure the spatiotemporal localization of the Par proteins at restricted membrane domains to orchestrate cell polarization in different developmental stages and different tissues.

In the past decades, the basic principles of the Par complex assembly and its functions in cell polarity in diverse cell types have been reasonably well established^2,3,4,8. However, how are the Par proteins themselves recruited and highly concentrated at very restricted membrane domains to set up the polarity remains unclear. Taking the ACD process of Drosophila neuroblasts (NBs) as an example, at the onset of mitosis, the uniformly distributed Baz/Par6/aPKC proteins are gradually concentrated and form a crescent on the apical cortex, whereas cell fate determinants and their adaptor proteins, including the Numb/Pon (Partner of Numb) complex and the Prospero/Miranda (Mira) complex, form crescents on the basal cortex, thus establishing the apical–basal polarity^{21,22,23,24,25,26,27}. During cell polarization in C. elegans zygotes, a similar Par crescent is observed on the anterior cortex^5,7. Recent studies on Drosophila epithelia development and C. elegans embryonic polarization demonstrated that such enriched Par crescent is actually an assembly of numerous micrometer-sized Par clusters, and formation of Par clusters requires the oligomerization of Par3 through its N-terminal domain (NTD)^{28,29,30,31,32}, which self-associates to form helical filaments³³. However, there is still a significant gap in understanding how the Par3 filaments in vitro establish the dynamic Par clusters in vivo that are capable to fuse with each other into larger ones^30,34. Interestingly, the Par proteins in the cortical clusters and other polarity complexes in the crescents are highly dynamic, and can rapidly exchange with the proteins in cytoplasm^{30,31,32,35,36,37,38}. It is not clear how these inner membrane-attached polarity complexes maintain highly localized concentration in context of the sharp concentration gradients between cell cortex and cytoplasm.

In this work, we uncover that endogenous Par proteins form discrete puncta-shaped condensates during the establishment of apical–basal polarity in Drosophila NBs. Mammalian Par3 PDZ3 specifically recognizes Par6 PBM, and the Par3/Par6 interaction can be significantly enhanced by Par3 NTD and Par6 PB1 through their oligomerization. Such multivalent interaction between Par3 and Par6 leads to the formation of self-organized, highly condensed, and dynamic droplets/puncta through liquid–liquid phase separation (LLPS) both in vitro and in vivo. Mutations that impair the LLPS of the Par complex led to defective assembly of the apical Par complex crescent during Drosophila NB asymmetric divisions, and consequently resulted in ACD defects and defective cell lineage. Thus, the local condensation of the Par complex during cell polarization is driven by LLPS mediated by their multivalent interactions.

Results

Par proteins exhibit cell cycle-dependent clustering in NBs

During the ACD of Drosophila NBs, Baz, Par6, and aPKC are found to form a co-localized, condensed crescent on the apical membrane at metaphase^25,26,27. However, careful analyses of microscopy images of dividing NBs showed that endogenous Baz, Par6, and aPKC displayed cell cycle-dependent puncta formation on the apical cortex (Fig. 1), just like their C. elegans counterparts during embryonic polarization^30,31. From prophase, Par proteins emerged as scattered puncta on the apical cortex, and these puncta concentrated to form a condensed crescent (from the apical–basal view) on the apical membrane at metaphase. Interestingly, when viewed from the lateral apical direction, the dim Par puncta grew into larger and brighter ones from prophase to metaphase, assembling into a highly concentrated cluster around the apical pole. From anaphase, the highly condensed puncta cluster began to disassemble into scattered small puncta, and finally the Par protein signal was distributed on the whole cortex of the apical daughter cell (Fig. 1).

**Fig. 1: Endogenous Par proteins form condensed puncta in *Drosophila* larval NBs in a cell cycle-dependent manner.**

The condensed Par puncta cluster was sensitive to 1,6-hexanediol treatment, a molecule known to disturb hydrophobic interaction-induced phase separation assemblies both in vitro and in vivo^39,40. Larval brain treated with 1,6-hexanediol, exhibited defective localization of these endogenous apical Par components, as well as the basal cortex component Mira, in a concentration-dependent manner (Supplementary Fig. 1a–c). In these NBs, Par proteins and Mira were partially localized with certain cytoplasmic distribution when treated with 5% 1,6-hexanediol, and fully diffused in cytoplasm with 10% 1,6-hexanediol. Importantly, removal of 1,6-hexanediol restored the crescent formation of Baz and Par6, as well as aPKC and Mira in dividing NBs, indicating that the cell cycle-dependent apical condensation of the Par complex is a reversible process and is likely driven by phase separation.

Par6β promotes Par3 puncta formation in living cells

The fact that the membrane-attached, highly concentrated Par3/Par6/aPKC assemblies are in open contact and fast protein equilibrium with the cytoplasm^30,31,32,35 led us to explore whether Par proteins can spontaneously form condensates in the aqueous cytoplasm. We overexpressed mammalian Par3, Par6β, or PKCι (aPKC in mammals) individually, or co-expressed multiple Par components in COS7 cells. To our surprise, COS7 cells transfected with green fluorescent protein (GFP)- or mCherry-tagged mammalian Par3, Par6β, or PKCι alone showed diffused cytoplasmic localization of these proteins (Fig. 2a). So did those co-expressed with two components of the complex (Fig. 2b). It was suggested that Par3 adopts an auto-inhibited closed conformation, with its C-terminal conserved 4N12 region packing with its N-terminal part covering NTD and PDZ1–3, and thus inhibiting its microtubule-binding property⁴¹. Interestingly, when ectopically expressed in COS7 cells, in addition to fibrillary structures⁴¹, the Par3 4N12 deletion mutant (Par3 Δ4N12; Supplementary Table 1) also formed scattered dim puncta in some cells (Fig. 2a). A truncated form of Par3 containing NTD and PDZ1–3 (referred to as Par3N) showed a similar dim puncta distribution as Par3 Δ4N12 did (Figs. 2a and 3a), indicating that domains within Par3N possess the ability to spontaneously form concentrated puncta in the cytoplasm.

**Fig. 2: Par proteins spontaneously form condensed and dynamic puncta in COS7 cells.**

**Fig. 3: Par6β promoted LLPS of Par3N in vitro.**

We further found that co-expression of Par6β together with Par3N dramatically promoted the puncta formation. GFP-Par3N and mCherry-Par6β spontaneously assembled into highly concentrated bright puncta in cytoplasm showing both GFP and mCherry signals (Fig. 2b). In sharp contrast, co-expression of PKCι with Par3N did not show such promotion effect. As no puncta was observed when co-expressing Par6β with full-length Par3 (Fig. 2b), we proposed that Par6β only promotes the puncta formation of Par3 in its open state. The Par3N and Par6β-enriched puncta were originally small and gradually fused into larger ones in a time-dependent manner (Fig. 2c, Supplementary Movie 1). Importantly, fuorescence recovery after photobleaching (FRAP) analysis revealed that both Par3N and Par6β in the condensed puncta rapidly exchanged with the proteins in the surrounding cytoplasm (~75% recovery with a halftime ~10 s; Fig. 2d–f, Supplementary Movies 2 and 3), reminiscent of the observations showing that Par proteins are in fast equilibrium between the anterior cortex and the cytoplasm during C.elegans embryonic polarization^30,31,32,35. This observation implies that the Par3N and Par6β-enriched puncta formed in cytoplasm may possess some of the properties of Par condensates beneath the membrane in polarized cells.

Phase separation of Par3N with Par6β in vitro

Next, we asked whether Par proteins undergo phase separation in vitro. Due to the poor protein behavior of Par3 Δ4N12, we chose Par3N for the LLPS assay. When assessing iFluor488-labeled Par3N with or without Par6β under fluorescence microscope, we observed numerous small spherical droplets with various diameters, a phenomenon characteristic of LLPS (Fig. 3a). The phase separation level of Par3N or Par3N/Par6β proteins was correlated to its or their concentration(s). When the protein concentration increased, the amount and the size of liquid droplets increased, and the Par3N/Par6β complex formed droplets with much larger amount and size than Par3N itself at the same protein concentration (Fig. 3a, b). The distributions of Par3N or Par3N/Par6β complex between the aqueous phase and the condensed droplets were quantified by a sedimentation-based assay. The LLPS level of Par3N/Par6β complex was constantly higher than that of Par3N alone at the same concentration before reaching saturation, with obvious LLPS at very low concentration in vitro (~0.5 µM; Fig. 3c, d), implying an important role of Par6β in promoting LLPS of Par3N.

Obvious fusion of the iFluor488-Par3N droplets could be observed under differential interference contrast (DIC) microscope at a protein concentration of 25 µM (Fig. 3e, Supplementary Movie 4). When mixed with Cy3-tagged Par6β at 1:1 molar ratio, every droplet was enriched with both Par6β and Par3N (Fig. 3f, Supplementary Movie 5). In sharp contrast, isolated solutions of Par6β remained clear at the same concentration (Supplementary Fig. 2a). Par3N either in the isolated or Par6β-bound droplets exchanged rapidly between the condensed phase and the surrounding aqueous solution (Fig. 3g, Supplementary Movies 6 and 7). Those Par3N/Par6β droplets transitioned into gel-like structures over time, and the droplets rarely fused together after 20 min (Supplementary Fig. 2b).

Par3 PDZ3 recognizes Par6β PBM

Next, we wanted to find out how Par6β promotes Par3N phase separation. Cell lysate glutathione S-transferase (GST) pull-down assay confirmed the interaction between Par6β and Par3N (Fig. 4a–c). Par6β selectively bound to Par3N PDZ3 though GST-Par3N pulled down more Par6β than GST-Par3 PDZ3 did (Fig. 4b), likely due to the oligomerization of NTD (ref. ³³), as it has been shown that Par6 preferentially binds to Par3 oligomers but not monomers³². Besides, Par3N selectively interacted with Par6β PBM (Fig. 4c). We confirmed the interaction between Par3 PDZ3 and Par6β PBM with a dissociation constant of ~1 µM, which was ~50-fold stronger than that in Drosophila¹⁷, whereas mammalian Par3 PDZ1 hardly bound to Par6β PBM (Fig. 4d).

**Fig. 4: Par3 recognizes Par6 via the interaction between Par3 PDZ3 and Par6 PBM.**

The crystal structure of Par3 PDZ3 in complex with a Par6β PBM peptide (Fig. 4a) revealed that the last three residues of Par6β PBM (⁻²ITL⁰) binds to the αB–βB groove of PDZ3 using a classical PDZ binding mode (Fig. 4e, f). The carboxyl terminal of L(0) from Par6β PBM forms extensive hydrogen bonds with the canonical “G⁶⁰⁰LG⁶⁰²F” loop of Par3 PDZ3. The sidechain of I(−2) from Par6β PBM inserts into a shallow hydrophobic pocket formed by the αB–βB groove of Par3 PDZ3. In line with the structure, the G600,602 A mutation of Par3 PDZ3 dramatically weakened the PDZ3–PBM interaction ~20-fold (Fig. 4d). We further found that addition of 1,6-hexanediol impaired the hydrophobic interaction between Par3 PDZ3 and Par6β PBM (Supplementary Fig. 2c). In line with this result, addition of 1,6-hexanediol led to the dispersion of the preformed Par3N/Par6β droplets (Supplementary Fig. 2d, e, Supplementary Movie 8), reminiscent the finding that it disturbed the apical localization of Par complex in dividing Drosophila NBs (Supplementary Fig. 1).

It is noted that Par6β Crib-PDZ slightly enhanced the binding between PBM and Par3N, and addition of PB1 dramatically strengthened the interaction (Fig. 4c). We observed that the recombinant Par6 PB1 was prone to aggregation, with a small elution volume in the size-exclusion chromatography (SEC) assay (Supplementary Fig. 3a). Different from Par3 NTD whose aggregation was sensitive to salt concentration³³, high salt concentration up to 2 M NaCl had negligible impact on Par6 PB1 oligomerization (Supplementary Fig. 3b). In line with the SEC result, the full-length Par6β could self-associate through its PB1 domain (Supplementary Fig. 3c, d).

Overall, Par3N specifically binds to Par6β via the PDZ3–PBM recognition, and both Par3 NTD and Par6β PB1 enhance the interaction likely through oligomerization-induced avidity increase.

Multivalency-dependent LLPS of Par3N with Par6β

We then investigated the specific role of the direct Par3N–Par6β interaction or oligomerization of each protein in promoting Par3N/Par6β phase separation, at a concentration relatively low (~3 µM) but with nearly saturating LLPS capacity (Fig. 3d). Consistent with the above biochemical data (Fig. 4), deletion of PDZ3 (∆PDZ3) but not PDZ1 (∆PDZ1) from Par3N effectively impaired LLPS of the Par3N/Par6β complex (Fig. 5a). Removing NTD (∆NTD) from Par3N significantly reduced phase separation efficiency of the Par3N/Par6β complex, as well as that of Par3N alone, demonstrating the important role of Par3 NTD self-association in increasing valency of Par3N for higher order architecture assembly. The LLPS of Par3N/Par6β was sensitive to salt concentration (Supplementary Fig. 3e), as the oligomerization of Par3 NTD is charge–charge interaction dependent³³.

**Fig. 5: Multivalent and specific protein–protein interactions drive LLPS of Par3N/Par6β complex.**

On the other hand, deletion of PBM (∆PBM) not only impaired LLPS of Par6β, but also reduced its promotion effect on LLPS of Par3N (Fig. 5b). Deletion of PB1 (∆PB1) from Par6β also dramatically decreased the condense phase of Par6β (Fig. 5b). Moreover, when we replaced PB1 of Par6β with another self-associating domain p62 PB1 (referred to as Par6β chimera)⁴², the phase separation efficiency of the Par3N/Par6β chimera can be restored to the similar extent as that of the Par3N/Par6β complex (Supplementary Fig. 3f), demonstrating the critical role of Par6β PB1 oligomerization in promoting LLPS. Due to the role of Par6β Crib-PDZ in increasing Par6β–Par3N interaction (Fig. 3c), deletion of Par6β Crib-PDZ (∆Crib-PDZ) slightly reduced LLPS efficiency of the Par3N/Par6β complex (Fig. 5b).

Together, Par3N undergoes weak phase separation in vitro, at least partially dependent on its NTD-mediated oligomerization. Par6β promotes Par3N LLPS, relying on the specific interaction between Par3 PDZ3 and Par6β PBM, as well as the high valency of both proteins.

Par puncta in living cells are phase separated droplets

Next, we wanted to test whether the multivalency-dependent LLPS of the Par3N/Par6β complex in solution accounts for the puncta formation in living cells (Fig. 2). Nicely correlated with in vitro phase separation results (Fig. 5a), deletion of Par3 PDZ1, PDZ2, or even PDZ12 had minor effects on the puncta formation of the co-expressed Par3N and Par6β, whereas deletion of the Par6β-binding Par3 PDZ3 led to a dramatic decrease of the puncta number (Fig. 5c, Supplementary Fig. 4a). When introducing a V13D, D70K mutation in Par3 NTD (referred to as NTDmu), which disrupts its oligomerization³³, only very few puncta could be detected. Similarly, fewer puncta were observed when Par3N was co-expressed with Par6β mutants with PB1 or Crib-PDZ deletions, and deletion of Par6β PBM eliminated the puncta formation (Fig. 5d, Supplementary Fig. 4b).

There are three isoforms of Par6 (Par6α, Par6β, and Par6γ) in mammals, which share ~60% sequence similarity (Fig. 4a) and exert distinct functions in cell polarization, e.g., tight junction formation⁴³. As a key driving factor for the specific interaction and LLPS of Par3N and Par6β, Par6β PBM is completely conserved in Par6γ but varies in Par6α (Fig. 4a). As expected, Par6γ PBM but not Par6α PBM bound to Par3 PDZ3 equally well as that of Par6β PBM (Fig. 4d). Many bright puncta were observed in the cytoplasm when co-expressing Par3N with Par6γ, and deletion of Par6γ PBM eliminated the puncta formation (Fig. 5d, Supplementary Fig. 4b). In sharp contrast, no puncta were observed in Par3N and Par6α co-expressed cells. We then constructed a Par6α chimera mutant, with its PBM replaced by the Par6β PBM, and the puncta formation property was gained in cells co-expressed with Par3N and the Par6α chimera (Fig. 5d, Supplementary Fig. 4b), further demonstrating the critical role of PBM–PDZ3 recognition in promoting puncta formation of Par3N in living cells. As LLPS of Par3N/Par6β was concentration dependent (Fig. 3), the expression level of each protein in above puncta assay was analyzed and adjusted to make sure that the imaging-based puncta formation assay of different constructs are directly comparable (Supplementary Fig. 4c, d).

The essential role of Par3 in promoting Par complex clustering and eventually the establishment of cell polarity is thought to be mediated by its NTD (refs. ^{28,29,30,31,32}). Par3 NTD can self-associate to form helical filaments in vitro³³, which may act as a central organizer for Par complex clustering in vivo³⁴. An intriguing question is, does NTD-mediated Par clustering rely on its self-association nature, or the oligomerization-dependent LLPS behavior? To answer this question, we replaced Par3 NTD with different fragments of the unstructured low-complexity domain (LCD) of fused in sarcoma (FUS), the driving factor for FUS LLPS (refs. ^44,45). The LLPS level of the longer fragment FUS_L (aa 1–214) is much higher than that of the shorter FUS_S (aa 1–141; Fig. 5e). Accordingly, FUS_S-Par3N chimera partially restored but FUS_L-Par3N chimera essentially restored the puncta formation property of Par3N when co-expressed with Par6β (Fig. 5f, g), indicating that Par3-mediated Par complex clustering is most likely driven by its LLPS.

PKCι activity may regulate LLPS of the Par complex

As it has been suggested that Par6 and aPKC form constitutive heterodimers^18,31, we next wondered whether aPKC, the known kinase in the Par complex, might participate in the phase separation of Par3 and Par6β. Under fluorescence microscopy, iFluor405-labeled PKCι PB1 was well co-localized with the condensed Par3N (without PKCι phosphorylation site)/Par6β droplets (Fig. 6a). Sedimentation assay further showed that though PKCι PB1 could be recruited into the Par3N/Par6β liquid phase, it did not enhance or decrease the LLPS extent of Par3N/Par6β proteins (Fig. 6b). Similarly, the full-length PKCι was enriched and co-localized within the Par3N/Par6β puncta in COS7 cells (Supplementary Fig. 5a), and co-expression of PKCι had negligible impact on Par3N/Par6β puncta formation (Fig. 6c), suggesting that PKCι is likely recruited into the Par3N/Par6β condensed phase as a client.

**Fig. 6: PKCι activity may regulate the LLPS of Par3N/Par6β.**

aPKC can phosphorylate Par3 in its CR3 region (Fig. 4a), leading to the release of Par3 from the Par6/aPKC complex^19,46. To probe the potential impact of aPKC kinase activity on Par3/Par6β LLPS, we co-expressed mCherry-Par6β with GFP-Par3 Δ4N12 (the open form of Par3 containing the aPKC phosphorylation sites) wild-type protein (WT) or the aPKC-mediated phospho-mimetic S827,829E mutant in COS7 cells. Fewer puncta were observed when Par6β was co-expressed with Par3 Δ4N12 S827,829E when compared with Par3 Δ4N12 (Fig. 6d, Supplementary Fig. 5b), indicating that PKCι-mediated Par3 phosphorylation at CR3 somehow interfered the Par3 Δ4N12/Par6β puncta formation. Then, we co-expressed GFP-Par3 Δ4N12 with Flag-Par6β and mCherry-PKCι WT, and various mutants in COS7 cells. To our surprise, PKCι WT, the kinase-dead K273R mutant⁴⁷, and the proposed constitutively active A120E mutant⁴⁸ were all enriched and co-localized within the Par3 Δ4N12/Par6β puncta (Fig. 6e, Supplementary Fig. 5c). Expression levels of each protein in above puncta assay were confirmed to be comparable (Supplementary Fig. 5d).

It has been proposed that association with Par3 and Par6 keeps aPKC in the inactive quiescent state in the Par complex^19,31, and this inhibitory interaction can be disrupted by other regulatory factors in a spatiotemporal manner^20,31. We then asked whether the recruitment of aPKC into Par3/Par6 condensates might keep aPKC in the inactive state. We conducted a sedimentation assay of Par3 1–854 (containing the aPKC phosphorylation sites) and Par6β with or without incubation of Flag-PKCι purified from HEK293 cells. Both PKCι WT and A120E efficiently phosphorylated Par3 1–854 (indicated by the upshifted phos-tag band) in the supernatant fraction, but not in the Par3/Par6β/PKCι condensates (Fig. 6f), indicating that though PKCι could be recruited into the Par3/Par6β condensates, its activity is suppressed.

In summary, aPKC can be recruited and concentrated into the Par3/Par6β condensates as an inactive client. However, activation of aPKC (e.g., cell cycle-dependent activation) will convert the kinase into an active Par3/Par6β condensate disperser likely via aPKC-mediated phosphorylation on Par3.

LLPS of Par proteins regulates their condensation in vivo

Both Par3 and Par6 have evolved into multiple isoforms in mammals with redundant functions. Drosophila contains only one copy of par3 (bazooka, baz) and par6 genes, making the organism desirable for functional studies on Par3/Par6 condensation. Importantly, Par6β PBM is highly conserved in Drosophila, e.g., the critical L(−2) of fly Par6 is a small hydrophobic residue, just like I(−2) in Par6β and Par6γ (Fig. 3a), though the binding between Baz PDZ3 and Par6 PBM is relatively weak in flies¹⁷. Thus, we employed type I NBs in the central brain region of Drosophila larvae as an in vivo model. We generated transgenic flies expressing the full-length Flag-tagged WT or LLPS-deficient mutant forms of Baz or Par6, respectively. Expressions of these fragments were further verified (Supplementary Fig. 6).

In a WT background under our experimental condition (see Methods section), Flag-Baz WT (n = 11), Flag-Par6 WT (n = 20), and Flag-Baz ∆PDZ2 (n = 19) that had high LLPS ability in vitro formed a crescent on the apical cortex in all or the majority of metaphase NBs (Fig. 7a–d). In contrast, the Par6-binding-deficient Flag-Baz PDZ3 G634,636 A (referred to as PDZ3mu, corresponding to rat Par3N PDZ3 G600,602 A; n = 17), and the oligomerization-deficient Flag-Baz NTD L13D,D68K (referred to as NTDmu, corresponding to rat Par3 NTD V13D,D70K; n = 23) and Flag-Par6 ∆PB1 (n = 19) lost their normal apical condensation in the majority of NBs examined. The localization of endogenous apical proteins Par6 and aPKC, and basal proteins Mira and Numb was also disrupted in these WT NBs expressing Flag-Baz NTDmu or Flag-Baz PDZ3mu (Supplementary Fig. 7a, d), suggesting a dominant-negative effect of both mutants. In contrast, though Flag-Par6 ∆PB1 diffused in cytoplasm, the localization of endogenous apical Baz and aPKC, and basal Mira and Numb seemed normal (Fig. 7b, Supplementary Fig. 7b, d).

**Fig. 7: LLPS of Baz/Par6 complex is required for their apical condensation during ACD of *Drosophila* larval NBs.**

To exclude the influence of endogenous proteins on these ectopically expressed variants, we addressed their localization in baz or par6 mutant NBs. As reported previously^26,27, endogenous Baz (n = 20) was apically localized on the cortex; Flag-Baz WT (n = 5), and Flag-Baz ΔPDZ2 (n = 5) with high LLPS ability also formed apical crescent in the majority of baz mutant NBs (Fig. 7e, f). However, majority baz mutant NBs expressing LLPS-deficient Flag-Baz NTDmu (n = 5) and PDZ3mu (n = 8) exhibited diffused cortex and cytoplasm localization (Fig. 7e, f). Similarly, Par6 was correctly localized on the apical cortex either in WT NBs (n = 20) or in par6 mutant NBs expressing Flag-Par6 WT (n = 5; Fig. 7g, h). However, Flag-Par6 ΔPB1 diffused in the whole cytoplasm in the majority of par6 mutant NBs (Fig. 7g, h; n = 9).

To further understand the function of these Flag-Baz and Flag-Par6 variants, we investigated whether they could rescue baz or par6 mutant phenotype. In the WT central brain, each type I NB lineage (marked by GFP) contains an average of ~73 cells, including NB, GMCs, and neurons (n = 20); however, baz NBs produced a much smaller lineage containing ~23 cells (n = 20; Fig. 8a, b). While restoring Flag-Baz WT or Flag-Baz ΔPDZ2 expression in baz mutant NBs largely or significantly restored the lineage defect, expression of LLPS-deficient Flag-Baz NTDmu and Flag-Baz PDZ3mu rescued the NB lineage defects to a lesser extent (Fig. 8a, b). As a negative control, ectopic expression of Flag-Par6 in baz NBs had no influence on the lineage defects. Similarly, a smaller lineage was generated in par6 mutant NBs, while restoring Flag-Par6 WT but not Flag-Par6 ΔPB1 expression in par6 mutant NBs could significantly rescue the defects (Fig. 8c, d). Ectopic expression of Flag-Baz was also included as a negative control.

**Fig. 8: LLPS of Baz/Par6 is critical for neuronal differentiation in *Drosophila* NBs.**

The rescuing efficiency of Baz or Par6 apical localization and NB defective lineage phenotype in baz or par6 mutant NBs by various Flag-Baz or Par6 variants was nicely correlated with their abilities to induce LLPS in heterologous cells and in vitro, demonstrating that the multivalent Baz-Par6 interaction-induced LLPS may be essential for the efficient Baz and Par6 enrichment in the apical crescent, and the correct ACD process in NBs.

The nature of the Par complex clustering is LLPS

Above biochemical, cellular, and genetic data had established a connection between in vitro Par proteins LLPS and in vivo Par complex local condensation; however, one thing hard to ignore is that, both the puncta formation assay in COS7 cells and the rescue assay in fly NBs were overexpression systems (Figs. 2 and 7). As Par3/Par6β LLPS was concentration dependent, though we have showed that Par3N/Par6β undergo LLPS at micromolar concentration (Fig. 3), we don’t know the actual protein levels in dividing NBs. Through a fluorescent microscope imaging-based measurement method⁴⁹, we measured the average protein concentration of Par3N (when co-expressed with Par6β) in COS7 cells to be ~5 μM (vs ~40 μM in puncta; Supplementary Fig. 8a), which was high enough to induce Par3 LLPS in the cytoplasm (Fig. 2). To measure the endogenous concentration of Baz protein, we used GFP::baz protein trap line that carries a GFP reporter gene inserted in the intronic region of baz locus and has been widely used in assaying Baz function⁵⁰. The estimated average Baz level in fly NBs was only 0.14 μM (vs 1.32 μM in the crescent; Supplementary Fig. 8b), consistent with the fact that Baz did not undergo LLPS in the cytoplasm. Compared with the cytoplasmic fraction, we assumed that spatiotemporal attachment of the Par complex to the membrane led to its enrichment and subsequent LLPS in the apical domain of dividing NBs.

The NTD domain is known for Baz self-association to promote Par complex clustering. Thus, to further address functional significance of LLPS of Baz at physiological level without interfering other protein–protein or protein–membrane interactions, we generated GFP knock-in Drosophila strains expressing endogenous levels of GFP-tagged Baz ΔNTD, FUSs-Baz, or FUS_L-Baz chimera mutant (Baz NTD was replaced by FUS_S or FUS_L, respectively; Supplementary Table 1). In sharp contrast to the early UAS/GAL4-based rescue assay results showing that Baz NTDmu lost its apical condensation in both WT and baz mutant backgrounds (Fig. 7), the knock-in mutant Baz ΔNTD showed obvious apical condensation in all NBs (Fig. 9a, b). We assumed that the distinct phenotypes of these variants arose from the different levels of protein expressed in these different assays (overexpression for Baz NTDmu vs endogenous level for Baz ΔNTD, see Discussion section for more details). However, we noticed that the asymmetrically localized proteins, including apical Baz ΔNTD, Par6 and aPKC, and basal Mira showed less condensed localization and significant cytoplasmic diffusion in Baz ΔNTD knock-in NBs compared to Baz WT knock-in NBs (Fig. 9a, b), implying that Baz LLPS-mediated by NTD (note that NTD oligomerization is only one driving force for Baz LLPS) at physiological condition indeed facilitates the efficient Baz apical localization and thus regulated local condensation of other proteins. In line with these defects, although Baz ΔNTD knock-in was viable and able to establish a homozygous stock, the L3 larval brain was however significantly smaller than that of Baz WT larvae (Fig. 9c). For some unidentified reason, FUS_L-Baz knock-in strain was lethal and excluded from this study. FUSs-Baz knock-in strain was viable. Though the LLPS less efficient FUS_S-Par3N only partially restored the puncta formation property of Par3N when co-expressed with Par6β in COS7 cells (Fig. 5e–g), FUSs-Baz exhibited apical localization in dividing NBs, similar to that of Baz WT knock-in (Fig. 9a, b). Importantly, the FUSs-Baz NBs showed largely normal localization of both apical (Par6 and aPKC) and basal (Mira) proteins. L3 larval brain of FUSs-Baz larvae was also significantly larger than that of Baz ΔNTD knock-in (Fig. 9c, d). In addition, we confirmed that the FUSs-Baz mutant had a comparable FRAP recovery rate as Baz WT (Fig. 9e–g). Collectively, the above data strongly demonstrated that NTD-mediated Par clustering in dividing NBs relied on its oligomerization-dependent LLPS behavior.

**Fig. 9: NTD-mediated Par clustering relies on its oligomerization-dependent LLPS behavior.**

Discussion

How the conserved Par (Par3/Par6/aPKC) complex is selectively recruited and concentrated on membranes for polarity establishment remains unclear. In this study, different from previously reported crescent localization patterning^25,26,27, we reveal that the endogenous Par complex exhibits cell cycle-dependent discrete puncta formation on the apical cortex in Drosophila NBs. The condensed Par puncta emerge from prophase, further condensate and enlarge as a clustered puncta structure in metaphase, then subsequently disassemble into scattered small puncta from anaphase (Fig. 9h). The cell cycle-dependent clustering of Par proteins in Drosophila NBs were also observed by two recent studies^51,52. Our in vitro biochemical data together with heterologous cell-based studies showed that the Par3/Par6 complex can undergo LLPS at very low protein concentrations (Fig. 3a, d), and mutations of Par3 or Par6 that impair LLPS were found to alter ACD in Drosophila NBs. It has been recently shown that the basal condensation of Numb in dividing NBs is also regulated by LLPS of the Numb/Pon complex⁵³. Thus, LLPS may be a common mechanism for the local condensation of apical and basal polarity determining protein complexes.

It is important to note that the Par proteins, each at their endogenous level, can form clustered puncta via LLPS on the cortex (Fig. 1). Though the measured endogenous Baz level in Drosophila NBs was too low to induce its LLPS in the cytoplasm, two-dimensional membrane attachment was expected to locally enrich the protein and lead to its LLPS (Supplementary Fig. 8b). In return, LLPS-mediated Par complex condensates formation acts as an effective way for cells to further concentrate limited amount of Par proteins to specific cell cortices for polarity establishment. We propose that apical Baz/Par3 localization is a balanced result of apical anchoring and LLPS-mediated local condensation (via multivalent protein–protein interaction, self-association, protein–membrane interaction, etc.). Thus, for the knock-in mutant Baz ΔNTD, partially impaired LLPS ability due to its defective oligomerization led to its less condensed localization and significant cytoplasmic diffusion. However, the situation was different for the overexpressed Baz NTDmu (driven by UAS/GAL4) in the rescue assay, which is ectopically localized. As LLPS is very sensitive to concentrations of biological components, an overexpression of Par proteins especially Baz/Par3, the core driving factor of LLPS, may cause artificial promotion of the Par complex condensation via LLPS. Whereas the apical anchoring capacity of NBs seems to have a limitation. In UAS/GAL4-based rescue assay, the overexpressed Baz WT phase condensates may just have reached the threshold of apical anchoring capacity, whereas the LLPS deficient, overexpressed Baz NTDmu broke the balance, and led to its cortical and cytoplasmic diffusion. If the expression level goes higher, even Baz WT can not be afforded apically. Consistent with this notion, high Flag-Baz expression in a WT background (driven by insc-gal4), has a dominant-negative effect and leads to ectopic localization of endogenous Par complex throughout the cortex, and consequently disrupts localization of basal proteins (Supplementary Fig. 7c, d). Similarly, ectopic Baz localization was observed when exogenous Baz is forcedly expressed in embryonic NBs (refs. ^25,54). It was recently shown that overexpression of Par3-induced cell polarity in apolar S2 cells by forming concentrated Par-dots that further fused into amorphous Par-islands⁵¹. According to a study of protein LLPS on lipid membrane bilayers, protein clusters gradually grew and fused into larger ones with irregular shapes, and finally coalesced into a mesh-like network⁴⁹. Thus, the amorphous structure of Par-islands in S2 cells may arise from the overexpression and overaccumulation of Par3 in the membrane region. Therefore, caution should be taken in interpreting the overexpression phenotypes of Par3.

Another key finding in this study is that aPKC can be recruited and concentrated in Par3/Par6 condensates as an inactive client. Such condensed phase droplets could be an efficient mechanism for local condensation of aPKC (ref. ³¹). Spatiotemporal activation of aPKC (e.g., by Cdc42) and consequent phosphorylation on Par3 CR3 leads to disassembly of the Par complex condensates (Fig. 9h). Another cell cycle regulator that might play a role in Par LLPS regulation is Plk1, which inhibits the oligomerization of Par3 by phosphorylating NTD in C. elegans³². A critical but currently unknown point is how the autoinhibition of Par3 is relieved, as the open conformation of Par3 is critical for the Par complex condensate formation. Nonetheless, it is likely that multilayered regulatory mechanisms can act concertedly to control the spatiotemporal assembly and disassembly of the Par complex phase separation, and hence the cell polarity regulations.

It is increasingly recognized that LLPS is a common strategy for cells to form membrane-less compartments by selectively recruiting and condensing proteins/RNAs/lipids^{55,56,57,58,59}. In a broader sense, the local condensation of other master polarity complexes, such as the conserved Lgl/Dlg/Scribble complex in the apical–basal polarity, and the Prickle/Vangl and Frizzled/Disheveled/Diego complexes in the planar cell polarity, may adopt a similar LLPS-driven mechanism to establish cell polarity in different tissues. Like the Par complex proteins, all these complexes share several common features: (1) these proteins contain multiple domains, which mutually interact with each other or self-associate in vitro to form complex platform, which further recruits other binding partners to assemble into higher order protein interaction network; (2) these complexes are found to form condensed patches or puncta attached to the inner surface of plasma membranes in vivo⁶⁰; and (3) proteins within these condensed patches or puncta are highly dynamic and rapidly exchange with corresponding proteins in the cytoplasm. The multivalent interaction-induced LLPS theory can perfectly explain above phenomena^61,62, allowing the stable existence of large concentration gradients of the proteins within the local protein condensates and those in the cytoplasm, and at the same time, keeping the proteins in the condensed phase highly dynamic. Such dynamic association may be essential for the fast assembly/disassembly of these polarity complexes in responding to extrinsic/intrinsic cues/signals to rearrange the cell polarity. We postulate that LLPS of polarity protein complexes induced by multivalent interactions is a general mechanism for the cell polarization.

Methods

Protein expression and purification

Various rat Par3 fragments (Uniprot ID: Q9Z340, Supplementary Tables 1 and 2), mouse Par6β fragments (Uniprot ID: Q9JK83), and the mouse PKCι PB1 (Uniprot ID: Q62074, aa 16–99) were individually cloned into pGEX-6P-1 or a modified version of pET32a vector⁵³. All the mutations used in this study were generated using the standard PCR-based mutagenesis method and confirmed by DNA sequencing. Human FUS LCD (Uniprot ID: P35637, aa 1–214 or 1–141) or p62 PB1 (Uniprot ID: Q13501, aa 1–102) was fused to Par3N ΔNTD using the standard PCR-based mutagenesis method and confirmed by DNA sequencing. Recombinant proteins were expressed in Escherichia coli BL21 (DE3) or Rosetta host cells in LB medium at 16 °C, and purified using a Ni²⁺-NTA agarose affinity column followed by SEC (using HiLoad 26/600 superdex 75/200 pg columns on an AKTA Fast Protein Liquid Chromatography (FPLC) system, GE Healthcare) with buffer A containing 50 mM Tris (pH 8.0), 100 mM NaCl (300 mM NaCl for Par3N), 1 mM EDTA, and 1 mM dithiothreitol (DTT). Par6α PBM peptide (MRGDVSGFSL), Par6β PBM peptide (LEEDGTIITL), and Par6γ PBM peptide (VEEHGPAITL) were commercially synthesized (Phtdpeptides).

GST pull-down assay

GST or GST fusion proteins (4 nmol) was first loaded onto 30 μl GSH-Sepharose 4B slurry beads and then incubated with 12 nmol indicated proteins in 500 μl buffer A at 4 °C for 1 h. After being washed three times with the same buffer, the above proteins captured by affinity beads were eluted by boiling with SDS-loading buffer, resolved by 12% SDS–polyacrylamide gel electrophoresis (SDS–PAGE), and detected by Coomassie blue staining.

Cell lysate GST pull-down assay and immunoblotting

Human HEK293T cells (from ATCC) were cultured in Dulbecco’s modified Eagle medium (Hyclone) containing 10% fetal bovine serum (FBS, Gibco). Cells were transiently transfected with 6 μg of Flag-tagged Par3N or Par6β using polyethylenimine transfection reagent (Polysciences). Cells were harvested 36 h post transfection and lysed in the buffer containing 50 mM Tris (pH 7.4), 150 mM sodium chloride, 1% Nonidet P-40, and protease inhibitor cocktail (APExBIO). Each lysate was incubated with GST fusion proteins at 4 °C for 2 h.

After extensive washing of the beads with the lysis buffer, bound proteins were boiled in SDS–PAGE loading buffer and subjected to SDS–PAGE. Proteins were transferred to a 0.45 µm polyvinylidene difluoride (PVDF) membrane (Millipore), which was blocked using 3% bovine serum albumin in TBST (20 mM Tris-HCl (pH 7.4), 137 mM NaCl, and 0.1% Tween-20) buffer at room temperature for 1 h, followed by incubation with the anti-Flag (ABclonal, AE005) at a 1/2000 dilution at 4 °C overnight. Membrane were washed three times with TBST buffer, incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (ABclonal, AS003) or anti-rabbit antibody (ABclonal, AS014), at a 1/5000 dilution, at room temperature for 1 h, and visualized on a LAS4000 chemiluminescent imaging system.

Fluorescence polarization assay

Fluorescence polarization assays were performed on a PerkinElmer LS-55 fluorimeter equipped with an automated polarizer at 25 °C. In a typical assay, a FITC (Molecular Probes)-labeled peptide (~1 μM) was titrated with a binding partner (Par3 PDZs) in buffer A. For the 1,6-hexanediol reversing experiment, Par3 PDZ3 was preincubated with indicated amounts of 1,6-hexanediol.

Crystallography

Freshly purified rat Par3 PDZ3 was concentrated to 7–15 mg/ml before adding Par6β PBM peptide (1 mM stock solution in buffer A) with a molar ratio of 1:3. Crystals of the Par3 PDZ3/Par6β PBM complex were grown by the hanging drop vapor diffusion method at 16 °C in a reservoir solution containing 2.4 M ammonium sulfate and 0.1 M citrate (pH 4.0). The crystals were soaked in crystallization solution containing 20% glycerol for cryoprotection. The diffraction data of the crystals was collected at the beamline BL17U1 at Shanghai Synchrotron Radiation Facility in China (SSRF) at wavelength of 0.9792 Å. The data were processed and scaled using HKL2000 (ref. ⁶³). The phase problem of the Par3 PDZ3/Par6β PBM complex was solved by molecular replacement using the program PHASER⁶⁴ with the solution structure of Par3 PDZ3 (PDB ID: 2K1Z) as the search model before was adjusted by COOT⁶⁵. The initial model was further rebuilt, adjusted manually with COOT, and refined by the phenix.refine program of PHENIX⁶⁶. The statistics of the data collection and final refinement statistics are summarized in Table 1. The PDB accession code is 6JUE.

Table 1 Data collection and refinement statistics.

Full size table

Analytical gel filtration chromatography

Analytical gel filtration chromatography was carried out on an AKTA FPLC system (GE Healthcare). Protein samples were pre-concentrated to indicated concentrations and then loaded on a Superdex^TM 200 increase 10/300 GL column (GE Healthcare) pre-equilibrated with the buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM (or indicated concentrations) NaCl, 1 mM DTT, and 1 mM EDTA.

In vitro phase transition assay

Various Par3, Par6β, and PKCι fragments were prepared in buffer A precleared via high-speed centrifugations. In this assay, the proteins were mixed at indicated molar radio at final concentrations spanning 0.5–25 μM. Formations of phase transition were assayed either directly by imaging-based methods or by sedimentation-based methods.

For imaging, mixtures were observed by being injected into a homemade flow chamber comprised of a glass slide sandwiched by a coverslip with one layer of double-sided tape as a spacer for DIC or fluorescent imaging (Leica TCS SP5). For the sedimentation assay, samples were subjected to centrifugation at 21,130 × g for 10 min. Supernatant was isolated from pellet into a clean tube immediately after centrifugation. The pellet fraction was washed once with buffer A and thoroughly resuspended with the same buffer to the equal volume as supernatant fraction. Proteins from both fractions were detected by 12% SDS–PAGE with Coomassie blue staining. Band intensities were quantified using the ImageJ software.

For fluorescence assay, Par3N, Par6β, and PKCι PB1 were purified in buffer containing 100 mM NaHCO₃ (pH 8.3), 100 mM NaCl (300 mM NaCl for Par3N), 1 mM EDTA, and 1 mM DTT. iFluor^TM 488 NHS ester, Cy3 NHS ester, or 405 NHS ester (AAT Bioquest) were incubated with Par3N, Par6β, or PKCι PB1, respectively, at room temperature for 1 h (fluorophore to protein molar ratio was 1:1). Reaction was quenched by 200 mM Tris (pH 8.0). Chemical-labeled proteins were further purified into buffer A by Hitrap desalting column. In flow chamber at room temperature, mixture of Par3N (25 µM, with iFluor^TM 488-labeled Par3N mixed with 60 molar ratios of unlabeled molecules) and Par6β (25 µM, with Cy3-labeled Par6β mixed with 300 molar ratios of unlabeled molecules) or PKCι PB1 (25 µM, with 405-labeled PKCι PB1 mixed with 100 molar ratios of unlabeled molecules) was observed with a Leica TCS SP5 confocal microscope.

Turbidity assay

Various protein concentrations of FUS LCD (1–214/1–141) were prepared in buffer A adding 10% PEG8000 in a 96-well microplate. The absorption (turbidity) of samples were measured at 600 nm in a SpectraMax microplate reader. Results were recorded in quintuplicate for each protein sample. All assays were performed in quintuplicate (n = 5).

COS7 cell imaging and data analysis

For each well in a six-well plate, various Par3, Par6, and PKCι plasmids were individually or co-transfected into COS7 cells (from ATCC) using polyethylenimine transfection reagent. Cells were fixed by 4% paraformaldehyde and mounted on glass slides for imaging using a Leica TCS SP5 confocal microscope by a 64X oil-immersion lens with DAPI staining. Confocal images were processed with ImageJ. For puncta-counting assay, data were collected from four to six independent batches of cultures as indicated in the figures. In each batch, at least 600 fluorescence-positive cells were counted for each group of experiments. A cell with more than two obvious fluorescence puncta was counted as a puncta-positive cell. Experiments were conducted in a blinded fashion.

FRAP assay

The in vitro FRAP analysis of iFluor488-Par3N droplets was carried out in a 1:1 mixture of Par3N and Par6β (25 µM) at room temperature. The 488 signal was bleached using a 488-nm laser beam with a Leica TCS SP5 confocal microscope.

COS7 cells were cultured in glass bottom dishes and transfected as described above. FRAP assay was performed on a Leica TCS SP5 confocal microscope. Puncta with diameters ~1.0 µm were assayed. GFP signal was bleached using a 488-nm laser beam. The fluorescence intensity difference between pre-bleaching and at time 0 (the time point right after photobleaching pulse) was normalized to 100%. The experimental control is to quantify fluorescence intensities of similar puncta/cytoplasm regions without photobleaching.

In vitro kinase assay

HEK293T cells transfected with the Flag-tagged PKCι WT or Α120Ε was lysed. After centrifugation, the lysate was mixed with anti-Flag M2 affinity gel (Sigma) at 4 °C for 3 h. The anti-Flag M2 affinity gel-captured PKCι was washed twice with the lysis buffer, once with the kinase assay buffer (50 mM Tris, 100 mM NaCl, 10 mM MgCl₂, 150 μM ATP, pH 8.0), and then released by adding the Flag peptide to a final concentration of 0.1 mg/ml. In each kinase reaction assay, Par3 1–854 (3 μM) and Par6β (3 μM) were mixed with PKCι in 100 μl kinase assay buffer at room temperature for 5 min before centrifugation at 21,130 × g for 10 min. Supernatant was isolated from pellet into a clean tube immediately after centrifugation. The pellet fraction was washed once with kinase assay buffer and thoroughly resuspended with the same buffer to the equal volume as supernatant fraction. Proteins from both fractions were detected by Phos-tag SDS–PAGE with Coomassie blue staining. Phos-tag SDS–PAGE was performed by using a resolving gel containing 50 μM phos-tag acrylamide (NARD, AAL-107) and 0.1 mM MnCl_2. The running buffer consisted of 25 mM Tris, 192 mM glycine, and 0.1% (w/v) SDS.

Quantification of protein concentrations in cells

To generate a standard calibration curve for directly converting the measured florescence intensity into absolute protein concentration, purified GFP at various indicated concentrations were injected into the homemade flow chamber, and the fluorescence intensity at each concentration was measured using the same imaging parameters (Olympus IX73 microscope, 12-bit image and exposure 60 ms for COS7 cells; Zeiss 780 upright confocal microscope, 16-bit image, laser power 2.0, and gain value 850 for NBs), as for the whole cell or puncta/crescent quantifications. With these calibration curves, the measured fluorescence intensity for GFP-Par3N in COS7 cells or GFP-Baz in NBs can be converted into absolute molar concentration, according to each standard calibration curve. Identical parameters (laser power, detector gain, bit depth, and exposure time) were used during the imaging processes for COS7 cells or NBs, respectively. Images were analyzed by the ImageJ software.

Drosophila S2 cell culture

Various Baz (WT gene is a gift from Fumio Matsuzaki and Daniel St Johnston) and Par6 (WT gene is a gift from Fumio Matsuzaki) fragments were subcloned into UASt.attB vector (a gift from Konrad Basler).

Drosophila S2 cells (from Drosophila Genomics Resource Center) were cultured with Shields and Sang M3 insect medium (Sigma) supplemented with 10% FBS at 25 °C. Effectene Reagent (Qiagen) was used in all transfections following the manufacturer’s instructions. In short, the ratio of DNA to effectene was sustained at 1:20. The S2 cells were co-transfected with 0.5 µg plasmids of interest (Baz or Par6 variants) together with act-gal4 plasmid. After 48 h post transfection, S2 cells were harvested and lysed in Nonidet P-40 lysis buffer containing 50 mM Tris (pH 8.0), 250 mM NaCl, 0.5% Nonidet P-40, 0.2 mM EDTA, protease inhibitor cocktail (complete, Roche), and phosphatase inhibitor. The cell lysate was spun by micro-centrifugation at maximum speed for 5 min at 4 °C. The samples were separated by 10% polyacrylamide SDS–PAGE gels followed by transferring to PVDF membrane (Millipore). Mouse anti-Flag antibody (Sigma, F1804, 1/2000), rabbit anti-Baz antibody (generated in our lab, 1/1000), and rabbit anti-Par6 (generated in our lab, 1/2000) were diluted in TBST with 5% non-fat dry milk.

Fly genetics

The fly stocks and crosses were maintained at 25 °C on standard medium. Detailed information of the fly stocks used in this study was provided in the following description or FlyBase (www.flybase.org). Fly stocks were attained from Blooming Drosophila Stock Center, unless otherwise stated below.

Stocks used in the study included FRT19A, elav-gal4, insc-gal4, Ay-gal4 (actin flip-out), Tub-gal80, hs-flp, UAS-CD8::GFP, baz[4] (a gift from Andreas Wodarz), and par6[Δ226] (a gift from Juergen A Knoblich), GFP::baz⁵⁰.

Baz and Par6 variants were cloned in UASt.attB vector and transgenic stocks with specific insertion site were obtained through target insertion utilizing attP landing site on III chromosome (Best Gene line 9732) or II chromosome (Best Gene strain 9723), respectively, by BestGene, Inc. (ChinoHills, CA).

Mosaic analysis with a repressible cell marker (MARCM) technique was used to positively mark mutant clones with a GFP signal according to published protocol⁶⁷. In brief, embryos were collected over a time window of 6 h, and larvae (24 h after larval hatching, ALH) was exposed to heat shock treatment for 1 h at 37 °C, and larvae with desired genotypes were dissected and examined.

Immunohistochemistry and imaging

Larvae of desired genotype were dissected at 96 h ALH and brains were fixed for 15 min in 3.7% formaldehyde in PBS with 0.1% Triton-X, and subsequently processed for immunochemistry analysis. The following antibodies were used: mouse anti-Flag (Sigma, F1804), 1/2000; rabbit anti-Mira⁶⁸ (generated in our lab), 1/1000; guinea-pig anti-Dpn (ref. ⁶⁸; generated in our lab), 1/1000; rabbit anti-Baz (generated in our lab), 1/2000; rabbit anti-Par6 (generated in our lab), 1/1000; rabbit anti-aPKCζ C20 (Santa Cruz Biotechnologies, SG-216-G), 1/1000; chicken anti-GFP (Abcam, ab13970), 1/5000; rabbit anti-Pon (generated in our lab), 1/2000; and rabbit anti-Numb (a gift from Xiaohang Yang), 1/1000. Secondary antibodies were conjugated to Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 633 (Molecular Probes) used at 1/500, 1/1000, and 1/250, respectively. TO-PRO-3 (Invitrogen) or Hoechst 33342 (Sigma) was used at 1/5000 for DNA staining, and samples were mounted in Vectashield (Vector Laboratories). Images were obtained using Leica SP8 confocal microscope or Zeiss LSM 780 laser scanning confocal microscope and processed in Adobe Photoshop CC2018.

Effect of 1,6-hexanediol on Par assemblies

To analyze the effect of 1,6-hexanediol on Baz/Par6 assemblies in vivo, larvae of w¹¹¹⁸ at 96 h ALH were dissected in ice cold Shields and Sang M3 insect medium (Sigma), and larvae brains were transferred to Shields and Sang M3 insect medium containing 0, 5, or 10% 1,6-hexanediol, and incubated for 2 min. The treated brains were fixed immediately for 15 min in 3.7% formaldehyde in PBS with 0.1% Triton-X, and then processed for immunochemistry analysis. For recovery experiment, the 1,6-hexanediol-treated brains were washed with M3 insect medium for several times, and incubated in Shields and Sang M3 insect medium for 20 min before fixation and staining. Baz and Par6 localization were analyzed under Leica Confocal microscopy SP8 system. Only mitotic NBs were analyzed.

Image analysis of ASI

The asymmetric index (ASI) for membrane-associated ACD proteins is defined by previous reference³¹ using the following equation (1):

$$\frac{{{\it{A}} - {\it{B}}}}{{2\left( {{\it{A}} + {\it{B}}} \right)}}$$

Where A and B are apical and basal signal intensity measured by ImageJ, respectively. For each experiment, the raw ASI values are normalized with the mean ASI of control. In this case, a value of 1 stands for WT asymmetry, whereas 0 stands for complete loss of asymmetry.

Image analysis of ACI

In some cases, apical or basal proteins have cytoplasmic localization. Then the measurement of apical or basal protein cortical intensity (ACI) follows previous reference³¹ and is calculated by the mean greyscale value of each protein on the apical or basal crescent normalized by the mean greyscale value of protein in the cytoplasmic region. A value of 1 means uniform distribution of the protein throughout cytoplasm and cortex, whereas a value >1 stands for its condensation on the membrane.

CRISPR CAS9 fly assay

CRISPR/CAS9 system was used to generate baz GFP knocked-in stocks according to published protocol⁶⁹. In brief, baz GFP knocked-in insertions were designed so that GFP tags all different splicing isoforms. gRNA sequence was cloned into pCFD4 vector and GFP knocked in baz donor templates were cloned into pHD-DsRed-attP vector. The gRNA constructs and donor templates were co-injected into vasa-Cas9 transgenic embryos (BDSC#51324). Injections were performed by Tsinghua Fly center. For GFP-Baz ΔNTD, nucleotides of baz at position 127–270 of the PA isoform was replaced with in frame GFP coding. For GFP-FUS_L-Baz and GFP-FUS_S-Baz, nucleotides of baz at position 127–270 of the PA isoform was replaced with in frame inserted GFP-linker-FUS_L and GFP-linker-FUSs sequence, respectively. gRNA construct and donor template was generated using primers listed in Supplementary Table 2.

Quantification and statistical analysis

Statistical parameters including the definitions and exact values of n (e.g., number of experiments, number of cells, number of droplets, etc.) are reported in the figures or corresponding figure legends. Data of COS7 cell culture were expressed as mean ± SEM; ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. Data of in vitro phase transition sedimentation assay and FRAP assay were expressed as mean ± SD. Data are judged to be statistically significant when p < 0.05 by one-way ANOVA with Tukey’s multiple comparison test. None of the data were removed from our statistical analysis as outliers. For protein localization in NBs of various genetic backgrounds, only mitotic NBs were imaged and counted. For counting of cell numbers in a MARCM lineage, z-series images were acquired and cells were counted. All statistical data were conducted in GraphPad Prism 6. All experiments related to cell cultures and imaging studies were performed in blinded fashion.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

The authors declare that all data supporting the findings of this study are available within the article and its Supplementary Information files or from the corresponding author upon request. Coordinates of the crystal structure of Par3 PDZ3/Par6β PBM complex have been deposited in the Protein Data Bank (http://www.rcsb.org/) under the accession code 6JUE. FlyBase dataset is available online at http://flybase.org. Source data file is available.

References

Nelson, W. J. Adaptation of core mechanisms to generate cell polarity. Nature 422, 766–774 (2003).
Article ADS CAS PubMed PubMed Central Google Scholar
St Johnston, D. & Ahringer, J. Cell polarity in eggs and epithelia: parallels and diversity. Cell 141, 757–774 (2010).
Article CAS Google Scholar
Rodriguez-Boulan, E. & Macara, I. G. Organization and execution of the epithelial polarity programme. Nat. Rev. Mol. Cell Biol. 15, 225–242 (2014).
Article CAS PubMed PubMed Central Google Scholar
Wen, W. & Zhang, M. Protein complex assemblies in epithelial cell polarity and asymmetric cell division. J. Mol. Biol. 430, 3504–3520 (2018).
Article CAS PubMed Google Scholar
Kemphues, K. PARsing embryonic polarity. Cell 101, 345–348 (2000).
Article CAS PubMed Google Scholar
Hoege, C. & Hyman, A. A. Principles of PAR polarity in Caenorhabditis elegans embryos. Nat. Rev. Mol. Cell Biol. 14, 315–322 (2013).
Article CAS PubMed Google Scholar
Gonczy, P. Mechanisms of asymmetric cell division: flies and worms pave the way. Nat. Rev. Mol. Cell Biol. 9, 355–366 (2008).
Article PubMed CAS Google Scholar
Knoblich, J. A. Asymmetric cell division: recent developments and their implications for tumour biology. Nat. Rev. Mol. Cell Biol. 11, 849–860 (2010).
Article CAS PubMed PubMed Central Google Scholar
Siller, K. H. & Doe, C. Q. Spindle orientation during asymmetric cell division. Nat. Cell Biol. 11, 365–374 (2009).
Article CAS PubMed Google Scholar
Nishimura, T. & Kaibuchi, K. Numb controls integrin endocytosis for directional cell migration with aPKC and PAR-3. Dev. Cell 13, 15–28 (2007).
Article CAS PubMed Google Scholar
Arimura, N. & Kaibuchi, K. Neuronal polarity: from extracellular signals to intracellular mechanisms. Nat. Rev. Neurosci. 8, 194–205 (2007).
Article CAS PubMed Google Scholar
Martin-Belmonte, F. & Perez-Moreno, M. Epithelial cell polarity, stem cells and cancer. Nat. Rev. Cancer 12, 23–38 (2011).
Article PubMed CAS Google Scholar
Tepass, U. The apical polarity protein network in drosophila epithelial cells: regulation of polarity, junctions, morphogenesis, cell growth, and survival. Annu. Rev. Cell Dev. Biol. 28, 655–685 (2012).
Article CAS PubMed Google Scholar
Kennedy, M. B. Origin of PDZ (DHR, GLGF) domains. Trends Biochem. Sci. 20, 350 (1995).
Article CAS PubMed Google Scholar
Joberty, G., Petersen, C., Gao, L. & Macara, I. G. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat. Cell Biol. 2, 531–539 (2000).
Article CAS PubMed Google Scholar
Lin, D. et al. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity. Nat. Cell Biol. 2, 540–547 (2000).
Article CAS PubMed Google Scholar
Renschler, F. A. et al. Structural basis for the interaction between the cell polarity proteins Par3 and Par6. Sci. Signal. 11, eaam9899 (2018).
Article PubMed CAS Google Scholar
Wilson, M. I., Gill, D. J., Perisic, O., Quinn, M. T. & Williams, R. L. PB1 domain-mediated heterodimerization in NADPH oxidase and signaling complexes of atypical protein kinase C with Par6 and p62. Mol. Cell 12, 39–50 (2003).
Article CAS PubMed Google Scholar
Soriano, E. V. et al. aPKC inhibition by Par3 CR3 flanking regions controls substrate access and underpins apical-junctional polarization. Dev. Cell 38, 384–398 (2016).
Article CAS PubMed PubMed Central Google Scholar
Wirtz-Peitz, F., Nishimura, T. & Knoblich, J. A. Linking cell cycle to asymmetric division: Aurora-A phosphorylates the Par complex to regulate Numb localization. Cell 135, 161–173 (2008).
Article CAS PubMed PubMed Central Google Scholar
Knoblich, J. A., Jan, L. Y. & Jan, Y. N. Asymmetric segregation of Numb and Prospero during cell division. Nature 377, 624–627 (1995).
Article ADS CAS PubMed Google Scholar
Lu, B., Rothenberg, M., Jan, L. Y. & Jan, Y. N. Partner of Numb colocalizes with Numb during mitosis and directs Numb asymmetric localization in Drosophila neural and muscle progenitors. Cell 95, 225–235 (1998).
Article CAS PubMed Google Scholar
Ikeshima-Kataoka, H., Skeath, J. B., Nabeshima, Y., Doe, C. Q. & Matsuzaki, F. Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions. Nature 390, 625–629 (1997).
Article ADS CAS PubMed Google Scholar
Shen, C. P., Jan, L. Y. & Jan, Y. N. Miranda is required for the asymmetric localization of Prospero during mitosis in Drosophila. Cell 90, 449–458 (1997).
Article CAS PubMed Google Scholar
Petronczki, M. & Knoblich, J. A. DmPAR-6 directs epithelial polarity and asymmetric cell division of neuroblasts in Drosophila. Nat. Cell Biol. 3, 43–49 (2001).
Article CAS PubMed Google Scholar
Schober, M., Schaefer, M. & Knoblich, J. A. Bazooka recruits Inscuteable to orient asymmetric cell divisions in Drosophila neuroblasts. Nature 402, 548–551 (1999).
Article ADS CAS PubMed Google Scholar
Wodarz, A., Ramrath, A., Kuchinke, U. & Knust, E. Bazooka provides an apical cue for Inscuteable localization in Drosophila neuroblasts. Nature 402, 544–547 (1999).
Article ADS CAS PubMed Google Scholar
Benton, R. & Johnston, D. S. A conserved oligomerization domain in Drosophila Bazooka/PAR-3 is important for apical localization and epithelial polarity. Curr. Biol. 13, 1330–1334 (2003).
Article CAS PubMed Google Scholar
McGill, M. A., McKinley, R. F. & Harris, T. J. Independent cadherin-catenin and Bazooka clusters interact to assemble adherens junctions. J. Cell Biol. 185, 787–796 (2009).
Article CAS PubMed PubMed Central Google Scholar
Wang, S. C. et al. Cortical forces and CDC-42 control clustering of PAR proteins for Caenorhabditis elegans embryonic polarization. Nat. Cell Biol. 19, 988–995 (2017).
Article CAS PubMed Google Scholar
Rodriguez, J. et al. aPKC cycles between functionally distinct PAR protein assemblies to drive cell polarity. Dev. Cell 42, 400–415 (2017).
Article CAS PubMed PubMed Central Google Scholar
Dickinson, D. J., Schwager, F., Pintard, L., Gotta, M. & Goldstein, B. A single-cell biochemistry approach reveals PAR complex dynamics during cell polarization. Dev. Cell 42, 416–434 (2017).
Article CAS PubMed PubMed Central Google Scholar
Feng, W., Wu, H., Chan, L. N. & Zhang, M. The Par-3 NTD adopts a PB1-like structure required for Par-3 oligomerization and membrane localization. EMBO J. 26, 2786–2796 (2007).
Article CAS PubMed PubMed Central Google Scholar
Harris, T. J. C. Protein clustering for cell polarity: Par-3 as a paradigm. F1000Res. 6, 1620 (2017).
Article PubMed PubMed Central Google Scholar
Cheeks, R. J. et al. C. elegans PAR proteins function by mobilizing and stabilizing asymmetrically localized protein complexes. Curr. Biol. 14, 851–862 (2004).
Article CAS PubMed Google Scholar
Slack, C., Overton, P. M., Tuxworth, R. I. & Chia, W. Asymmetric localisation of Miranda and its cargo proteins during neuroblast division requires the anaphase-promoting complex/cyclosome. Development 134, 3781–3787 (2007).
Article CAS PubMed Google Scholar
Lu, B., Ackerman, L., Jan, L. Y. & Jan, Y. N. Modes of protein movement that lead to the asymmetric localization of partner of Numb during Drosophila neuroblast division. Mol. Cell 4, 883–891 (1999).
Article CAS PubMed Google Scholar
Mayer, B., Emery, G., Berdnik, D., Wirtz-Peitz, F. & Knoblich, J. A. Quantitative analysis of protein dynamics during asymmetric cell division. Curr. Biol. 15, 1847–1854 (2005).
Article CAS PubMed Google Scholar
Patel, S. S., Belmont, B. J., Sante, J. M. & Rexach, M. F. Natively unfolded nucleoporins gate protein diffusion across the nuclear pore complex. Cell 129, 83–96 (2007).
Article CAS PubMed Google Scholar
Ribbeck, K. & Gorlich, D. The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion. EMBO J. 21, 2664–2671 (2002).
Article CAS PubMed PubMed Central Google Scholar
Chen, S. et al. Regulation of microtubule stability and organization by mammalian Par3 in specifying neuronal polarity. Dev. Cell 24, 26–40 (2013).
Article CAS PubMed Google Scholar
Ciuffa, R. et al. The selective autophagy receptor p62 forms a flexible filamentous helical scaffold. Cell Rep. 11, 748–758 (2015).
Article CAS PubMed Google Scholar
Gao, L. & Macara, I. G. Isoforms of the polarity protein par6 have distinct functions. J. Biol. Chem. 279, 41557–41562 (2004).
Article CAS PubMed Google Scholar
Patel, A. et al. A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162, 1066–1077 (2015).
Article CAS PubMed Google Scholar
Murakami, T. et al. ALS/FTD mutation-induced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function. Neuron 88, 678–690 (2015).
Article CAS PubMed PubMed Central Google Scholar
Morais-de-Sa, E., Mirouse, V. & St Johnston, D. aPKC phosphorylation of Bazooka defines the apical/lateral border in Drosophila epithelial cells. Cell 141, 509–523 (2010).
Article CAS PubMed PubMed Central Google Scholar
Wang, C., Shang, Y., Yu, J. & Zhang, M. Substrate recognition mechanism of atypical protein kinase Cs revealed by the structure of PKCiota in complex with a substrate peptide from Par-3. Structure 20, 791–801 (2012).
Article CAS PubMed Google Scholar
Spitaler, M., Villunger, A., Grunicke, H. & Uberall, F. Unique structural and functional properties of the ATP-binding domain of atypical protein kinase C-iota. J. Biol. Chem. 275, 33289–33296 (2000).
Article CAS PubMed Google Scholar
Zeng, M. et al. Reconstituted postsynaptic density as a molecular platform for understanding synapse formation and plasticity. Cell 174, 1172–1187 (2018).
Article CAS PubMed Google Scholar
Buszczak, M. et al. The carnegie protein trap library: a versatile tool for Drosophila developmental studies. Genetics 175, 1505–1531 (2007).
Article CAS PubMed PubMed Central Google Scholar
Kono, K. et al. Reconstruction of Par-dependent polarity in apolar cells reveals a dynamic process of cortical polarization. Elife 8, e45559 (2019).
Article PubMed PubMed Central Google Scholar
Oon, C. H. & Prehoda, K. E. Asymmetric recruitment and actin-dependent cortical flows drive the neuroblast polarity cycle. Elife 8, e45815 (2019).
Article PubMed PubMed Central Google Scholar
Shan, Z. et al. Basal condensation of Numb and Pon complex via phase transition during Drosophila neuroblast asymmetric division. Nat. Commun. 9, 737 (2018).
Article ADS PubMed PubMed Central CAS Google Scholar
Izumi, Y., Ohta, N., Itoh-Furuya, A., Fuse, N. & Matsuzaki, F. Differential functions of G protein and Baz-aPKC signaling pathways in Drosophila neuroblast asymmetric division. J. Cell Biol. 164, 729–738 (2004).
Article CAS PubMed PubMed Central Google Scholar
Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).
Article CAS PubMed PubMed Central Google Scholar
Boeynaems, S. et al. Protein phase separation: a new phase in cell biology. Trends Cell Biol. 28, 420–435 (2018).
Article CAS PubMed PubMed Central Google Scholar
Shin, Y. & Brangwynne, C. P. Liquid phase condensation in cell physiology and disease. Science 357, 1253 (2017). eaaf4382.
Article CAS Google Scholar
Hyman, A. A., Weber, C. A. & Julicher, F. Liquid-liquid phase separation in biology. Annu. Rev. Cell Dev. Biol. 30, 39–58 (2014).
Article CAS PubMed Google Scholar
Brangwynne, C. P. et al. Germline P granules are liquid droplets that localize by controlled dissolution/condensation. Science 324, 1729–1732 (2009).
Article ADS CAS PubMed Google Scholar
Strutt, H., Warrington, Samantha J. & Strutt, D. Dynamics of core planar polarity protein turnover and stable assembly into discrete membrane subdomains. Dev. Cell 20, 511–525 (2011).
Li, P. et al. Phase transitions in the assembly of multivalent signalling proteins. Nature 483, 336–340 (2012).
Article ADS CAS PubMed PubMed Central Google Scholar
Wen, W. Phase separation in asymmetric cell division. Biochemistry 59, 47–56 (2020).
Article CAS PubMed Google Scholar
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Method Enzymol. 276, 307–326 (1997).
Article CAS Google Scholar
McCoy, A. J. et al. Phasercrystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).
Article CAS PubMed PubMed Central Google Scholar
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Article PubMed CAS Google Scholar
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).
Article CAS PubMed PubMed Central Google Scholar
Lee, T. & Luo, L. Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22, 451–461 (1999).
Article CAS PubMed Google Scholar
Chai, P. C., Liu, Z., Chia, W. & Cai, Y. Hedgehog signaling acts with the temporal cascade to promote neuroblast cell cycle exit. PLoS Biol. 11, e1001494 (2013).
Article CAS PubMed PubMed Central Google Scholar
Gratz, S. J. et al. Highly specific and efficient CRISPR/Cas9-catalyzed homology-directed repair in Drosophila. Genetics 196, 961–971 (2014).
Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We thank the staff of beamlines BL17U1 and BL18U1 at SSRF and –National Center for Protein Sciences Shanghai for data collection; Jiayi Zhang and Yanjun Liu for helping with DIC and fluorescence imaging; and Fumio Matsuzaki, Daniel St Johnston, Konrad Basler, Andreas Wodarz, Juergen A Knoblich, Xiaohang Yang, the Bloomington Stock Center for antibodies, vector and stocks. This work was supported by grants from the Ministry of Science and Technology of China (2019YFA0508401) and the National Natural Science Foundation of China (31871394 and 31670730) to W.W., Shanghai Municipal Science and Technology Major Project (2018SHZDZX01) and ZJLab, the National Natural Science Foundation of China (31972893) and Municipal Natural Science Foundation of Beijing (KZ201910028040) to Z.Li, RGC of Hong Kong (T13-605/18W) to M.Z., and Temasek Life Sciences Laboratory and Singapore Millennium Foundation to Y.C.

Author information

These authors contributed equally: Ziheng Liu, Ying Yang, Aihong Gu.

Authors and Affiliations

Department of Neurosurgery, Huashan Hospital, the Shanghai Key Laboratory of Medical Epigenetics, the International Co-laboratory of Medical Epigenetics and Metabolism, Ministry of Science and Technology, Institutes of Biomedical Sciences, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, China
Ziheng Liu, Aihong Gu, Jiawen Xu, Ying Mao, Haojie Lu, Weiguo Hu & Wenyu Wen
Temasek Life Sciences Laboratory, Department of Biological Sciences, National University of Singapore, Singapore, 117604, Singapore
Ying Yang & Yu Cai
Fudan University Shanghai Cancer Center and Cancer Metabolism Laboratory, Fudan University, Shanghai, 200032, China
Weiguo Hu & Qun-Ying Lei
College of Life Sciences, Capital Normal University, Beijing, 100048, China
Zhouhua Li
Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
Mingjie Zhang

Authors

Ziheng Liu
View author publications
You can also search for this author in PubMed Google Scholar
Ying Yang
View author publications
You can also search for this author in PubMed Google Scholar
Aihong Gu
View author publications
You can also search for this author in PubMed Google Scholar
Jiawen Xu
View author publications
You can also search for this author in PubMed Google Scholar
Ying Mao
View author publications
You can also search for this author in PubMed Google Scholar
Haojie Lu
View author publications
You can also search for this author in PubMed Google Scholar
Weiguo Hu
View author publications
You can also search for this author in PubMed Google Scholar
Qun-Ying Lei
View author publications
You can also search for this author in PubMed Google Scholar
Zhouhua Li
View author publications
You can also search for this author in PubMed Google Scholar
Mingjie Zhang
View author publications
You can also search for this author in PubMed Google Scholar
Yu Cai
View author publications
You can also search for this author in PubMed Google Scholar
Wenyu Wen
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

Z.Liu, Y.Y., A.G., Y.C., and W.W. conceived the research and analyzed data. Z.Liu, Y.Y., A.G., J.X., H.L., W.H., Q.-Y.L., and Z.Li performed experiments. Z.Liu, Y.Y., A.G., Y.C., and W.W. created figures. Z.Liu, Y.M., M.Z., Y.C., and W.W. wrote the manuscript. W.W. coordinated the research.

Corresponding authors

Correspondence to Yu Cai or Wenyu Wen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Communications thanks Daniel St Johnston and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Peer Review File

Description of Additional Supplementary Files

Supplementary Movie 1

Supplementary Movie 2

Supplementary Movie 3

Supplementary Movie 4

Supplementary Movie 5

Supplementary Movie 6

Supplementary Movie 7

Supplementary Movie 8

Reporting Summary

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Liu, Z., Yang, Y., Gu, A. et al. Par complex cluster formation mediated by phase separation. Nat Commun 11, 2266 (2020). https://doi.org/10.1038/s41467-020-16135-6

Download citation

Received: 28 July 2019
Accepted: 16 April 2020
Published: 08 May 2020
DOI: https://doi.org/10.1038/s41467-020-16135-6

This article is cited by

Asymmetric division of stem cells and its cancer relevance
- Shanshan Chao
- Huiwen Yan
- Pengcheng Bu
Cell Regeneration (2024)
Revisiting bicoid function: complete inactivation reveals an additional fundamental role in Drosophila egg geometry specification
- Stefan Baumgartner
Hereditas (2024)
Apical–basal polarity and the control of epithelial form and function
- Clare E. Buckley
- Daniel St Johnston
Nature Reviews Molecular Cell Biology (2022)
Myosin 1D and the branched actin network control the condensation of p62 bodies
- Xuezhao Feng
- Wanqing Du
- Na Mi
Cell Research (2022)
Structural Organization of Human Full-Length PAR3 and the aPKC–PAR6 Complex
- Le T. M. Le
- Srdja Drakulic
- Bjoern Sander
Molecular Biotechnology (2022)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.