Homer condensates orchestrate YAP-Wnt signaling crosstalk downstream of the Crumbs polarity complex

This study reveals that Homer scaffolding proteins function as polarity-sensitive, phase-separating condensates that integrate Crumbs complex cues to differentially regulate YAP and Wnt signaling pathways through distinct interactions with FRYL and PATJ, thereby coordinating cell growth and transcriptional output in both development and cancer.

The Gist

The Big Picture: The Cell's "Control Room"

Imagine a cell as a busy city. For the city to function, it needs to know two things: how big it is (so it doesn't grow too crowded) and what the weather is like (so it knows when to build new roads or repair old ones).

In biology, these "rules" are controlled by two major signaling pathways:

1. The Hippo/YAP Pathway: The "Stop/Go" sign for cell growth. If the city is too crowded, this pathway tells the cells to stop dividing.
2. The Wnt Pathway: The "Construction Crew" that tells cells where to build and how to move.

Usually, scientists think these two pathways work separately. But this paper discovers that they actually talk to each other, and the conversation happens inside a special, gooey "cloud" inside the cell.

The Main Characters

1. Homer Proteins (The Cloud Builders):
   Think of Homer proteins as LEGO bricks that have a special superpower. They can snap together to form a giant, floating cloud (called a biomolecular condensate) inside the cell. This isn't a solid object; it's more like a drop of oil in water—a "liquid-like" blob where other proteins can gather.

2. The Crumbs Complex (The City Planners):
   This is a group of proteins that sit at the very top of the cell (the "roof"). They act like the city planners, telling the cell, "We are organized and polarized; keep things in order." One of their key members is PATJ.

3. FRYL (The Traffic Cop):
   This is a protein that acts like a traffic cop for the Hippo pathway. Its job is usually to slow down cell growth.

The Story: How the Cloud Controls the City

1. The Cloud Forms a Meeting Place

The researchers found that Homer proteins naturally clump together to form these liquid-like clouds. Inside these clouds, important signaling proteins gather to have a meeting. It's like a pop-up coffee shop where the most important business deals happen.

2. The "Roof" vs. The "Cloud"

In a healthy, organized cell (like in the kidney), the "City Planners" (PATJ) grab the Homer bricks and pull them up to the roof (the cell junction).

• What happens? When PATJ pulls the Homers up, the cloud stays small and organized. The "Traffic Cop" (FRYL) is kept busy, and the cell growth is kept in check. The cell knows it's time to stay put and not grow too fast.

3. What Happens in Cancer?

In cancer cells (or when the cell loses its shape), the "City Planners" (PATJ) are missing or broken.

• The Result: The Homer bricks are left floating in the middle of the city (the cytoplasm). They form huge, chaotic clouds.
• The Effect: Inside these big clouds, the "Traffic Cop" (FRYL) gets trapped and silenced. Without the traffic cop, the "Growth Signal" (YAP) goes wild. The cell starts dividing uncontrollably and moving around aggressively.

4. The Twist: Two Different Jobs

The paper found something really cool: The Homer cloud doesn't just do one thing. It acts like a smart switchboard.

• For Growth (Hippo/YAP): The cloud hides the Traffic Cop (FRYL), letting growth happen.
• For Construction (Wnt): The cloud helps the Construction Crew (Wnt) work better.

It's as if the cloud is a dual-purpose hub: It turns off the "Stop" sign while simultaneously turning on the "Build" sign. This explains why cancer cells grow so fast and move so easily—they have a cloud that is constantly screaming "BUILD MORE!"

The Material Science Part: Why the Cloud Matters

The researchers also looked at what these clouds are made of.

• With FRYL: The cloud becomes more like liquid water. It flows easily, and the proteins inside can move around quickly. This helps the cell react fast.
• With PATJ: The cloud becomes more like a jelly or a net. It's stickier and more rigid. This rigidity helps keep the cell organized and stops the growth signals from going crazy.

The Takeaway

This paper tells us that cells don't just use chemical signals to talk; they use physical structures (like these liquid clouds) to organize their conversations.

• Healthy Cells: The "Roof" (PATJ) keeps the "Cloud" (Homer) organized and small, keeping growth under control.
• Cancer Cells: The "Roof" is broken. The "Cloud" explodes into a giant, sticky mess that silences the brakes and hits the gas pedal, leading to tumor growth.

In short: The cell uses a liquid cloud as a control center. If the cloud is the right shape and size, the cell is healthy. If the cloud gets too big and messy, the cell becomes cancerous. This discovery opens up new ways to treat cancer: maybe we can design drugs that break up these bad clouds or force them to become the right shape again!

Technical Summary

1. Problem Statement

The Hippo/YAP and Wnt/β-catenin signaling pathways are critical regulators of cell growth, differentiation, and tissue homeostasis, and their deregulation is a hallmark of cancer. While both pathways respond to epithelial cell architecture and polarity, the molecular mechanisms coordinating their crosstalk remain poorly understood. Specifically, it is unclear how apico-basal polarity cues (mediated by the Crumbs complex) integrate with the phase separation dynamics of signaling molecules to regulate the transcriptional output of YAP and Wnt. Previous studies suggested that biomolecular condensates play a role in Hippo regulation, but the specific scaffolds linking polarity to these condensates and their functional consequences on pathway crosstalk were undefined.

2. Methodology

The authors employed a multidisciplinary approach combining cell biology, biochemistry, biophysics, and transcriptomics:

• Cell Models: Used polarized Madin-Darby Canine Kidney (MDCK) cells, non-polarized HEK293T cells, and colorectal cancer (HCT116) cells.
• Genetic Manipulation: Generated single and triple Homer knockout (KO) MDCK cells, PATJ KO cells, and FRYL KO cells using CRISPR/Cas9. Utilized siRNA knockdowns in 293T and HCT116 cells.
• Protein Interaction Analysis: Performed Co-immunoprecipitation (Co-IP) and Immunoprecipitation-Mass Spectrometry (IP-MS) to identify Homer interactors. Used site-directed mutagenesis to disrupt specific PxxF motifs in PATJ and FRYL to validate binding sites.
• Phase Separation Characterization:
  • Live-cell Imaging: Used spinning disk confocal microscopy to visualize GFP-tagged Homer condensates.
  • FRAP (Fluorescence Recovery After Photobleaching): Measured the mobility and material properties (liquid-like vs. gel-like) of condensates.
  • Osmotic Stress: Applied 0.4 M sorbitol to induce hyperosmotic stress and observe condensate dynamics.
  • Electron Microscopy: Used APEX2-TEM to visualize the ultrastructure of Homer assemblies.
• Functional Assays:
  • Reporter Assays: Luciferase assays for TEAD (YAP) and TOPFlash (Wnt/β-catenin) activity.
  • qPCR & RNA-seq: Analyzed gene expression changes in Homer TKO cells to identify downstream targets.
  • Wound Healing: Assessed collective cell migration.

3. Key Contributions

• Identification of Homer as a Polarity-Linked Scaffold: Established that Homer proteins (1, 2, and 3) are recruited to apical cell junctions by the Crumbs complex component PATJ via a conserved PxxF motif interaction.
• Discovery of FRYL as a Novel Homer Interactor: Identified Furry-like (FRYL), an NDR kinase scaffold, as a direct binding partner of Homers via C-terminal PxxF motifs.
• Mechanism of Phase Separation: Demonstrated that Homers form biomolecular condensates in the cytoplasm. Crucially, the material properties of these condensates are tunable:
  • FRYL promotes spherical, liquid-like condensates and increases Homer mobility.
  • PATJ drives the formation of irregular, network-like (gel-like) assemblies, particularly under osmotic stress.
• Pathway-Selective Regulation: Revealed that Homers act as a signaling hub that differentially regulates YAP and Wnt pathways:
  • Homers promote YAP/TEAD signaling by antagonizing FRYL-mediated suppression.
  • Homers cooperate with FRYL to enhance Wnt/β-catenin signaling.
• Polarity-Dependent Signaling Hub: Proposed a model where the Crumbs complex (via PATJ) restrains Homer-driven YAP activation in polarized cells, while loss of polarity (as in cancer) or altered condensate properties leads to hyperactivation of YAP and Wnt.

4. Key Results

• Homer-PATJ Interaction: Homer EVH1 domains bind to a specific PxxF motif (PKGF) in the linker region between PDZ5 and PDZ6 of PATJ. Loss of PATJ displaces Homers from apical junctions.
• Homer-FRYL Interaction: Homers bind FRYL via two conserved C-terminal PxxF motifs. This interaction is specific (no binding to Furry) and cooperative.
• Signaling Effects in Polarized Cells (MDCK):
  • Homer Loss: Triple KO of Homers reduced YAP/TEAD reporter activity and Wnt reporter activity. It increased YAP S127 phosphorylation (inactivation) and downregulated target genes (e.g., CTGF, HAS2).
  • PATJ Loss: Mimicked Homer loss at junctions, reducing YAP/Wnt signaling, but paradoxically increased endogenous YAP target gene expression, suggesting PATJ acts as a brake on Homer-driven YAP activation.
  • FRYL Loss: Surprisingly, FRYL KO increased YAP/TEAD activity (indicating FRYL normally suppresses YAP) but decreased Wnt target gene expression.
• Condensate Dynamics:
  • Homers form cytoplasmic puncta that exhibit liquid-like properties (fusion, coalescence) and are sensitive to osmotic stress.
  • FRYL enhances the liquid nature of Homer condensates (higher mobile fraction).
  • PATJ promotes the formation of irregular, percolated networks (lower mobility, gel-like state).
• Cancer Context (HCT116): In colorectal cancer cells, Homer knockdown suppressed both YAP and Wnt signaling and impaired cell migration, suggesting Homers are pro-tumorigenic in this context.
• Mechanism of Crosstalk: The data supports a model where Homers sequester the FRYL/NDR complex. In the absence of polarity cues (or when Homers are upregulated), this sequestration prevents NDR from phosphorylating/inhibiting YAP, leading to YAP activation. Simultaneously, Homers and FRYL converge to enhance Wnt signaling through a mechanism independent of YAP sequestration (potentially involving Src).

5. Significance

• Novel Regulatory Mechanism: The study establishes biomolecular phase separation as a tunable mechanism for integrating cell polarity cues with growth signaling. It demonstrates that the material state of a condensate (liquid vs. gel) can dictate signaling output.
• Resolution of Pathway Crosstalk: It provides a molecular explanation for how YAP and Wnt pathways are coordinated. Homers act as a central hub that can simultaneously suppress YAP inhibition (via FRYL sequestration) and promote Wnt signaling, explaining their co-activation in cancer.
• Therapeutic Implications: Given that Homers are upregulated in various tumors and correlate with poor prognosis, the "Homer condensate" represents a potential therapeutic target. Modulating the phase separation properties of Homers or their interaction with PATJ/FRYL could restore normal polarity-dependent signaling and inhibit oncogenic YAP/Wnt activation in epithelial cancers.
• Paralogue Specificity: The work highlights that Homer paralogues (1, 2, 3) are not functionally redundant; Homer3 appears dominant in promoting YAP/Wnt signaling in MDCK cells, while their effects vary by cell type and expression level.

In summary, this paper defines Homer proteins as polarity-sensitive, phase-separating scaffolds that orchestrate the crosstalk between the Hippo/YAP and Wnt pathways, offering a new paradigm for understanding how cell architecture controls transcriptional programs in development and cancer.