Wnt signalling controls abscission dynamics in mouse embryonic stem cells

This study reveals that the Wnt signaling pathway regulates the timing of cell abscission in mouse embryonic stem cells through two distinct mechanisms involving Aurora B stability and GSK-3β-mediated microtubule dynamics, demonstrating that the pathway's impact on cell division is context-dependent and influenced by the cell's pluripotent state.

The Gist

Imagine a cell dividing like a baker splitting a loaf of bread. Usually, once the dough is cut in half, the two new loaves separate quickly. But in mouse embryonic stem cells (the "master builders" of the body), there's a weird delay. After the cut is made, the two halves stay connected by a tiny, stretchy bridge for a very long time—sometimes up to 12 hours—before finally snapping apart.

This paper is about figuring out why these stem cells take so long to let go of each other, and how a specific chemical signal called Wnt acts as the "traffic controller" for this process.

Here is the story of the research, explained simply:

1. The Setting: The Sticky Bridge

When a cell divides, it forms a narrow bridge between the two new daughter cells. Think of this bridge like a piece of taffy or a rubber band.

• Normal cells: Cut the taffy quickly (1–2 hours).
• Stem cells: Let the taffy stretch out for a long time (up to 12 hours).

The scientists wanted to know: What keeps the stem cells holding on so tight?

2. The Main Character: The Wnt Signal

The researchers discovered that a chemical pathway called Wnt is the boss here.

• When Wnt is "ON" (Active): The stem cells hold on tight. The bridge stays long and stretchy.
• When Wnt is "OFF" (Inactive): The stem cells let go quickly. The bridge snaps fast.

It's like Wnt is a "Do Not Disturb" sign on the door of the dividing cell. As long as the sign is up, the cell refuses to finish the job.

3. How Wnt Pulls the Strings (The Two Mechanisms)

The scientists found that Wnt uses two different tricks to keep the bridge from snapping:

Trick #1: The "Anchor" (Microtubules)

Inside the bridge, there are tiny structural rods called microtubules. Think of these as the steel cables holding the bridge together.

• The Problem: If these cables are wobbly, the bridge breaks easily. If they are stiff and strong, the bridge holds firm.
• Wnt's Move: Wnt turns off a chemical enzyme called GSK-3b. When GSK-3b is turned off, it stops attacking the "stabilizer proteins" (like CLASP2) that wrap around the steel cables.
• The Result: The cables become super-stable and stiff. The bridge becomes a fortress that is hard to cut.

Trick #2: The "Guardian" (Aurora B)

There is another protein called Aurora B that acts like a security guard at the bridge. Its job is to tell the cell, "Wait! Don't cut yet!"

• The Problem: Usually, the cell has a garbage disposal system (the proteasome) that eats up excess proteins. If Aurora B gets eaten, the guard leaves, and the bridge snaps.
• Wnt's Move: When Wnt is active, it stops the garbage disposal from eating Aurora B. It keeps the guard on duty.
• The Result: The guard stays at the bridge, shouting "Hold on!" for a long time, delaying the final cut.

4. The Plot Twist: Context Matters!

Here is the most interesting part. The scientists tried to turn Wnt "ON" in cells that were leaving their stem cell state (getting ready to become skin, muscle, or nerve cells).

• In pure stem cells: Turning Wnt ON makes the bridge stay long.
• In differentiating cells: Turning Wnt ON actually makes the bridge snap faster!

The Analogy: Imagine a "Stop" sign.

• In a school zone (Stem Cells), the Stop sign works as intended: cars stop and wait.
• In a race track (Differentiating Cells), the same "Stop" sign might be interpreted differently, or the drivers ignore it, causing them to speed up.

This shows that the same chemical signal (Wnt) can have opposite effects depending on the "mood" or "state" of the cell.

5. The Big Picture

This research is important because:

1. Timing is Everything: The length of time cells stay connected affects how they develop. If they separate too fast or too slow, it can cause birth defects or diseases.
2. Stem Cell Identity: It helps us understand how stem cells know they are still "special" and haven't turned into regular cells yet.
3. New Tools: By understanding how to control this bridge, scientists might be able to manipulate stem cells better for regenerative medicine (growing new organs or repairing tissues).

Summary

Think of cell division as a dance. The Wnt signal is the music.

• When the music is playing (Wnt active), the stem cells dance slowly, holding hands (the bridge) for a long time to ensure everything is perfect.
• When the music stops (Wnt inactive), they let go quickly.
• But if the dancers change their style (exit the stem cell state), the same music might make them dance faster instead of slower.

The scientists figured out that Wnt keeps the "hand-holding" strong by reinforcing the structural cables and keeping the "security guard" on duty, ensuring the stem cells take their time before becoming something new.

Technical Summary

1. Problem Statement

Cell division concludes with abscission, the physical separation of two daughter cells via the severing of the intercellular bridge. While abscission typically occurs 1–2 hours after chromosome segregation in most mammalian cells, it is significantly delayed (up to 12 hours) in naïve mouse embryonic stem cells (mESCs). This delay is crucial for cell fate transitions but the molecular mechanisms linking cell state (pluripotency) to abscission dynamics remain unclear. Specifically, it is unknown how pluripotency-maintaining signaling pathways, such as Wnt signaling, regulate the stability of the intercellular bridge and the timing of its severing.

2. Methodology

The study utilized mouse embryonic stem cells (mESCs) as a model system, comparing cells in a naïve pluripotent state versus cells exiting pluripotency (differentiating).

• Cell Culture Conditions:
  • Naïve State: Cultured in 2i-Lif media (containing GSK-3 inhibitor CHIR-99021 and MEK inhibitor PD0325901) or 1i-Lif (lacking CHIR-99021, allowing active GSK-3).
  • Exiting/Differentiating State: Cultured in N2B27 media.
• Live-Cell Imaging: Cells were incubated with SiR-tubulin to visualize microtubules. Abscission timing was measured from bridge formation to microtubule severing (a proxy for membrane scission).
• Genetic and Pharmacological Manipulations:
  • Wnt Modulation: Use of CHIR-99021 (GSK-3 inhibitor) to activate Wnt; WAY-2626211 (Dkk-1 inhibitor) to activate Wnt; and Wnt3A ligand addition.
  • Optogenetics: Use of Opto-Wnt (LRP6-Cry2 fusion) to activate Wnt signaling locally in single cells using blue light.
  • Knockouts: $\beta$-Catenin KO mESCs to test canonical Wnt dependence.
  • Proteasome Inhibition: Treatment with MG132 to block protein degradation.
  • Kinase Inhibition: Use of ZM447439 (Aurora B inhibitor).
  • Constructs: Expression of Aurora B-USP28-eGFP (to prevent Aurora B ubiquitination/degradation) and CLASP2-GFP.
• Immunofluorescence & Quantification: Analysis of protein localization (GSK-3$\beta$, Aurora B, CLASP1/2, ubiquitin, acetylated tubulin) at the midbody/bridge using confocal microscopy.

3. Key Contributions and Results

A. Wnt Signaling Delays Abscission via Non-Canonical Mechanisms

• Observation: In naïve mESCs (2i-Lif), where Wnt is active (GSK-3 inhibited), abscission is slow (10 hours). Removing the GSK-3 inhibitor (1i-Lif) or activating GSK-3 accelerates abscission (5 hours).
• Activation: Adding Wnt3A or inhibiting Dkk-1 in exiting cells (N2B27) slows down abscission, mimicking the naïve state.
• Single-Cell Resolution: Optogenetic activation of Wnt signaling in single exiting cells was sufficient to delay abscission in those specific cells, proving this is a cell-autonomous property.
• Canonical vs. Non-Canonical: $\beta$-Catenin KO cells showed normal abscission dynamics compared to WT, indicating that canonical Wnt signaling ($\beta$-catenin dependent) is not required. The regulation is mediated by non-canonical Wnt signaling involving GSK-3$\beta$.

B. GSK-3$\beta$ Regulates Microtubule Stability via CLASP2

• Microtubule Stability: Active GSK-3 (in 1i-Lif or N2B27) leads to less acetylated tubulin (a marker of stability) and shorter microtubules at the bridge. Inactive GSK-3 (2i-Lif) results in highly stable, acetylated microtubules.
• Role of CLASP2: GSK-3$\beta$ localizes to the bridge. In the absence of GSK-3 activity (2i-Lif), CLASP2 (a microtubule-stabilizing protein) accumulates at the bridge.
• Mechanism: GSK-3$\beta$ likely phosphorylates CLASP2, reducing its affinity for microtubules and causing it to detach. When GSK-3 is inactive, CLASP2 binds and stabilizes microtubules, delaying abscission. Overexpression of CLASP2 in 2i-Lif further delays abscission, confirming its role.

C. Wnt Signaling Controls Aurora B Levels via Protein Degradation

• Proteasome Dependence: Inhibiting the proteasome (MG132) delays abscission, increases bridge numbers, and increases Aurora B levels at the bridge.
• Ubiquitination: Active GSK-3 (1i-Lif/N2B27) correlates with higher levels of ubiquitin-remnant motifs at the bridge, suggesting active protein degradation. Active Wnt (2i-Lif) reduces ubiquitination.
• Aurora B Regulation: High Aurora B levels at the bridge are associated with delayed abscission.
  • Inhibiting GSK-3 increases Aurora B levels.
  • Expressing Aurora B-USP28 (preventing ubiquitination) keeps Aurora B at the bridge and delays abscission.
  • Inhibiting Aurora B rescues the delayed abscission caused by proteasome inhibition.
• Conclusion: Wnt signaling (via GSK-3 inhibition) prevents the ubiquitination and subsequent degradation of Aurora B, maintaining high Aurora B activity which stabilizes microtubules and delays abscission.

D. Context-Dependence of Wnt Signaling

• Cell State Switch: The effect of GSK-3 inhibition is context-dependent.
  • Early Exit (0–16h): Adding CHIR-99021 (inhibiting GSK-3) delays abscission.
  • Late Exit (48h+): Adding CHIR-99021 to cells that have been exiting for 48 hours accelerates abscission.
• Significance: This demonstrates that the Wnt pathway's output on abscission is dynamically reprogrammed as cells transition from naïve pluripotency to a primed/differentiated state.

4. Proposed Model

The authors propose a dual mechanism where active Wnt signaling (via GSK-3 inhibition) delays abscission in naïve mESCs:

1. Microtubule Stabilization: Inactive GSK-3$\beta$ allows CLASP2 to bind and stabilize intercellular bridge microtubules.
2. Aurora B Stabilization: Inactive GSK-3$\beta$ prevents the phosphorylation and subsequent ubiquitination/degradation of Aurora B kinase. High Aurora B levels further reinforce microtubule stability and delay the recruitment of the severing machinery (MCAK/ESCRT).

5. Significance

• Mechanistic Insight: This study identifies protein degradation (specifically of Aurora B) and microtubule-associated proteins (CLASP2) as the direct effectors linking Wnt signaling to cytokinesis timing.
• Cell Fate Coupling: It establishes a direct molecular link between the pluripotency state and the duration of cell division, suggesting that abscission timing is a readout of cell identity.
• Non-Canonical Wnt: It highlights that non-canonical Wnt signaling is also context-dependent, capable of promoting differentiation or maintaining pluripotency depending on the cell state, similar to canonical Wnt.
• Therapeutic Implications: Understanding how stem cells regulate division timing could inform strategies for controlling stem cell expansion or differentiation in regenerative medicine.

In summary, the paper demonstrates that Wnt signaling controls abscission dynamics in mESCs by inhibiting GSK-3$\beta$, which simultaneously stabilizes CLASP2-mediated microtubules and prevents Aurora B degradation, thereby delaying cell separation in a cell-state-dependent manner.