Septin crosstalk with microtubules and actin is regulated by a GSK3-dependent phosphoswitch

This study reveals that glycogen synthase kinase 3 (GSK3) regulates septin 9 (SEPT9) distribution between actin and microtubule cytoskeletons through a phosphorylation-dependent switch, thereby controlling neuronal polarization and linking cytoskeletal dynamics to broader cellular signaling pathways.

The Gist

Imagine your cell as a bustling construction site. Inside, there are two major types of scaffolding holding everything together: actin (which is like flexible, stretchy rope) and microtubules (which are like rigid, steel beams).

Now, meet the Septins. Think of them as the "smart connectors" or "traffic cops" that can attach to either the rope or the steel beams. They help organize the construction site, but scientists didn't fully understand how they decided which one to grab onto at any given moment.

This paper reveals that the decision-making process is controlled by a specific manager called GSK3. Here is how the story unfolds:

The "Switch" Mechanism

The researchers discovered that GSK3 acts like a light switch or a molecular toggle for the Septins (specifically a type called SEPT9). It does this by adding a tiny chemical tag (a phosphate group) to the Septin.

• When the switch is ON (GSK3 is active): It tags the Septin. This tag acts like a magnet that pushes the Septin away from the steel beams (microtubules) and pulls it closer to the stretchy rope (actin).
• When the switch is OFF (GSK3 is inactive): The tag is removed. Without the tag, the Septin feels a strong pull toward the steel beams (microtubules) and lets go of the rope.

Testing the Theory

To prove this, the scientists played a few tricks with the Septins:

1. The "Fake Tag" Trick: They created Septins that always looked like they had the tag on them (even when they didn't). These "fake tagged" Septins refused to touch the steel beams and stuck only to the rope.
2. The "No Tag" Trick: They created Septins that could never get the tag. These "tag-free" Septins loved the steel beams and helped them grow longer.

What Happens in a Real Brain Cell?

The team tested this in actual brain cells (hippocampal neurons). These cells need to grow long, thin arms called neurites to talk to other cells. This growth needs to be uneven and specific (asymmetric) for the cell to work correctly.

• When they turned off the GSK3 manager, the Septins moved to the steel beams, and the cells grew their arms normally.
• When they forced the Septins to stay on the rope (using the "fake tag" trick), the cells got confused. They couldn't grow their arms properly, and the "polarization" (the process of deciding which way to grow) failed.

The Big Picture

In simple terms, this paper shows that GSK3 is the boss that decides whether Septins hang out with the flexible ropes or the rigid beams. By flipping this chemical switch, the cell can instantly reorganize its internal structure. This is a big deal because GSK3 is already known to be involved in many other important jobs in the body, like metabolism and general cell health, meaning it's a central hub for how cells manage their shape and movement.

Technical Summary

Problem
Septins are cytoskeletal filaments known to associate with both the actin and microtubule networks. However, the specific mechanisms governing the crosstalk between septins and these distinct cytoskeletal systems, as well as how septin function is regulated within these networks, remain largely unknown.

Methodology
The study investigates the role of glycogen synthase kinase 3 (GSK3) in regulating septin 9 (SEPT9) distribution. The researchers employed a multi-faceted approach including:

• Pharmacological intervention: Using GSK3 inhibitors to observe changes in endogenous SEPT9 distribution across multiple cell types.
• Mutagenesis: Generating specific SEPT9 mutants, including phosphomimetic mutations at serines 82 and 85 (to simulate phosphorylation) and phosphonull mutations (to prevent phosphorylation).
• In vitro and in vivo assays: Assessing the binding affinity of these mutants to microtubules and actin in cell-free systems and within cells.
• Neuronal models: Utilizing primary hippocampal neurons to examine the effects of GSK3 inactivation and SEPT9 mutations on neuronal polarization and asymmetric neurite growth.

Key Contributions and Results
The paper identifies GSK3 as a direct regulator of septin function through the phosphorylation of SEPT9. Key findings include:

• GSK3 as a Molecular Switch: GSK3 directly phosphorylates SEPT9, creating a bidirectional switch that controls the partitioning of septins between actin and microtubules.
• Effect of Inhibition: Inhibiting GSK3 leads to a redistribution of endogenous SEPT9 toward microtubules in various cell types.
• Role of Specific Phosphorylation Sites:
  • Phosphomimetic mutations (S82/S85): These mutations reduce the binding of SEPT9 to microtubules while enhancing its association with actin, both in cellular and in vitro contexts.
  • Phosphonull mutations: These mutations promote microtubule binding and growth.
• Neuronal Impact: In primary hippocampal neurons, GSK3 inactivation promotes the association of SEPT9 with microtubules. Conversely, phosphomimetic mutations impair asymmetric neurite growth, a critical process during neuronal polarization.

Significance
This work reveals a phosphorylation-dependent mechanism that dictates how septins partition between actin and microtubule networks. By placing the cytoskeletal functions of septins under the control of GSK3, the study links septin regulation to broader signaling pathways involved in cell physiology and metabolism. The findings provide a mechanistic understanding of how a single kinase can orchestrate the interaction of septins with distinct cytoskeletal elements to influence cellular morphology and function.