Long range mutual activation establishes Rho and Rac polarity during cell migration

Background and hypothesis.

In multicellular organisms, cells need to have the ability to translocate to different places. As such, they need to have a system in place that regulates the various parameters required for efficient migration and directionality. Previous studies have established the crucial roles of Rac and Rho GTPase in cell polarity by organizing actin cytoskeleton dynamics. This is necessary for mitosis, morphogenesis, migration, and development. Rac is localized to front-promoting protrusions, while RhoA is at the rear end of migrating cells driving contractions. Although studies have shown that local inhibition of Rac and RhoA occurs, it is insufficient to explain long-range coordination.

This preprint explores if mechanical forces and the actin cortex could act together for long-range coordination of the front and the back. The authors utilized optogenetic tools to locally manipulate Rac and RhoA in unpolarized immune human T-cells and examined the activation impact on one another at opposite ends. The resulting findings described in this preprint beautifully reveal that the plasma membrane and actin cortex act as a mechanochemical system to coordinate long-range cell polarity.

Key findings.

Rac stimulation triggers long-range RhoA activation. Using optogenetics (Opto Pi3K tool), Rac was stimulated at one end of an unpolarized neutrophil cell, driving cell protrusion. Simultaneous measurements with the biosensor (Anillin-RBD) reveal a rapid long-range increase in RhoA activity at the cell rear (opposite end of Rac stimulation). This confirms that Rac not only inhibits Rho locally but also stimulates it at a distance.

Rac triggers RhoA by protrusion-mediated elevation in membrane tension. Two possibilities could explain long-range RhoA activation: (i) local Rac-dependent biochemical inhibition, and (ii) indirect long-range mechanical signal. The first possibility was ruled out as successful Rac stimulation in the presence of Arp2/3 inhibitor (CK666) did not elicit RhoA activation. Increased membrane tension alone could promote Rho activation independently of Rac or actin dynamics in neutrophil cells. This underscores that Rac activates Rho by long-range propagation of membrane tension, independently of actin dynamics.

mTORC2 links membrane tension to Rho activation. mTORC2, a mechanosensor known to regulate cell polarity, was explored as a potential molecular link between membrane tension and Rho activation. Cells devoid of mTORC2 or treated with a small molecule inhibitor (Rictor) were ineffective in activating Rho regardless of protrusions generated by Rac stimulation, validating mTORC2’s mechanosensitive position in Rho activation.

Rho activates Rac at a distance through blebbing. Local activation of RhoA (through Opto-LARG) also induced long-range activation of Rac at the opposite end of neutrophil cells. This effect was observed when blebbing was blocked by an actomyosin inhibitor (Blebbistatin).  This indicated that myosin-driven contractions and blebbing are essential for this long-range signaling. Additionally, the presence of actin inhibitor Latrunculin B (which induces stable blebbing), could alone activate Rac, demonstrating the complex interplay between these two GTPases during cell polarity and cell migration.

Contraction-induced asymmetry pushes Rac stimulation via PIP2 release. Next, the authors established a mechanism where local Rho activation led to membrane-to-cortex depletion and subsequent release of PIP2. This facilitated Pi3K-dependent Rac activation at the opposite side of the cell, illustrating a multifaceted relationship between contraction, membrane dynamics, and cell signaling.

Theoretical model explains Rho and Rac coordination in sustaining cell polarity. A mathematical theoretical model successfully simulated local Rac-Rho inhibition together with long-range mutual activation to describe how cells sustain polarity. The model accurately demonstrated that long-range activation is fundamental for robust Rho and Rac partitioning across the cell. These findings underscore the impact of coordinated front-back signaling for efficient immune cell movement.

Long-range mutual activation supports sustained polarity in T-cell migration. Using local optogenetics and chemoattractant CCL19 in primary unpolarized T cells, the authors further verified that Rac and Rho reinforce each other over distances. Blocking either Rho or Rac led to transient polarization, while both pathways were required for effective, sustained polarization and chemotaxis. These findings highlight the importance of coordinated front-back signaling for efficient immune cell movement.

What do I like most about the preprint?

What I like most about this preprint is its demonstration of a novel mechanism supporting effective cell polarization and how it depends on both local inhibition and long-range facilitation between Rac and Rho GTPase at the front and back polarity programs (Figure 1). The long-range activation complements the local inhibition, corroborating the accurate distribution of Rac and Rho at opposite poles of the cell.

Significance of the study.

These findings could potentially offer insights into how cancer cells polarize and migrate in the fluctuating tumor microenvironment. Further, targeting these pathways could lead to new strategies to inhibit metastasis by interrupting coordinated cell movement. Additionally, this preprint emphasizes how strategies focused on targeting the mechanisms regulating immune cell polarity and movement could advance therapeutic approaches aimed at metastasis.

The outcome from this preprint could also be applied to the nervous system, where similar mechanisms of long-range signaling are involved in processes like axon branching and synapse formation. Deregulation in Rac and Rho signaling pathways might be connected to neurodegenerative diseases, or impaired neuronal repair after injury.

Tags: #cellmigration, cellpolarity, rhogtpase

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