Actin dynamics sustains spatial gradients of membrane tension in adherent cells

Background.

Membrane tension is crucial for coordinating various cellular functions such as exocytosis and endocytosis, migration, signaling, and actin polymerization. Although pure lipid membranes show rapid tension spread, which might impede noticeable gradients, adhered supported bilayers and migrating cells have been described to exhibit tension gradients. Previous studies’ calculation of tension gradients relies on tether-pulling experiments, which have participation from plasma membrane tension, membrane-cytoskeleton, and lipid bilayer bending rigidity, all of which are composition-dependent, and limited in spatial resolution. In this preprint, an advanced tension probe, Flipper-TR, has been used to accurately measure tension gradients and the underlying mechanism that sustains them in the cells. Flipper-TR is a fluorescent probe that targets the plasma membrane, reporting tension changes through alterations in its fluorescence lifetime. It integrates into the plasma membrane and fluoresces only within a lipid environment, allowing quantitative analysis of spatial tension distribution and propagation upon mechanical alteration (Colom A et.al; Nature Chem. 2018). In this preprint branched actin and membrane-cortex attachment are proposed to establish membrane tension gradients in all adherent cells: migrating and non-migrating

Key findings of the study.

(a) Flipper-TR detects tension gradients in supported membranes and cells. Flipper-TR can efficiently report membrane tension gradients in reconstituted model membranes as well as in migrating cells by measuring variations in lifetime values. In an expanding model membrane system, Flipper-TR lifetime values exhibit tension gradients dependent on lipid composition and spreading speed. Furthermore, several migrating cell types including keratinocytes, U2OS, RPE1, HeLa, and Cos7 demonstrate large lifetime differences between the front (high tension) and rear (low tension), with robust differences for persistently migrating cells. To identify the underlying mechanism, the authors examined cell edge dynamics and Flipper-TR lifetime correlation.  They found that front protrusion speed, and therefore actin polymerization, are positively correlated with high tension in the front.

(b) Tension gradients exist in non-migratory cells. No significant difference was observed between different shapes of micropatterned cells, and tension gradients exist between the dorsal (high) and ventral (low) planes across shapes. This confirms that cell edge and micropattern boundary are the principal determinants of Flipper-TR lifetime values. Actin protrusion at the cell edge is crucial for high tension and micropattern boundaries decrease tension. These two effects are accountable for gradient formation in non-migratory cells.

(c) Lipid Composition and diffusion. Patterned cells have a uniform distribution of main lipids like phosphatidylcholine (PC) and phosphatidylethanolamine (PE) suggesting tension gradient is not due to variations in these abundant main lipids. Less abundant lipids such as ceramide (Cer), sphingomyelin (SM), and globotriaosylceramide (Gb3) show moderate spatial gradients. Overall, the tension gradient is independent of lipid composition. Fluorescence recovery after photobleaching (FRAP) confirms no significant lipid flow across different cell regions, indicating gradients are maintained despite the absence of lipid flow. Moreover, lipid diffusion barriers at the cell edges suggested by fluorescence fluctuation spectroscopy (FFS) measurements may contribute to the persistence of tension gradients.

 (d) Actin cytoskeleton and membrane tension. Latrunculin A and JLY-induced inhibition of actin dynamics abolished membrane ruffles and led to uniform distribution of membrane tension, signifying actin polymerization is necessary to maintain tension gradients. Pharmacological treatments promoting lamellipodia formation increased membrane tension, whereas treatments promoting filopodia decreased membrane tension. Ezrin inhibition weakened membrane-to-cortex attachment, resulting in higher tension at protrusion and faster tension dissipation away from protrusions.

 (e) Substrate Rigidity and Adhesion. Less rigid substrates displayed weakened tension gradients leading to nearly uniform tension, demonstrating that substrate stiffness impacts the magnitude of membrane tension gradients. Reducing adhesion strength through specific inhibitors (e.g., Cilengitide) also leads to a more uniform distribution of membrane tension, emphasizing the contribution of adhesion in creating and maintaining tension gradients. Bleb-based migrating cells, devoid of adhesion, display a lack of tension gradients. This implies that a membrane tension gradient is not imperative for migration. Taken together, this preprint highlights the multifaceted interplay between lipid composition, actin cytoskeleton dynamics, and substrate properties responsible for membrane tension gradients.

Significance of the study.

 

Figure 1: (Reproduced from the preprint Fig.5H). Conceptual model of membrane tension gradient maintenance. The diagram shows tension variation across the cell, highlighting high tension on the dorsal side and gradients on the ventral side. Key factors include branched actin filaments (red), cellular adhesions, and clathrin plaques (blue) acting as barriers, creating tension gradients, and directing lipid flow, crucial for cellular processes like movement and signaling.

Diffusion barriers are key in sustaining membrane tension gradients, as no significant lipid flow was observed. This contradicts previous studies that imply lipid flow regulates tension gradients, underscoring the context-specific mechanisms in cell biology. The dependence of tension gradients on cell-ECM adhesion strength and substrate rigidity further reinforces that adhesion is crucial for tension distribution (Figure 1).

What do I like most about the preprint?

The novel application of Flipper-TR as a real-time quantitative membrane tension probe is the most striking feature of the preprint. The study depicts a comprehensive and dynamic assessment of the formation of tension gradients within adherent cells, migrating and non-migrating by integrating innovative tools with advanced imaging and analytical approaches. The conclusions from the preprint not only advance our understanding of the membrane mechanical properties but also resolve former theories on tension propagation and its reliance on adhesion and cytoskeletal dynamics.

Tags: actin dynamics, actomyosin, biophysics, cell mechanics, cell migration, cytoskeleton, membrane tension

doi: https://doi.org/10.1242/prelights.37980

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