Mechanical stretch regulates macropinocytosis in Hydra vulgaris

Taylor D. Skokan, Bert Hobmayer, Kara L. McKinley, Ronald D. Vale

Posted on: 22 February 2022 , updated on: 23 March 2022

Preprint posted on 4 December 2021

Article now published in Molecular Biology of the Cell at http://dx.doi.org/10.1091/mbc.E22-02-0065

Hydra stretches and Hydra gulps

Selected by Mugdha Sathe

Categories: cell biology, developmental biology

Background:

The role of vesicular trafficking in regulating membrane tension has been explored for several different types of endocytic pathways. Depending upon the cell type and context, both clathrin-dependent and independent pathways have been shown to be either sensitive to membrane tension or actively regulate it^[1]. However, most of the studies exploring this relationship have been performed in an in vitro context. Thus, it remains an open question if one can actively tune the mechanical properties of an animal and see a response in the endocytic pathway.

In this study, the authors have focused on a clathrin-independent endocytic pathway: macropinocytosis, a highly versatile pathway in terms of vesicles sizes and regulating cellular functions. This property of macropinocytosis enables it to quickly respond and elicit change in a cell’s surface area. The functions and mechanisms behind macropinocytosis have been studied mostly in mammalian tissue culture models. Here, the authors report a serendipitous discovery wherein unlike mammalian cells that exhibit growth factor-dependent macropinocytosis, Hydra’s superficial ectoderm exhibits constitutive macropinocytosis. Furthermore, the macropinocytosis was regulated by tissue-level mechanics.

Key Findings:

The ectoderm of Hydra exhibits constitutive macropinocytosis: Initially, the authors describe that Lifeact (an F-actin reporter), localizes to dynamic ring-shaped structures enriched at the apical surface of the ectoderm cells relative to the rest of the body. Inspired by the considerable contractile movements displayed by Hydra, they developed an experimental preparation where the head and the foot of the animals were amputated, the remaining body columns threaded onto filaments to constrain tissue movements along one plane. This made it possible to now visualize the actin-rich rings in live imaging and describe its lifecycle. Actin rings formed as an expanding ring (around 11 µm diameter) and constricted to a point and disappeared. This process took around 110s on average. The rings also accumulated externally added fluorescent dextran. These findings suggest that the ectoderm of Hydra exhibits constitutive macropinocytosis.

Hydra’s macropinocytosis is regulated by stretch-activated channels: Curiously, when the authors compared the numbers of macropinocytic cups in fixed, intact vs live but amputated animals they observed higher numbers in the latter. This, along with the presence of putative stretch-activated channels (TRP, Piezo) hinted towards a potential role of tissue mechanics. The authors treated amputated Hydra preparations with a broad spectrum stretch-activated channel gadolinium chloride (GdCl₃) and found no further increase in macropinocytosis indicating that it is already elevated to its maximum. Conversely, when authors used Jedi1 and Jedi2, drugs that activate Piezo1, it resulted in a huge depletion of macropinocytic cups. These results support the hypothesis that stretch-activated channels are involved in regulating macropinocytosis in Hydra.

Hydra’s macropinocytosis is inhibited by mechanical stretch: The authors also used an alternate way of stretching Hydra to directly test the effects of tissue stretch. When left alone after dissection, the fragments of the body column regenerate back to healthy Hydra. During this process, they form intermediate hollow tissue spheres which undergo cyclic swelling and rupturing. By injecting Hydra medium into their lumen, the authors were able to induce ‘spheroids’ to show enlarged apical surface area (planar ectodermal stretch). As expected, macropinocytic cups were depleted in inflated spheroids relative to their pre-inflation state. Furthermore, Ca2+ signalling that is known to increase in response to the mechanical stretch also showed a rise upon inflation.

What I liked about the preprint: The paper has leveraged two clever preparations that allows one to directly investigate/manipulate tissue mechanics directly which is hard to do in vivo. The relationship between membrane tension and vesicular trafficking pathways in single cells is being studied by several groups, but here they investigate the process in live animal models. Furthermore, using Hydra as an emerging model system of tissue mechanics regulation outside of regeneration is exciting. Constitutive macropinocytosis in the absence of growth factors is underappreciated and these model systems could help the field pin down molecules involved in macropinosome formation in a comprehensive fashion.

Future directions and questions for the authors:

Is there a role of constitutive macropinocytosis in Hydra during steady-state and during regeneration outside of tissue mechanics?
Did the authors investigate the presence of other endocytic pathways in Hydra and their role in this mechano-biochemical crosstalk?
Is the level of constitutive macropinocytosis proportional to the size of Hydra?
Was the temporal dynamics of macropinosome formation similar between isolated body column preparation and spheroid inflation approaches?
What happens to tissue mechanics when macropinocytosis is blocked directly using a Rac1 inhibitor or EIPA drug?

^[1] Thottacherry et al., “Mechanochemical Feedback Control of Dynamin Independent Endocytosis Modulates Membrane Tension in Adherent Cells”; Boulant et al., “Actin Dynamics Counteract Membrane Tension during Clathrin-Mediated Endocytosis.”

doi: https://doi.org/10.1242/prelights.31471

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Author's response

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Is there a role of constitutive macropinocytosis in Hydra during steady state and during regeneration outside of tissue mechanics?

This is the million-dollar question. Unfortunately, transiently inhibiting macropinocytosis yields no gross phenotypes, and, using a variety of strategies, we have yet to permanently inhibit macropinocytosis in Hydra, rendering long-term loss-of-function studies currently out of reach. Therefore, we can only speculate on what physiological role macropinocytosis is playing here. Given that Hydra exhibits predatory feeding behaviors and inhabits a relatively dilute freshwater environment, we suspect that macropinocytosis is unlikely to play a significant role in nutrient acquisition. Prior electron microscopy studies have revealed vacuoles—likely macropinosomes resulting from the process we describe—containing bacteria and fragments of Hydra’s proteinaceous outer layer (cuticle). These observations raise the possibility that this process may play a role in sampling or remodeling the microbiome, cuticle, or other contents on the animal’s body surface. Of course, given the influence of stretch on this process, we are also intrigued by the possibility that macropinocytosis may play a direct role in membrane tensioning, but much work remains to be done to establish this functional connection.

Did authors investigate the presence of other endocytic pathways in Hydra and their role in this mechano-biochemical crosstalk?

Given the power of this platform to directly visualize macropinocytosis in real time, we quickly narrowed in on this endocytic pathway and have not investigated potential interactions with additional pathways. That said, we hope that this work, along with the many questions it raises, can further highlight the strengths of Hydra as a system for dissecting the interactions between basic cell biological processes and tissue mechanics in vivo, and will encourage others to explore its wealth of uncharted biology.

Is the level of constitutive macropinocytosis proportional to the size of Hydra?

This is a fascinating question, but one that we have not explored in depth. For the sake of consistency, we sought to use intact Hydra or tissue fragments of comparable sizes throughout this study. Given similar rates of macropinocytosis in cells scattered across the body plan and the persistence of macropinocytosis even in regenerating tissues, we expect that levels of macropinocytosis likely scale with cell number across a range of body sizes, but this remains to be explored. Furthermore, whether levels of macropinocytosis are altered in stages and/or tissues in which mechanics may be globally or locally affected, for instance, at the site of new bud growth, is an interesting aspect worth investigating.

Was the temporal dynamics of macropinosome formation similar between isolated body column preparation and spheroid inflation approaches?

From cup formation to closure, the timing of macropinocytosis is generally comparable in isolated body columns and unperturbed spheroids. However, the technical limitations of our current inflation approach, which is applied only for a short duration (several minutes), preclude more nuanced quantification of the temporal dynamics over a range of inflated states; whether intermediate states of inflation may alter these dynamics, for instance, is an important question worthy of further investigation. We find that the natural swelling of spheroids, which has previously been reported, can similarly inhibit macropinocytosis, and the more gradual nature of this swelling may provide a practical inroad to address this question.

What happens to tissue mechanics when macropinocytosis is blocked directly using a Rac1 inhibitor or EIPA drug?

Unfortunately, we have had little success inhibiting macropinocytosis with a number of common inhibitors, including EIPA and NSC23766. Whether these observations point to interesting divergences in the underlying biology or mere technical limitations remains unclear. For instance, EIPA and NSC23766 have also been reported to be ineffective for inhibiting macropinocytosis in Dictyostelium, despite efficacy in mammalian cells. We hope that additional screening of potential inhibitors/activators will shed light on this matter and afford new insights into the physiological role for macropinocytosis in Hydra.

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