Glycocalyx-mediated Cell Adhesion and Migration

Samuel Schmidt, Bettina Weigelin, Joost te Riet, Veronika te Boekhorst, Mariska te Lindert, Mietske Wijers-Rouw, Barbara Lelli, Lorenz Rognoni, Marina Krause-Vortmeyer, Anthea Messent, Luisa Bracci, Kay-Eberhard Gottschalk, Stephan Kissler, Martin J. Humphries, Dirk J. Lefeber, Jack Fransen, Peter Friedl

Posted on: 9 July 2020

Preprint posted on 13 June 2020

Getting a grip: a role for the glycocalyx

Selected by Mariana De Niz

Categories: biophysics, cancer biology, cell biology

Background

Cell shape, polarity, anchorage, and migration across surfaces, depend upon the function of integrin adhesion receptors, which form transient focalized actin- containing adhesion complexes that control cytoskeletal organization, mechano-transduction, and intracellular signaling. Little is known, however, about mechanisms mediating cell-substrate coupling and migration when integrin availability is low or absent, mediated by “friction”, physical intercalation, or alternative adhesion systems. In their work, Schmidt and colleagues aimed to identify the cellular and molecular mechanisms of integrin-independent cell-matrix interaction, force generation, and migration within collagen-rich interstitial tissue in vitro and in vivo when integrin-mediated adhesion is marginalized or absent (Figure 1).

Figure 1. The glycocalyx is implicated in mediating generic stickiness to support nanoscale interactions between the cell surface and ECM, in mechano-coupling, and in migration.

Key findings and developments

Mesenchymal-to-amoeboid transition after interference with integrins

Fibrillar collagen is the predominant extracellular matrix (ECM) structure in mammalian tissues, and is recognized with high affinity by various β1 integrins, and weakly by aVβ3. During migration through 3D fibrillar collagen, the vast majority of melanoma MV3 cells adopted an elongated, spindle-shaped morphology and were capable of migration. Interference with β1 and β3 integrins (either individually (β1) or dually (β1/ aVβ3)) vastly reduced migration speed but failed to achieve complete cell immobilization. Moreover, the spindle-shaped, elongated morphology converted to an ellipsoid cell shape with multiple dynamic blebs and occasional filopodia in contact with collagen fibrils. Equally, focalizations of β1 integrin and filamentous actin at contact sites to collagen fibers converted to diffuse distribution. These data suggest that metastatic melanoma cells transition from mesenchymal to amoeboid migration when integrin availability is limited.

To determine whether the same occurs in non-cancer cells, the authors used β1-deficientmurine embryonic fibroblasts (MEFs). They observed an amoeboid migration type with increased speed, directional persistence, and reduced cell elongation, suggesting overall that integrin deficiency did not compromise effective migration.

The authors went on to investigate motility in integrin-expressing but integrin-independent migrating Molt-4 human T lymphoma cells. These cells developed an amoeboid rounded shape, showed directional persistence, and were capable of migration at significant speed, despite interference with β1 and aVβ3 integrins. These results confirm for a 3D model, that moving cells possess integrin-independent interaction mechanisms with fibrillar collagen to maintain migration.

Alternative collagen receptors that might compensate for the loss of integrin-mediated adhesion and migration in melanoma MV3 cells were explored, but none appeared to play an important role in mediating amoeboid movement in MV3 cells. To determine if these observations remained valid in vivo, the authors injected MV3 cells with depleted integrins, into the deep dermis of nude mice and monitored them by intravital microscopy. Integrin targeting caused rounded morphology and blebbing movement in vivo. Overall, the authors conclude that lowering integrin availability in mesenchymal cells results in amoeboid movement irrespective of 3D environments in various in vitro setups, and in vivo.

Surface-glycan dependent cell migration

Additional to cell surface receptors, the surface glycocalyx can interact with proteins and other materials through carbohydrate-binding domains, or unclassified ionic and non-ionic bonds providing general stickiness. To explore whether low-affinity interactions of mammalian cells with collagen fibres could be mediated by the glycocalyx, the authors enzymatically removed from the surface of live cells (all models previously introduced), protein- and, possibly, lipid-linked glycoconjugates. This treatment resulted inunperturbed migration speeds, cell viability, and cytoskeletal activity compared to controls, when integrin systems were intact. However, a combination of glycan removal and interference with integrin expression severely compromised cell migration and persistence in 3D collagen lattices across cell models. Glycan removal immobilized all cells, and was accompanied by compromised directional persistence and cell elongation. In vivo, cells underwent near-complete migration arrest, but maintained oscillatory shape change, indicating unperturbed viability. The authors concluded that an intact surface glycocalyx is required to maintain amoeboid migration in collagen-rich tissue in vitro and in vivo when integrin functions are perturbed.

Glycan-mediated attachment forces in the pN range

The authors next explored whether the glycocalyx functions as adhesion system using atomic force spectroscopy and plasmon resonance detection. Besides background-level attachments (40-120 pN), amylose or cellulose also enabled stronger bonds. After engagement, bonds between amylose and monomeric collagen were stable for >15 min, irrespective of amylose concentration. The authors then went on to determine binding forces between the glycocalyx and fibrillar collagen by a live-cell strategy. They showthat the glycocalyx supports fast binding (MV3 cells) and slightly prolonged binding (Molt-4 cells) to collagen in the pN range independent of integrin-collagen interactions. They also show that the glycocalyx engages with substrates other than collagen, such as bovine serum albumin, and that binding occurs with similar forces.

Glycan-mediated interactions to collagen fibrils

Finally, the authors went on to explore the cell contact structures mediated by surface glycans towards collagen fibres, in control cells and upon limiting integrin and glycan availability. Cell morphology analysis of the various cell types used in the study, indicated that filopod-like and bleb-like protrusions observed in the different cell types, both depend on an intact glycocalyx. Scanning electron microscopy further showed that blebs in direct contact with a collagen fiber formed complex-shaped indentations, while contact-free blebs remained spherical and without indentation, and these indentations required an intact glycocalyx. Altogether, the data indicates a scaffold function of the glycocalyx, mediating grip-like membrane topologies towards irregular-shaped extracellular structures.

Overall, the data implicate the glycocalyx in mediating generic stickiness to support nanoscale interactions (nanogrips) between the cell surface and ECM, mechano-coupling, and migration.

What I like about this preprint

I think there has been a lot of effort aiming to understand cell adhesion and migration specifically in terms of protein receptor-ligand interactions. I found the work presented here very solid, exploring multiple details to great extent to understand glycocalyx-mediated cell adhesion and migration. I found the questions and the methods ‘out-of-the-box’.

Open questions

You mention in your introduction, that exploring integrin function in 2D and 3D environments results in different outcomes. You explain that 3D environments provide a complex geometry and confining interfaces. Could you expand a bit further on this seemingly well-established discrepancy?
A question as future perspective I am curious about is whether you think that there are other contributors to migration, beyond the integrins and the glycocalyx?
In in vivo situations, a lot has been explored in terms of cell migration based on cytokine/chemokine-mediated endothelial receptor up/down-regulation. Is there anything known on how the glycocalyx can be affected in homeostasis and disease conditions to influence the movement of cells within and across tissues?
Also referring to in vivo, the extracellular matrix varies across organs- is it your future perspective to better understand how the glycocalyx contributes to cell movement in specific niches of the mammalian body?
Beyond the molecular findings you have provided, are you interested in exploring further the biophysical component of the questions your finding raises?