Natascha Leijnse

Helical buckling of actin inside filopodia generates traction.

Significance Filopodia are essential membrane protrusions that facilitate cellular sensing and interaction with the environment. The mechanical properties of filopodia are crucial for their ability to push and pull on external objects and are attributed to actin dynamics. We confirm the presence of F-actin inside extended filopodia and reveal a new mechanism by which actin can exert traction forces on external objects. This mechanism is mediated by rotation and helical buckling, which cause shortening and retraction of the actin shaft. By imaging of F-actin and simultaneous force spectroscopy, we reveal and detail how force propagates through this spiral actin structure and show how torsional twist of the actin shaft is translated into a traction force at the filopodial tip. Cells can interact with their surroundings via filopodia, which are membrane protrusions that extend beyond the cell body. Filopodia are essential during dynamic cellular processes like motility, invasion, and cell–cell communication. Filopodia contain cross-linked actin filaments, attached to the surrounding cell membrane via protein linkers such as integrins. These actin filaments are thought to play a pivotal role in force transduction, bending, and rotation. We investigated whether, and how, actin within filopodia is responsible for filopodia dynamics by conducting simultaneous force spectroscopy and confocal imaging of F-actin in membrane protrusions. The actin shaft was observed to periodically undergo helical coiling and rotational motion, which occurred simultaneously with retrograde movement of actin inside the filopodium. The cells were found to retract beads attached to the filopodial tip, and retraction was found to correlate with rotation and coiling of the actin shaft. These results suggest a previously unidentified mechanism by which a cell can use rotation of the filopodial actin shaft to induce coiling and hence axial shortening of the filopodial actin bundle.

An updated look at actin dynamics in filopodia

Cells dynamically interact with and probe their environment by growing finger‐like structures named filopodia. The dynamics of filopodia are mainly caused by the actin rich core or shaft which sits inside the filopodial membrane and continuously undergoes changes like growth, shrinking, bending, and rotation. Recent experiments combining advanced imaging and manipulation tools have provided detailed quantitative data on the correlation between mechanical properties of filopodia, their molecular composition, and the dynamic architecture of the actin structure. These experiments have revealed how retrograde flow and twisting of the actin shaft within filopodia can generate traction on external substrates. Previously, the mechanism behind filopodial pulling was mainly attributed to retrograde flow of actin, but recent experiments have shown that rotational dynamics can also contribute to the traction force. Although force measurements have indicated a step‐like behavior in filopodial pulling, no direct evidence has been provided to link this behavior to a molecular motor like myosin. Therefore, the underlying biochemical and mechanical mechanisms behind filopodial force generation still remain to be resolved.

Membrane curvature regulates ligand-specific membrane sorting of GPCRs in living cells

The targeted spatial organization (sorting) of Gprotein-coupled receptors (GPCRs) is essential for their biological function and often takes place in highly curved membrane compartments such as filopodia, endocytic pits, trafficking vesicles or endosome tubules. However, the influence of geometrical membrane curvature on GPCR sorting remains unknown. Here we used fluorescence imaging to establish a quantitative correlation between membrane curvature and sorting of three prototypic class A GPCRs (the neuropeptide Y receptor Y2, the β1 adrenergic receptor and the β2 adrenergic receptor) in living cells. Fitting of a thermodynamic model to the data enabled us to quantify how sorting is mediated by an energetic drive to match receptor shape and membrane curvature. Curvature-dependent sorting was regulated by ligands in a specific manner. We anticipate that this curvature-dependent biomechanical coupling mechanism contributes to the sorting, trafficking and function of transmembrane proteins in general.

A cytoskeletal clutch mediates cellular force transmission in a soft, three-dimensional extracellular matrix

Quantitative analysis of the pairwise dynamics of the actin cytoskeleton, focal adhesions, and ECM fibrils reveals how cytoskeletal dynamics drive matrix deformation and cell motility for primary human fibroblasts embedded in a 3D fibrin matrix.

Dynamics of cancerous tissue correlates with invasiveness

Two of the classical hallmarks of cancer are uncontrolled cell division and tissue invasion, which turn the disease into a systemic, life-threatening condition. Although both processes are studied, a clear correlation between cell division and motility of cancer cells has not been described previously. Here, we experimentally characterize the dynamics of invasive and non-invasive breast cancer tissues using human and murine model systems. The intrinsic tissue velocities, as well as the divergence and vorticity around a dividing cell correlate strongly with the invasive potential of the tissue, thus showing a distinct correlation between tissue dynamics and aggressiveness. We formulate a model which treats the tissue as a visco-elastic continuum. This model provides a valid reproduction of the cancerous tissue dynamics, thus, biological signaling is not needed to explain the observed tissue dynamics. The model returns the characteristic force exerted by an invading cell and reveals a strong correlation between force and invasiveness of breast cancer cells, thus pinpointing the importance of mechanics for cancer invasion.

Dynamic buckling of actin within filopodia.

Abstract Filopodia are active tubular structures protruding from the cell surface which allow the cell to sense and interact with the surrounding environment through repetitive elongation-retraction cycles. The mechanical behavior of filopodia has been studied by measuring the traction forces exerted on external substrates.1 These studies have revealed that internal actin flow can transduce a force across the cell surface through transmembrane linkers like integrins. In addition to the elongation-retraction behavior filopodia also exhibit a buckling and rotational behavior. Filopodial buckling in conjunction with rotation enables the cell to explore a much larger 3-dimensional space and allows for more complex, and possibly stronger, interactions with the external environment.2 Here we focus on how bending of the filopodial actin dynamically correlates with pulling on an optically trapped microsphere which acts like an external substrate attached to the filopodial tip. There is a clear correlation between presence of actin near the tip and exertion of a traction force, thus demonstrating that the traction force is transduced along the actin shaft inside the filopodium. By extending a filopodium and holding it while measuring the cellular response, we also monitor and analyze the waiting times for the first buckle observed in the fluorescently labeled actin shaft.

Diffusion inside Living Human Cells

Naturally occurring lipid granules diffuse in the cytoplasm and can be used as tracers to map out the viscoelastic landscape inside living cells. Using optical trapping and single particle tracking we found that lipid granules exhibit anomalous diffusion inside human umbilical vein endothelial cells. For these cells the exact diffusional pattern of a particular granule depends on the physiological state of the cell and on the localization of the granule within the cytoplasm. Granules located close to the actin rich periphery of the cell move less than those located towards to the center of the cell or within the nucleus. Also, granules in cells which are stressed or have attached to a surface for a long period of time move in a more restricted fashion than within healthy cells. For granules diffusing in healthy cells, in regions away from the cell periphery, occurrences of weak ergodicity breaking are observed, similar to the recent observations inside living fission yeast cells [1].[1] J.-H. Jeon, V. Tejedor, S. Burov, E. Barkai, C. Selhuber-Unkel, K. Berg-Sorensen, L. B. Oddershede, and R. Metzler, Phys. Rev. Lett. 106, 048103 (2011).