Cell motility and cytoskeleton dynamics play a fundamental role in many biological events, including embryonic development, wound healing and immune responses, such as phagocytosis and T-cell activation. Regulation of actin cytoskeleton remodelling depends on the activities of several proteins that co-ordinate events, such as actin filament nucleation, elongation, capping and cross-linking, both in space and time. Our studies aim at unravelling molecular mechanisms underlying cytoskeleton-dependent processes and the impact of biomaterials on them.
Impact of LSP1 on the Regulation of Actin-based Processes
We have characterised the impact of the actin-associated protein leukocyte-specific protein 1 (LSP1) on Fcγ receptor-driven phagocytosis. LSP1 localises to nascent phagocytic cups and displays the same spatial and temporal distribution as the actin cytoskeleton. Down regulation of LSP1 severely reduced the phagocytic activity of macrophages (Maxeiner et al., 2015). LSP1 binds to SH3 domain of the molecular motor myosin1e and both proteins co-localise at nascent phagocytic cups. Thus, the LSP1-myosin1e bi-molecular complex plays a pivotal role in the regulation of actin cytoskeleton remodelling during Fcγ receptor-driven phagocytosis.
Dynamics of myosin1e and LSP1 during Fcγ receptor–mediated phagocytosis. Note the simultaneous accumulation of GFP-myosin1e and RFP-LSP1 around opsonized beads (arrows; asterisks indicate beads). Lower panels show corresponding phase contrast images. Numbers represent elapsed time in seconds. Scale bar, 5 μm.
Schematic representation of the role of LSP1 and myosin1e in receptor–mediated phagocytosis. Engagement of Fcγ receptors leads to LSP1 recruitment that will interact with myosin1e at nascent phagocytic cups. LSP1 and myosin1e work in concert to coordinate actin assembly, lipid transport, and actin–membrane linkage, finally leading to efficient phagocytosis.
Recognition, Segmentation and Tracking of Focal Adhesions
Cell adhesion is a highly coordinated process and its understanding requires the precise analysis of focal adhesions (FAs) turnover and of the relative occupancy of the individual components present in FAs. We have developed a dedicated computational approach for the recognition, segmentation and tracking of FAs (Würflinger et al. 2011). Our algorithm can also corrects segmentation errors that may arise from the analysis of poorly defined FAs. By achieving accurate and consistent FA segmentation and tracking, our work establishes the basis for a comprehensive analysis of FA dynamics under various experimental regimes and the future development of mathematical models that simulate FA behaviour.
Segmentation of individual focal adhesions in clusters (1-8) results in optimal separation of single focal adhesions (A). Geometry-based criterion for focal adhesion merging, which takes into account distance, overlap and relative orientation of adjacent focal adhesions (B).
Synthetic and natural biomaterials play a central role in therapeutic strategies aimed at directing cell behaviour and function. The guidance provided by biomaterials is exerted at multiple levels. For instance, cells sense biomaterial topography and respond by regulating cellular processes such as adhesion and migration. We focus on understanding the molecular mechanisms underlying the impact of biomaterials on cell motility and adhesion.
We study the impact of polymeric fibres composed of PLA or PLA/PEG blends on DC function (Paschoalin et al., 2017; in collaboration with L. Mattoso, EMBRAPA, São Carlos, Brazil). DC interact with fibres resulting in the accumulation of actin and vinculin at sites of cell-fibre interactions. Moreover, DC movement was guided along fibres and actin and zyxin showed a highly dynamic behaviour at cell-fibre interfaces. Importantly, fibres did not elicit DC activation, which them particularly attractive for biomedical applications (Paschoalin et al., 2017).
Current studies also employ surface-grafted microgels arrays to specifically direct cell adhesion and migration and study the molecular basis underlying these processes (Sechi et al., 2016; in collaboration with A. Pich, DWI Leibniz-Institute for Interactive Materials, RWTH Aachen University, Aachen, Germany). Microgel array spacing and stiffness serve as an effective tool to modulate cell adhesion and motility. Thus, microgels could be used as precise and tuneable systems to understand and control cell migration in the context, for instance, wound healing and tissue regeneration.
Schematic representation of solution blow spinning (SBS) for production of fibres and fibre interaction with immature dendritic cells (iDC, Paschoalin et al., 2017).
|Solution blow spinning fibres: New immunologically inert substrates for the analysis of cell adhesion and motility.|
Paschoalin, R.T., Traldi, B., Aydin, G., Oliveira, J.E., Rütten, S., Mattoso, L.H.C., Zenke, M. and Sechi, A. (2017).
Acta Biomaterialia, 51: 161-174. doi: 10.1016/j.actbio.2017.01.020.
|Surface-grafted nanogel arrays direct cell adhesion and motility.|
Sechi, A, Freitas, J, Wünnemann, P, Töpel, A, Paschoalin, RT, Ullmann, S, Schröder, R, Aydin, G, Rütten, S, Böker, A, Zenke, M and Pich, A. (2016).
Adv Mater Int, 1600455, doi: 10.1002/admi.201600455.
|Crucial role for the LSP1-myosin1e bi-molecular complex in the regulation of Fcγ receptor-driven phagocytosis.|
Maxeiner, S., Shi, N., Schalla, C., Aydin, G., Hoss, M., Vogel, S., Zenke, M., and Sechi, A. S. (2015).
Mol. Biol. Cell 26, 1652-1664.
|Automated segmentation and tracking for large scale analysis of focal adhesion dynamics.|
Würflinger, T., Gamper, I., Aach, T., and Sechi, A. S. (2011).
J. Microsc. 241, 37-53.
|The role of multiple Toll-like receptor signalling cascades on interactions between biomedical polymers and dendritic cells.|
Shokouhi, B., Coban, C., Hasirci, V., Aydin, E., Dhanasingh, A., Shi, N., Koyama, S., Akira, S., Zenke, M., and Sechi, A. S. (2010).
Biomaterials 31, 5759-5771.
|Listeria monocytogenes exploits ERM protein functions to efficiently spread from cell-to-cell.|
Pust, S., Morrison, H., Wehland, J., Sechi, A. S., and Herrlich, P. (2005).
EMBO J. 24, 1287-1300.
|Interplay between TCR signalling and actin cytoskeleton dynamics.|
Sechi, A. S. and Wehland, J. (2004).
Trends Immunol. 25, 257-265.
|Crucial role for profilin:actin in the intracellular motility of Listeria monocytogenes.|
Grenklo, S., Geese, M., Lindberg, U., Wehland, J., Karlsson, R., and Sechi, A. S. (2003).
EMBO Reports 4, 523-529.
|The actin cytoskeleton and plasma membrane connection: PtdIns(4,5)P2 influences cytoskeletal protein activity at the plasma membrane.|
Sechi, A. S., and Wehland, J. (2000).
J. Cell Sci. 113, 3685-3695.
|The isolated comet tail pseudopodium of Listeria monocytogenes: a tail of two actin filaments populations; long and axial and short and random.|
Sechi, A. S., Wehland, J. and Small, J. V. (1997)
|J. Cell Biol. 137, 155-167.||Abstract | Full|