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.

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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).

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Impact of Biomaterials on Dendritic Cell Function

Biomaterials play a central role in therapeutic strategies aimed at directing cell behavior and functions such as immune response and cell migration. In our studies, we focus on the molecular mechanisms underlying the impact of biomaterials on dendritic cell (DC) function and cell motility and adhesion.

We have found that DC can sense biomedical polymers through multiple TLR/MyD88-dependent signaling pathways. TLR-biomaterial interactions induce the expression of activation markers and pro-inflammatory cytokines and are sufficient to activate DC (Shokouhi et al., 2010). TLR-biomaterial interaction also profoundly alters DC cell morphology and distribution of podosomes (Shokouhi et al., 2010), suggesting a direct involvement of the actin cytoskeleton.

We currently study the impact of polymeric fibers composed of PLA or PLA/PEG blends on DC function. DC easily interact with fibers resulting in the accumulation of actin and vinculin at sites of cell-fiber interactions. Moreover, DC movement was guided along SBS fibers and actin and zyxin showed a highly dynamic behavior at cell-fiber interfaces (Paschoalin et al., 2017; in collaboration with L. Mattoso, EMBRAPA, São Carlos, Brazil). Importantly, SBS fibers did not elicit any substantial increase of activation markers and inflammatory cytokines in DC (Paschoalin et al., 2017).

Current studies also employ nanostructured hydrogel materials 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).

Potential role of DC in the development of the foreign body reaction (FBR). DC-biomaterial interaction leads to alterations of surface markers, adhesion and cytokine secretion in DC.  At implantation sites, stimulated DC may directly affect the immune system homeostasis, thus initiating or sustaining the events leading to FBR and implant malfunction. FBR could also be the consequence of an altered function of macrophages, neutrophils and T cells induced by stimulated DC.

 

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Selected Publications 

ITIH5 mediates epigenetic reprogramming of breast cancer cells.
Rose, M., Kloten, V., Noetzel, E., Gola, L., Ehling, J., Heide, T., Meurer, S.K., Gaiko-Shcherbak, A., Sechi, A.S., Huth, S., Weiskirchen, R., Klaas, O., Antonopoulos, W., Lin, Q., Wagner, W., Veeck, J., Gremse, F., Steitz, J., Knüchel, R. and Dahl, E. (2017).
Mol Cancer, doi: 10.1186/s12943-017-0610-2.
 
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 contribution of Ena/VASP proteins to the intracellular motility of Listeria requires phosphorylation and the proline-rich core but not F-actin binding or multimerisation.
Geese, M, Loureiro, J. J., Bear, J. E., Wehland, J., Gertler, F. B., and Sechi, A. S. (2002).
Mol. Biol. Cell. 13, 2383-2396.
 
Evidence for a molecular complex consisting of Fyb/SLAP, SLP-76, Nck, VASP and WASP that links the actin cytoskeleton to Fcγ receptor signalling during phagocytosis.
Coppolino, M. G., Krause, M., Hagendorff, P., Monner, D. A., Trimble, W., Grinstein, S., Wehland, J., and Sechi A. S. (2001).
J. Cell Sci. 114, 4307-4318.
 
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 Arp2/3 complex is essential for the actin-based motility of Listeria monocytogenes.
May, R. C., Hall, M. E., Higgs, H. N., Pollard, T. D., Chakraborty, T., Wehland, J., Machesky, L. M., and Sechi A. S. (1999).
Curr. Biol. 9, 759-762.
 
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