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Abstract

Clathrin-mediated endocytosis (CME) is one of the main uptake processes in cells. The biomechanical study of the formation of clathrin-coated structures has been attracting more attention since the first discovery of the clathrin protein more than 50 years ago. For CME to occur, adhesion energy between the cell and cargo must overcome an energy barrier produced by turgor pressure, membrane tension, and bending energy. While the molecular force mechanism of CME is relatively better understood, the role of cellular forces by rigidity sensing in supporting CME from the ventral side is less studied. My doctoral thesis aimed to elucidate how the cellular adhesion forces to the extracellular matrix (ECM) contribute to the CME of nanoparticles at the cellular ventral side. The experimental setup entails the immobilization of nanoparticles on substrates coated with an ECM protein such as fibronectin. Next, cells expressing CME adaptor protein 2 (AP2) tagged with e-GFP (i.e., MEF, HeLa) were seeded on the substrate and were subsequently analyzed at around 4 hours post-seeding. Traction force microscopy (TFM) was used to quantify the ability of cells to generate traction forces. As TFM requires a substrate that can be deformed by adherent cells, I described in the first part the preparation of elastic polyacrylamide (PA)/viscoelastic polydimethylsiloxane (PDMS) substrates and their mechanical characterization using nanoindentation experiments. Additionally, I investigated the response of materials upon the exposure to ultraviolet light (both PA and PDMS) and oxygen plasma (only PDMS elastomer). Next, I described the surface patterning on the substrate, including micropatterning (both PA and PDMS) using the maskless photolithography method and nanopatterning (only PA hydrogel) using the block-copolymer micellar nanolithography (BCMN) method. Finally, I described the development of an integrated bioengineering toolbox that includes a novel method called ”Local Ultraviolet Illumination Traction Force Microscopy (LUVI-TFM)” that could be combined with micro- and nanopatterned sub- strates to perform 2D- and 2.5D-TFM on a single cell or multicellular clusters. This toolbox was devised so users could choose a single tool or a combination of multiple tools to study cell-matrix interaction. In the second part, I described the use of the bioengineering tools established in the previous part to study the role of traction forces in initiating CME of nanoparticles at the cellular ventral side. First, I observed that electrostatically immobilized nanoparticles were removed under cellular traction forces by using live 2D-LUVI-TFM. However, it was unclear if traction forces caused the particle removal. To elucidate it, I employed micropatterning to control the size and shape, thereby the traction force hotspots of a single cell. I could also immobilize nanoparticles in regions where the cell exerted high traction forces. On micropatterned cells, I observed a low spatial correlation between the clathrin adaptor protein 2 (AP2) clusters and focal adhesion marker clusters (paxillin, β1 integrin, and β3 integrin). Furthermore, I observed that traction forces alone were insufficient to remove nanoparticles from the surface. Despite no removal, I observed immobilized nanoparticles initiated CME in high traction force regions. Concerning the role of traction force in initiating CME of nanoparticles at the cellular ventral side, I focused on cells that were transitioning between the early adhesion phase and the initial contractility at around 4 hours post-seeding. In this phase, cells exhibited the shape of a symmetric ”bulls-eye egg” and were easier to be compared. I observed that AP2 clusters were highly distributed at the cell periphery, irrespective of the presence of covalently immobilized nanoparticles and substrate rigidity. Furthermore, I observed no significant difference between cells growing on the 3 kPa substrate and the glass concerning the cluster area and the signal lifetime of AP2. Lastly, I observed that the cell pushed down the substrate at the cell periphery, and AP2 clusters were found highly distributed inside the indentation area. Eventually, AP2 clusters were recruited above covalently immobilized nanoparticles in the indentation area. This result strongly suggested that the normal component of traction forces plays an undeniably important role in initiating CME of nanoparticles at the cellular ventral side.