|
|
Reorganization of in vitro reconstituted actin-based networks
Sabó, Ján ; Lánský, Zdeněk (advisor) ; Varga, Vladimír (referee) ; Cifra, Michal (referee)
The cytoskeleton provides living organisms with machinery to move. On the molecular scale, the same cytoskeletal components undergo constant reorganization to contribute to distinct cellular processes, such as the navigation of neuronal growth cones in brain development or the creation of chiral actin flows during cellular division. During brain development, neuronal growth cones navigate by employing cytoskeletal crosstalk between actin filament networks and microtubules. Crosstalk in the growth cones between these cytoskeletal components was linked with known microtubule polymerase human cytoskeleton associated protein 5 (CKAP5; homolog of XMAP215, MSPS, Zyg9), while the details of the direct molecular mechanism of CKAP5-provided remodeling of both microtubules and actin networks are lacking. Therefore, we used total internal reflection fluorescence (TIRF) microscopy together with state- of-the-art in vitro reconstituted assays combining microtubules, actin filament networks, and recombinant proteins to understand cytoskeleton-provided molecular mechanisms underlying cellular motion. In this thesis, we show that CKAP5 alone bundles both supposedly randomly oriented and parallel actin filaments, crosslinks actin filaments to microtubules regardless of their polarity, positions prevailing actin...
|
|
|
The role of CKAP5 in mediating crosstalk between actin and microtubule cytoskeleton
Dujava Ždímalová, Michaela ; Lánský, Zdeněk (advisor) ; Middelkoop, Teije Corneel (referee)
Dynamic changes in cytoskeletal architecture are essential for many crucial events throughout lives of all cells. One of the examples is neuronal pathfinding, ensured by specialized axonal structures enriched in actin and microtubule cytoskeleton, known as neuronal growth cones. Growth cones act as motile sensors and navigators, as the tight regulation of their actin and microtubule cytoskeleton results in directed axonal outgrowth guided towards the proper targets, which is crucial for processes such as synaptogegnesis or regeneration. Recently, protein CKAP5 (previously described as a microtubule + tip polymerase) has been proposed to facilitate actin-microtubule crosstalk in growth cones, indispensable for their proper functioning. Here, we combine the power of in vitro reconstitution assays with total internal reflection fluorescence microscopy to explore the underlying mechanism of CKAP5 actions. Our findings confirm that CKAP5 also associates with actin filaments and indeed, recruits them to the microtubule lattice. Furthermore, we describe a remarkable behavior of dynamic system containing CKAP5 and both, microtubules and actin filaments, wherein actin bundles are templated along dynamic microtubules by CKAP5. Importantly, upon microtubule depolymerization, the corresponding actin bundle can...
|
guest ::
