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Role of tau phosphorylation in formation of tau envelopes
Karhanová, Adéla ; Lánský, Zdeněk (advisor) ; Štěpánek, Luděk (referee)
Tau is an intrinsically-disordered microtubule-associated protein important for axonal development and a critical regulator of microtubule functions in axons. Tau activity is controlled by phosphorylation and its deregulation resulting in tau hyperphosphorylation and aggregation has been linked to multiple neurodegenerative disorders, collectively termed tauopathies. On microtubules, tau molecules segregate into two kinetically distinct phases, consisting of either independently diffusing molecules or interacting molecules that form cohesive "envelopes" around microtubules. Tau envelopes regulate the action of other microtubule-associated proteins, such as the motility of molecular motors, and protect microtubules against degradation by microtubule-severing enzymes. How the formation, dynamics, and function of tau envelopes are regulated, however, is unknown. Here we show that tau phosphorylation impedes the formation and functioning of protective tau envelopes. Using a combination of reconstitution experiments and live cell imaging, we show that phosphorylated tau incorporates into tau envelopes and that it slows down the envelope growth. Importantly, we demonstrate that phosphorylated tau also destabilizes already existing envelopes leading to their disassembly. Together, our results demonstrate...
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Regulation of microtubule dynamics revealed by single-molecule TIRF and IRM microscopy
Zhernov, Ilia ; Lánský, Zdeněk (advisor) ; Cifra, Michal (referee) ; Varga, Vladimír (referee)
The microtubular cytoskeleton is a ubiquitous and highly diverse biopolymer network present in all eukaryotic cells. Microtubules stochastically alternate between phases of growth and shrinkage. Cells take advantage of this dynamicity to generate forces for essential processes, such as cell division, motility or morphogenesis. Regulating the microtubule dynamics enables cells to adaptively respond to a wide range of tasks and conditions. Molecular mechanisms underpinning the regulation are not fully understood. Using a bottom-up approach and the combination of single molecule total internal reflection fluorescence (TIRF) microscopy and interference reflection microscopy (IRM), we here reconstituted and explored two dynamic cytoskeletal systems. (i) Microtubule doublets, comprising incomplete B-microtubule on the surface of a complete A- microtubule, provide an essential structural scaffold for flagella. Despite the fundamental role of microtubule doublets, the molecular mechanism governing their formation is unknown. We here demonstrate an inhibitory role of tubulin C-terminus in microtubule doublet assembly. By partial enzymatic digestion of polymerized microtubules followed by the addition of free tubulin in the presence of a stabilizing agent, we assembled microtubule doublets and revealed the B-...
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