TY - THES AB - Plants maintain the capacity to develop new organs e.g. lateral roots post-embryonically throughout their whole life and thereby flexibly adapt to ever-changing environmental conditions. Plant hormones auxin and cytokinin are the main regulators of the lateral root organogenesis. Additionally to their solo activities, the interaction between auxin and cytokinin plays crucial role in fine-tuning of lateral root development and growth. In particular, cytokinin modulates auxin distribution within the developing lateral root by affecting the endomembrane trafficking of auxin transporter PIN1 and promoting its vacuolar degradation (Marhavý et al., 2011, 2014). This effect is independent of transcription and translation. Therefore, it suggests novel, non-canonical cytokinin activity occuring possibly on the posttranslational level. Impact of cytokinin and other plant hormones on auxin transporters (including PIN1) on the posttranslational level is described in detail in the introduction part of this thesis in a form of a review (Semeradova et al., 2020). To gain insights into the molecular machinery underlying cytokinin effect on the endomembrane trafficking in the plant cell, in particular on the PIN1 degradation, we conducted two large proteomic screens: 1) Identification of cytokinin binding proteins using chemical proteomics. 2) Monitoring of proteomic and phosphoproteomic changes upon cytokinin treatment. In the first screen, we identified DYNAMIN RELATED PROTEIN 2A (DRP2A). We found that DRP2A plays a role in cytokinin regulated processes during the plant growth and that cytokinin treatment promotes destabilization of DRP2A protein. However, the role of DRP2A in the PIN1 degradation remains to be elucidated. In the second screen, we found VACUOLAR PROTEIN SORTING 9A (VPS9A). VPS9a plays crucial role in plant’s response to cytokin and in cytokinin mediated PIN1 degradation. Altogether, we identified proteins, which bind to cytokinin and proteins that in response to cytokinin exhibit significantly changed abundance or phosphorylation pattern. By combining information from these two screens, we can pave our way towards understanding of noncanonical cytokinin effects. AU - Semerádová, Hana ID - 10135 SN - 2663-337X TI - Molecular mechanisms of the cytokinin-regulated endomembrane trafficking to coordinate plant organogenesis ER - TY - THES AB - Most real-world flows are multiphase, yet we know little about them compared to their single-phase counterparts. Multiphase flows are more difficult to investigate as their dynamics occur in large parameter space and involve complex phenomena such as preferential concentration, turbulence modulation, non-Newtonian rheology, etc. Over the last few decades, experiments in particle-laden flows have taken a back seat in favour of ever-improving computational resources. However, computers are still not powerful enough to simulate a real-world fluid with millions of finite-size particles. Experiments are essential not only because they offer a reliable way to investigate real-world multiphase flows but also because they serve to validate numerical studies and steer the research in a relevant direction. In this work, we have experimentally investigated particle-laden flows in pipes, and in particular, examined the effect of particles on the laminar-turbulent transition and the drag scaling in turbulent flows. For particle-laden pipe flows, an earlier study [Matas et al., 2003] reported how the sub-critical (i.e., hysteretic) transition that occurs via localised turbulent structures called puffs is affected by the addition of particles. In this study, in addition to this known transition, we found a super-critical transition to a globally fluctuating state with increasing particle concentration. At the same time, the Newtonian-type transition via puffs is delayed to larger Reynolds numbers. At an even higher concentration, only the globally fluctuating state is found. The dynamics of particle-laden flows are hence determined by two competing instabilities that give rise to three flow regimes: Newtonian-type turbulence at low, a particle-induced globally fluctuating state at high, and a coexistence state at intermediate concentrations. The effect of particles on turbulent drag is ambiguous, with studies reporting drag reduction, no net change, and even drag increase. The ambiguity arises because, in addition to particle concentration, particle shape, size, and density also affect the net drag. Even similar particles might affect the flow dissimilarly in different Reynolds number and concentration ranges. In the present study, we explored a wide range of both Reynolds number and concentration, using spherical as well as cylindrical particles. We found that the spherical particles do not reduce drag while the cylindrical particles are drag-reducing within a specific Reynolds number interval. The interval strongly depends on the particle concentration and the relative size of the pipe and particles. Within this interval, the magnitude of drag reduction reaches a maximum. These drag reduction maxima appear to fall onto a distinct power-law curve irrespective of the pipe diameter and particle concentration, and this curve can be considered as the maximum drag reduction asymptote for a given fibre shape. Such an asymptote is well known for polymeric flows but had not been identified for particle-laden flows prior to this work. AU - Agrawal, Nishchal ID - 9728 KW - Drag Reduction KW - Transition to Turbulence KW - Multiphase Flows KW - particle Laden Flows KW - Complex Flows KW - Experiments KW - Fluid Dynamics SN - 2663-337X TI - Transition to turbulence and drag reduction in particle-laden pipe flows ER - TY - JOUR AB - Biological membranes can dramatically accelerate the aggregation of normally soluble protein molecules into amyloid fibrils and alter the fibril morphologies, yet the molecular mechanisms through which this accelerated nucleation takes place are not yet understood. Here, we develop a coarse-grained model to systematically explore the effect that the structural properties of the lipid membrane and the nature of protein–membrane interactions have on the nucleation rates of amyloid fibrils. We identify two physically distinct nucleation pathways—protein-rich and lipid-rich—and quantify how the membrane fluidity and protein–membrane affinity control the relative importance of those molecular pathways. We find that the membrane’s susceptibility to reshaping and being incorporated into the fibrillar aggregates is a key determinant of its ability to promote protein aggregation. We then characterize the rates and the free-energy profile associated with this heterogeneous nucleation process, in which the surface itself participates in the aggregate structure. Finally, we compare quantitatively our data to experiments on membrane-catalyzed amyloid aggregation of α-synuclein, a protein implicated in Parkinson’s disease that predominately nucleates on membranes. More generally, our results provide a framework for understanding macromolecular aggregation on lipid membranes in a broad biological and biotechnological context. AU - Krausser, Johannes AU - Knowles, Tuomas P. J. AU - Šarić, Anđela ID - 10336 IS - 52 JF - Proceedings of the National Academy of Sciences SN - 0027-8424 TI - Physical mechanisms of amyloid nucleation on fluid membranes VL - 117 ER - TY - JOUR AB - The blood-brain barrier is made of polarized brain endothelial cells (BECs) phenotypically conditioned by the central nervous system (CNS). Although transport across BECs is of paramount importance for nutrient uptake as well as ridding the brain of waste products, the intracellular sorting mechanisms that regulate successful receptor-mediated transcytosis in BECs remain to be elucidated. Here, we used a synthetic multivalent system with tunable avidity to the low-density lipoprotein receptor–related protein 1 (LRP1) to investigate the mechanisms of transport across BECs. We used a combination of conventional and super-resolution microscopy, both in vivo and in vitro, accompanied with biophysical modeling of transport kinetics and membrane-bound interactions to elucidate the role of membrane-sculpting protein syndapin-2 on fast transport via tubule formation. We show that high-avidity cargo biases the LRP1 toward internalization associated with fast degradation, while mid-avidity augments the formation of syndapin-2 tubular carriers promoting a fast shuttling across. AU - Tian, Xiaohe AU - Leite, Diana M. AU - Scarpa, Edoardo AU - Nyberg, Sophie AU - Fullstone, Gavin AU - Forth, Joe AU - Matias, Diana AU - Apriceno, Azzurra AU - Poma, Alessandro AU - Duro-Castano, Aroa AU - Vuyyuru, Manish AU - Harker-Kirschneck, Lena AU - Šarić, Anđela AU - Zhang, Zhongping AU - Xiang, Pan AU - Fang, Bin AU - Tian, Yupeng AU - Luo, Lei AU - Rizzello, Loris AU - Battaglia, Giuseppe ID - 10342 IS - 48 JF - Science Advances KW - multidisciplinary SN - 2375-2548 TI - On the shuttling across the blood-brain barrier via tubule formation: Mechanism and cargo avidity bias VL - 6 ER - TY - JOUR AB - In this study, we investigate the role of the surface patterning of nanostructures for cell membrane reshaping. To accomplish this, we combine an evolutionary algorithm with coarse-grained molecular dynamics simulations and explore the solution space of ligand patterns on a nanoparticle that promote efficient and reliable cell uptake. Surprisingly, we find that in the regime of low ligand number the best-performing structures are characterized by ligands arranged into long one-dimensional chains that pattern the surface of the particle. We show that these chains of ligands provide particles with high rotational freedom and they lower the free energy barrier for membrane crossing. Our approach reveals a set of nonintuitive design rules that can be used to inform artificial nanoparticle construction and the search for inhibitors of viral entry. AU - Forster, Joel C. AU - Krausser, Johannes AU - Vuyyuru, Manish R. AU - Baum, Buzz AU - Šarić, Anđela ID - 10344 IS - 22 JF - Physical Review Letters SN - 0031-9007 TI - Exploring the design rules for efficient membrane-reshaping nanostructures VL - 125 ER - TY - JOUR AB - Tracing the motion of macromolecules, viruses, and nanoparticles adsorbed onto cell membranes is currently the most direct way of probing the complex dynamic interactions behind vital biological processes, including cell signalling, trafficking, and viral infection. The resulting trajectories are usually consistent with some type of anomalous diffusion, but the molecular origins behind the observed anomalous behaviour are usually not obvious. Here we use coarse-grained molecular dynamics simulations to help identify the physical mechanisms that can give rise to experimentally observed trajectories of nanoscopic objects moving on biological membranes. We find that diffusion on membranes of high fluidities typically results in normal diffusion of the adsorbed nanoparticle, irrespective of the concentration of receptors, receptor clustering, or multivalent interactions between the particle and membrane receptors. Gel-like membranes on the other hand result in anomalous diffusion of the particle, which becomes more pronounced at higher receptor concentrations. This anomalous diffusion is characterised by local particle trapping in the regions of high receptor concentrations and fast hopping between such regions. The normal diffusion is recovered in the limit where the gel membrane is saturated with receptors. We conclude that hindered receptor diffusivity can be a common reason behind the observed anomalous diffusion of viruses, vesicles, and nanoparticles adsorbed on cell and model membranes. Our results enable direct comparison with experiments and offer a new route for interpreting motility experiments on cell membranes. AU - Debets, V. E. AU - Janssen, L. M. C. AU - Šarić, Anđela ID - 10341 IS - 47 JF - Soft Matter KW - condensed matter physics KW - general chemistry SN - 1744-683X TI - Characterising the diffusion of biological nanoparticles on fluid and cross-linked membranes VL - 16 ER - TY - JOUR AB - One of the most robust examples of self-assembly in living organisms is the formation of collagen architectures. Collagen type I molecules are a crucial component of the extracellular matrix, where they self-assemble into fibrils of well-defined axial striped patterns. This striped fibrillar pattern is preserved across the animal kingdom and is important for the determination of cell phenotype, cell adhesion, and tissue regulation and signaling. The understanding of the physical processes that determine such a robust morphology of self-assembled collagen fibrils is currently almost completely missing. Here, we develop a minimal coarse-grained computational model to identify the physical principles of the assembly of collagen-mimetic molecules. We find that screened electrostatic interactions can drive the formation of collagen-like filaments of well-defined striped morphologies. The fibril axial pattern is determined solely by the distribution of charges on the molecule and is robust to the changes in protein concentration, monomer rigidity, and environmental conditions. We show that the striped fibrillar pattern cannot be easily predicted from the interactions between two monomers but is an emergent result of multibody interactions. Our results can help address collagen remodeling in diseases and aging and guide the design of collagen scaffolds for biotechnological applications. AU - Hafner, Anne E. AU - Gyori, Noemi G. AU - Bench, Ciaran A. AU - Davis, Luke K. AU - Šarić, Anđela ID - 10346 IS - 9 JF - Biophysical Journal KW - biophysics SN - 0006-3495 TI - Modeling fibrillogenesis of collagen-mimetic molecules VL - 119 ER - TY - JOUR AB - The misfolding and aberrant aggregation of proteins into fibrillar structures is a key factor in some of the most prevalent human diseases, including diabetes and dementia. Low molecular weight oligomers are thought to be a central factor in the pathology of these diseases, as well as critical intermediates in the fibril formation process, and as such have received much recent attention. Moreover, on-pathway oligomeric intermediates are potential targets for therapeutic strategies aimed at interrupting the fibril formation process. However, a consistent framework for distinguishing on-pathway from off-pathway oligomers has hitherto been lacking and, in particular, no consensus definition of on- and off-pathway oligomers is available. In this paper, we argue that a non-binary definition of oligomers' contribution to fibril-forming pathways may be more informative and we suggest a quantitative framework, in which each oligomeric species is assigned a value between 0 and 1 describing its relative contribution to the formation of fibrils. First, we clarify the distinction between oligomers and fibrils, and then we use the formalism of reaction networks to develop a general definition for on-pathway oligomers, that yields meaningful classifications in the context of amyloid formation. By applying these concepts to Monte Carlo simulations of a minimal aggregating system, and by revisiting several previous studies of amyloid oligomers in light of our new framework, we demonstrate how to perform these classifications in practice. For each oligomeric species we obtain the degree to which it is on-pathway, highlighting the most effective pharmaceutical targets for the inhibition of amyloid fibril formation. AU - Dear, Alexander J. AU - Meisl, Georg AU - Šarić, Anđela AU - Michaels, Thomas C. T. AU - Kjaergaard, Magnus AU - Linse, Sara AU - Knowles, Tuomas P. J. ID - 10350 IS - 24 JF - Chemical Science KW - general chemistry SN - 2041-6520 TI - Identification of on- and off-pathway oligomers in amyloid fibril formation VL - 11 ER - TY - JOUR AB - Sulfolobus acidocaldarius is the closest experimentally tractable archaeal relative of eukaryotes and, despite lacking obvious cyclin-dependent kinase and cyclin homologs, has an ordered eukaryote-like cell cycle with distinct phases of DNA replication and division. Here, in exploring the mechanism of cell division in S. acidocaldarius, we identify a role for the archaeal proteasome in regulating the transition from the end of one cell cycle to the beginning of the next. Further, we identify the archaeal ESCRT-III homolog, CdvB, as a key target of the proteasome and show that its degradation triggers division by allowing constriction of the CdvB1:CdvB2 ESCRT-III division ring. These findings offer a minimal mechanism for ESCRT-III–mediated membrane remodeling and point to a conserved role for the proteasome in eukaryotic and archaeal cell cycle control. AU - Tarrason Risa, Gabriel AU - Hurtig, Fredrik AU - Bray, Sian AU - Hafner, Anne E. AU - Harker-Kirschneck, Lena AU - Faull, Peter AU - Davis, Colin AU - Papatziamou, Dimitra AU - Mutavchiev, Delyan R. AU - Fan, Catherine AU - Meneguello, Leticia AU - Arashiro Pulschen, Andre AU - Dey, Gautam AU - Culley, Siân AU - Kilkenny, Mairi AU - Souza, Diorge P. AU - Pellegrini, Luca AU - de Bruin, Robertus A. M. AU - Henriques, Ricardo AU - Snijders, Ambrosius P. AU - Šarić, Anđela AU - Lindås, Ann-Christin AU - Robinson, Nicholas P. AU - Baum, Buzz ID - 10349 IS - 6504 JF - Science KW - multidisciplinary SN - 0036-8075 TI - The proteasome controls ESCRT-III–mediated cell division in an archaeon VL - 369 ER - TY - JOUR AB - Understanding the mechanism of action of compounds capable of inhibiting amyloid-fibril formation is critical to the development of potential therapeutics against protein-misfolding diseases. A fundamental challenge for progress is the range of possible target species and the disparate timescales involved, since the aggregating proteins are simultaneously the reactants, products, intermediates, and catalysts of the reaction. It is a complex problem, therefore, to choose the states of the aggregating proteins that should be bound by the compounds to achieve the most potent inhibition. We present here a comprehensive kinetic theory of amyloid-aggregation inhibition that reveals the fundamental thermodynamic and kinetic signatures characterizing effective inhibitors by identifying quantitative relationships between the aggregation and binding rate constants. These results provide general physical laws to guide the design and optimization of inhibitors of amyloid-fibril formation, revealing in particular the important role of on-rates in the binding of the inhibitors. AU - Michaels, Thomas C. T. AU - Šarić, Anđela AU - Meisl, Georg AU - Heller, Gabriella T. AU - Curk, Samo AU - Arosio, Paolo AU - Linse, Sara AU - Dobson, Christopher M. AU - Vendruscolo, Michele AU - Knowles, Tuomas P. J. ID - 10347 IS - 39 JF - Proceedings of the National Academy of Sciences KW - multidisciplinary SN - 0027-8424 TI - Thermodynamic and kinetic design principles for amyloid-aggregation inhibitors VL - 117 ER -