Biology subjects

Sajko, S.

Publications and source records attributed to Sajko, S..

4 recordsLinked to original sources

bioRxiv2022

Endocytosis is required for access of surface-bound cargo to the flagellar pocket of trypanosomes

All endo- and exocytosis in the African trypanosome Trypanosoma brucei occurs at a single subdomain of the plasma membrane. This subdomain, the flagellar pocket, is a small vase-shaped invagination containing the root of the cells single flagellum. Several cytoskeleton-associated multiprotein complexes are coiled around the neck of the flagellar pocket on its cytoplasmic face. One of these, the hook complex, was proposed to affect macromolecule entry into the flagellar pocket lumen. In previous work, knockdown of the hook complex component TbMORN1 resulted in larger cargo being unable to enter the flagellar pocket. In this study, the hook complex component TbSmee1 was characterised in bloodstream form Trypanosoma brucei and was found to be essential for cell viability. TbSmee1 knockdown resulted in flagellar pocket enlargement and impaired access to the flagellar pocket membrane by surface-bound cargo, similar to depletion of TbMORN1. Unexpectedly, inhibition of endocytosis by knockdown of clathrin phenocopied TbSmee1 knockdown, suggesting that endocytic activity itself is a prerequisite for the entry of surface-bound cargo into the flagellar pocket. SummaryCharacterisation of the essential trypanosome protein TbSmee1 suggests that endocytosis is required for flagellar pocket access of surface-bound cargo.

cell biology↗

bioRxiv2020

Structural insights into F-actin regulation and sarcomere assembly via myotilin

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain containing protein myotilin provides structural integrity to Z-discs - the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and -actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. We used a combination of small angle X-ray scattering, cross-linking mass spectrometry, biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilins structural role in Z-discs. Significance StatementSarcomeres are the primary structural and functional unit of striated muscles, conferring movement in all animals. The Z-disk is the boundary between adjacent sarcomeres, where actin filaments (F-actin) are anchored. Z-disc protein myotilin, is a scaffold protein, which provides structural integrity to the Z-disc by multiple interactions to its central components, including F-actin and -actinin-2. Here we provide the structure of myotilin, revealing its structural plasticity in solution and the first integrative structural model of its complex with F-actin. We further show that myotilin displaces tropomyosin from F-actin, implying a novel role of myotilin in sarcomere biogenesis beyond being an interaction hub for Z-disk partners. Highlights Myotilin is structurally described as a dynamic ensemble Flanking regions enhance F-acting binding to tandem Ig domains Integrative structural model of myotilin bound to F-actin Myotilin displaces tropomyosin from F-actin, suggesting an organisational role in Z-disc

biochemistry↗

bioRxiv2019

Structures of three MORN repeat proteins and a re-evaluation of the proposed lipid-binding properties of MORN repeats

MORN (Membrane Occupation and Recognition Nexus) repeat proteins have a wide taxonomic distribution, being found in both prokaryotes and eukaryotes. Despite this ubiquity, they remain poorly characterised at both a structural and a functional level compared to other common repeats. In functional terms, they are often assumed to be lipid-binding modules that mediate membrane targeting. We addressed this putative activity by focusing on a protein composed solely of MORN repeats - Trypanosoma brucei MORN1. Surprisingly, no evidence for binding to membranes or lipid vesicles by TbMORN1 could be obtained either in vivo or in vitro. Conversely, TbMORN1 did interact with individual phospholipids. High- and low-resolution structures of the MORN1 protein from Trypanosoma brucei and homologous proteins from the parasites Toxoplasma gondii and Plasmodium falciparum were obtained using a combination of macromolecular crystallography, small-angle X-ray scattering, and electron microscopy. This enabled a first structure-based definition of the MORN repeat itself. Furthermore, all three structures dimerised via their C-termini in an antiparallel configuration. The dimers could form extended or V-shaped quaternary structures depending on the presence of specific interface residues. This work provides a new perspective on MORN repeats, showing that they are protein-protein interaction modules capable of mediating both dimerisation and oligomerisation.

biochemistry↗

bioRxiv2019

Using a novel web application for thermodynamic characterization of Tel22 G-quadruplex unfolding

Measuring and quantifying thermodynamic parameters that determine stability of and interactions between biological macromolecules is an essential and necessary complement to structural studies. Although several laboratories are able to obtain basic thermodynamic parameters for the observed process, the data interpretation and analysis quality of reported data can be extremely tedious. We have started to develop a web application that will help users to perform thermodynamic characterization of G-quadruplex unfolding. The application can perform global fitting of calorimetric and spectroscopic data, and it uses a three-state equilibrium model to obtain thermodynamic parameters for each transition step: the Gibbs energy, the enthalpy, and the heat capacity. As well as these, the application can define the number of K+ ions and the number of water molecules being released or taken up during the unfolding. To test our application, we used UV spectroscopy, circular dichroism, and differential scanning calorimetry, to monitor folding and unfolding of a model 22-nucleotide-long sequence of human 3-telomeric overhang, known as Tel22. The obtained data was fed to the web application and global fit revealed that unfolding of Tel22 involves at least one intermediate state, and that K+ ions are released during the unfolding, whereas water molecules are taken up.\n\nSTATEMENT OF SIGNIFICANCEThe laws of thermodynamics provide tools for the use of elegant mathematical expressions to describe stabilities and interactions of biological macromolecule. Even though thermodynamic profiles of simple transitions (e.g., two state) can be obtained in a relatively straightforward manner, performing thermodynamic analysis of complex/ multistep transitions or global analysis of several experimental data requires some experiences and skills. In the present study we are demonstrating how newly developed web application can be used to provide better understanding of driving forces responsible for the structural interconversion of G-quadruplex structures. We have tested this web application with experimental data obtained from monitoring thermal folding/ unfolding of the 5-AG3T2AG3T2AG3T2AG3-3 (Tel22) DNA sequence. We believe that this application can be used as a research and/or teaching tool, and it will allow comparisons of the thermodynamic parameters obtained between different laboratories.

biochemistry↗