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Identifier 000443470
Title Investigating the link between phase separation in RBP1 and its response to stress (Chapter I.). Unraveling the role of Kin7.3 in blue light signaling (Chapter II.)
Alternative Title Διερεύνηση της σχέσης της RBP1 με την αλλαγή φάσης σε συνθήκες καταπόνησης (Κεφάλαιο Ι.). Ο ρόλος της Κινεσίνης 7.3 στην απόκριση στο μπλε φως (Κεφάλαιο ΙΙ.)
Author Κατίδου, Βασιλική Ν.
Thesis advisor Μόσχου, Παναγιώτης
Reviewer Καλαντίδης, Κρίτων
Κοκκινίδης, Μιχαήλ
Abstract Chapter I: Plant signaling responses can be triggered by diverse forms of abiotic stress, which are increasingly escalated by climate change. Therefore, deciphering how plants perceive and react to stress is becoming a highly necessary field of study. NtRBP1, a previously suggested to phaseseparate in heat stress glycine-rich protein, provides a possible link between stress response and phase separation. The aim of my thesis was to investigate the properties of RBP1 that drive phase separation in stress response, employing (i) an in silico approach to assess its phase separation propensity and to identify possible post-translational modifications or disorder-to-order substitutions that disturb biomolecular condensation, (ii) a site-directed mutagenesis approach for the in vitro characterization of these predictions and (iii) a cell biology approach to uncover how phase separation is functionally linked to stress response and how it affects subcellular localization. My results showed that the predicted SUMOylation-deficient mutant RBP1K55R did not show strikingly different subcellular localization patterns during heat and cold stress, nor it affected alternative splicing, but was successfully purified with RBP1 for further in vitro phase separation experiments. Additionally, in silico disorder prediction tools highlighted its phaseseparation propensity and similarity to prion-like examples of amino acid composition. Finally, substitutions that alter predicted disorder-to-order (RBP1Y114F) transition and phosphorylation site conservation (RBP1Y129F), showed irregular subcellular localization patterns both in room temperature conditions and after heat stress, underlining the importance of further investigation for their functional impact. Overall, my findings provide insights to how phase separation and stress response are potentially linked, and highlight the importance of a more in-depth investigation of structural and functional changes in RBP1. Chapter II: Blue light (BL) photoreceptors are necessary components of signaling processes in plants, regulating the growth of plants to the most optimal position for survival and growth. Modern vertical farms utilize light-emitting diode lights with customized light to maximize efficiency, underlining the importance of deciphering the inner workings of BL response. My study aimed to contribute to the understanding of how Kin 7.3, a motor-based microtubule protein that was found to associate with BL photoreceptor PHOT1, is involved in blue light (BL) induced signaling response. Specifically, my thesis combined two approaches (i) genetic studies to explore the impact and the mechanism of PHOT1- dependent response to BL and (ii) cell biology experiments to study the effect of Kin7.3 on PHOT1 localization patterns and microtubule reorganization. Results showed that loss-of-function mutants display insensitivity to BL-induced bending, which poses a key phototropic response. Furthermore, PHOT1 distribution patterns at the plasma membrane (PM) appeared altered by the lack of Kin7.3, with PHOT1-GFP to present increased levels and retardation in internalization. Additionally, lack of Kin7.3 seems to affect microtubule BL-induced organization, with microtubules showing resistance to revert from a longitudinal to a transverse pattern of organization after BL-induced reorientation. Combined with experimental evidence suggesting that Kin7.3 affects the phosphorylation status of PHOT1, these assays collectively highlight Kin7.3 as a novel component of the PHOT1-dependent signaling.
Language English
Subject Kinesin 7.3
Phototropin 1
Site mutagenesis
Subcellular Localization
Σηματοδότηση στο μπλε φως
Σημειακές μεταλλάξεις
Υποκυτταρικός εντοπισμός
Φωτοτροπίνη 1
Issue date 2021-11-26
Collection   School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses
  Type of Work--Post-graduate theses
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