Journal articles
-
Cosma M-A, Curtis NL, Pain C, Kriechbaumer V, Bolanos-Garcia VM, 'Biochemical, biophysical, and functional characterisation of the E3 Ubiquitin ligase APC/C regulator CDC20 from Arabidopsis thaliana'
Frontiers in Physiology In press (2022)
ISSN: 1664-042X eISSN: 1664-042X
Abstract The Anaphase Promoting Complex (APC/C), a large cullin-RING E3-type ubiquitin ligase, constitutes the ultimate target of the Spindle Assembly Checkpoint (SAC), an intricate regulatory circuit that ensures the high fidelity of chromosome segregation in eukaryotic organisms by delaying the onset of anaphase until each chromosome is properly bi-oriented on the mitotic spindle. Cell-division cycle protein 20 homologue (CDC20) is a key regulator of APC/C function in mitosis. The formation of the APC/CCDC20 complex is required for the ubiquitination and degradation of select substrates and this is necessary to maintain the mitotic state. In contrast to the roles of CDC20 in animal species, little is known about CDC20 roles in the regulation of chromosome segregation in plants. Here we address this gap in knowledge and report the expression in insect cells; the biochemical and biophysical characterisation of Arabidopsis thaliana (AtCDC20) WD40 domain; and the nuclear and cytoplasmic distribution of full-length AtCDC20 when transiently expressed in tobacco plants. We also show that most AtCDC20 degrons share a high sequence similarity to other eukaryotes, arguing in favour of conserved degron functions in AtCDC20. However, important exceptions were noted such as the lack of a canonical MAD1 binding motif; a fully conserved RRY-box in all six AtCDC20 isoforms instead of a CRY-box motif as well as a low conservation of key residues known to be phosphorylated by BUB1 and PLK1 in other species to ensure a robust SAC response. Taken together, our studies provide insights into AtCDC20 structure and function and the evolution of SAC signalling in plants.
-
Kerselidou D, Dohai BS, Nelson DR, Daakour S, De Cock N, Al Oula Hassoun Z, Kim D-K, Olivet J, El Assal DC , Jaiswal A, Alzahmi A, Saha D, Pain C, Matthijssens F, Lemaitre P, Herfs M, Chapuis J, Ghesquiere B, Vertommen D, Kriechbaumer V, Knoops K, Lopez-Iglesias C, van Zandvoort M, Lambert J-C, Hanson J, Desmet C ,Thiry M, Lauersen K, Vidal M, Van Vlierberghe P, Dequiedt F, Salehi-Ashtiani K, Twizere J-C, 'Alternative glycosylation controls endoplasmic reticulum dynamics and tubular extension in mammalian cells'
Science Advances 7 (19) (2021)
ISSN: 2375-2548 eISSN: 2375-2548
Abstract The endoplasmic reticulum (ER) is a central eukaryotic organelle with a tubular network made of hairpin proteins linked by hydrolysis of GTP nucleotides. Among post-translational modifications initiated at the ER level, glycosylation is the most common reaction. However, our understanding of the impact of glycosylation on the ER structure remains unclear. Here, we show that Exostosin-1 (EXT1) glycosyltransferase, an enzyme involved in N-glycosylation, is a key regulator of the ER morphology and dynamics. We have integrated multi-omics data and super-resolution imaging to characterize the broad effect of EXT1 inactivation, including the ER shape-dynamics-function relationships in mammalian cells. We have observed that inactivating EXT1 induces cell enlargement and enhances metabolic switches such as protein secretion. In particular, suppressing EXT1 in mouse thymocytes causes developmental dysfunctions associated with the ER network extension. Finally, our data illuminate the physical and functional aspects of the ER proteome-glycome-lipidome-structure axis, with implications in biotechnology and medicine.
Website
-
Zang J, Klemm S, Pain C, Duckney P, Bao Z, Stamm G, Kriechbaumer V, Bürstenbinder K, Hussey PJ, Wang P, 'A novel plant actin-microtubule bridging complex regulates cytoskeletal and ER structure at Endoplasmic Reticulum-Plasma Membrane Contact Sites (EPCS) '
Current Biology 31 (6) (2021) pp.1251-1260
ISSN: 0960-9822
Abstract In plants, the cortical ER network is connected to the plasma membrane through the ER-PM contact sites (EPCS), whose structures are maintained by EPCS resident proteins and the cytoskeleton [1-7] . Strong co-alignment between EPCS and the cytoskeleton is observed in plants [1, 8], but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by FRET-FLIM analysis, to identify two microtubule binding proteins, KLCR1 (Kinesin Light Chain Related protein 1) and IQD2 (IQ67-Domain 2) that interact with the actin binding protein NET3C and form a component of plant EPCS, that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology and EPCS structure. Their loss of function mutants, net3a/NET3C RNAi, klcr1 or iqd2, exhibit defects in pavement cell morphology which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal associated complex, which is essential for the maintenance and organization of cytoskeletal structure and ER morphology at the EPCS, and for normal plant cell morphogenesis.
Website
-
Pain C, Kriechbaumer V, 'Defining the dance: quantification and classification of ER dynamics'
Journal of Experimental Botany 71 (6) (2019) pp.1757-1762
ISSN: 0022-0957 eISSN: 1460-2431
Abstract The availability of quantification methods for sub-cellular organelle dynamic analysis has increased rapidly over the last 20 years. The application of these techniques to contiguous sub-cellular structures that exhibit dynamic re-modelling over a range of scales and orientations is challenging as quantification of ‘movement’ rarely corresponds to traditional, qualitative classifications of types of organelle movement. The plant endoplasmic reticulum represents a particular challenge for dynamic quantification as it itself is an entirely contiguous organelle that is in a constant state of flux and gross remodelling, controlled by the actinomyosin cytoskeleton.
Website
-
Pain C, Tolmie F, Wojcik S, Wang P, Kriechbaumer V, 'intER-ACTINg: the structure and dynamics of ER and actin are interlinked'
Journal of Microscopy [in press]
ISSN: 0022-2720 eISSN: 1365-2818
Abstract The actin cytoskeleton is the driver of gross ER remodelling and the movement and positioning of other membrane-bound organelles such as Golgi bodies. Rapid ER membrane remodelling is a feature of most plant cells and is important for normal cellular processes, including targeted secretion, immunity and signalling. Modifications to the actin cytoskeleton, through pharmacological agents such as Latrunculin B and phalloidin, or disruption of normal myosin function also affect ER structure and/or dynamics. Here, we investigate the impact of changes in the actin cytoskeleton on structure and dynamics on the ER as well as in return the impact of modified ER structure on the architecture of the actin cytoskeleton. By expressing actin markers that affect actin dynamics, or expressing of ER-shaping proteins that influence ER architecture, we found that the structure of ER-actin networks is closely inter-related; affecting one component is likely to have a direct effect on the other. Therefore, our results indicate that a complicated regulatory machinery and cross-talk between these two structures must exist in plants to co-ordinate the function of ER-actin network during multiple subcellular processes. In addition, when considering organelle structure and dynamics, the choice of actin marker is essential in preventing off-target organelle structure and dynamics modifications.
Pain, C. Kriechbaumer, V. Kittelmann, M. Hawes, C. & Fricker, M. (2019). Quantitative analysis of plant ER architecture and dynamics. Nature Communications, 10(1), 984.
Kriechbaumer, V. Breeze, E. Pain, C. Tolmie, F. Frigerio, L. & Hawes, C. (2018). Arabidopsis Lunapark proteins are involved in ER cisternae formation. New Phytologist, 219(3), 990–1004.