Structural Analysis of Arabidopsis thaliana Cellulose Synthase A1 Catalytic Domain

Abstract Background Cortical microtubules regulate cell expansion by determining cellulose microfibril orientation in the root apex of Arabidopsis thaliana. While the regulation of cell wall properties by cortical microtubules is well studied, the data on the influence of cell wall to cortical microtubule organization and stability remain scarce. Studies on cellulose biosynthesis mutants revealed that cortical microtubules depend on Cellulose Synthase A (CESA) function and/or cell expansion. Furthermore, it has been reported that cortical microtubules in cellulose-deficient mutants are hypersensitive to oryzalin. In this work, the persistence of cortical microtubules against anti-microtubule treatment was thoroughly studied in the roots of several cesa mutants, namely thanatos, mre1, any1, prc1-1 and rsw1, and the Cellulose Synthase Interacting 1 protein (csi1) mutant pom2-4. In addition, various treatments with drugs affecting cell expansion were performed on wild-type roots. Whole mount tubulin immunolabeling was applied in the above roots and observations were performed by confocal microscopy. Results Cortical microtubules in all mutants showed statistically significant increased persistence against anti-microtubule drugs, compared to those of the wild-type. Furthermore, to examine if the enhanced stability of cortical microtubules was due to reduced cellulose biosynthesis or to suppression of cell expansion, treatments of wild-type roots with 2,6-dichlorobenzonitrile (DCB) and Congo red were performed. After these treatments, cortical microtubules appeared more resistant to oryzalin, than in the control. Conclusions According to these findings, it may be concluded that inhibition of cell expansion, irrespective of the cause, results in increased microtubule stability in A. thaliana root. In addition, cell expansion does not only rely on cortical microtubule orientation but also plays a regulatory role in microtubule dynamics, as well. Various hypotheses may explain the increased cortical microtubule stability under decreased cell expansion such as the role of cell wall sensors and the presence of less dynamic cortical microtubules.

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Cryo-EM structural analysis of FADD:Caspase-8 complexes defines the catalytic dimer architecture for co-ordinated control of cell fate

Nature Communications ◽

10.1038/s41467-020-20806-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Joanna L. Fox ◽

Michelle A. Hughes ◽

Xin Meng ◽

Nikola A. Sarnowska ◽

Ian R. Powley ◽

...

Keyword(s):

Cell Death ◽

Structural Analysis ◽

Cell Fate ◽

Catalytic Domain ◽

Caspase 8 ◽

Type I ◽

Signaling Complex ◽

Catalytic Domains ◽

Regulated Cell Death ◽

Death Effector

AbstractRegulated cell death is essential in development and cellular homeostasis. Multi-protein platforms, including the Death-Inducing Signaling Complex (DISC), co-ordinate cell fate via a core FADD:Caspase-8 complex and its regulatory partners, such as the cell death inhibitor c-FLIP. Here, using electron microscopy, we visualize full-length procaspase-8 in complex with FADD. Our structural analysis now reveals how the FADD-nucleated tandem death effector domain (tDED) helical filament is required to orientate the procaspase-8 catalytic domains, enabling their activation via anti-parallel dimerization. Strikingly, recruitment of c-FLIPS into this complex inhibits Caspase-8 activity by altering tDED triple helix architecture, resulting in steric hindrance of the canonical tDED Type I binding site. This prevents both Caspase-8 catalytic domain assembly and tDED helical filament elongation. Our findings reveal how the plasticity, composition and architecture of the core FADD:Caspase-8 complex critically defines life/death decisions not only via the DISC, but across multiple key signaling platforms including TNF complex II, the ripoptosome, and RIPK1/RIPK3 necrosome.

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FRA1 modulates cortical microtubule localization of CMU proteins

10.1101/760389 ◽

2019 ◽

Author(s):

Anindya Ganguly ◽

Chuanmei Zhu ◽

Weizu Chen ◽

Ram Dixit

Keyword(s):

Arabidopsis Thaliana ◽

Cell Wall ◽

Molecular Mechanisms ◽

Cortical Microtubule ◽

Cellulose Synthase ◽

Lateral Stability ◽

Cortical Microtubules ◽

Tail Region ◽

Opposing Effect ◽

Growth And Reproduction

ABSTRACTConstruction of the cell wall demands harmonized deposition of cellulose and matrix polysaccharides. Cortical microtubules orient the deposition of cellulose by guiding the trajectory of plasma membrane-embedded cellulose synthase complexes. Vesicles containing matrix polysaccharides are thought to be transported by the FRA1 kinesin to facilitate their secretion along cortical microtubules. The cortical microtubule cytoskeleton thus provides a platform to coordinate the delivery of cellulose and matrix polysaccharides, but the underlying molecular mechanisms remain unknown. Here, we show that the tail region of the FRA1 kinesin physically interacts with CMU proteins which are important for the microtubule-dependent guidance of cellulose synthase complexes. Interaction with CMUs did not affect microtubule binding or motility of the FRA1 kinesin but had an opposing effect on the cortical microtubule localization of CMU1 and CMU2 proteins, thus regulating the lateral stability of cortical microtubules. Phosphorylation of the FRA1 tail region by CKL6 inhibited binding to CMUs and consequently reversed the extent of cortical microtubule decoration by CMU1 and CMU2. Genetic experiments demonstrated the significance of this interaction to the growth and reproduction of Arabidopsis thaliana plants. We propose that modulation of CMU’s microtubule localization by FRA1 provides a mechanism to control the coordinated deposition of cellulose and matrix polysaccharides.

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Auxin and Cell Wall Crosstalk as Revealed by the Arabidopsis thaliana Cellulose Synthase Mutant Radially Swollen 1

Plant and Cell Physiology ◽

10.1093/pcp/pcz055 ◽

2019 ◽

Vol 60 (7) ◽

pp. 1487-1503 ◽

Cited By ~ 3

Author(s):

Thiel A. Lehman ◽

Karen A Sanguinet

Keyword(s):

Arabidopsis Thaliana ◽

Cell Wall ◽

Cellulose Synthase ◽

Cellulose Microfibrils ◽

Single Amino Acid ◽

Cell Wall Biosynthesis ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Anisotropic Expansion ◽

Single Amino Acid Change

AbstractPlant cells sheath themselves in a complex lattice of polysaccharides, proteins and enzymes forming an integral matrix known as the cell wall. Cellulose microfibrils, the primary component of cell walls, are synthesized at the plasma membrane by CELLULOSE SYNTHASE A (CESA) proteins throughout cellular growth and are responsible for turgor-driven anisotropic expansion. Associations between hormone signaling and cell wall biosynthesis have long been suggested, but recently direct links have been found revealing hormones play key regulatory roles in cellulose biosynthesis. The radially swollen 1 (rsw1) allele of Arabidopsis thaliana CESA1 harbors a single amino acid change that renders the protein unstable at high temperatures. We used the conditional nature of rsw1 to investigate how auxin contributes to isotropic growth. We found that exogenous auxin treatment reduces isotropic swelling in rsw1 roots at the restrictive temperature of 30ï¿½C. We also discovered decreases in auxin influx between rsw1 and wild-type roots via confocal imaging of AUX1-YFP, even at the permissive temperature of 19ï¿½C. Moreover, rsw1 displayed mis-expression of auxin-responsive and CESA genes. Additionally, we found altered auxin maxima in rsw1 mutant roots at the onset of swelling using DII-VENUS and DR5:vYFP auxin reporters. Overall, we conclude disrupted cell wall biosynthesis perturbs auxin transport leading to altered auxin homeostasis impacting both anisotropic and isotropic growth that affects overall root morphology.

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