Biology subjects

Pohl, E.

Publications and source records attributed to Pohl, E..

4 recordsLinked to original sources

bioRxiv2024

A receptor kinase complex refines cambium activity in Arabidopsis

In plant development, key receptor kinases are often active in disparate cell types, with each requiring vastly different signalling outputs. The ERECTA (ER) receptor kinase and its homologs ERL1 and ERL2 exemplify this pleiotropy. In Arabidopsis, they influence stomatal patterning, shoot meristem function, ovule morphogenesis, xylem fiber differentiation, and cell division in the vascular cambium1-6. Such diverse expression and functionality raises the question of how ER signalling can specify such distinct cell behaviours. One mechanism is via cell-type specific interactions with co-receptors, ligands, or other proteins that modulate signalling. However, little is known about ER interactors in the vascular cambium, a bifacial stem cell niche that generates phloem and xylem (Figure 1A). Combinatorial mutations between ER, ERL1 and ERL2 and receptor kinases of a second family, PXY, PXL1, and PXL2, show severe cambial defects5,7, but the mechanism underpinning these phenotypes is not known. Here we discovered that PXY proteins form protein complexes with ER and ERL2. PXY signalling can be manipulated by altering levels of its cognate ligand, TDIF. In genetic analysis, plant lines in which TDIF levels were altered had dramatic phenotypic changes that required the presence of ER or ERL2. Our results demonstrate that PXY signalling mediated cambium regulation depends on ER signalling and explains ER function in the cambium. Because the cambium produces xylem, which constitutes the wood in vascular plants, our findings position PXY-ER complexes at the centre of the accumulation of this versatile biomaterial and essential carbon sink. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/594489v1_fig1.gif" ALT="Figure 1"> View larger version (79K): org.highwire.dtl.DTLVardef@289bb6org.highwire.dtl.DTLVardef@6d0fdorg.highwire.dtl.DTLVardef@1829836org.highwire.dtl.DTLVardef@d70b3b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO PXY forms heterodimers with ER and ERL2 (A) Transverse sections of a wild type hypocotyl showing the region of secondary growth. Black rectangle marks the area of cambium shown in the diagrammatic representation on the lower left. (B-D) Localisation of PXY-CFP (B), ER-YFP (C) and overlap (D) in Nicotiana epidermal cells. (E) FRET signal. (F) Graph showing FRET efficiencies of PXY-CFP and ER/ERL1/ERL2-YFP pairs. (G-H) Coimmunoprecipitation of PXY-FLAG with ER-HA or ERL2-HA and vice versa in Arabidopsis cells. Scales (A) are 50 M. C_FIG

plant biology↗

bioRxiv2022

Toxoplasma ceramide synthases: a curious case of gene duplication, divergence and key functionality

Toxoplasma gondii is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause foetal damage and abortion in both animals and humans. Sphingolipids have indispensable functions as signaling molecules and are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Ceramide is the precursor for all sphingolipids, and here we report the identification, localisation and analyses of the Toxoplasma ceramide synthases TgCerS1 and TgCerS2 and, using a conditional gene regulation approach, establish their roles in pathogenicity and parasite fitness. Interestingly, we observed that whilst TgCerS1 was a fully functional orthologue of the yeast Lag1p capable of catalysing the conversion of sphinganine to ceramide, in contrast TgCerS2 was catalytically inactive. Furthermore, genomic deletion of TgCerS1 using CRISPR/Cas-9 led to viable but slow growing parasites indicating its importance but not indispensability. In contrast, genomic knock out of TgCerS2 was only accessible utilising the rapamycin-inducible Cre recombinase system. Surprisingly, the results demonstrated that this pseudo ceramide synthase, TgCerS2, has an even greater role in parasite fitness than its catalytically active orthologue (TgCerS1). Phylogenetic analyses indicated that, as in humans and plants, the ceramide synthase isoforms found in Toxoplasma and other Apicomplexa arose through gene duplication. However, in the Apicomplexa the duplicated copy subsequently evolved into a non-functional pseudo ceramide synthase. This arrangement is unique to the Apicomplexa and further illustrates the unusual biology that characterize these protozoan parasites, a feature that could potentially be exploited in the development of new antiprotozoals. Author SummarySphingolipids, essential and ubiquitous lipids in the Eukaryota, are both synthesized and scavenged by the parasitic apicomplexan protozoa, including Toxoplasma gondii. Ceramide is the precursor for all sphingolipids and here we report the identification, localisation and analyses of the Toxoplasma ceramide synthases TgCerS1 and TgCerS2. Surprisingly, whilst TgCerS1 was fully functional, catalysing the conversion of sphinganine to ceramide, TgCerS2 was catalytically inactive. However, we demonstrated that this pseudo ceramide synthase has an even greater role in parasite fitness than the catalytically active TgCerS1. Phylogenetic analyses indicated that these isoforms arose through gene duplication and the duplicated copy subsequently evolved into the pseudo ceramide synthase. This arrangement is unique to the Apicomplexa and further illustrates the highly unusual biology that characterizes these protozoan parasites, a feature that could potentially be exploited for the development of new antiprotozoals.

microbiology↗

bioRxiv2019

A tertiary structure protein model for the ab-initio interpretation of small angle X-ray scattering data

Small angle X-ray scattering (SAXS) has become an important tool to investigate the structure of proteins in solution. In this paper we present a novel ab-initio method to represent polypeptide chains as discrete curves that can be used to derive a meaningful three-dimensional model from only the primary sequence and experimental SAXS data. High resolution crystal structures were used to generate probability density functions for each of the common secondary structural elements found in proteins. These are used to place realistic restraints on the model curves geometry. To evaluate the quality of potential models and demonstrate the efficacy of this novel technique we developed a new statistic to compare the entangled geometry of two open curves, based on mathematical techniques from knot theory. The chain model is coupled with a novel explicit hydration shell model in order derive physically meaningful 3D models by optimizing configurations against experimental SAXS data using a monte-caro based algorithm. We show that the combination of our ab-initio method with spatial restraints based on contact predictions successfully derives a biologically plausible model of the coiled-coil component of the human synaptonemal complex central element protein.\n\nSIGNIFICANCESmall-angle X-ray scattering allows for structure determination of biological macromolecules and their complexes in aqueous solution. Using a discrete curve representation of the polypeptide chain and combining it with empirically determined constraints and a realistic solvent model we are now able to derive realistic ab-initio 3-dimensional models from BioSAXS data. The method only require a primary sequence and the scattering data form the user.

molecular biology↗

bioRxiv2018

The protein architecture of the endocytic coat analyzed by FRET

Endocytosis is a fundamental cellular trafficking pathway, which requires an organized assembly of the multiprotein endocytic coat to pull the plasma membrane into the cell. Although the protein composition of the endocytic coat is known, its functional architecture is not well understood. Here we determine the nanoscale organization of the endocytic coat by FRET microscopy in yeast. We assessed proximities of 18 conserved coat-associated proteins and used clathrin subunits and protein truncations as molecular rulers to obtain a high-resolution protein map of the coat. Furthermore, we followed rearrangements of coat proteins during membrane invagination and their binding dynamics at the endocytic site. We show that the endocytic coat is stratified into several functional layers situated above and below the clathrin lattice with key proteins transversing through the lattice deeply into the cytoplasm. We propose that this conserved design enables an efficient and regulated function of the endocytic coat during endocytosis.

cell biology↗