Heinkow, P. | Search bioRxiv

bioRxiv2023

A bi-kinase module sensitizes and potentiates plant immune signaling

Systemic signaling is an essential hallmark of multicellular life. Pathogen encounter occurs locally but triggers organ-scale and organismic immune responses. In plants, elicitor perception provokes systemically expanding Ca2+ and H2O2 signals conferring immunity. Here, we identify a Ca2+ sensing bi-kinase module as becoming super-activated through mutual trans-phosphorylation and imposing synergistically enhanced NADPH oxidase activation. A combined two-layer bi-kinase/substrate phospho-code allows for sensitized signaling initiation already by near-resting elevations of Ca2+ concentration at the infection site. Subsequently, it facilitates further signal wave proliferation with minimal amplitude requirement, triggering protective defense responses throughout the plant. Our study reveals how plants build and perpetuate trans-cellular immune signal proliferation while avoiding disturbance of ongoing cellular signaling along the path of response dissemination. One sentence summaryMutual trans-activation of a Ca2+ sensing bi-kinase module potentiates NADPH oxidase activation to facilitate systemic immunity.

plant biology↗

bioRxiv2021

A versatile mitochondria isolation- and analysis-pipeline generates 3D nano-topographies and mechano-physical surface maps of single organelles

Living eukaryotic cells typically contain large quantities of highly dynamic mitochondria, which sustain the cells energy and redox homeostasis. Growing evidence suggests that mitochondria can functionally differ among but also within cells. The extent and biological significance of mitochondrial diversity is still largely unexplored, due to technical limitations that hamper profiling of individual organelles. Previous measurements of the cells interior have shown that membrane-bound compartments respond to metabolic manipulation by changes in their surface stiffness, suggesting that mechano-physical properties are a valuable readout of mitochondrial function. We here present the establishment of a robust multi-step analysis pipeline that allows one to profile mechano-physical properties of single mitochondria at the nanoscale using Atomic Force Microscopy (AFM). Firstly, we developed a rapid cell-type specific isolation protocol (mRACE), which selectively functionalizes mitochondria with biotin, facilitating isolation by streptavidin decorated microbeads. We established the technique for human and rat cell cultures, the invertebrate Caenorhabditis elegans, and the model plant Arabidopsis thaliana. Based on this versatile tool, we detected diversity of mitochondrially associated proteins among different tissues, reflecting the trophic condition of the source material. Secondly, a rapid filtration-based mitochondria isolation protocol was established, which was combined with mRACE. Lastly, we established an AFM analysis platform, which generates 3D maps of the nano-topography and mechano-physical properties of individual mitochondria. The comparison of mitochondria with each other revealed an unprecedented diversity in their mechano-physical properties and suggests that shape is not the sole determining parameter for outer membrane stiffness. We expect our results to not only introduce a new dimension for basic mitochondrial research, but in addition to open the door for the exploitation of individual mitochondria for diagnostic characterization.

plant biology↗