Membrane-anchored calpains – hidden regulators of growth and development beyond plants?

Calpains are modulatory proteases that modify diverse cellular substrates and play essential roles in eukaryots. The best studied are animal cytosolic calpains. Here, we focus on enigmatic membrane-anchored calpains, their structural and functional features as well as phylogenetic distribution. Based on domain composition, we identified four types of membrane-anchored calpains. Type 1 and 2 show broad phylogenetic distribution among unicellular protists and streptophytes suggesting their ancient evolutionary origin. Type 3 and 4 diversified early and are present in brown algae and oomycetes. The plant DEK1 protein is the only representative of membrane-anchored calpains that has been functionally studied. Here, we present up to date knowledge about its structural features, putative regulation, posttranslational modifications, and biological role. Finally, we discuss potential model organisms and available tools for functional studies of membrane-anchored calpains with yet unknown biological role. Mechanistic understanding of membrane-anchored calpains may provide important insights into fundamental principles of cell polarization, cell fate control, and morphogenesis beyond plants

Endosidin 2 accelerates PIN2 endocytosis and disturbs intracellular trafficking of PIN2, PIN3, and PIN4 but not of SYT1.

We established that Endosidin2 (ES2) affected the trafficking routes of both newly synthesized and endocytic pools of PIN-FORMED2 (PIN2) in Arabidopsis root epidermal cells. PIN2 populations accumulated in separated patches, which gradually merged into large and compact ES2 aggregates (ES2As). FM4-64 endocytic tracer labeled ES2As as well. Both PIN2 pools also appeared in vacuoles. Accelerated endocytosis of PIN2, its aggregation in the cytoplasm, and redirection of PIN2 flows to vacuoles led to a substantial reduction of the abundance of this protein in the plasma membrane. Whereas PIN-FORMED3 and PIN-FORMED4 also aggregated in the cytoplasm, SYT1 was not sensitive to ES2 treatment and did not appear either in the cytoplasmic aggregates or vacuoles. Ultrastructural analysis revealed that ES2 affects the Golgi apparatus so that stacks acquired cup-shape and even circular shape surrounded by several vesicles. Abnormally shaped Golgi stacks, stack remnants, multi-lamellar structures, separated Golgi cisterna rings, tubular structures, and vesicles formed discrete clusters

<i>Actin3</i> promoter reveals undulating F-actin bundles at shanks and dynamic F-actin meshworks at tips of tip-growing pollen tubes

<p>The dynamic actin cytoskeleton of pollen tubes is both the driver of the tip growth and the organizer of cell polarity. In order to understand this fast re-arranging cytoskeletal system, we need reliable constructs expressed under relevant promoters. Here we are reporting that the Lifeact reporter, expressed under the pollen-specific <i>Actin3</i> promoter, visualizes very dynamic F-actin elements both in germinating pollen grains and tip-growing pollen tubes. Importantly, we have documented very active actin polymerization at the cell periphery, especially in the bulging area during pollen germination and in the apical clear zone. Expression of the Lifeact reporter under control of the pollen-specific <i>Actin3</i> promoter revealed 2 new aspects: (i) long F-actin bundles in pollen tube shanks are dynamic, showing undulating movements, (ii) subapical ‘actin collars’ or ‘fringes’ are absent.</p

SKP1–SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase

Arabidopsis Snf1-related protein kinases (SnRKs) are implicated in pleiotropic regulation of metabolic, hormonal and stress responses through their interaction with the kinase inhibitor PRL1 WD-protein. Here we show that SKP1/ASK1, a conserved SCF (Skp1-cullin-F-box) ubiquitin ligase subunit, which suppresses the skp1-4 mitotic defect in yeast, interacts with the PRL1-binding C-terminal domains of SnRKs. The same SnRK domains recruit an SKP1/ASK1-binding proteasomal protein, α4/PAD1, which enhances the formation of a trimeric SnRK complex with SKP1/ASK1 in vitro. By contrast, PRL1 reduces the interaction of SKP1/ASK1 with SnRKs. SKP1/ASK1 is co-immunoprecipitated with a cullin SCF subunit (AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts. SKP1/ASK1, cullin and proteasomal α-subunits show nuclear co-localization in differentiated Arabidopsis cells, and are observed in association with mitotic spindles and phragmoplasts during cell division. Detection of SnRK in purified 26S proteasomes and co-purification of epitope- tagged SKP1/ASK1 with SnRK, cullin and proteasomal α-subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and α4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis

Darkness treatment diminishes PIN2 abundance in the membrane mostly by suppressing its delivery.

<p>Relocation of seedlings from the light to darkness significantly affected green and red signal intensities in photoconverting experiments and also green signal intensity of unconverted samples (in all cases p≤0.001). In photoconverting experiments, differences in green signal were detected at time point 6 h (p≤0.05) and 12 h (p≤0.001), in red signal at time point 1.5 h (p≤0.001) and in experiments without photoconversion at every time point from 3 to 12 h (p≤0.001). For others, the differences were statistically not significant.</p

Turnover of PIN2 in the membranes of root tips under normal and anaerobic conditions.

<p>(<b>A</b>) The cover slips were either removed after every imaging (labeled as uncovered) or roots were permanently covered by cover slips (anaerobic conditions). Converted red in the labeling means red signal intensity in transversal membranes after photoconversion; converted green in the labeling means green signal intensity after photoconversion; unconverted in the labeling means green signal in the membrane without photoconversion. (<b>B</b>) Images of the same roots taken in green and red channels characterizing the decrease of red signal intensity and increase of green signal intensity in membranes within 12 h after photoconversion.</p

Jasmonates influence PIN2 plasma membrane dynamic.

<p>Both JA and MeJA at the concentrations 5 µM and 50 µM affect green signal intensity in transversal plasma membranes of unconverted samples (p≤0.01 for 5 µM JA and 5 µM MeJA, p≤0.001 for 50 µM JA and 50 µM MeJA,). Differences were significant at time point 6 h (p≤0.05) and 12 h (p≤0.001) for 5 µM JA, at time point 6 h (p≤0.001) and 12 h (p≤0.001) for 50 µM JA, at time point 6 h (p≤0.05) and 12 h (p≤0.01) for 5 µM MeJA, and at time point 6 h (p≤0.05) and 12 h (p≤0.001) for 50 µM MeJA. For others differences were statistically not significant. Both jasmonates at the concentrations examined slowed-down the disappearance of red signals from the membrane (labeled as converted red; for each jasmonate and concentration p≤0.001) For both jasmonates used at a concentration of 5 µM differences were significant at time point 1.5 h (p≤0.01), 3 h (p≤0.001) and 6 h (p≤0.001), for 50 µM JA at time point 3 h (p≤0.05) and 6 h (p≤0.001), for 50 µM MeJA at time point 3 h (p≤0.05) and 6 h (p≤0.05). For others, the differences were statistically not significant. Both jasmonates at the concentrations examined delayed membrane green signal recovery (labeled as converted green; for each jasmonate and concentration p≤0.001)). Differences were significant at time point 6 h (p≤0.001) and 12 h (p≤0.001) for 5 µM JA, at time point 6 h (p≤0.01) and 12 h (p≤0.01) for 5 µM MeJA, at time point 6 h (p≤0.001) and 12 h (p≤0.001) for 50 µM JA and 50 µM MeJA. For others, the differences were statistically not significant.</p

Time-dependent photoconversion of PIN2-Dendra2 fusion protein.

<p>(<b>A</b>) The same root was analyzed for green and red fluorescence before (time 0) and after 3 s and 20 s conversion. (<b>B</b>) Efficiency of photoconversion depends on the time of illumination with blue-violet light. Green and red fluorescence signals were collected and the intensities of 150 transversal membranes within 5 roots were assessed with the ImageJ software. The interrupted line with open circles represents changes in red signal intensities, the solid line with closed circles represents decreasing in green signal intensities, bars represent SE. (<b>C</b>) Emission spectra of green and red signals of unconverted and photoconverted membranes were analyzed in three roots (represented by three lines in the graph). The HFT UV/488/543/633 was used as a beam splitter. Each point on the graph with SE bar represents the mean of 30 transversal membranes.</p

Representative spectra of Dendra2 before and after photoconversion.

<p>Dendra2 was expressed under the control of the 35S promoter and data for guard cells and root tips are presented. PIN2-Dendra2 driven by the endogenous promoter was analyzed in the membranes of root epidermis. Spectra were measured with a Zeiss LSM-510 Meta microscope before (labeled as unconverted) and after photoconversion (labeled as converted) for regions of interest (ROI) drawn in the coded images (left pictures in panel). Intact Dendra2 was analyzed in the nuclei (ROI1 drawn in the red color in the coded images) and in the cytoplasm (ROI2 drawn in green color in the coded images) of the guard cells and the cells of the root cap. Red lines in graphs represent the spectra emitted by nuclei, green lines represent the cytoplasm. The lower row in the panel shows the spectra emitted by the membrane-localized PIN2-Dendra2 (region drawn in red color in the coded image). The 458 nm excitation was combined with the HFT 458 beam splitter, the 488 nm excitation with the HFT 488 beam splitter and the 543 nm excitation with the HFT UV/488/543/633 beam splitter.</p