Stem cell number in shoot and floral meristems of Arabidopsis (Arabidopsis thaliana) is regulated by the CLAVATA3 (CLV3) signaling pathway. Perception of the CLV3 peptide requires the receptor kinase CLV1, the receptor-like protein CLV2, and the kinase CORYNE (CRN). Genetic analysis suggested that CLV2 and CRN act together and in parallel with CLV1. We studied the intracellular localization of receptor fusions with fluorescent protein tags and their capacities for interaction via efficiency of fluorescence resonance energy transfer. We found that CLV2 and CRN require each other for export from the endoplasmic reticulum and localization to the plasma membrane (PM). CRN readily forms homomers and interacts with CLV2 through the transmembrane domain and adjacent juxtamembrane sequences. CLV1 forms homomers independently of CLV2 and CRN at the PM. We propose that the CLV3 signal is perceived by a tetrameric CLV2/CRN complex and a CLV1 homodimer that localize to the PM and can interact via CRN.In Arabidopsis (Arabidopsis thaliana), the stem cell number in the shoot apical meristem is regulated by negative feedback regulation. Stem cell induction and maintenance are controlled by the homeodomain protein WUSCHEL (WUS), and WUS expression is in turn repressed by CLAVATA3 (CLV3; Brand et al., 2000;Schoof et al., 2000), which encodes a 13-amino acid arabinosylated glycopeptide that is secreted from stem cells (Ohyama et al., 2009). Three genes have been identified that encode receptors for CLV3 signaling. Mutations in CLV1 (Clark et al., 1997), encoding a leucine-rich repeat (LRR) receptor kinase, CLV2, encoding a LRR receptor-like protein (Jeong et al., 1999), and CORYNE (CRN), encoding a receptor-like kinase, disrupt CLV3 signaling and allow the stem cell domain to expand (Sablowski, 2007;Mü ller et al., 2008). Binding of CLV3 to the LRR domains of CLV1 was recently shown (Ogawa et al., 2008).A simple readout for CLV3 signaling is carpel number. Stem cells of floral meristems are normally consumed with the production of two central carpels. Any reduction in CLV3 signaling, which results in increased WUS expression and production of more stem cells, causes an increase in carpel number. Mutations in CLV1, CLV2, or CRN showed an intermediate carpel number phenotype and reduced CLV3 signaling (Mü ller et al., 2008). Double mutants of clv2 with crn were epistatic, but double mutants of clv1 with clv2 or crn were synergistic and abolished CLV3 signaling. This indicated that CLV1 acts independently from, and in parallel with, CLV2 and CRN to transmit the CLV3 signal. Furthermore, clv2 and crn mutants showed additional phenotypes, such as elongated pedicels and defects in stamen development, suggesting that CLV2 and CRN act in a common pathway (Mü ller et al., 2008). Both CRN and CLV2 were proposed to be membrane localized and may physically interact via their transmembrane domains or immediately adjacent sequences (Fig. 1A). A loss-of-function mutation of CRN, crn-1, is caused by an amino acid exchange within the predicted tra...

We hypothesize that these homo- and heteromeric complexes may differentially regulate distal root meristem maintenance. We conclude that essential components of the ancestral shoot stemness regulatory system also act in the root and that the specific interaction of CLV1 with ACR4 serves to moderate and control stemness homeostasis in the root meristem. The structural differences between these two meristem types may have necessitated this recruitment of ACR4 for signaling by CLV1.

Abbreviations used in this paper: CENP, centromere protein; FCS, fl uorescence correlation spectroscopy; mRFP, monomeric red fl uorescent protein; PCNA, proliferating cell nuclear antigen.The online version of this paper contains supplemental material.

GBPs are essential for immunity against intracellular pathogens, especially for Toxoplasma gondii control. Here, the molecular interactions of murine GBPs (mGBP1/2/3/5/6), homo- and hetero-multimerization properties of mGBP2 and its function in parasite killing were investigated by mutational, Multiparameter Fluorescence Image Spectroscopy, and live cell microscopy methodologies. Control of T. gondii replication by mGBP2 requires GTP hydrolysis and isoprenylation thus, enabling reversible oligomerization in vesicle-like structures. mGBP2 undergoes structural transitions between monomeric, dimeric and oligomeric states visualized by quantitative FRET analysis. mGBPs reside in at least two discrete subcellular reservoirs and attack the parasitophorous vacuole membrane (PVM) as orchestrated, supramolecular complexes forming large, densely packed multimers comprising up to several thousand monomers. This dramatic mGBP enrichment results in the loss of PVM integrity, followed by a direct assault of mGBP2 upon the plasma membrane of the parasite. These discoveries provide vital dynamic and molecular perceptions into cell-autonomous immunity.DOI: http://dx.doi.org/10.7554/eLife.11479.001

PML nuclear bodies (NBs) are involved in the regulation of key nuclear pathways but their biochemical function in nuclear metabolism is unknown. In this study PML NB assembly dynamics were assessed by live cell imaging and mathematic modeling of its major component parts. We show that all six nuclear PML isoforms exhibit individual exchange rates at NBs and identify PML V as a scaffold subunit. SP100 exchanges at least five times faster at NBs than PML proteins. Turnover dynamics of PML and SP100 at NBs is modulated by SUMOylation. Exchange is not temperature-dependent but depletion of cellular ATP levels induces protein immobilization at NBs. The PML-RARα oncogene exhibits a strong NB retention effect on wild-type PML proteins. HIPK2 requires an active kinase for PML NB targeting and elevated levels of PML IV increase its residence time. DAXX and BLM turn over rapidly and completely at PML NBs within seconds. These findings provide a kinetics model for factor exchange at PML NBs and highlight potential mechanisms to regulate intranuclear trafficking of specific factors at these domains.