When brassinosteroid (BR) levels are low, the GSK3-like kinase BIN2 phosphorylates and inactivates the BZR1 transcription factor to inhibit growth in plants. BR promotes growth by inducing dephosphorylation of BZR1, but the phosphatase that dephosphorylates BZR1 has remained unknown. Here we identified protein phosphatase 2A (PP2A) as BZR1-interacting proteins using tandem affinity purification. Genetic analyses demonstrated a positive role of PP2A in BR signalling and BZR1 dephosphorylation. Members of the B'regulatory subunits of PP2A directly interact with BZR1's putative PEST domain containing the site of the bzr1-1D mutation. Interaction with and dephosphorylation by PP2A are enhanced by the bzr1-1D mutation, reduced by two intragenic bzr1-1D suppressor mutations, and abolished by deletion of the PEST domain. This study reveals a crucial function of PP2A in dephosphorylating and activating BZR1 and completes the set of core components of the BR-signalling cascade from cell surface receptor kinase to gene regulation in the nucleus.

SUMMARYNeuroblasts generate neurons with different functions by asymmetric cell division, cell cycle exit and differentiation. The underlying transcriptional regulatory pathways remain elusive. Here, we performed genetic screens in C. elegans and identified three evolutionarily conserved transcription factors (TFs) essential for Q neuroblast lineage progression. Through live cell imaging and genetic analysis, we showed that the storkhead TF HAM-1 regulates spindle positioning and myosin polarization during asymmetric cell division and that the PAR-1-like kinase PIG-1 is a transcriptional regulatory target of HAM-1. The TEAD TF EGL-44, in a physical association with the zinc-finger TF EGL-46, instructs cell cycle exit after the terminal division. Finally, the Sox domain TF EGL-13 is necessary and sufficient to establish the correct neuronal fate. Genetic analysis further demonstrated that HAM-1, EGL-44/EGL-46 and EGL-13 form three transcriptional regulatory pathways. We have thus identified TFs that function at distinct developmental stages to ensure appropriate neuroblast lineage progression and suggest that their vertebrate homologs might similarly regulate neural development.

Directional cell migration is a fundamental process in neural development. In Caenorhabditis elegans , Q neuroblasts on the left (QL) and right (QR) sides of the animal generate cells that migrate in opposite directions along the anteroposterior body axis. The homeobox (Hox) gene lin-39 promotes the anterior migration of QR descendants (QR.x), whereas the canonical Wnt signaling pathway activates another Hox gene, mab-5 , to ensure the QL descendants’ (QL.x) posterior migration. However, the regulatory targets of LIN-39 and MAB-5 remain elusive. Here, we showed that MIG-13, an evolutionarily conserved transmembrane protein, cell-autonomously regulates the asymmetric distribution of the actin cytoskeleton in the leading migratory edge. We identified mig-13 as a cellular target of LIN-39 and MAB-5. LIN-39 establishes QR.x anterior polarity by binding to the mig-13 promoter and promoting mig-13 expression, whereas MAB-5 inhibits QL.x anterior polarity by associating with the lin-39 promoter and downregulating lin-39 and mig-13 expression. Thus, MIG-13 links the Wnt signaling and Hox genes that guide migrations, to the actin cytoskeleton, which executes the motility response in neuronal migration.

Autophagy genes are not only essential for exposing the engulfment signal on apoptotic cells but also function in phagocytes to promote apoptotic cell removal.

Postembryonic development is an important process of organismal maturation after embryonic growth. Despite key progress in recent years in understanding embryonic development via fluorescence time-lapse microscopy, comparatively less live-cell imaging of postembryonic development has been done. Here we describe a protocol to image larval development in the nematode Caenorhabditis elegans. Our protocol describes the construction of fluorescent transgenic C. elegans, immobilization of worm larvae and time-lapse microscopy analysis. To improve the throughput of imaging, we developed a C. elegans triple-fluorescence imaging approach with a worm-optimized blue fluorescent protein (TagBFP), green fluorescent protein (GFP) and mCherry. This protocol has been previously applied to time-lapse imaging analysis of Q neuroblast asymmetric division, migration and apoptosis, and we show here that it can also be used to image neuritogenesis in the L1 larvae. Other applications are also possible. The protocol can be completed within 3 h and may provide insights into understanding postembryonic development.

Independent of their role in apoptosis, cell engulfment proteins are essential for midbody internalization and degradation after cell division.

Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14 -3-3, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipidbinding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response. Molecular & Cellular Proteomics 12:

Cell-cell fusion generally requires cellular fusogenic proteins and actin-propelled membrane protrusions. However, the molecular connections between fusogens and the actin cytoskeleton remain unclear. Here, we show that the Caenorhabditis elegans fusogen EFF-1 and F-actin are enriched at the cortex of the post-embryonic fusing cells, and conditional mutations of WASP and Arp2/3 delay cell-cell fusion by impairing EFF-1 localization. Our affinity purification and mass spectrometry analyses determined that an actin-binding protein, spectraplakin/VAB-10A, binds to EFF-1. VAB-10A promotes cell-cell fusion by linking EFF-1 to the actin cytoskeleton. Conversely, EFF-1 enhanced the F-actin bundling activity of VAB-10A in vitro, and actin dynamics in the cortex were reduced in eff-1 or vab-10a mutants. Thus, cell-cell fusion is promoted by a positive feedback loop in which actin filaments that are crosslinked by spectraplakin to recruit fusogens to fusion sites are reinforced via fusogens, thereby increasing the probability of further fusogen accumulation to form fusion synapses.