A Multimodal Strategy Used by a Large c-di-GMP Network.

The Pseudomonas fluorescens genome encodes more than 50 proteins predicted to be involved in c-di-GMP signaling. Here, we demonstrated that, tested across 188 nutrients, these enzymes and effectors appeared capable of impacting biofilm formation. Transcriptional analysis of network members across ∼50 nutrient conditions indicates that altered gene expression can explain a subset of but not all biofilm formation responses to the nutrients. Additional organization of the network is likely achieved through physical interaction, as determined via probing ∼2,000 interactions by bacterial two-hybrid assays. Our analysis revealed a multimodal regulatory strategy using combinations of ligand-mediated signals, protein-protein interaction, and/or transcriptional regulation to fine-tune c-di-GMP-mediated responses. These results create a profile of a large c-di-GMP network that is used to make important cellular decisions, opening the door to future model building and the ability to engineer this complex circuitry in other bacteria.IMPORTANCE Cyclic diguanylate (c-di-GMP) is a key signaling molecule regulating bacterial biofilm formation, and many microbes have up to dozens of proteins that make, break, or bind this dinucleotide. A major open issue in the field is how signaling specificity is conferred in the unpartitioned space of a bacterial cell. Here, we took a systems approach, using mutational analysis, transcriptional studies, and bacterial two-hybrid analysis to interrogate this network. We found that a majority of enzymes are capable of impacting biofilm formation in a context-dependent manner, and we revealed examples of two or more modes of regulation (i.e., transcriptional control with protein-protein interaction) being utilized to generate an observable impact on biofilm formation.

Isoform-selective chemical inhibition of mDia-mediated actin assembly.

Formins are potent actin assembly factors. Diaphanous formins, including mDia1, mDia2, and mDia3 in mammals, are implicated in mitosis and cytokinesis, but no chemical interactors have been reported. We developed an in vitro screen for inhibitors of actin assembly by mDia1 and identified an inhibitor of mDia1 and mDia2 that does not inhibit mDia3 at the concentrations tested. These results establish the druggability of mDia formins and introduce a first-generation inhibitor.

Single-molecule dynamics of the P granule scaffold MEG-3 in the Caenorhabditis elegans zygote.

During the asymmetric division of the Caenorhabditis elegans zygote, germ (P) granules are disassembled in the anterior cytoplasm and stabilized/assembled in the posterior cytoplasm, leading to their inheritance by the germline daughter cell. P granule segregation depends on MEG (maternal-effect germline defective)-3 and MEG-4, which are enriched in P granules and in the posterior cytoplasm surrounding P granules. Here we use single-molecule imaging and tracking to characterize the reaction/diffusion mechanisms that result in MEG-3::Halo segregation. We find that the anteriorly enriched RNA-binding proteins MEX (muscle excess)-5 and MEX-6 suppress the retention of MEG-3 in the anterior cytoplasm, leading to MEG-3 enrichment in the posterior. We provide evidence that MEX-5/6 may work in conjunction with PLK-1 kinase to suppress MEG-3 retention in the anterior. Surprisingly, we find that the retention of MEG-3::Halo in the posterior cytoplasm surrounding P granules does not appear to contribute significantly to the maintenance of P granule asymmetry. Rather, our findings suggest that the formation of the MEG-3 concentration gradient and the segregation of P granules are two parallel manifestations of MEG-3's response to upstream polarity cues.

PIE-1 Translation in the Germline Lineage Contributes to PIE-1 Asymmetry in the Early Caenorhabditis elegans Embryo.

In the C. elegans embryo, the germline lineage is established through successive asymmetric cell divisions that each generate a somatic and a germline daughter cell. PIE-1 is an essential maternal factor that is enriched in embryonic germline cells and is required for germline specification. We estimated the absolute concentration of PIE-1::GFP in germline cells and find that PIE-1::GFP concentration increases by roughly 4.5 fold, from 92 nM to 424 nM, between the 1 and 4-cell stages. Previous studies have shown that the preferential inheritance of PIE-1 by germline daughter cells and the degradation of PIE-1 in somatic cells are important for PIE-1 enrichment in germline cells. In this study, we provide evidence that the preferential translation of maternal PIE-1::GFP transcripts in the germline also contributes to PIE-1::GFP enrichment. Through an RNAi screen, we identified Y14 and MAG-1 (Drosophila tsunagi and mago nashi) as regulators of embryonic PIE-1::GFP levels. We show that Y14 and MAG-1 do not regulate PIE-1 degradation, segregation or synthesis in the early embryo, but do regulate the concentration of maternally-deposited PIE-1::GFP. Taken together, or findings point to an important role for translational control in the regulation of PIE-1 levels in the germline lineage.

The formin FMNL3 assembles plasma membrane protrusions that participate in cell-cell adhesion.

FMNL3 is a vertebrate-specific formin protein previously shown to play a role in angiogenesis and cell migration. Here we define the cellular localization of endogenous FMNL3, the dynamics of GFP-tagged FMNL3 during cell migration, and the effects of FMNL3 suppression in mammalian culture cells. The majority of FMNL3 localizes in a punctate pattern, with >95% of these puncta being indistinguishable from the plasma membrane by fluorescence microscopy. A small number of dynamic cytoplasmic FMNL3 patches also exist, which enrich near cell-cell contact sites and fuse with the plasma membrane at these sites. These cytoplasmic puncta appear to be part of larger membranes of endocytic origin. On the plasma membrane, FMNL3 enriches particularly in filopodia and membrane ruffles and at nascent cell-cell adhesions. FMNL3-containing filopodia occur both at the cell-substratum interface and at cell-cell contacts, with the latter being 10-fold more stable. FMNL3 suppression by siRNA has two major effects: decrease in filopodia and compromised cell-cell adhesion in cells migrating as a sheet. Overall our results suggest that FMNL3 functions in assembly of actin-based protrusions that are specialized for cell-cell adhesion.

A role for myosin II in mammalian mitochondrial fission.

Mitochondria are dynamic organelles, undergoing both fission and fusion regularly in interphase cells. Mitochondrial fission is thought to be part of a quality-control mechanism whereby damaged mitochondrial components are segregated from healthy components in an individual mitochondrion, followed by mitochondrial fission and degradation of the damaged daughter mitochondrion. Fission also plays a role in apoptosis. Defects in mitochondrial dynamics can lead to neurodegenerative diseases such as Alzheimer's disease. Mitochondrial fission requires the dynamin GTPase Drp1, which assembles in a ring around the mitochondrion and appears to constrict both outer and inner mitochondrial membranes. However, mechanisms controlling Drp1 assembly on mammalian mitochondria are unclear. Recent results show that actin polymerization, driven by the endoplasmic reticulum-bound formin protein INF2, stimulates Drp1 assembly at fission sites. Here, we show that myosin II also plays a role in fission. Chemical inhibition by blebbistatin or small interfering RNA (siRNA)-mediated suppression of myosin IIA or myosin IIB causes an increase in mitochondrial length in both control cells and cells expressing constitutively active INF2. Active myosin II accumulates in puncta on mitochondria in an actin- and INF2-dependent manner. In addition, myosin II inhibition decreases Drp1 association with mitochondria. Based on these results, we propose a mechanistic model in which INF2-mediated actin polymerization leads to myosin II recruitment and constriction at the fission site, enhancing subsequent Drp1 accumulation and fission.

FMNL3 FH2-actin structure gives insight into formin-mediated actin nucleation and elongation.

Formins are actin-assembly factors that act in a variety of actin-based processes. The conserved formin homology 2 (FH2) domain promotes filament nucleation and influences elongation through interaction with the barbed end. FMNL3 is a formin that induces assembly of filopodia but whose FH2 domain is a poor nucleator. The 3.4-Å structure of a mouse FMNL3 FH2 dimer in complex with tetramethylrhodamine-actin uncovers details of formin-regulated actin elongation. We observe distinct FH2 actin-binding regions; interactions in the knob and coiled-coil subdomains are necessary for actin binding, whereas those in the lasso-post interface are important for the stepping mechanism. Biochemical and cellular experiments test the importance of individual residues for function. This structure provides details for FH2-mediated filament elongation by processive capping and supports a model in which C-terminal non-FH2 residues of FMNL3 are required to stabilize the filament nucleus.

Assembly of filopodia by the formin FRL2 (FMNL3).

Actin-dependent finger-like protrusions such as filopodia and microvilli are widespread in eukaryotes, but their assembly mechanisms are poorly understood. Filopodia assembly requires at least three biochemical activities on actin: actin filament nucleation, prolonged actin filament elongation, and actin filament bundling. These activities are shared by several mammalian formin proteins, including mDia2, FRL1 (also called FMNL1), and FRL2 (FMNL3). In this paper, we compare the abilities of constructs from these three formins to induce filopodia. FH1-FH2 constructs of both FRL2 and mDia2 stimulate potent filopodia assembly in multiple cell types, and enrich strongly at filopodia tips. In contrast, FRL1 FH1-FH2 lacks this activity, despite possessing similar biochemical activities and being highly homologous to FRL2. Chimeric FH1-FH2 experiments between FRL1 and FRL2 show that, while both an FH1 and an FH2 are needed, either FH1 domain supports filopodia assembly but only FRL2's FH2 domain allows this activity. A mutation that compromises FRL2's barbed end binding ability abolishes filopodia assembly. FRL2's ability to stimulate filopodia assembly is not altered by additional domains (GBD, DID, DAD), but is significantly reduced in the full-length construct, suggesting that FRL2 is subject to inhibitory regulation. The data suggest that the FH2 domain of FRL2 possesses properties not shared by FRL1 that allow it to generate filopodia.