Summary 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 [1]. Fission also plays a role in apoptosis [2]. Defects in mitochondrial dynamics can lead to neurodegenerative diseases such as Alzheimer’s [3]. 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 [4]. 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 [5]. Here, we show that myosin II also plays a role in fission. Chemical inhibition by blebbistatin or 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.

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.

The 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. 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.

FMNL3 localizes broadly over the plasma membrane as discrete puncta, with particular enrichment in filopodia and ruffles and at cell–cell contacts. In addition, a population of FMNL3-containing vesicles of endocytic origin can fuse with the plasma membrane. FMNL3 suppression causes reductions in filopodia and cell–cell adhesion.

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.