The apical extracellular matrix can form elaborate three-dimensional structures on animal surfaces. To better understand the mechanisms that pattern and shape these structures, we focus on development of collagen-rich cuticle ridges (alae) in adult C. elegans. Previous studies suggested that longitudinal actin filament bundles (AFBs) in the lateral seam epidermis specify alae position through a mechanism that involves post-secretory matrix delamination. Here we identify additional components of this highly organized cortical actin network and show that loss of the apical βH-spectrin SMA-1 specifically disrupts organization of the two AFBs that would normally flank the site where the middle alae ridge forms. Correspondingly, sma-1 loss, or mutation of its actin binding domains, also disrupts formation of the middle alae ridge. Ultrastructurally, sma-1 mutants have expanded regions of matrix delamination that can explain middle ridge loss. Together, these data highlight the importance of apical spectrin for organizing a patterned actin network within epithelia and show that, via its effects on actin organization, spectrin can also change the extracellular matrix and its patterns on animal surfaces.

Proper contractility of the Caenorhabditis elegans spermatheca relies on the coordinated developmental assembly of actomyosin bundles, yet the mechanisms that organize these structures remain poorly understood. From an RNAi screen targeting 117 F-actin regulators, we uncovered an essential role for ANI-1/anillin in spermatheca morphogenesis. Live imaging revealed that ANI-1 depletion can prevent organ contractility due to disrupted F-actin organization, impaired Rho activation, and absent calcium signaling, leading to ovulation failure. We show that a primary defect of ANI-1 depletion is furrow regression during spermatheca cell cytokinesis, which leads to multinucleation during early spermatheca development. Following failed cytokinesis, F-actin bundle formation is severely impaired, preventing robust F-actin network assembly especially in the spermatheca distal neck. Depletion of the cytokinetic regulators AIR-2/Aurora B and the centralspindlin component ZEN-4 produced identical F-actin defects in the mature spermatheca. We conclude that successful cell division is a prerequisite for subsequent F-actin network assembly that supports organ contractility, and identify ANI-1 as a key regulator of spermatheca function.

The microtubule motor cytoplasmic dynein-1 transports and positions various organelles, but the molecular basis of this functional diversity is not fully understood. Cargo adaptors of the Hook protein family recruit dynein to early endosomes (EE) in fungi and human cells by forming the FTS-Hook-FHIP (FHF) complex. By contrast, the Caenorhabditis elegans Hook homologue ZYG-12 recruits dynein to the nuclear envelope (NE) in the meiotic gonad and mitotic early embryo by forming a Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Here, we demonstrate that ZYG-12 recruits dynein to EE in epithelia. We identify and functionally characterize the homologues of FTS (UBC-19) and FHIP (FHIP-1) that constitute the C. elegans FHF complex, validate the predicted FHIP-1-RAB-5 binding interface in vivo, and show that ZYG-12 forms FHF via a conserved segment that precedes, and is distinct from, its C-terminal NE targeting domain. Finally, we show that C-terminal ZYG-12 splice isoforms differ in their ability to target to the NE and EE. We conclude that the C. elegans Hook adaptor evolved to recruit dynein to two distinct organelles, and that cargo specificity of ZYG-12 is regulated by alternative splicing.

During unilateral furrow ingression, one side of the cytokinetic ring (leading edge) ingresses before the opposite side (lagging edge). Anillin mediates unilateral furrowing during cytokinesis in the one-cell C. elegans zygote by limiting myosin II accumulation in the ring. Here, we address the role of anillin in this process and show that anillin inhibits not only the accumulation of myosin II but also of other RhoA effectors by binding and blocking the RhoA effector site. The interaction between the anillin's RhoA-binding domain (RBD) and active RhoA is enhanced by the disordered linker region and differentially regulated at the leading and lagging edge, which together results in asymmetric RhoA signaling and accumulation of myosin II. In summary, we discover a RhoA GEF- and GAP-independent mechanism, where RhoA activity is limited by anillin binding to the RhoA effector site. Spatial fine-tuning of anillin's inhibitory role on RhoA signaling enables unilateral furrow ingression and contributes to animal development.

During cytokinesis, a contractile ring consisting of unbranched filamentous actin (F-actin) and myosin II con-stricts at the cell equator. Unbranched F-actin is generated by formin, and without formin no cleavage furrow forms. In Caenorhabditis elegans, depletion of septin restores furrow ingression in formin mutants. How the cleavage furrow ingresses without a detectable unbranched F-actin ring is unknown. We report that, in this setting, anillin (ANI-1) forms a meshwork of circumferentially aligned linear structures decorated by non -mus-cle myosin II (NMY-2). Analysis of ANI-1 deletion mutants reveals that its disordered N-terminal half is required for linear structure formation and sufficient for furrow ingression. NMY-2 promotes the circumferen-tial alignment of the linear ANI-1 structures and interacts with various lipids, suggesting that NMY-2 links the ANI-1 network with the plasma membrane. Collectively, our data reveal a compensatory mechanism, medi-ated by ANI-1 linear structures and membrane-bound NMY-2, that promotes furrowing when unbranched F-actin polymerization is compromised.

Cytokinesis requires the constriction of an actomyosin-based contractile ring and involves multiple F-actin crosslinkers. We show that partial depletion of the C. elegans cytokinetic formin generates contractile rings with low F-actin levels that constrict but are structurally fragile, and we use this background to investigate the roles of the crosslinkers plastin/PLST-1 and β-heavy-spectrin/SMA-1 during ring constriction. We show that the removal of PLST-1 or SMA-1 has opposite effects on the structural integrity of fragile rings. PLST-1 loss reduces cortical tension that resists ring constriction and makes fragile rings less prone to ruptures and regressions, whereas SMA-1 loss exacerbates structural defects, leading to frequent ruptures and cytokinesis failure. Fragile rings without SMA-1 or containing a shorter SMA-1, repeatedly rupture at the same site, and SMA-1::GFP accumulates at repair sites in fragile rings and in rings cut by laser microsurgery. These results establish that β-heavy-spectrin stabilizes the constricting ring and reveals the importance of β-heavy-spectrin size for network connectivity at low F-actin density.

The C. elegans vulva is a polarized epithelial tube that has been studied extensively as a model for cell-cell signaling, cell fate specification, and tubulogenesis. Here we used endogenous fusions to show that the spectrin cytoskeleton is polarized in this organ, with conventional beta-spectrin ( UNC-70 ) found only at basolateral membranes and beta heavy spectrin ( SMA-1 ) found only at apical membranes. The sole alpha-spectrin ( SPC-1 ) is present at both locations but requires SMA-1 for its apical localization. Thus, beta spectrins are excellent markers for vulva cell membranes and polarity.

Cytokinesis, the process that partitions the mother cell into two daughter cells, requires the assembly and constriction of an equatorial actomyosin network. Different types of non-motor F-actin crosslinkers localize to the network, but their functional contribution remains poorly understood. Here, we describe a synergy between the small rigid crosslinker plastin and the large flexible crosslinker spectrin in the C. elegans one-cell embryo. In contrast to single inhibitions, co-inhibition of plastin and the βH-spectrin (SMA-1) results in cytokinesis failure due to progressive disorganization and eventual collapse of the equatorial actomyosin network. Cortical localization dynamics of non-muscle myosin II in co-inhibited embryos mimic those observed after drug-induced F-actin depolymerization, suggesting that the combined action of plastin and spectrin stabilizes F-actin in the contractile ring. An in silico model predicts that spectrin is more efficient than plastin at stabilizing the ring and that ring formation is relatively insensitive to βH-spectrin length, which is confirmed in vivo with a sma-1 mutant that lacks 11 of its 29 spectrin repeats. Our findings provide the first evidence that spectrin contributes to cytokinesis and highlight the importance of crosslinker interplay for actomyosin network integrity.

Cytokinesis is the last step of cell division that physically partitions the mother cell into two daughter cells. Cytokinesis requires the assembly and constriction of a contractile ring, a circumferential array of filamentous actin (F-actin), non-muscle myosin II motors (myosin), and actin-binding proteins that forms at the cell equator. Cytokinesis is accompanied by long-range cortical flows from regions of relaxation toward regions of compression. In the C. elegans one-cell embryo, it has been suggested that anterior-directed cortical flows are the main driver of contractile ring assembly. Here, we use embryos co-expressing motor-dead and wild-type myosin to show that cortical flows can be severely reduced without major effects on contractile ring assembly and timely completion of cytokinesis. Fluorescence recovery after photobleaching in the ingressing furrow reveals that myosin recruitment kinetics are also unaffected by the absence of cortical flows. We find that myosin cooperates with the F-actin crosslinker plastin to align and compact F-actin bundles at the cell equator, and that this cross-talk is essential for cytokinesis. Our results thus argue against the idea that cortical flows are a major determinant of contractile ring assembly. Instead, we propose that contractile ring assembly requires localized concerted action of motor-competent myosin and plastin at the cell equator.

Cytokinesis completes cell division by constriction of an actomyosin contractile ring that separates the two daughter cells. Here we use the early Caenorhabditis elegans embryo to explore how the actin filament network in the ring and the surrounding cortex is regulated by the single cytokinesis formin CYK-1 and the ARP2/3 complex, which nucleate nonbranched and branched filaments, respectively. We show that CYK-1 and the ARP2/3 complex are the predominant F-actin nucleators responsible for generating distinct cortical F-actin architectures and that depletion of either nucleator affects the kinetics of cytokinesis. CYK-1 is critical for normal F-actin levels in the contractile ring, and acute inhibition of CYK-1 after furrow ingression slows ring constriction rate, suggesting that CYK-1 activity is required throughout ring constriction. Surprisingly, although the ARP2/3 complex does not localize in the contractile ring, depletion of the ARP2 subunit or treatment with ARP2/3 complex inhibitor delays contractile ring formation and constriction. We present evidence that the delays are due to an excess in formin-nucleated cortical F-actin, suggesting that the ARP2/3 complex negatively regulates CYK-1 activity. We conclude that the kinetics of cytokinesis are modulated by interplay between the two major actin filament nucleators.

Cytokinesis is the process that completes cell division by partitioning the contents of the mother cell between the two daughter cells. It involves the highly regulated assembly and constriction of an actomyosin contractile ring, whose function is to pinch the mother cell in two. Research on the contractile ring has particularly focused on the signaling mechanisms that dictate when and where the ring is formed. In vivo studies of ring constriction are however scarce and its mechanistic understanding is therefore limited. Here we present several experimental approaches for monitoring ring constriction in vivo, using the four-cell C. elegans embryo as model. These approaches allow for the ring to be perturbed only after it forms and include the combination of live imaging with acute drug treatments, temperature-sensitive mutants and rapid temperature shifts, as well as laser microsurgery. In addition, we explain how to combine these with RNAi-mediated depletion of specific components of the cytokinetic machinery.

Cytokinesis in animal cells requires the assembly and constriction of a contractile actomyosin ring. Non-muscle myosin II is essential for cytokinesis, but the role of its motor activity remains unclear. Here, we examine cytokinesis in C. elegans embryos expressing non-muscle myosin motor mutants generated by genome editing. Two nonmuscle motor-dead myosins capable of binding F-actin do not support cytokinesis in the one-cell embryo, and two partially motor-impaired myosins delay cytokinesis and render rings more sensitive to reduced myosin levels. Further analysis of myosin mutants suggests that it is myosin motor activity, and not the ability of myosin to crosslink F-actin, that drives the alignment and compaction of F-actin bundles during contractile ring assembly, and that myosin motor activity sets the pace of contractile ring constriction. We conclude that myosin motor activity is required at all stages of cytokinesis. Finally, characterization of the corresponding motor mutations in C. elegans major muscle myosin shows that motor activity is required for muscle contraction but is dispensable for F-actin organization in adult muscles. This article has an associated 'The people behind the papers' interview.

Cytokinesis in animal cells requires the assembly of a contractile actomyosin ring, whose subsequent constriction physically separates the two daughter cells. Non-muscle myosin II (myosin) is essential for cytokinesis, but the role of its motor activity remains poorly defined. Here, we examine cytokinesis in C. elegans one-cell embryos expressing myosin motor mutants generated by genome editing. Motor-dead myosin, which is capable of binding F-actin, does not support cytokinesis, and embryos co-expressing motor-dead and wild-type myosin are delayed in cytokinesis. Partially motor-impaired myosin also delays cytokinesis and renders contractile rings more sensitive to reduced myosin levels. Thus, myosin motor activity, rather than its ability to cross-link actin filaments, drives contractile ring assembly and constriction. We further demonstrate that myosin motor activity is required for long-range cortical actin flows, but that flows per se play a minor role in contractile ring assembly. Our results suggest that flow-independent recruitment of motor-competent myosin to the cell equator is both essential and rate-limiting for cytokinesis.

The sorting nexins family of proteins (SNXs) plays pleiotropic functions in protein trafficking and intracellular signaling and has been associated with several disorders, namely Alzheimer's disease and Down's syndrome. Despite the growing association of SNXs with neurodegeneration, not much is known about their function in the nervous system. The aim of this work was to use the nematode Caenorhabditis elegans that encodes in its genome eight SNXs orthologs, to dissect the role of distinct SNXs, particularly in the nervous system. By screening the C. elegans SNXs deletion mutants for morphological, developmental and behavioral alterations, we show here that snx-3 gene mutation leads to an array of developmental defects, such as delayed hatching, decreased brood size and life span and reduced body length. Additionally, ∆snx-3 worms present increased susceptibility to osmotic, thermo and oxidative stress and distinct behavioral deficits, namely, a chemotaxis defect which is independent of the described snx-3 role in Wnt secretion. ∆snx-3 animals also display abnormal GABAergic neuronal architecture and wiring and altered AIY interneuron structure. Pan-neuronal expression of C. elegans snx-3 cDNA in the ∆snx-3 mutant is able to rescue its locomotion defects, as well as its chemotaxis toward isoamyl alcohol. Altogether, the present work provides the first in vivo evidence of the SNX-3 role in the nervous system.

We have recently identified a new class of filamenting temperature-sensitive mutant Z (FtsZ)-interacting compounds that possess a 2,4,6-trisubstituted pyrimidine-quinuclidine scaffold with moderate antibacterial activity. Employing this scaffold as a molecular template, a compound library of amine-linked 2,4,6-trisubstituted pyrimidines with 99 candidates was successfully established by employing an efficient convergent synthesis designed to explore their structure-activity relationship. The results of minimum inhibitory concentration (MIC) assay against Staphylococcus aureus strains and cytotoxicity assay against the mouse L929 cell line identified those compounds with potent antistaphylococcal properties (MIC ranges from 3 to 8 μg/mL) and some extent of cytotoxicity against normal cells (IC50 ranges from 6 to 27 μM). Importantly, three compounds also exhibited potent antibacterial activities against nine clinically isolated methicillin-resistant S. aureus (MRSA) strains. One of the compounds, 14av_amine16, exhibited low spontaneous frequency of resistance, low toxicity against Galleria mellonella larvae, and the ability to rescue G. mellonella larvae (20% survival rate at a dosage of 100 mg/kg) infected with a lethal dose of MRSA ATCC 43300 strain. Biological characterization of compound 14av_amine16 by saturation transfer difference NMR, light scattering assay, and guanosine triphosphatase hydrolysis assay with purified S. aureus FtsZ protein verified that it interacted with the FtsZ protein. Such a property of FtsZ inhibitors was further confirmed by observing iconic filamentous cell phenotype and mislocalization of the Z-ring formation of Bacillus subtilis. Taken together, these 2,4,6-trisubstituted pyrimidine derivatives represent a novel scaffold of S. aureus FtsZ inhibitors.

The prevalence of multidrug resistance among clinically significant bacteria calls for the urgent development of new antibiotics with novel mechanisms of action. In this study, a new small molecule exhibiting excellent inhibition of bacterial cell division with potent antibacterial activity was discovered through cell-based screening. The compound exhibits a broad spectrum of bactericidal activity, including the methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and NDM-1 Escherichia coli. The in vitro and in vivo results suggested that this compound disrupts the dynamic assembly of FtsZ protein and Z-ring formation through stimulating FtsZ polymerization. Moreover, this compound exhibits no activity on mammalian tubulin polymerization and shows low cytotoxicity on mammalian cells. Taken together, these findings could provide a new chemotype for development of antibacterials with FtsZ as the target.

Peptidoglycan glycosyltransferase (PGT) has been shown to be an important pharmacological target for the inhibition of bacterial cell wall biosynthesis. Structure‐based virtual screening of about 3 000 000 commercially available compounds against the crystal structure of the glycosyltransferase (GT) domain of the Staphylococcus aureus penicillin‐binding protein 2 (S. aureus PBP2) resulted in identification of an isatin derivative, 2‐(3‐(2‐carbamimidoylhydrazono)‐2‐oxoindolin‐1‐yl)‐N‐(m‐tolyl)acetamide (4) as a novel potential GT inhibitor. A series of 4 derivatives were synthesized. Several compounds showed more active antimicrobial activity than the initial hit compound 4, in particular 2‐(3‐(2‐carbamimidoylhydrazono)‐2‐oxoindolin‐1‐yl)‐N‐(3‐nitrophenyl)acetamide (4l), against Gram‐positive Bacillus subtilis and S. aureus with MIC values of 24 and 48 μg/mL, respectively. Saturation transfer difference (STD) NMR study revealed that there is a binding contact between 4l and the GT domain of S. aureus PBP2. Competitive STD‐NMR further proved that 4l and moenomycin A bind to GT domain in a competitive manner. Molecular docking study suggests a potential binding pocket of 4l in the GT domain of S. aureus PBP2. Taken together, compound 4l would provide a new scaffold for further development of potent GT inhibitors.