Type 2 conventional dendritic cells (cDC2s) are functionally and phenotypically heterogenous. Previous work in mice and humans identified two cDC2 subsets (cDC2As and cDC2Bs) and a monocytic DC3 subset. However, the microenvironmental cues governing their distinct differentiation pathways remain unclear. Here, we delineate mouse cDC2 lineage relationships and the sequential signals required for cDC2A maintenance. We show that cDC2s, arising from the CLEC9A+ cDC progenitor, encompass T-BET-expressing cDC2As and two cDC2B subsets distinguished by MGL2 expression, with monocytic DC3s exhibiting transcriptional overlap with Mgl2− cDC2Bs. Among these subsets, T-BET+ cDC2As dominate the spleen where they require cell-intrinsic retinoic acid signaling to sustain their differentiation via Notch signals. Lymphotoxin-β receptor signaling on splenic cDC2s limits F-actin content retaining cDC2s at sites of retinol delivery. In summary, these data establish the developmental and transcriptional relationships between diverse cDC2 subsets and identify signals that regulate their prevalence in specific lymphoid tissues. Nguyen et al. show that the splenic environment provides sequential signals via lymphotoxin and retinoic acid that guide cDC2A development and retention.

ABSTRACT Bacteria often make initial contact with their hosts through the ligand-binding domains of large adhesin proteins. Recent analyses of repeats-in-toxin (RTX) adhesins in Gram-negative bacteria suggest that ligand-binding domains can be identified by the way they emerge from “split” domains within the adhesin. Here, using this criterion and an AlphaFold3 model of a 5047-residue RTX adhesin from Aeromonas hydrophila, we identified three different ligand-binding domains in this fibrillar protein. The crystal structures of the two novel domains were solved to 1.4 and 1.95 Å resolution, respectively, and demonstrate excellent agreement with their modeled structures. The other domain was recognized as a carbohydrate-binding module based on its beta-strand topology and confirmed by its micromolar affinity for fucosylated glycans, including the Lewis B and Y antigens. This lectin-like module, which was recombinantly produced with its companion split domain and nearby extender domain, bound to a wide variety of cells including yeasts, diatoms, erythrocytes, and human endothelial cells. In each case, 50 mM free fucose prevented this binding and may offer some protection from infection. The carbohydrate-binding module with its neighboring domains also caused aggregation of yeast and erythrocytes, which was again blocked by the addition of free fucose. The second putative ligand-binding domain has a beta-roll structure supported by a parallel alpha-helix, and the third is a homolog of a von Willebrand Factor A domain. These two domains bind to a more limited range of cell types, and their ligands have yet to be identified.IMPORTANCECharacterizing the ligand-binding domains of fibrillar adhesins is important for understanding how bacteria can colonize host surfaces and how this colonization might be blocked. Here, we show that the opportunistic pathogen, Aeromonas hydrophila, uses a carbohydrate-binding module (CBM) to attach to several different cell types. The CBM is one of three ligand-binding domains at the distal tip of the adhesin. Identifying the glycans bound by the CBM as Lewis B and Y antigens has helped explain the range of cell types that the bacterium will bind and colonize, and it has suggested sugars that might interfere with these processes. Indeed, fucose, which is a constituent of the Lewis B and Y antigens, is effective at 50 mM concentrations in blocking the attachment of the CBM to host cells. This will lead to the design of more effective inhibitors against bacterial infections.

Kinesin-8 motors regulate kinetochore-microtubule dynamics and control spindle length and positioning. Certain isoforms achieve this by traversing microtubules, accumulating at plus-ends, and depolymerizing terminal αβ-tubulin subunits. While the kinesin-8 motor domain is well characterized, the tail domain regions are less understood. Using the Candida albicans Kip3 protein as a model for fungal kinesin-8, we present an X-ray crystal structure and hydrodynamic analysis of its motor-proximal tail segment, revealing its role in motor dimerization. This segment forms a compact, 92 Å-long four-helix bundle, rather than an elongated coiled-coil stalk seen in most kinesins. The bundle is stabilized primarily by interactions between helices one and three, with additional support from helices two and four. A flexible hinge bisects the bundle into two lobules, imparting mechanical pliability and asymmetric exterior surfaces. These unique features may facilitate interactions with regulatory elements or contribute to the functional versatility of kinesin-8 motors.

IntroductionThe fungal G protein-coupled receptors Ste2 and Ste3 are vital in mediating directional hyphal growth of the agricultural pathogen Fusarium graminearum towards wheat plants. This chemotropism is induced by a catalytic product of peroxidases secreted by the wheat. Currently, the identity of this product, and the substrate it is generated from, are not known.Methods and resultsWe provide evidence that a peroxidase substrate is derived from F. graminearum conidia and report a simple method to extract and purify the FgSte2-activating ligand for analyses by mass spectrometry. The mass spectra arising from t he ligand extract are characteristic of a 400 Da carbohydrate moiety. Consistent with this type of molecule, glycosidase treatment of F. graminearum conidia prior to peroxidase treatment significantly reduced the amount of ligand extracted. Interestingly, availability of the peroxidase substrate appears to depend on the presence of both FgSte2 and FgSte3, as knockout of one or the other reduces the chemotropism-inducing effect of the extracts.ConclusionsWhile further characterization is necessary, identification of the F. graminearum-derived peroxidase substrate and the FgSte2-activating ligand will unearth deeper insights into the intricate mechanisms that underlie fungal pathogenesis in cereal crops, unveiling novel avenues for inhibitory interventions.

Actin barbed end-binding macrolides have been shown to inhibit cancer cell motility and invasion of extracellular matrix (ECM), evoking their potential utility as therapies for metastatic cancers. Unfortunately, the direct use of these compounds in clinical settings is impeded by their limited natural abundance, challenging total synthesis, and detrimental effects on normal tissues. To develop potent analogues of these compounds that are simpler to synthesize and compatible with cell-specific targeting systems, such as antibodies, we designed over 20 analogues of the acyclic side chain (tail) of the macrolide Mycalolide B. These analogues probed the contributions of four distinct regions of the tail towards the inhibition of actin polymerization and ECM invasion by human lung cancer A549 cells. We observed that two of these regions tolerate considerable substituent variability, and we identified a specific combination of substituents that leads to the optimal inhibition of the ECM invasion activity of A549 cells.

Budding yeast must position the mitotic spindle at the mother-bud junction during anaphase to ensure proper chromosome segregation. Pushing and pulling forces on astral microtubules from dynein motors in the cell cortex are crucial for spindle positioning. In some higher eukaryotes and filamentous fungi, dynein also accelerates spindle elongation during anaphase, but in yeast model systems spindle elongation primarily relies on intra-spindle pushing forces from kinesin-5 motors. The pathogenic yeast Candida albicans is unique because its spindle can elongate without a functional kinesin-5. Our study helps explain C. albicans ' diminished kinesin-5 requirement by showing that its dynein, Dyn1, collaborates with its kinesin-5, Kip1, to facilitate spindle elongation. Cells lacking Kip1 activity by either KIP1 deletion or Kip1 inhibition have more Dyn1 and astral microtubules, indicating spindle elongation happens through enhanced pulling on spindle pole body-bound astral microtubules by cortical Dyn1. When Dyn1 activity is depleted, spindle elongation speed is normal, but anaphase spindles persist for extended periods, and the number and length of astral microtubules increases. Depletion of both motors prevents spindle elongation and is lethal. The significance of astral microtubules in cells lacking either motor is highlighted by the lethal effects of microtubule-destabilizing drugs and exposure to low temperatures (8°C) that compromise microtubule stability. These findings demonstrate that C. albicans spindle elongation involves cooperative forces from Kip1 and Dyn1 to ensure chromosome separation during cell division and that C. albicans adapts to changes in abundance of these motors through alterations in their astral microtubule content. ### Competing Interest Statement The authors have declared no competing interest.

Kinesin motor proteins couple mechanical movements in their motor domain to the binding and hydrolysis of ATP in their nucleotide-binding pocket. Forces produced through this ‘mechanochemical’ coupling are typically used to mobilize kinesin-mediated transport of cargos along microtubules or microtubule cytoskeleton remodeling. This review discusses the recent high-resolution structures (<4 Å) of kinesins bound to microtubules or tubulin complexes that have resolved outstanding questions about the basis of mechanochemical coupling, and how family-specific modifications of the motor domain can enable its use for motility and/or microtubule depolymerization.

Fusarium head blight of wheat, caused by the filamentous fungus Fusarium graminearum, leads to devastating global food shortages and economic losses. Fungal hyphal chemotropism has been shown to be a major contributor to host-pathogen interactions. Here, the role of the opposite mating type GPCR, Ste3, is characterized with respect to F. graminearum chemotropism and pathogenicity. These findings contribute to our understanding of the mechanisms underlying fungal chemotropism and pathogenesis.

Kinesin-8s are dual-activity motor proteins that can move processively on microtubules and depolymerize microtubule plus-ends, but their mechanism of combining these distinct activities remains unclear. We addressed this by obtaining cryo-EM structures (2.6-3.9 Å) of Candida albicans Kip3 in different catalytic states on the microtubule lattice and on a curved microtubule end mimic, as well as a microtubule-unbound CaKip3-ADP crystal structure (2.0 Å). Together with biochemical analyses of CaKip3 and kinesin-1 mutants, we define a model that explains the kinesin-8 mechanism. The microtubule depolymerization activity originates in conformational changes of the kinesin-8 motor core that are amplified by its dynamic loop-2. On curved microtubule ends, loop-1 assists depolymerization by inserting into preceding motor domains, forming head-to-tail arrays of kinesin-8s that complement loop-2 contacts with curved tubulin. On straight tubulin protofilaments in the microtubule lattice, extended loop-2-tubulin contacts inhibit conformational changes in the motor core, but in the ADP-Pi state these contacts are relaxed, allowing neck-linker docking for motility. These tubulin shape-induced alternations between pro-microtubule-depolymerization and pro-motility kinesin states, regulated by loop-2, are the key to the dual activity of kinesin-8 motors.

Cancer metastasis is a complex process involving highly motile tumor cells that breach tissue barriers, enter the bloodstream and lymphatic system, and disseminate throughout the body as circulating tumor cells. The primary cellular mechanism contributing to these critical events is the reorganization of the actin cytoskeleton. Mycalolide B (MycB) is an actin-targeting marine macrolide that can suppress proliferation, migration, and invasion of breast and ovarian cancer cells at low nanomolar doses. Through structure-activity relationship studies focused on the actin-binding tail region (C24-C35) of MycB, we identified a potent truncated derivative that inhibits polymerization of G-actin and severs F-actin by binding to actin's barbed end cleft. Biological analyses of this miniature MycB derivative demonstrate that it causes a rapid collapse of the actin cytoskeleton in ovarian cancer cells and impairs cancer cell motility and invasion of the extracellular matrix (ECM) by inhibiting invadopodia-mediated ECM degradation. These studies provide essential proof-of-principle for developing actin-targeting therapeutic agents to block cancer metastasis and establish a synthetically tractable barbed end-binding pharmacophore that can be further improved by adding targeting groups for precision drug design.

Kinesins are a diverse group of adenosine triphosphate (ATP)-dependent motor proteins that transport cargos along microtubules (MTs) and change the organization of MT networks. Shared among all kinesins is a ~40 kDa motor domain that has evolved an impressive assortment of motility and MT remodeling mechanisms as a result of subtle tweaks and edits within its sequence. Several elegant studies of different kinesin isoforms have exposed the purpose of structural changes in the motor domain as it engages and leaves the MT. However, few studies have compared the sequences and MT contacts of these kinesins systematically. Along with clever strategies to trap kinesin-tubulin complexes for X-ray crystallography, new advancements in cryo-electron microscopy have produced a burst of high-resolution structures that show kinesin-MT interfaces more precisely than ever. This review considers the MT interactions of kinesin subfamilies that exhibit significant differences in speed, processivity, and MT remodeling activity. We show how their sequence variations relate to their tubulin footprint and, in turn, how this explains the molecular activities of previously characterized mutants. As more high-resolution structures become available, this type of assessment will quicken the pace toward establishing each kinesin's design-function relationship.

By using the Cre-mediated genetic switch technology, we were able to successfully generate a conditional knock-in mouse, bearing the KIF2A p.His321Asp missense point variant, identified in a subject with malformations of cortical development. These mice present with neuroanatomical anomalies and microcephaly associated with behavioral deficiencies and susceptibility to epilepsy, correlating with the described human phenotype. Using the f lexibility of this model, we investigated Rosa(Cre)-, Nestin(Cre)- and Nex(Cre)- driven expression of the mutation to dissect the pathophysiological mechanisms underlying neurodevelopmental cortical abnormalities. We show that the expression of the p.His321Asp pathogenic variant increases apoptosis and causes abnormal multipolar to bipolar transition in newborn neurons, providing therefore insights to better understand cortical organization and brain growth defects that characterize KIF2A-related human disorders. We further demonstrate that the observed cellular phenotypes are likely to be linked to deficiency in the microtubule depolymerizing function of KIF2A.

Fusarium Head Blight of wheat, caused by the filamentous fungus Fusarium graminearum, leads to devastating global food shortages and economic losses. While many studies have addressed the responses of both wheat and F. graminearum during their interaction, the possibility of fungal chemotropic sensing enabling pathogenicity remains unexplored. Based on recent findings linking the pheromone-sensing G-protein-coupled receptor Ste2 to host-directed chemotropism in Fusarium oxysporum , we investigated the role of the Ste2 receptor and its downstream signaling pathways in mediating chemotropism of F. graminearum . Interestingly, a chemotropic response of growing hyphae towards catalytically active Triticum aestivum ‘Roblin’ cultivar secreted peroxidases was detected, with deletion of STE2 in F. graminearum leading to loss of the observed response. At the same time, deletion of STE2 significantly decreased infection on germinating wheat coleoptiles, highlighting an association between Ste2, chemotropism and infection by F. graminearum . Further characterization revealed that the peroxidase-directed chemotropism is associated with stimulation of the fungal cell wall integrity mitogen-activated protein kinase signaling cascade. Altogether, this study demonstrates conservation of Ste2-mediated chemotropism by Fusarium species, and its important role in mediating pathogenicity.

Mutations in KIF14 have previously been associated with either severe, isolated or syndromic microcephaly with renal hypodysplasia (RHD). Syndromic microcephaly-RHD was strongly reminiscent of clinical ciliopathies, relating to defects of the primary cilium, a signalling organelle present on the surface of many quiescent cells. KIF14 encodes a mitotic kinesin, which plays a key role at the midbody during cytokinesis and has not previously been shown to be involved in cilia-related functions. Here, we analysed four families with fetuses presenting with the syndromic form and harbouring biallelic variants in KIF14. Our functional analyses showed that the identified variants severely impact the activity of KIF14 and likely correspond to loss-of-function mutations. Analysis in human fetal tissues further revealed the accumulation of KIF14-positive midbody remnants in the lumen of ureteric bud tips indicating a shared function of KIF14 during brain and kidney development. Subsequently, analysis of a kif14 mutant zebrafish line showed a conserved role for this mitotic kinesin. Interestingly, ciliopathy-associated phenotypes were also present in mutant embryos, supporting a potential direct or indirect role for KIF14 at cilia. However, our in vitro and in vivo analyses did not provide evidence of a direct role for KIF14 in ciliogenesis and suggested that loss of kif14 causes ciliopathy-like phenotypes through an accumulation of mitotic cells in ciliated tissues. Altogether, our results demonstrate that KIF14 mutations result in a severe syndrome associating microcephaly and RHD through its conserved function in cytokinesis during kidney and brain development.

Mitotic spindles assume a bipolar architecture through the concerted actions of microtubules, motors, and cross-linking proteins. In most eukaryotes, kinesin-5 motors are essential to this process, and cells will fail to form a bipolar spindle without kinesin-5 activity. Remarkably, inactivation of kinesin-14 motors can rescue this kinesin-5 deficiency by reestablishing the balance of antagonistic forces needed to drive spindle pole separation and spindle assembly. We show that the yeast form of the opportunistic fungus Candida albicans assembles bipolar spindles in the absence of its sole kinesin-5, CaKip1, even though this motor exhibits stereotypical cell-cycle-dependent localization patterns within the mitotic spindle. However, cells lacking CaKip1 function have shorter metaphase spindles and longer and more numerous astral microtubules. They also show defective hyphal development. Interestingly, a small population of CaKip1-deficient spindles break apart and reform two bipolar spindles in a single nucleus. These spindles then separate, dividing the nucleus, and then elongate simultaneously in the mother and bud or across the bud neck, resulting in multinucleate cells. These data suggest that kinesin-5-independent mechanisms drive assembly and elongation of the mitotic spindle in C. albicans and that CaKip1 is important for bipolar spindle integrity. We also found that simultaneous loss of kinesin-5 and kinesin-14 (CaKar3Cik1) activity is lethal. This implies a divergence from the antagonistic force paradigm that has been ascribed to these motors, which could be linked to the high mitotic error rate that C. albicans experiences and often exploits as a generator of diversity.IMPORTANCECandida albicans is one of the most prevalent fungal pathogens of humans and can infect a broad range of niches within its host. This organism frequently acquires resistance to antifungal agents through rapid generation of genetic diversity, with aneuploidy serving as a particularly important adaptive mechanism. This paper describes an investigation of the sole kinesin-5 in C. albicans, which is a major regulator of chromosome segregation. Contrary to other eukaryotes studied thus far, C. albicans does not require kinesin-5 function for bipolar spindle assembly or spindle elongation. Rather, this motor protein associates with the spindle throughout mitosis to maintain spindle integrity. Furthermore, kinesin-5 loss is synthetically lethal with loss of kinesin-14-canonically an opposing force producer to kinesin-5 in spindle assembly and anaphase. These results suggest a significant evolutionary rewiring of microtubule motor functions in the C. albicans mitotic spindle, which may have implications in the genetic instability of this pathogen.