Biology subjects

Kage, F.

Publications and source records attributed to Kage, F..

7 recordsLinked to original sources

bioRxiv2025

Convergent evolution of a fungal effector enabling phagosome membrane penetration

The ability of pathogens to evade phagosomal killing is critical for their pathogenicity. Previously, we had identified the HscA effector protein in the clinically important fungal pathogen Aspergillus fumigatus, which redirects conidia-containing phagosomes from the degradative to the non-degradative pathway. Here, we discovered a pathogenic form of this surface protein, determined by a single tyrosine residue (Y) at position 596, which is lacking in most fungi analyzed, that have a leucine (L) instead. Y596 enables HscA to penetrate the phagosomal membrane. In line, the introduction of a single L- to-Y exchange in the orthologous Ssb protein of Saccharomyces cerevisiae enabled the protein to penetrate phagosomal membranes that was reduced by deletion of one of the two Y-encoding SSB genes in the pathogenic fungus Candida glabrata. These data suggest a convergent evolution of HscA/Ssb proteins among human-pathogenic fungi and that a single amino acid exchange determines a virulence factor. HighlightsO_LIHscA with Y596 penetrates host cell membranes and causes phagosomal damage C_LIO_LIMost fungi (96%) have a leucine at the respective position C_LIO_LIAn L-to-Y mutation in Ssb enables S. cerevisiae to damage phagosomes C_LIO_LIRecruitment of ESCRT complex to the phagosomal membrane requires human p11 protein C_LI

microbiology↗

bioRxiv2024

Identification of a fungal antibacterial endopeptidase that modulates immune responses

Aspergillus fumigatus is a saprophytic fungus dwelling in soil and on decaying plant material, but also an opportunistic pathogen in immunocompromised patients. In its environmental niche, A. fumigatus faces competition from other microorganisms including bacteria. Here, we describe the discovery of the first secreted antibacterial protein in A. fumigatus. We identified a secreted fungal endopeptidase, designated CwhA, that cleaves peptidoglycan of Gram-positive bacteria at specific residues within the peptidoglycan stem peptide. Cleavage leads to bacterial lysis and the release of peptidoglycan cleavage products. Expression of cwhA is induced by the presence of bacteria. Furthermore, CwhA is highly abundant in murine lungs during invasive pulmonary aspergillosis and peptidoglycan cleavage products generated by CwhA stimulate cytokine production of human immune cells. Although CwhA does not affect human cells directly, this novel player in fungal-bacterial interactions could affect A. fumigatus infections by inhibiting Gram-positive bacteria in its vicinity, and modulating the immune system.

microbiology↗

bioRxiv2023

Long-chain fatty acyl-coenzyme A activates the mitochondrial fission factors MiD49 and MiD51 by inducing their oligomerization

Mitochondrial fission occurs in many cellular processes, but the regulation of fission is poorly understood. We show that long-chain acyl coenzyme A (LCACA) activates two related mitochondrial fission proteins, MiD49 and MiD51, by inducing their oligomerization, activating their ability to stimulate DRP1 GTPase activity. The 1:1 stoichiometry of LCACA:MiD in the oligomer suggests interaction in the previously identified nucleotide-binding pocket, and a point mutation in this pocket reduces LCACA binding and LCACA-induced oligomerization for MiD51. In cells, this LCACA binding mutant does not assemble into puncta on mitochondria or rescue MiD49/51 knock-down effects on mitochondrial length and DRP1 recruitment. Furthermore, cellular treatment with the fatty acid analogue 2-bromopalmitate, which causes increased acyl-CoA, promotes mitochondrial fission in an MiD49/51-dependent manner. These results suggest that LCACA is an endogenous ligand for MiDs, inducing mitochondrial fission and providing a potential mechanism for fatty acid-induced mitochondrial fragmentation. Finally, MiD49 or MiD51 oligomers synergize with MFF, but not with actin filaments, in DRP1 activation, suggesting distinct pathways for DRP1 activation.

cell biology↗

bioRxiv2021

Lamellipodia-like actin networks in cells lacking WAVE Regulatory Complex

Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases mediating activation of WAVE Regulatory Complex (WRC) driving Arp2/3 complex-dependent actin assembly. Previous genome editing studies solidified the view of an essential, linear pathway employing aforementioned components. Using disruption of the WRC subunit Nap1 and its paralogue Hem1 followed by serum and growth factor stimulation or expression of active GTPases now revealed a pathway to formation of Arp2/3 complex-dependent, lamellipodia-like structures (LLS) that require both Rac and Cdc42, but not WRC. These observations were independent of WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from the latter, induced LLS contained all common lamellipodial regulators tested, including cortactin, the Ena/VASP ligand lamellipodin or FMNL subfamily formins. Our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery operating without WRC and Ena/VASP.

cell biology↗

bioRxiv2020

Induced Arp2/3 complex depletion increases FMNL2/3 formin expression and filopodia formation

The Arp2/3 complex generates branched actin filament networks operating in cell edge protrusion and vesicle trafficking. Here we employ a novel, conditional knockout mouse model permitting tissue- or cell-type specific deletion of the murine Actr3 gene (encoding Arp3). A functional Actr3 gene appeared essential for fibroblast viability and growth. Thus, we developed cell lines for exploring the consequences of acute, tamoxifen-induced Actr3 deletion causing near-complete loss of Arp/3 complex function as well as abolished lamellipodia formation and membrane ruffling, as expected. However, Arp3-depleted cells displayed enhanced rather than reduced cell spreading, employing numerous filopodia, and showed little defects in individual cell migration. Reduction of collective cell migration as observed for instance in wound healing assays likely derived from defects in maintaining directionality during migration, while the principal ability to chemotax was only moderately affected. Analyses of actin turnover at the cell periphery revealed reduced actin turnover rates in Arp2/3-deficient cells, clearly deviating from previous sequestration approaches. Most surprisingly, induced removal of Arp2/3 complexes reproducibly increased FMNL formin expression, which correlated with the explosive induction of filopodia formation. Our results thus highlight both direct and indirect effects of acute Arp2/3 complex removal on actin cytoskeleton regulation.

cell biology↗

bioRxiv2020

Loss of Hem1 disrupts macrophage function and impacts on migration, phagocytosis and integrin-mediated adhesion

The hematopoietic-specific protein 1 (Hem1) comprises an essential subunit of the WAVE Regulatory Complex (WRC) in immune cells. WRC has a fundamental role in Arp2/3 complex activation and the protrusion of branched actin networks in motile cells. Hem1 deficiency leads to suppression of the entire WRC in immune cells. Defective WRC function in macrophages results in loss of lamellipodia and migration defects. Moreover, phagocytosis, commonly accompanied by lamellipodium protrusion during cup formation, is altered in Hem1 null cells concerning frequency and efficacy. When analyzing cell spreading, adhesion and podosome formation, we found that Hem1 null cells are capable, in principle, of podosome formation and consequently, do not show any quantitative differences in extracellular matrix degradation. Their adhesive behavior, however, was significantly altered. Specifically, adhesion as well as de-adhesion of Hem1 null cells was strongly compromised, likely contributing to the observed reduced efficiency of phagocytosis. In line with this, phosphorylation of the prominent adhesion component paxillin was diminished. Non-hematopoietic somatic cells disrupted in expression for both Hem1 and its ubiquitous orthologue Nck-associated protein 1 (Nap1) or the essential WRC components Sra-1/PIR121 did not only confirm defective paxillin phosphorylation, but also revealed that paxillin turnover in focal adhesions is accelerated in the absence of WRC. Finally, adhesion assays using platelets lacking functional WRC as model system unmasked radically decreased IIb{beta}3 integrin activation. Our results thus demonstrate that WRC-driven actin networks impact on integrin-dependent processes controlling formation and dismantling of different types of cell-substratum adhesion. One sentence summaryInterference of Hem1 function in mice and cells uncovers a hitherto unrecognized role in integrin-mediated cell adhesion that is crucial for macrophage function and connects to recently discovered immunodeficiencies in patients carrying Hem1 mutations.

cell biology↗

bioRxiv2019

RhoG and Cdc42 can contribute to Rac-dependent lamellipodia formation through WAVE Regulatory Complex-binding

Cell migration frequently involves the formation of lamellipodial protrusions, the initiation of which requires Rac GTPases signalling to heteropentameric WAVE regulatory complex (WRC). While Rac-related RhoG and Cdc42 can potently stimulate lamellipodium formation, so far presumed to occur by upstream signalling to Rac activation, we show here that the latter can be bypassed by RhoG and Cdc42 given that WRC has been artificially activated. This evidence arises from generation of B16-F1 cells simultaneously lacking both Rac GTPases and WRC, followed by reconstitution of lamellipodia formation with specific Rho-GTPase and differentially active WRC variant combinations. We conclude that formation of canonical lamellipodia requires WRC activation through Rac, but can possibly be tuned, in addition, by WRC interactions with RhoG and Cdc42.

cell biology↗