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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, which activates their ability to stimulate the DRP1 GTPase. 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 knockdown effects on mitochondrial length and DRP1 recruitment. Furthermore, cellular treatment with BSA-bound oleic acid, which causes increased LCACA, 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 division. Finally, MiD49 or MiD51 oligomers synergize with Mff, but not with actin filaments, in DRP1 activation, suggesting distinct pathways for DRP1 activation., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Cell migration frequently involves the formation of lamellipodia induced by Rac GTPases activating WAVE regulatory complex (WRC) to drive Arp2/3 complex-dependent actin assembly. Previous genome editing studies in B16-F1 melanoma cells solidified the view of an essential, linear pathway employing the aforementioned components. Here, disruption of the WRC subunit Nap1 (encoded by Nckap1) and its paralog Hem1 (encoded by Nckap1l) followed by serum and growth factor stimulation, or active GTPase expression, revealed a pathway to formation of Arp2/3 complex-dependent lamellipodia-like structures (LLS) that requires both Rac and Cdc42 GTPases, but not WRC. These phenotypes were independent of the WRC subunit eliminated and coincided with the lack of recruitment of Ena/VASP family actin polymerases. Moreover, aside from Ena/VASP proteins, LLS contained all lamellipodial regulators tested, including cortactin (also known as CTTN), the Ena/VASP ligand lamellipodin (also known as RAPH1) and FMNL subfamily formins. Rac-dependent but WRC-independent actin remodeling could also be triggered in NIH 3T3 fibroblasts by growth factor (HGF) treatment or by gram-positive Listeria monocytogenes usurping HGF receptor signaling for host cell invasion. Taken together, our studies thus establish the existence of a signaling axis to Arp2/3 complex-dependent actin remodeling at the cell periphery that operates without WRC and Ena/VASP., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The formin INF2 polymerizes a calcium-activated cytoplasmic network of actin filaments, which we refer to as calcium-induced actin polymerization (CIA). CIA plays important roles in multiple cellular processes, including mitochondrial dynamics and vesicle transport. Here, we show that nonmuscle myosin II (NMII) is activated within 60 s of calcium stimulation and rapidly recruited to the CIA network. Knockout of any individual NMII in U2OS cells affects the organization of the CIA network, as well as three downstream effects: endoplasmic-reticulum-to-mitochondrial calcium transfer, mitochondrial Drp1 recruitment, and mitochondrial division. Interestingly, while NMIIC is the least abundant NMII in U2OS cells (>200-fold less than NMIIA and >10-fold less than NMIIB), its knockout is equally deleterious to CIA. On the basis of these results, we propose that myosin II filaments containing all three NMII heavy chains exert organizational and contractile roles in the CIA network. In addition, NMIIA knockout causes a significant decrease in myosin regulatory light chain levels, which might have additional effects.

The human gut acts as the main reservoir of microbes and a relevant source of life-threatening infections, especially in immunocompromised patients. There, the opportunistic fungal pathogen Candida albicans adapts to the host environment and additionally interacts with residing bacteria. We investigated fungal-bacterial interactions by coinfecting enterocytes with the yeast Candida albicans and the Gram-negative bacterium Proteus mirabilis resulting in enhanced host cell damage. This synergistic effect was conserved across different P. mirabilis isolates and occurred also with non- albicans Candida species and C. albicans mutants defective in filamentation or candidalysin production. Using bacterial deletion mutants, we identified the P. mirabilis hemolysin HpmA to be the key effector for host cell destruction. Spatially separated coinfections demonstrated that synergism between Candida and Proteus is induced by contact, but also by soluble factors. Specifically, we identified Candida -mediated glucose consumption and farnesol production as potential triggers for Proteus virulence. In summary, our study demonstrates that coinfection of enterocytes with C. albicans and P. mirabilis can result in increased host cell damage which is mediated by bacterial virulence factors as a result of fungal niche modification via nutrient consumption and production of soluble factors. This supports the notion that certain fungal-bacterial combinations have the potential to result in enhanced virulence in niches such as the gut and might therefore promote translocation and dissemination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Niemiec, Kapitan, Himmel, Döll, Krüger, Köllner, Auge, Kage, Alteri, Mobley, Monsen, Linde, Nietzsche, Kniemeyer, Brakhage and Jacobsen.)

Mitochondrial damage (MtD) represents a dramatic change in cellular homeostasis, necessitating metabolic changes and stimulating mitophagy. One rapid response to MtD is a rapid peri-mitochondrial actin polymerization termed ADA (acute damage-induced actin). The activation mechanism for ADA is unknown. Here, we use mitochondrial depolarization or the complex I inhibitor metformin to induce ADA. We show that two parallel signaling pathways are required for ADA. In one pathway, increased cytosolic calcium in turn activates PKC-β, Rac, WAVE regulatory complex, and Arp2/3 complex. In the other pathway, a drop in cellular ATP in turn activates AMPK (through LKB1), Cdc42, and FMNL formins. We also identify putative guanine nucleotide exchange factors for Rac and Cdc42, Trio and Fgd1, respectively, whose phosphorylation states increase upon mitochondrial depolarization and whose suppression inhibits ADA. The depolarization-induced calcium increase is dependent on the mitochondrial sodium-calcium exchanger NCLX, suggesting initial mitochondrial calcium efflux. We also show that ADA inhibition results in enhanced mitochondrial shape changes upon mitochondrial depolarization, suggesting that ADA inhibits these shape changes. These depolarization-induced shape changes are not fragmentation but a circularization of the inner mitochondrial membrane, which is dependent on the inner mitochondrial membrane protease Oma1. ADA inhibition increases the proteolytic processing of an Oma1 substrate, the dynamin GTPase Opa1. These results show that ADA requires the combined action of the Arp2/3 complex and formin proteins to polymerize a network of actin filaments around mitochondria and that the ADA network inhibits the rapid mitochondrial shape changes that occur upon mitochondrial depolarization., Competing Interests: Declaration of interests H.N.H. is a member of the Advisory Board of Current Biology., (Copyright © 2022 Elsevier Inc. All rights reserved.)