Much remains to be learned about the molecular mechanisms underlying a class of human disorders called actinopathies. These genetic disorders are characterized by loss-of-function mutations in actin-associated proteins that affect immune cells, leading to human immunopathology. However, much remains to be learned about how cytoskeletal dysregulation promotes immunological dysfunction. The current study reveals that the macrophage actin cytoskeleton responds to LPS/IFNγ stimulation in a biphasic manner that involves cellular contraction followed by cellular spreading. Myosin II inhibition by blebbistatin blocks the initial contraction phase and lowers iNOS protein levels and nitric oxide secretion. Conversely, conditional deletion of Arp2/3 complex in macrophages attenuates spreading and increases nitric oxide secretion. However, iNOS transcription is not altered by loss of myosin II or Arp2/3 function, suggesting post-transcriptional regulation of iNOS by the cytoskeleton. Consistent with this idea, proteasome inhibition reverses the effects of blebbistatin and rescues iNOS protein levels. Arp2/3-deficient macrophages demonstrate two additional phenotypes: defective MHCII surface localization, and depressed secretion of the T cell chemokine CCL22. These data suggest that interplay between myosin II and Arp2/3 influences macrophage activity, and potentially impacts adaptive-innate immune coordination. Disrupting this balance could have detrimental impacts, particularly in the context of Arp2/3-associated actinopathies.

Trauma triggers critical molecular and cellular signaling cascades that drive biological outcomes and recovery. Variations in the gene expression of common endogenous reference housekeeping genes (HKGs) used in data normalization differ between tissue types and pathological states. Systematically, we investigated the gene stability of nine HKGs (Actb, B2m, Gapdh, Hprt1, Pgk1, Rplp0, Rplp2, Tbp, and Tfrc) from tissues prone to remote organ dysfunction (lung, liver, kidney, and muscle) following extremity trauma. Computational algorithms (geNorm, Normfinder, ΔCt, BestKeeper, RefFinder) were applied to estimate the expression stability of each HKG or combinations of them, within and between tissues, under both steady-state and systemic inflammatory conditions. Rplp2 was ranked as the most suitable in the healthy and injured lung, kidney, and skeletal muscle, whereas Rplp2 and either Hprt1 or Pgk1 were the most suitable in the healthy and injured liver, respectively. However, the geometric mean of the three most stable genes was deemed the most stable internal reference control. Actb and Tbp were the least stable in normal tissues, whereas Gapdh and Tbp were the least stable across all tissues post-trauma. Ct values correlated poorly with the translation from mRNA to protein. Our results provide a valuable resource for the accurate normalization of gene expression in trauma-related experiments.

Post-translational regulations of Shaker-like voltage-gated K⁺ channels were reported to be essential for rapid responses to environmental stresses in plants. In particular, it has been shown that calcium-dependent protein kinases (CPKs) regulate Shaker channels in plants. Here, the focus was on KAT2, a Shaker channel cloned in the model plant Arabidopsis thaliana, where is it expressed namely in the vascular tissues of leaves. After co-expression of KAT2 with AtCPK6 in Xenopuslaevis oocytes, voltage-clamp recordings demonstrated that AtCPK6 stimulates the activity of KAT2 in a calcium-dependent manner. A physical interaction between these two proteins has also been shown by Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM). Peptide array assays support that AtCPK6 phosphorylates KAT2 at several positions, also in a calcium-dependent manner. Finally, K⁺ fluorescence imaging in planta suggests that K⁺ distribution is impaired in kat2 knock-out mutant leaves. We propose that the AtCPK6/KAT2 couple plays a role in the homeostasis of K⁺ distribution in leaves.

Arthropod-borne pathogens, many of which are neurotropic, can disseminate beyond the central nervous system to infect peripheral organs. In recent years, an increasing number of cardiac dysfunctions have been reported following arthropod-borne viral infections; however, the mechanism underlying these cardiac manifestations remains poorly understood. In this study, we investigated the impact of Venezuelan Equine Encephalitis Virus (VEEV) TC-83 infection on cardiac function and immune-response of human induced-pluripotent stem cell (hIPSC)-derived cardiomyocytes (hIPSC-CMs). We first confirmed the successful differentiation of hIPSCs into spontaneously beating hIPSC-CMs. We then demonstrated that these cells are highly susceptible to VEEV TC-83 infection, which induced pronounced arrhythmias and complete cessation of beating within 24 hours post-infection. To quantify these functional changes, we developed a segmentation-free computational pipeline that converts frame-to-frame motion in brightfield time-lapse movies into a one-dimensional signal reflecting contractile activity and extracts beat timing, beat rate, and rhythm-regularity features in the time and frequency domains. This analysis revealed progressive disruption of beating dynamics following VEEV TC-83 infection, with early rhythm instability and complete loss of coordinated beating by 24 hours post-infection. Furthermore, mass spectrometry analysis of VEEV TC-83-infected hIPSC-CMs supernatants revealed the presence of biomarkers typically associated with heart failure in patients, underscoring a virus-induced cardiac functional impairment. Together, these findings provide new insight into cardiac complications associated with arthropod-borne viral infections and may support advances in preventive medicine.