Formation of neutrophil extracellular traps requires actin cytoskeleton rearrangements

Neutrophils are important effector cells in the host defense against invading micro-organisms. One of the mechanisms they employ to eliminate pathogens is the release of neutrophil extracellular traps (NETs). Although NET release and subsequent cell death known as NETosis have been intensively studied, the cellular components and factors determining or facilitating the formation of NETs remain incompletely understood. Using various actin polymerization and myosin II modulators on neutrophils from healthy individuals, we show that intact F-actin dynamics and myosin II function are essential for NET formation when induced by different stimuli, i.e. phorbol 12-myristate 13-acetate, monosodium urate crystals and Candida albicans. The role of actin polymerization in NET formation could not be explained by the lack of reactive oxygen species production or granule release, which were normal or enhanced under the given conditions. Neutrophils from patients with very rare inherited actin polymerization defects by either ARPC1B- or MKL1-deficiency also failed to show NETosis. We found that upon inhibition of actin dynamics there is a lack of translocation of NE to the nucleus, which may well explain the impaired NET formation. Collectively, our data illustrate the essential requirement of an intact and active actin polymerization process, as well as active myosin II to enable the release of nuclear DNA by neutrophils during NET formation.

Bioactive compounds from Lactarius deterrimus interfere with the invasive potential of gastric cancer cells

Stomach cancer is the 4th most common cancer diagnosed worldwide. Despite intensive research on its etiopathology, its treatment strategies have not changed in the last 50 years. Mushrooms have recently attracted much attention as the source of bioactive compounds that can potentially complement cancer therapies. Here, we extracted a phenolic fraction from Lactarius deterrimus and analyzed its composition and bioactivity against the gastric cancer (AGS) cells. The complexity of L. deterrimus compounds was revealed by an HPLC assay, and was accompanied by cytostatic, cytotoxic and anti-invasive effects of the L. deterrimus extract (LDE). These are illustrated by inhibition of the AGS cells’ proliferation, metabolic activity and motility, and by induction of the cytoskeleton rearrangements. Apparently, these effects are exerted via activation of intracellular oxidative stress and decreased ATP production in AGS cells that could not be compensated by induction of autophagy. Less severe LDE effects were seen on physiology of normal gastric fibroblasts; however, inhibition of their motility indicates that LDE can interfere with gastric cancer development via an effect on stromal cells. Along with the observed synergy of LDE and cisplatin/5-fluorouracil effects on AGS cells, our data show the potential of LDE for supplementation of the gastric cancer therapy.

Both sides of the story How bacteria transfer through the blood–brain barrier?

ABSTRACT Infections of central nervous system (CNS) still represent an important cause of mortality although globally abundance of antibiotics usage. Sepsis, bacterial invasion and microbial transversal of the Blood–Brain Barrier (BBB) are required to infect CNS. The latest research showed up that bacterial translocation through the BBB includes cytoskeleton rearrangements. Three different mechanism were described: paracellular, transcellular and Trojan-horse mechanism (in phagocytes). The consequences may be dramatic – disruptions of structure and loss of functionality of the BBB causing increased permeability, inflammatory and encephalopathy. Further experimental research should lead us to gain complete understanding of the host-bacteria interaction within microbial transversal of the BBB.

Zika virus employs the host antiviral RNase L protein to support replication factory assembly

Infection with the flavivirus Zika virus (ZIKV) can result in tissue tropism, disease outcome, and route of transmission distinct from those of other flaviviruses; therefore, we aimed to identify host machinery that exclusively promotes the ZIKV replication cycle, which can inform on differences at the organismal level. We previously reported that deletion of the host antiviral ribonuclease L (RNase L) protein decreases ZIKV production. Canonical RNase L catalytic activity typically restricts viral infection, including that of the flavivirus dengue virus (DENV), suggesting an unconventional, proviral RNase L function during ZIKV infection. In this study, we reveal that an inactive form of RNase L supports assembly of ZIKV replication factories (RFs) to enhance infectious virus production. Compared with the densely concentrated ZIKV RFs generated with RNase L present, deletion of RNase L induced broader subcellular distribution of ZIKV replication intermediate double-stranded RNA (dsRNA) and NS3 protease, two constituents of ZIKV RFs. An inactive form of RNase L was sufficient to contain ZIKV genome and dsRNA within a smaller RF area, which subsequently increased infectious ZIKV release from the cell. Inactive RNase L can interact with cytoskeleton, and flaviviruses remodel cytoskeleton to construct RFs. Thus, we used the microtubule-stabilization drug paclitaxel to demonstrate that ZIKV repurposes RNase L to facilitate the cytoskeleton rearrangements required for proper generation of RFs. During infection with flaviviruses DENV or West Nile Kunjin virus, inactive RNase L did not improve virus production, suggesting that a proviral RNase L role is not a general feature of all flavivirus infections.

Role of γ-Adducin in Actin Cytoskeleton Rearrangements in Podocyte Pathophysiology

We recently reported that the enhanced susceptibility to chronic kidney disease (CKD) in the FHH rat is caused, at least in part, by a mutation in γ-adducin (ADD3) that attenuates renal vascular function. The present study explored whether Add3 contributes to the modulation of podocyte structure and function using FHH and FHH.Add3 transgenic rats. The expression of ADD3 on the membrane of primary podocytes isolated from FHH was reduced compared with FHH.Add3 transgenic rats. We found that F-actin nets, which are typically localized in the lamellipodia, replaced unbranched stress fibers in conditionally immortalized mouse podocytes transfected with Add3 DsiRNA and primary podocytes isolated from FHH rats. There were increased F/G-actin ratio and expression of the Arp2/3 complexes throughout FHH podocytes in association with reduced synaptopodin and RhoA but enhanced Rac1 and CDC42 expression in the renal cortex, glomeruli and podocytes of FHH rats. The expression of Nephrin at the slit diaphragm and the levels of focal adhesion proteins ITGA3 and ITGB1 were decreased in the glomeruli of FHH rats. Cell migration was enhanced and adhesion was reduced in podocytes of FHH rats, as well as in immortalized mouse podocyte transfected with Add3 DsiRNA. Mean arterial pressures were similar in FHH and FHH.Add3 transgenic rats at 16-week of age; however, FHH rats exhibited enhanced proteinuria associated with podocyte foot process effacement. These results demonstrate that reduced ADD3 function in FHH rats alters baseline podocyte pathophysiology by rearrangement of the actin cytoskeleton at the onset of proteinuria in young animals.

Autophagy inhibition rescues structural and functional defects caused by the loss of mitochondrial chaperone Hsc70-5/mortalin in Drosophila

We investigate in larval and adult Drosophila models whether loss of the mitochondrial chaperone Hsc70-5/mortalin is sufficient to cause pathological alterations commonly observed in Parkinson disease. At affected larval neuromuscular junctions, no effects on terminal size, bouton size or number, synapse size, or number were observed, suggesting that we study an early stage of pathogenesis. At this stage, we noted a loss of synaptic vesicle proteins and active zone components, delayed synapse maturation, reduced evoked and spontaneous excitatory junctional potentials, increased synaptic fatigue, and cytoskeleton rearrangements. The adult model displays ATP depletion, altered body posture, and susceptibility to heat-induced paralysis. Adult phenotypes could be suppressed by knockdown of DJ-1b, LRRK, p50, p150, Atg1, Atg101, Atg5, Atg7, and Atg12. The knockdown of components of the autophagy machinery or overexpression of human mortalin broadly rescued larval and adult phenotypes, while disease-associated HSPA9 variants did not. Overexpression of Pink1 or promotion of autophagy exacerbated defects.

The role of Rho GTPases’ substrates Rac and Cdc42 in osteoclastogenesis and relevant natural medicinal products study

Recently, Rho GTPases substrates include Rac (Rac1 and Rac2) and Cdc42 that have been reported to exert multiple cellular functions in osteoclasts, the most prominent of which includes regulating the dynamic actin cytoskeleton rearrangements. In addition, natural products and their molecular frameworks have a long tradition as valuable starting points for medicinal chemistry and drug discovery. Although currently, there are reports about the natural product, which could play a therapeutic role in bone loss diseases (osteoporosis and osteolysis) through the regulation of Rac1/2 and Cdc42 during osteoclasts cytoskeletal structuring. There have been several excellent studies for exploring the therapeutic potentials of various natural products for their role in inhibiting cancer cells migration and function via regulating the Rac1/2 and Cdc42. Herein in this review, we try to focus on recent advancement studies for extensively understanding the role of Rho GTPases substrates Rac1, Rac2 and Cdc42 in osteoclastogenesis, as well as therapeutic potentials of natural medicinal products for their properties on the regulation of Rac1, and/or Rac2 and Cdc42, which is in order to inspire drug discovery in regulating osteoclastogenesis.