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IntroductionKugeln are a recently characterised endothelial cell (EC) membrane behaviour in zebrafish, exclusive to the cerebral vasculature and of unknown function. Kugeln are highly dynamic structures that alter their shape and size throughout their lifetime. Early studies revealed that the formation of kugeln is regulated by filamentous actin (F-actin) cytoskeletal dynamics, with F-actin localised predominantly at the kugel neck. However, the spatiotemporal dynamics of F-actin recruitment and redistribution remain to be studied. Therefore, we characterised kugel F-actin dynamics in relation to the kugel membrane in the zebrafish cerebral vasculature, using in vivo time-lapse light sheet imaging.MethodsDouble-transgenic RFP-tagged EC membrane Tg(kdrl:HRASmCherry)s916 and GFP-tagged EC F-actin Tg(fli1a:LifeAct-mClover)sh467 3–4 dpf embryos were anaesthetised and imaged with a Zeiss Z.1 light sheet fluorescence microscope to capture 2-hour time-lapse recordings (time interval – 1 minute) of up to 100μm x 100μm regions of interest at 1μm z-intervals within the midbrain. Raw data was processed with FIJI (v1.51) using custom macro plugins created by our group. Each kugel observed was analysed to evaluate the kugel EC F-actin and EC membrane dynamics over time.ResultsWe analysed 45 kugeln from 15 zebrafish embryos (3 experimental repeats) and observed three distinct kugel membrane behaviours. These included rounded kugeln where the kugel membrane maintains a spherical shape, a ‘snowman’-like behaviour where spherical kugeln domes occur on top of one another, and an undulating wave-like kugel membrane behaviour (Figure 1). A fourth, mixed kugel behaviour was also observed, where the kugel membrane dynamically transitions between the previously defined kugel membrane behaviours (Figure1). 78% of the kugeln exhibited the rounded morphology and the remaining 22% displayed either the wave-like, ‘snowman’ or mixed behaviours (Figure 2). Each behaviour was associated with a distinct pattern of F-actin dynamics. F-actin in rounded kugeln was initially localised to the kugel neck and subsequently to the top of the dome. In the wave-like kugel behaviour, F-actin was localised to the kugel neck throughout its lifespan. In the ‘snowman’-like kugeln, F-actin was predominantly localised at the neck of each successive kugel dome.Abstract BS42 Figure 1Mixed type Kugel membrane (red) behaviour transitioning between wave-like, rounded and ‘snowman‘- like behaviours and characteristic F-actin dynamics (Green)Abstract BS42 Figure 2The proportion of distinct kugel behaviours observed in the 3–4dpf zebrafish midbrain vasculatureConclusionWe have further elucidated the spatiotemporal dynamics of kugel behaviour and linked discrete kugel membrane behaviours to specific patterns of F-actin rearrangement. These results provide more insight into the cytoskeletal mechanisms of kugel formation, maintenance and demonstrate the complexity of kugel behaviour.

The Hippo tumour suppressor pathway is a conserved signalling pathway that controls organ size. The core of the Hpo pathway is a kinase cascade, which in Drosophila involves the Hpo and Warts kinases that negatively regulate the activity of the transcriptional coactivator Yorkie. Although several additional components of the Hippo pathway have been discovered, the inputs that regulate Hippo signalling are not fully understood. Here, we report that induction of extra F‐actin formation, by loss of Capping proteins A or B, or caused by overexpression of an activated version of the formin Diaphanous, induced strong overgrowth in Drosophila imaginal discs through modulating the activity of the Hippo pathway. Importantly, loss of Capping proteins and Diaphanous overexpression did not significantly affect cell polarity and other signalling pathways, including Hedgehog and Decapentaplegic signalling. The interaction between F‐actin and Hpo signalling is evolutionarily conserved, as the activity of the mammalian Yorkie‐orthologue Yap is modulated by changes in F‐actin. Thus, regulators of F‐actin, and in particular Capping proteins, are essential for proper growth control by affecting Hippo signalling. This study identifies actin organization as an upstream regulator of the Hippo pathway: F‐actin accumulation promotes Yorkie‐dependent transcriptional activation. This modulation of Hippo signalling by actin regulators controls organ growth in Drosophila.

• In plants, the actin cytoskeleton plays a central role in regulating intracellular transport and trafficking in the endomembrane system. Work in legumes suggested that during nodulation, the actin cytoskeleton coordinates numerous cellular processes in the development of nitrogen-fixing nodules. However, we lacked live-cell visualizations demonstrating dynamic remodeling of the actin cytoskeleton during infection droplet release and symbiosome development. • Here, we generated transgenic Medicago truncatula lines stably expressing the fluorescent actin marker ABD2-GFP, and utilized live-cell imaging to reveal the architecture and dynamics of the actin cytoskeleton during nodule development. • Live-cell observations showed that different zones in nitrogen-fixing nodules exhibit distinct actin architectures and infected cells display five characteristic actin architectures during nodule development. Live-cell imaging combined with three-dimensional reconstruction demonstrated that dense filamentous-actin (F-actin) arrays channel the elongation of infection threads and the release of infection droplets, an F-actin network encircles freshly-released rhizobia, and short F-actin fragments and actin dots around radially distributed symbiosomes. • Our findings suggest an important role of the actin cytoskeleton in infection droplet release, symbiosome development and maturation, and provide significant insight into the cellular mechanisms underlying nodule development and nitrogen fixation during legume–rhizobia interactions.

Postoperative neurocognitive disorders (pNCD) are a common neurological complication, especially in elderly following anesthesia and surgery. Yet, the underlying mechanisms of pNCD remain elusive. This study aimed to investigate the molecular mechanisms that compromise synaptic metaplasticity in pNCD development with a focus on the involvement of Nogo‐66 receptor 1 (NgR1) in the pathogenesis of pNCD in aged mice. Aged mice subjected to anesthesia and laparotomy surgery exhibited anxiety‐like behavior and contextual fear memory impairment. Moreover, the procedure significantly increased NogoA and NgR1 expressions, particularly in the hippocampal CA1 and CA3 regions. This increase led to the depolymerization of F‐actin, attributed to the activation of the RhoA‐GTPase, resulting in a reduction of dendritic spines and changes in their morphology. Additionally, these changes hindered the efficient postsynaptic delivery of the subunit GluA1 and GluA2 of AMPA receptors (AMPARs), consequently diminishing excitatory neurotransmission in the hippocampus. Importantly, administering the competitive NgR1 antagonist peptide NEP1‐40 (Nogo‐A extracellular peptide residues 1–40 amino acids of Nogo‐66) and Fasudil (a Rho‐kinase inhibitor) effectively mitigated synaptic impairments and reversed neurocognitive deficits in aged mice following anesthesia and surgery. Our work indicates that high hippocampal Nogo66‐NgR1 signaling disrupts postsynaptic AMPA receptor surface delivery due to F‐actin depolymerization in the pathophysiology of pNCD. Hippocampal Nogo66‐NgR1 signaling activation, disrupting postsynaptic AMPA receptor surface delivery due to F‐actin depolymerization, leads to anxiety‐like behavior and contextual fear memory impairment in aged mice following anaesthesia and surgery.

Podocyte injury is an early pathological change characteristic of various glomerular diseases, and apoptosis and F‐actin cytoskeletal disruption are typical features of podocyte injury. In this study, we found that adriamycin (ADR) treatment resulted in typical podocyte injury and repressed plectin expression. Restoring plectin expression protected against ADR‐induced podocyte injury whereas siRNA‐mediated plectin silencing produced similar effects as ADR‐induced podocyte injury, suggesting that plectin plays a key role in preventing podocyte injury. Further analysis showed that plectin repression induced significant integrin α6β4, focal adhesion kinase (FAK) and p38 MAPK phosphorylation. Mutating Y1494, a key tyrosine residue in the integrin β4 subunit, blocked FAK and p38 phosphorylation, thereby alleviating podocyte injury. Inhibitor studies demonstrated that FAK Y397 phosphorylation promoted p38 activation, resulting in podocyte apoptosis and F‐actin cytoskeletal disruption. In vivo studies showed that administration of ADR to rats resulted in significantly increased 24‐hour urine protein levels along with decreased plectin expression and activated integrin α6β4, FAK, and p38. Taken together, these findings indicated that plectin protects podocytes from ADR‐induced apoptosis and F‐actin cytoskeletal disruption by inhibiting integrin α6β4/FAK/p38 pathway activation and that plectin may be a therapeutic target for podocyte injury‐related glomerular diseases.

Summary As a multifunctional hormone‐like molecule, melatonin exhibits a pleiotropic role in plant salt stress tolerance. While actin cytoskeleton is essential to plant tolerance to salt stress, it is unclear if and how actin cytoskeleton participates in the melatonin‐mediated alleviation of plant salt stress. Here, we report that melatonin alleviates salt stress damage in pigeon pea by activating a kinase‐like protein, which interacts with an actin‐depolymerizing factor. Cajanus cajan Actin‐Depolymerizing Factor 9 (CcADF9) has the function of severing actin filaments and is highly expressed under salt stress. The CcADF9 overexpression lines (CcADF9‐OE) showed a reduction of transgenic root length and an increased sensitivity to salt stress. By using CcADF9 as a bait to screen an Y2H library, we identified actin depolymerizing factor‐related phosphokinase 1 (ARP1), a novel protein kinase that interacts with CcADF9. CcARP1, induced by melatonin, promotes salt resistance of pigeon pea through phosphorylating CcADF9, inhibiting its severing activity. The CcARP1 overexpression lines (CcARP1‐OE) displayed an increased transgenic root length and resistance to salt stress, whereas CcARP1 RNA interference lines (CcARP1‐RNAi) presented the opposite phenotype. Altogether, our findings reveal that melatonin‐induced CcARP1 maintains F‐actin dynamics balance by phosphorylating CcADF9, thereby promoting root growth and enhancing salt tolerance.

The immune system has to cope with a wide range of irregularly shaped pathogens that can actively move (e.g., by flagella) and also dynamically remodel their shape (e.g., transition from yeast-shaped to hyphal fungi). The goal of this review is to draw general conclusions of how the size and geometry of a pathogen affect its uptake and processing by phagocytes of the immune system. We compared both theoretical and experimental studies with different cells, model particles, and pathogenic microbes (particularly fungi) showing that particle size, shape, rigidity, and surface roughness are important parameters for cellular uptake and subsequent immune responses, particularly inflammasome activation and T cell activation. Understanding how the physical properties of particles affect immune responses can aid the design of better vaccines.

Although the phagocytic activity of macrophages has long been studied, the involvement of microtubules in the process is not well understood. In this study, we improved the fixation protocol and revealed a dynamically rearranging microtubule network in macrophages, consisting of a basal meshwork, thick bundles at the cell edge, and astral microtubules. Some astral microtubules extended beneath the cell cortex and continued to form bundles at the cell edge. These microtubule assemblies were mutually exclusive of actin accumulation during membrane ruffling. Although the stabilization of microtubules with paclitaxel did not affect the resting stage of the macrophages, it reduced the phagocytic activity and membrane ruffling of macrophages activated with serum-MAF, which induced rapid phagocytosis. In contrast, the destabilization of microtubules with nocodazole enhanced membrane ruffling and the internalization of phagocytic targets suggesting an inhibitory effect of the microtubule network on the remodeling of the actin network. Meanwhile, the microtubule network was necessary for phagosome maturation. Our detailed analyses of cytoskeletal filaments suggest a phagocytosis control system involving Ca2+ influx, the destabilization of microtubules, and activation of actin network remodeling, followed by the translocation and acidification of phagosomes on the microtubule bundles.

Recent evidence suggests that capillary pericytes are contractile and play a crucial role in the regulation of microcirculation. However, failure to detect components of the contractile apparatus in capillary pericytes, most notably α-smooth muscle actin (α-SMA), has questioned these findings. Using strategies that allow rapid filamentous-actin (F-actin) fixation (i.e. snap freeze fixation with methanol at −20°C) or prevent F-actin depolymerization (i.e. with F-actin stabilizing agents), we demonstrate that pericytes on mouse retinal capillaries, including those in intermediate and deeper plexus, express α-SMA. Junctional pericytes were more frequently α-SMA-positive relative to pericytes on linear capillary segments. Intravitreal administration of short interfering RNA (α-SMA-siRNA) suppressed α-SMA expression preferentially in high order branch capillary pericytes, confirming the existence of a smaller pool of α-SMA in distal capillary pericytes that is quickly lost by depolymerization. We conclude that capillary pericytes do express α-SMA, which rapidly depolymerizes during tissue fixation thus evading detection by immunolabeling. Blood vessels in animals’ bodies are highly organized. The large blood vessels from the heart branch to smaller vessels that are spread throughout the tissues. The smallest vessels, the capillaries, allow oxygen and nutrients to pass from the blood to nearby cells in tissues. Some capillaries, including those at the back of the eye (in the retina) and those in the brain, change their diameter in response to activity in the nervous system. This allows more or less oxygen and nutrients to be delivered to match these tissues’ demands. However, unlike for larger blood vessels, how capillaries constrict or dilate is debated. While large vessels are encircled by smooth muscle cells, capillaries are instead surrounded by muscle-like cells called pericytes, and some scientists have suggested that it is these cells that contract to narrow the diameter of a capillary or relax to widen it. However, other researchers have questioned this explanation. This is mostly because several laboratories could not detect the proteins that would be needed for contraction within these pericytes – the most notable of which is a protein called α-smooth muscle actin (or α-SMA for short). Alarcon-Martinez, Yilmaz-Ozcan et al. hypothesized that the way samples are usually prepared for analysis was causing the α-SMA to be degraded before it could be detected. To test this hypothesis, they used different methods to fix and preserve capillaries and pericytes in samples taken from the retinas of mice. When the tissue samples were immediately frozen with ice-cold methanol instead of a more standard formaldehyde solution, α-SMA could be detected at much higher levels in the capillary pericytes. Treating samples with a toxin called phalloidin, which stabilizes filaments of actin, also made α-SMA more readily visible. When α-SMA was experimentally depleted from the mouse retinas, the capillary pericytes were more affected than the larger blood vessels. This finding supports the idea that the pericytes contain, and rely upon, only a small amount of α-SMA. Finding α-SMA in capillary pericytes may explain how these small blood vessels can change their diameter. Future experiments will clarify how these pericytes regulate blood flow at the level of individual capillaries, and may give insights into conditions such as stroke, which is caused by reduced blood flow to the brain.

Acute liver failure (ALF) is a rare and critical medical condition. This study was designed to investigate the protective effects and underlying mechanism of ACY1215 in ALF mice. Our findings suggested that ACY1215 treatment ameliorates the pathological hepatic damage of ALF and decreases the serum levels of ALT and AST. Furthermore, ACY1215 pretreatment increased the level of ATM, γ‐H2AX, Chk2, p53, p21, F‐actin and vinculin in ALF. Moreover, ACY1215 inhibited the level of NLRP3, ASC, caspase‐1, IL‐1β and IL‐18 in ALF. The ATM inhibitor KU55933 could decrease the level of ATM, γ‐H2AX, Chk2, p53, p21, F‐actin and vinculin in ALF with ACY1215 pretreatment. The F‐actin inhibitor cytochalasin B decreased the level of F‐actin and vinculin in ALF with ACY1215 pretreatment. However, cytochalasin B had no effect on protein levels of ATM, Chk2, p53 and p21 in ALF with ACY1215 pretreatment. Cytochalasin B could dramatically increase the level of NLRP3, ASC, caspase‐1, IL‐1β and IL‐18 in ALF with ACY1215 pretreatment. These results indicated that ACY1215 exhibited hepatoprotective properties, which was associated with the inhibition of NLRP3 inflammasome, and this effect of ACY1215 was connected with upregulation of the ATM/F‐actin mediated signalling pathways.