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The cytoskeleton, a dynamic network of structural proteins within cells, is essential for cellular deformation and responds to external mechanical cues. Here, based on the structure of the cytoskeleton, combined with the biochemical reactions of the activator RhoA and the inhibitor F-actin, we develop a novel mechanochemical cytoskeleton model to investigate the mechanical behavior of cells. Interestingly, we find that active stress fibers exhibit diverse dynamical modes at specific inhibitor concentration thresholds. The existence of concentration differences and sustained mechanochemical feedback in activators and inhibitors trigger a global oscillation of isolated cells. In addition, under uniaxial and biaxial stretches, activators and inhibitors preferentially diffuse toward the more significantly deformed cytoskeletal elements, and their dynamic interactions regulate the cell to align with the main stretching direction. Our findings, consistent with many experimental results, provide fundamental insights into cytoskeletal remodeling and cellular mechanosensing mechanisms. [ABSTRACT FROM AUTHOR]

Our understanding of the transitions of human embryonic stem cells (hESCs) between distinct stages of pluripotency relies predominantly on regulation by transcriptional and epigenetic programs with limited insight on the role of established morphological changes. We report remodeling of the actin cytoskeleton of hESCs as they transition from primed to naïve pluripotency which includes assembly of a ring of contractile actin filaments encapsulating colonies of naïve hESCs. Activity of the Arp2/3 complex is required for formation of the actin ring, to establish uniform cell mechanics within naïve colonies, to promote nuclear translocation of the Hippo pathway effectors YAP and TAZ, and for effective transition to naïve pluripotency. RNA-sequencing analysis confirms that Arp2/3 complex activity regulates Hippo signaling in hESCs, and impaired naïve pluripotency with inhibited Arp2/3 complex activity is rescued by expressing a constitutively active, nuclear-localized YAP-S127A. Moreover, expression of YAP-S127A partially restores the actin filament fence with Arp2/3 complex inhibition, suggesting that actin filament remodeling is both upstream and downstream of YAP activity. These new findings on the cell biology of hESCs reveal a mechanism for cytoskeletal dynamics coordinating cell mechanics to regulate gene expression and facilitate transitions between pluripotency states.

The cell cortex is a dynamic assembly formed by the plasma membrane and underlying cytoskeleton. As the main determinant of cell shape, the cortex ensures its integrity during passive and active deformations by adapting cytoskeleton topologies through yet poorly understood mechanisms. The spectrin meshwork ensures such adaptation in erythrocytes and neurons by adopting different organizations. Erythrocytes rely on triangular-like lattices of spectrin tetramers, whereas in neurons they are organized in parallel, periodic arrays. Since spectrin is ubiquitously expressed, we exploited Expansion Microscopy to discover that, in fibroblasts, distinct meshwork densities co-exist. Through biophysical measurements and computational modeling, we show that the non-polarized spectrin meshwork, with the intervention of actomyosin, can dynamically transition into polarized clusters fenced by actin stress fibers that resemble periodic arrays as found in neurons. Clusters experience lower mechanical stress and turnover, despite displaying an extension close to the tetramer contour length. Our study sheds light on the adaptive properties of spectrin, which participates in the protection of the cell cortex by varying its densities in response to key mechanical features.

Several actin-binding proteins (ABPs) phase separate to form condensates capable of curating the actin network shapes. Here, we use computational modeling to understand the principles of actin network organization within VASP condensate droplets. Our simulations reveal that the different actin shapes, namely shells, rings, and mixture states are highly dependent on the kinetics of VASP-actin interactions, suggesting that they arise from kinetic trapping. Specifically, we show that reducing the residence time of VASP on actin filaments reduces degree of bundling, thereby promoting assembly of shells rather than rings. We validate the model predictions experimentally using a VASP-mutant with decreased bundling capability. Finally, we investigate the ring opening within deformed droplets and found that the sphere-to-ellipsoid transition is favored under a wide range of filament lengths while the ellipsoid-to-rod transition is only permitted when filaments have a specific range of lengths. Our findings highlight key mechanisms of actin organization within phase-separated ABPs.

How a developing organ robustly coordinates the cellular mechanics and growth to reach a final size and shape remains poorly understood. Through iterations between experiments and model simulations that include a mechanistic description of interkinetic nuclear migration, we show that the local curvature, height, and nuclear positioning of cells in the Drosophila wing imaginal disc are defined by the concurrent patterning of actomyosin contractility, cell-ECM adhesion, ECM stiffness, and interfacial membrane tension. We show that increasing cell proliferation via different growth-promoting pathways results in two distinct phenotypes. Triggering proliferation through insulin signaling increases basal curvature, but an increase in growth through Dpp signaling and Myc causes tissue flattening. These distinct phenotypic outcomes arise from differences in how each growth pathway regulates the cellular cytoskeleton, including contractility and cell-ECM adhesion. The coupled regulation of proliferation and cytoskeletal regulators is a general strategy to meet the multiple context-dependent criteria defining tissue morphogenesis.

Myelination of neuronal axons is essential for nervous system development. Myelination requires dramatic cytoskeletal dynamics in oligodendrocytes, but how actin is regulated during myelination is poorly understood. We recently identified serum response factor (SRF)-a transcription factor known to regulate expression of actin and actin regulators in other cell types-as a critical driver of myelination in the aged brain. Yet, a major gap remains in understanding the mechanistic role of SRF in oligodendrocyte lineage cells. Here, we show that SRF is required cell autonomously in oligodendrocytes for myelination during development. Combining ChIP-seq with RNA-seq identifies SRF-target genes in oligodendrocyte precursor cells and oligodendrocytes that include actin and other key cytoskeletal genes. Accordingly, SRF knockout oligodendrocytes exhibit dramatically reduced actin filament levels early in differentiation, consistent with its role in actin-dependent myelin sheath initiation. Surprisingly, oligodendrocyte-restricted loss of SRF results in upregulation of gene signatures associated with aging and neurodegenerative diseases. Together, our findings identify SRF as a transcriptional regulator that controls the expression of cytoskeletal genes required in oligodendrocytes for myelination. This study identifies an essential pathway regulating oligodendrocyte biology with high relevance to brain development, aging, and disease.

The polymerization of myosin molecules into thick filaments in muscle sarcomeres is essential for cardiac contractility, with the attenuation of interactions between the heads of myosin molecules within the filaments being proposed to result in hypercontractility, as observed in hypertrophic cardiomyopathy (HCM). However, experimental evidence demonstrates that the structure of these giant macromolecular complexes is highly dynamic, with molecules exchanging between the filaments and a pool of soluble molecules on the minute timescale. Therefore, we sought to test the hypothesis that the enhancement of interactions between the heads of myosin molecules within thick filaments limits the mobility of myosin by taking advantage of mavacamten, a small molecule approved for the treatment of HCM. Myosin molecules were labeled in vivo with a green fluorescent protein (GFP) and imaged in intact hearts using multiphoton microscopy. Treatment of the intact hearts with mavacamten resulted in an unexpected > 5-fold enhancement in GFP-myosin mobility within the sarcomere. In vitro biochemical assays suggested that mavacamten enhanced the mobility of GFP-myosin by increasing the solubility of myosin molecules, through the stabilization of a compact/folded conformation of the molecules, once disassociated from the thick filaments. These findings provide alternative insight into the mechanisms by which molecules exchange into and out of thick filaments and have implications for how mavacamten may affect cardiac contractility.

Fluid viscosity and osmolarity are among some of the underappreciated mechanical stimuli that cells can detect. Abnormal changes of multiple fluidic factors such as viscosity and osmolarity have been linked with diseases such as cystic fibrosis, cancer, and coronary heart disease. Changes in viscosity have been recently suggested as a regulator of cell locomotion. These novel studies focus on cell migration and spreading on glass substrates and through microchannels, and it remains a question whether viscosity impacts the cellular remodeling of extracellular matrices (ECMs). Here, we demonstrate that elevated viscosity induces cellular remodeling of collagen substrates and enhances cell spreading on ECM‐mimetic substrates. Our results expand on recent work showing that viscosity induces increased cellular forces and demonstrates that viscosity can drive local ECM densification. Our data further show that microtubules, Ras‐related C3 botulinum toxin substrate 1 (Rac1), actin‐related protein 2/3 (Arp2/3) complex, Rho‐associated protein kinase 1 (ROCK), and myosin are important regulators of viscosity‐induced ECM remodeling. In the context of viscosity‐induced cell spreading, cells cultured on glass and collagen substrates exhibit markedly different responses to pharmacological treatments, indicating that microtubules, Rac1, and Arp2/3 play distinct roles in regulating cellular spreading depending on the substrate. In addition, our results demonstrate that high osmotic pressures override viscosity‐induced cell spreading by suppressing membrane ruffling. Our results demonstrate viscosity as a regulator of ECM remodeling and cell spreading in a fibrillar microenvironment. We also reveal a complex interplay between viscosity and osmolarity. We anticipate that our research can pave the way for future investigations into the crucial roles played by viscosity in both physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

Fibrosis is the process whereby cells at a damaged site are transformed into fibrotic tissue, comprising fibroblasts and an extracellular matrix rich in collagen and fibronectin, following damage to organs or tissues that exceeds their repair capacity. Depending on the affected organs or tissues, fibrosis can be classified into types such as pulmonary fibrosis, hepatic fibrosis, renal fibrosis, and cardiac fibrosis. The primary pathological features of fibrotic diseases include recurrent damage to normal cells and the abnormal activation of fibroblasts, leading to excessive deposition of extracellular matrix and collagen in the intercellular spaces. However, the etiology of certain specific fibrotic diseases remains unclear. Recent research increasingly suggests that the cytoskeleton plays a significant role in fibrotic diseases, with structural changes in the cytoskeleton potentially influencing the progression of organ fibrosis. This review examines cytoskeletal remodeling and its impact on the transformation or activation of normal tissue cells during fibrosis, potentially offering important insights into the etiology and therapeutic strategies for fibrotic diseases. [ABSTRACT FROM AUTHOR]

Osteocytes' response to dynamic loading plays a crucial role in regulating the bone mass but quickly becomes saturated such that downstream induction of bone formation plateaus. The underlying mechanisms that downregulate osteocytes' sensitivity and overall response to loading remain unknown. In other cell types, purinergic signaling through the P2Y2 receptor has the potential to downregulate the sensitivity to loading by modifying cell stiffness through actin polymerization and cytoskeleton organization. Herein, we examined the role of P2Y2 activation in regulating osteocytes' mechanotransduction using a P2Y2 knockout cell line alongside conditional knockout mice. Our findings demonstrate that the absence of P2Y2 expression in MLO-Y4 cells prevents actin polymerization while increasing the sensitivity to fluid flow-induced shear stress. Deleting osteocytes' P2Y2 expression in conditional-knockout mice enabled bone formation to increase when increasing the duration of exercise. Overall, P2Y2 activation under loading produces a negative feedback loop, limiting osteocytes' response to continuous loading by shifting the sensitivity to mechanical strain through actin stress fiber formation. [ABSTRACT FROM AUTHOR]

Membrane remodeling drives a broad spectrum of cellular functions, and it is regulated through mechanical forces exerted on the membrane by cytoplasmic complexes. Here, we investigate how actin filaments dynamically tune their structure to control the active transfer of membranes between cellular compartments with distinct compositions and biophysical properties. Using intravital subcellular microscopy in live rodents we show that a lattice composed of linear filaments stabilizes the granule membrane after fusion with the plasma membrane and a network of branched filaments linked to the membranes by Ezrin, a regulator of membrane tension, initiates and drives to completion the integration step. Our results highlight how the actin cytoskeleton tunes its structure to adapt to dynamic changes in the biophysical properties of membranes. [ABSTRACT FROM AUTHOR]

Autoimmunity underlies many painful disorders, such as enthesopathies, which localize to the enthesis. From infiltration of the synovium and axial skeleton by B cells, to disturbances in the ratio of M1/M2 enthesis macrophages, to CD8 + T cell mediated inflammation, autoimmune dysregulation is becoming increasingly well characterized in enthesopathies. Tissue resident B cells, macrophages, neutrophils, and T cells have also been localized in healthy human entheses. However, the potential developmental origins, presence, and role of immune cells (ICs) in enthesis development is not known. Here, we use single-cell RNA-sequencing analysis to describe IC subtypes present in the enthesis before, during, and after mineralization, and to infer regulatory interactions between ICs and mesenchymal cells (MCs). We report the presence of nine phenotypically distinct IC subtypes, including B cells, macrophages, neutrophils, and T cells. We find that specific IC subtypes may promote MC-proliferation and differentiation, and that MCs may regulate IC phenotype and autoimmunity. Our findings suggest that bidirectional regulatory interactions between ICs and MCs may be important to enthesis mineralization, and suggest that progenitor MCs have a unique ability to limit autoimmunity during development. [ABSTRACT FROM AUTHOR]

The cellular cortex provides crucial mechanical support and plays critical roles during cell division and migration. The proteins of the ERM family, comprised of ezrin, radixin, and moesin, are central to these processes by linking the plasma membrane to the actin cytoskeleton. To investigate the contributions of the ERM proteins to leukocyte migration, we generated single and triple ERM knockout macrophages. Surprisingly, we found that even in the absence of ERM proteins, macrophages still form the different actin structures promoting cell migration, such as filopodia, lamellipodia, podosomes, and ruffles. Furthermore, we discovered that, unlike every other cell type previously investigated, the single or triple knockout of ERM proteins does not affect macrophage migration in diverse contexts. Finally, we demonstrated that the loss of ERMs in macrophages does not affect the mechanical properties of their cortex. These findings challenge the notion that ERMs are universally essential for cortex mechanics and cell migration and support the notion that the macrophage cortex may have diverged from that of other cells to allow for their uniquely adaptive cortical plasticity. Synopsis: Ezrin, radixin, and moesin (collectively known as ERM proteins) serve as crucial cytoskeletal linker proteins connecting the actin cytoskeleton to the plasma membrane. This study shows that a complete loss of ERM proteins in macrophages does not affect the mechanics of their actin cortex and their capacity to migrate. Macrophage actin structures are still correctly formed in the absence of ERM proteins. Macrophage migration in vitro, ex vivo and in vivo is not affected by ERM depletion. The mechanical properties of the macrophage cortex are independent of ERM proteins. Macrophages do not require the ERM family proteins for phagocytosis and cell migration, suggesting divergence of macrophage cortical properties. [ABSTRACT FROM AUTHOR]

Conventional cell culture techniques generally fail to recapitulate the expression profiles or functional phenotypes of the in vivo equivalents they are meant to model. These cell culture models are indispensable for preclinical drug discovery and mechanistic studies. However, if our goal is to develop effective therapies that work as intended in the human body, we must revise our cell culture models to recapitulate normal and disease physiology to ensure that we identify compounds that are useful and effective beyond our in vitro models. [ABSTRACT FROM AUTHOR]

Eukaryotic cells depend on dynamic changes in shape to fulfill a wide range of cellular functions, maintain essential biological processes, and regulate cellular behavior. The single-celled, predatory ciliate Lacrymaria exhibits extraordinary dynamic shape-shifting using a flexible "neck" that can stretch 7–8 times the length of its body to capture prey. The molecular mechanism behind this morphological change remains a mystery. We have observed that when in an active state, Lacrymaria repeatedly extends and contracts its neck to enable 360-degree space search and prey capture. This remarkable morphological change involves a unique actin-myosin system rather than the Ca2+-dependent system found in other contractile ciliates. Two cytoskeletons are identified in the cortex of the Lacrymaria cell, namely the myoneme cytoskeleton and the microtubule cytoskeleton. The myoneme cytoskeleton is composed of centrin-myosin proteins, exhibiting distinct patterns between the neck and body, with their boundary seemingly associated with the position of the macronucleus. A novel giant protein forming a ladder-like structure was discovered as a component of the microtubule cytoskeleton. Thick centrin-myosin fibers are situated very close to the right side of the ladders in the neck but are far away from such structures in the body. This arrangement enables the decoupling of the neck and body. Plasmodium -like unconventional actin has been discovered in Lacrymaria , and this may form highly dynamic short filaments that could attach to the giant protein and myosin, facilitating coordination between the two cytoskeletons in the neck. In summary, this fascinating organism employs unconventional cytoskeletal components to accomplish its extraordinary dynamic shape-shifting. [Display omitted] • Lacrymaria uses a unique actin-myosin system for neck morphology changes • Two cytoskeletal structures with unconventional components work together • The centrin-myosin-based myoneme shows distinct patterns in neck and body • A giant protein forms a ladder structure in the microtubule cytoskeleton Qin et al. discover the mechanism behind the remarkable shape-shifting of the unicellular predator, Lacrymaria. They identify two key cytoskeletal structures: a centrin-myosin-based myoneme and a giant protein-containing microtubule cytoskeleton. These structures work together to decouple the neck and body, enabling its dynamic neck morphology. [ABSTRACT FROM AUTHOR]

Focal adhesions (FAs) strengthen their link with the actin cytoskeleton to resist force. Talin-vinculin association could reinforce actin anchoring to FAs by controlling actin polymerization. However, the actin polymerization activity of the talin-vinculin complex is not known because it requires the reconstitution of the mechanical and biochemical activation steps that control the association of talin and vinculin. By combining kinetic and binding assays with single actin filament observations in TIRF microscopy, we show that the association of talin and vinculin mutants, mimicking mechanically stretched talin and activated vinculin, triggers a sequential mechanism in which filaments are nucleated, capped and released to elongate. In agreement with these observations, FRAP experiments in cells co-expressing the same constitutive mutants of talin and vinculin revealed accelerated growth of stress fibers. Our findings suggest a versatile mechanism for the regulation of actin assembly in FAs subjected to various combinations of biochemical and mechanical cues. In cells, focal adhesions (FAs) strengthen their connection with the actin cytoskeleton to resist force. By combining biochemistry and cell biology, the authors show that the proteins talin and vinculin control actin assembly, thus reinforcing the anchoring of actin to FAs. [ABSTRACT FROM AUTHOR]

The linker of nucleoskeleton and cytoskeleton (LINC) complex, which spans the nuclear envelope, physically connects nuclear components to the cytoskeleton and plays a pivotal role in various cellular processes, including nuclear positioning, cell migration, and chromosomal configuration. Studies have revealed that the LINC complex is essential for different aspects of the nervous system, particularly during development. The significance of the LINC complex in neural lineage cells is further corroborated by the fact that mutations in genes associated with the LINC complex have been implicated in several neurological diseases, including neurodegenerative and psychiatric disorders. In this review, we aimed to summarize the expanding knowledge of LINC complex-related neuronal functions and associated neurological diseases. [ABSTRACT FROM AUTHOR]

Red blood cells respond to fluctuations in blood plasma pH by changing the rate of biochemical and physical processes that affect the specific functions of individual cells. This study aimed to analyze the effect of pH changes on red blood cell morphology and structure. The findings revealed that an increase or decrease in pH above or below the physiological level of pH 7.4 results in the transformation of discocytes into echinocytes and causes significant alterations in the membrane, including its roughness, cytoskeleton structure, and the cell's elastic modulus. Furthermore, the study shown a strong connection between critical acidosis and alkalosis with increased intracellular reactive oxygen species production. [ABSTRACT FROM AUTHOR]

With the popularization of wireless communication, radar, and electronic devices, the hidden harm of electromagnetic radiation is becoming increasingly serious. The design of green biomass carbon-based interface heterojunctions based on lightweight porous materials can effectively protect against electromagnetic radiation hazards. In this work, we constructed an anisotropic heterojunction interface with magnetic and dielectric coupling based on a honeycomb-like periodic matrix multi-layer array repeating unit. The removal of lignin components from bamboo through oxidation enriches the impregnation pores and uniform adsorption sites of the magnetic medium. Further, in situ pyrolysis promotes the formation of a large number of electric dipoles at the interface between the magnetic medium and dielectric coupling inside the periodic cell carbon skeleton, enhancing interface polarization and relaxation. Local carrier traps and uneven electromagnetic density enhance dielectric and hysteresis losses, resulting in excellent impedance matching. Therefore, the obtained bamboo-based carbon multiphase composite absorbent has satisfactory electromagnetic loss characteristics. At a thickness of 1.55 mm, the effective absorption bandwidth reaches 5.1 GHz, and the minimum reflection loss (RL) value reaches −54.7 dB. In addition, the far-field radar simulation results show that the sample has an excellent RCS (radar cross-section) reduction of 33.3 dB·m2. This work provides new directions for the diversified development of green biomass and the optimization of the design of magnetic and dielectric coupling in periodic array structures. [ABSTRACT FROM AUTHOR]

Small Heat Shock Proteins are considered as the first line of defense when proteostasis fails. Among them, αB-crystallin is expressed in striated muscles in which it interacts with desmin intermediate filaments to stabilize them, maintaining cytoskeleton's integrity and muscular functionalities. Desmin is a key actor for muscle health; its targeting by αB-crystallin is thus crucial, especially in stress conditions. αB-crystallin is phosphorylated and O-GlcNAcylated. Its phosphorylation increases consecutively to various stresses, correlated with its recruitment for cytoskeleton's safeguarding. However, phosphorylation as unique signal for cytoskeleton translocation remains controversial; indeed, O-GlcNAcylation was also proposed to be involved. Thus, there are still some gaps for a deeper comprehension of how αB-crystallin functions are finely regulated by post-translational modifications. Furthermore, desmin also bears both post-translational modifications; while desmin phosphorylation is closely linked to desmin intermediates filaments turnover, it is unclear whereas its O-GlcNAcylation could impact its proper function. In the herein paper, we aim at identifying whether phosphorylation and/or O-GlcNAcylation are involved in αB-crystallin targeting towards cytoskeleton in proteotoxic stress induced by proteasome inhibition in C2C12 myotubes. We demonstrated that proteotoxicity led to αB-crystallin's phosphorylation and O-GlcNAcylation patterns changes, both presenting a dynamic interplay depending on protein subfraction. Importantly, both post-translational modifications showed a spatio-temporal variation correlated with αB-crystallin translocation towards cytoskeleton. In contrast, we did not detect any change of desmin phosphorylation and O-GlcNAcylation. All together, these data strongly support that αB-crystallin phosphorylation/O-GlcNAcylation interplay rather than changes on desmin is a key regulator for its cytoskeleton translocation, preserving it towards stress. [Display omitted] • Small Heat Shock Proteins (sHSPs) are the first line of defense when proteostasis failed in stressful conditions. • The MG132-induced proteotoxicity modifies O-GlcNAcylation/phosphorylation patterns of αB-crystallin. • The MG132-induced proteotoxicity does not modify the O-GlcNAcylation/phosphorylation patterns of desmin. • Phosphorylation/O-GlcNAcylation of αB-crystallin spatio-temporally varies along proteotoxic stress. • Phosphorylation/O-GlcNAcylation interplay is a key regulator of αB-crystallin cytoskeleton translocation. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 mainly infects the respiratory tract but can also target other organs, including the central nervous system. While it was recently shown that cells of the blood-brain-barrier are permissive to SARS-CoV-2 infection in vitro , it remains debated whether neurons can be infected. In this study, we demonstrate that vesicular stomatitis virus particles pseudotyped with the spike protein of SARS-CoV-2 variants WT, Alpha, Delta and Omicron enter the neuronal model cell line SH-SY5Y. Cell biological analyses of the pseudo-virus treated cultures showed marked alterations in microtubules of SH-SY5Y cells. Because the changes in β-tubulin occurred in most cells, but only few were infected, we further asked whether interaction of the cells with spike protein might be sufficient to cause molecular and structural changes. For this, SH-SY5Y cells were incubated with trimeric spike proteins for time intervals of up to 24 h. CellProfiler™-based image analyses revealed changes in the intensities of microtubule staining in spike protein-incubated cells. Furthermore, expression of the spike protein-processing protease cathepsin L was found to be up-regulated by wild type, Alpha and Delta spike protein pseudotypes and cathepsin L was found to be secreted from spike protein-treated cells. We conclude that the mere interaction of the SARS-CoV-2 with neuronal cells can affect cellular architecture and proteolytic capacities. The molecular mechanisms underlying SARS-CoV-2 spike protein induced cytoskeletal changes in neuronal cells remain elusive and require future studies. [Display omitted] • ACE2, Basigin and Neuropilin-1 are detected at the plasma membrane of SH-SY5Y cells. • SARS-CoV-2 spike protein mediates entry of pseudotyped particles into SH-SY5Y cells. • SARS-CoV-2 spike variants rearrange the cytoskeleton of SH-SY5Y cells. • SARS-CoV-2 spike variants have differential effects on cathepsin L expression. [ABSTRACT FROM AUTHOR]

Background Information: Microvilli are finger‐like, straight, and stable cellular protrusions that are filled with F‐actin and present a stereotypical length. They are present in a broad range of cell types across the animal tree of life and mediate several fundamental functions, including nutrient absorption, photosensation, and mechanosensation. Therefore, understanding the origin and evolution of microvilli is key to reconstructing the evolution of animal cellular form and function. Here, we review the current state of knowledge on microvilli evolution and perform a bioinformatic survey of the conservation of genes encoding microvillar proteins in animals and their unicellular relatives. Results: We first present a detailed description of mammalian microvilli based on two well‐studied examples, the brush border microvilli of enterocytes and the stereocilia of hair cells. We also survey the broader diversity of microvilli and discuss similarities and differences between microvilli and filopodia. Based on our bioinformatic survey coupled with carefully reconstructed molecular phylogenies, we reconstitute the order of evolutionary appearance of microvillar proteins. We document the stepwise evolutionary assembly of the "molecular microvillar toolkit" with notable bursts of innovation at two key nodes: the last common filozoan ancestor (correlated with the evolution of microvilli distinct from filopodia) and the last common choanozoan ancestor (correlated with the emergence of inter‐microvillar adhesions). Conclusion and Significance: We conclude with a scenario for the evolution of microvilli from filopodia‐like ancestral structures in unicellular precursors of animals. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) ranks among the most aggressive types of cancer globally. Currently, clinical tumor prognostic biomarkers still lack accuracy. Mitotic spindle positioning (MISP) protein connects microtubules to the actin cytoskeleton and adhesive plaques, playing a critical role in spindle positioning, orientation, and the process of cell division. MISP can regulate the malignant biological functions of pancreatic cancer and intrahepatic cholangiocarcinoma and it acts as biomarker for prognosis, but its role in CRC remains unclear. Methods: This study has collected 37 CRC tissue samples and 37 corresponding adjacent nontumor tissue samples, and 57 additional CRC tissues samples. Clinical data were obtained from the patients with CRC. MISP mRNA and protein expression levels were analyzed in normal control and CRC tissues using the GEPIA and Human Protein Atlas website. MISP protein levels in the collected tissues were analyzed using immunohistochemistry. Results: MISP mRNA and protein expression levels were significantly increased in CRC tissues compared to adjacent nontumor tissues. Higher MISP protein levels were associated with distant metastasis, recurrence, and lower survival rates. Kaplan–Meier analysis showed that high expression levels of MISP protein were associated with recurrence and death in CRC patients. In addition, a high expression level of MISP protein, lymph node metastasis, and distance metastasis were risk factors for recurrence and a poor prognosis in patients with CRC. Conclusion: Elevated MISP protein correlated with tumor metastasis, recurrence, and lower survival rates in patients with CRC, and thus, MISP has the potential to become a prognostic marker for CRC. [ABSTRACT FROM AUTHOR]

Autophagy regulates the formation of primary cilia, which in turn affects autophagy. The relationship between autophagy and cilia is known to be bidirectional although the specific mechanisms involved have yet to be elucidated. In this study, we found for the first time that ATG8 protein localizes in the basal body of the dorsal kineties and the base of the ventral cirri in Euplotes amieti. ATG8 protein maintains the structural integrity of cilia and plays a role in the construction of the cortical ciliature and microtubule cytoskeleton associated with cilia. ATG8 gene interference leads to the degradation of IFT88, the transport protein in cilia, thus inhibiting the generation of cilia, and affecting the swing of cilia. This influences the swimming speed and cilia pattern, leading to death in Euplotes amieti. [ABSTRACT FROM AUTHOR]

In plants, robust defense depends on the efficient and resilient trafficking supply chains to the site of pathogen attack. Though the importance of intracellular trafficking in plant immunity has been well established, a lack of clarity remains regarding the contribution of the various trafficking pathways in transporting immune‐related proteins. We have recently identified a trans‐Golgi network protein, TGN‐ASSOCIATED PROTEIN 1 (TGNap1), which functionally links post‐Golgi vesicles with the cytoskeleton to transport immunity‐related proteins in the model plant species Arabidopsis thaliana. We propose new hypotheses on the various functional implications of TGNap1 and then elaborate on the surprising heterogeneity of TGN vesicles during immunity revealed by the discovery of TGNap1 and other TGN‐associated proteins in recent years. [ABSTRACT FROM AUTHOR]

Cell-based therapies have shown significant promise for treating many diseases, including cancer. Current cell therapy manufacturing processes primarily utilize viral transduction to insert genomic material into cells, which has limitations, including variable transduction efficiency and extended processing times. Non-viral transfection techniques are also limited by high variability or reduced molecular delivery efficiency. Novel 3D-printed acoustofluidic devices are in development to address these challenges by delivering biomolecules into cells within seconds via sonoporation. In this study, we assessed biological parameters that influence the ultrasound-mediated delivery of fluorescent molecules (i.e. , calcein and 150 kDa FITC-Dextran) to human T cells using flow cytometry and confocal imaging. Low cell plating densities (100,000 cells/mL) enhanced molecular delivery compared to higher cell plating densities (p < 0.001), even though cells were resuspended at equal concentrations for acoustofluidic processing. Additionally, cells in the S phase of the cell cycle had enhanced intracellular delivery compared to cells in the G2/M phase (p < 0.001) and G0/G1 phase (p < 0.01), while also maintaining higher viability compared to G0/G1 phase (p < 0.001). Furthermore, the calcium chelator (EGTA) decreased overall molecular delivery levels. Confocal imaging indicated that the actin cytoskeleton had important implications on plasma membrane recovery dynamics after sonoporation. In addition, confocal imaging indicates that acoustofluidic treatment can permeabilize the nuclear membrane, which could enable rapid intranuclear delivery of nucleic acids. The results of this study demonstrate that a 3D-printed acoustofluidic device can enhance molecular delivery to human T cells, which may enable improved techniques for non-viral processing of cell therapies. [ABSTRACT FROM AUTHOR]

Background: Alcohol abuse, a prevalent global health issue, is associated with the onset of cognitive impairment and neurodegeneration. Actin filaments (F-actin) and microtubules (MTs) polymerized from monomeric globular actin (G-actin) and tubulin form the structural basis of the neuronal cytoskeleton. Precise regulation of the assembly and disassembly of these cytoskeletal proteins, and their dynamic balance, play a pivotal role in regulating neuronal morphology and function. Nevertheless, the effect of prolonged alcohol exposure on cytoskeleton dynamics is not fully understood. This study investigates the chronic effects of alcohol on cognitive ability, neuronal morphology and cytoskeleton dynamics in the mouse hippocampus. Methods: Mice were provided ad libitum access to 5% (v/v) alcohol in drinking water and were intragastrically administered 30% (v/v, 6.0 g/kg/day) alcohol for six weeks during adulthood. Cognitive functions were then evaluated using the Y maze, novel object recognition and Morris water maze tests. Hippocampal histomorphology was assessed through hematoxylin-eosin (HE) and Nissl staining. The polymerized and depolymerized states of actin cytoskeleton and microtubules were separated using two commercial assay kits and quantified by Western blot analysis. Results: Mice chronically exposed to alcohol exhibited significant deficits in spatial and recognition memory as evidenced by behavioral tests. Histological analysis revealed notable hippocampal damage and neuronal loss. Decreased ratios of F-actin/G-actin and MT/tubulin, along with reduced levels of polymerized F-actin and MTs, were found in the hippocampus of alcohol-treated mice. Conclusions: Our findings suggest that chronic alcohol consumption disrupted the assembly of the actin cytoskeleton and MTs in the hippocampus, potentially contributing to the cognitive deficits and pathological injury induced by chronic alcohol intoxication. [ABSTRACT FROM AUTHOR]

The Daam1 protein regulates Wnt-induced cytoskeletal changes during vertebrate gastrulation though its full mode of action and binding partners remain unresolved. Here we identify Reversion Induced LIM domain protein (RIL) as a new interacting protein of Daam1. Interaction studies uncover binding of RIL to the C-terminal actin-nucleating portion of Daam1 in a Wnt-responsive manner. Immunofluorescence studies showed subcellular localization of RIL to actin fibers and co-localization with Daam1 at the plasma membrane. RIL gain- and loss-of-function approaches in Xenopus produced severe gastrulation defects in injected embryos. Additionally, a simultaneous loss of Daam1 and RIL synergized to produce severe gastrulation defects indicating RIL and Daam1 may function in the same signaling pathway. RIL further synergizes with another novel Daam1-interacting protein, Formin Binding Protein 1 (FNBP1), to regulate gastrulation. Our studies altogether show RIL mediates Daam1-regulated non-canonical Wnt signaling that is required for vertebrate gastrulation. [Display omitted] • Reversion Induced LIM domain protein (RIL) is a new interacting protein of Daam1. • RIL co-localizes with actin fibers. • RIL regulates non-canonical Wnt signaling during gastrulation. • RIL interacts with FNBP1 to regulate gastrulation. [ABSTRACT FROM AUTHOR]

The Lassa virus (LASV) is a widely recognized virulent pathogen that frequently results in lethal viral hemorrhagic fever (VHF). Earlier research has indicated that macroautophagy/autophagy plays a role in LASV replication, but, the precise mechanism is unknown. In this present study, we show that LASV matrix protein (LASV-Z) is essential for blocking intracellular autophagic flux. LASV-Z hinders actin and tubulin folding by interacting with CCT2, a component of the chaperonin-containing T-complexes (TRiC). When the cytoskeleton is disrupted, lysosomal enzyme transit is hampered. In addition, cytoskeleton disruption inhibits the merge of autophagosomes with lysosomes, resulting in autophagosome accumulation that promotes the budding of LASV virus-like particles (VLPs). Inhibition of LASV-Z-induced autophagosome accumulation blocks the LASV VLP budding process. Furthermore, it is found that glutamine at position 29 and tyrosine at position 48 on LASV-Z are important in interacting with CCT2. When these two sites are mutated, LASV-mut interacts with CCT2 less efficiently and can no longer inhibit the autophagic flux. These findings demonstrate a novel strategy for LASV-Z to hijack the host autophagy machinery to accomplish effective transportation. Abbreviation: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; Baf-A1: bafilomycin A1; CCT2: chaperonin containing TCP1 subunit 2; co-IP: co-immunoprecipitation; CTSD: cathepsin D; DAPI: 4',6-diamidino-2'-phenylindole; DMSO: dimethyl sulfoxide; EGFR: epidermal growth factor receptor; GFP: green fluorescent protein; hpi: hours post-infection; hpt: hours post-transfection; LAMP1: lysosomal-associated membrane protein 1; LASV: lassa virus; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry: red fluorescent protein; PM: plasma membrane; SQSTM1/p62: sequestosome 1; STX6: syntaxin 6; VLP: virus-like particle; TEM: transmission electron microscopy; TRiC: chaperonin-containing T-complex; WB: western blotting; μm: micrometer; μM: micromole. [ABSTRACT FROM AUTHOR]

Main conclusion: Our studies reveal the involvement of SPI in cytoskeleton-associated trichome morphogenesis, expanding the roles of SPI in regulating plant epidermal cell development. Acquisition of distinct shapes is crucial for cells to perform their biological functions in multicellular organisms. Trichomes are specialized epidermal cells of plant aerial parts, offering an excellent paradigm for dissecting the underlying regulatory mechanism of plant cell shape development at the single-cell level. SPIRRIG (SPI) that encodes a BEACH domain-containing protein was initially identified to regulate trichome branch extension, but the possible pathway(s) through which SPI regulates trichome morphogenesis remain unclear. Here, we report that SPI facilitates microtubule-associated regulation on trichome branching in Arabidopsis. Functional loss of SPI results in trichome morphogenesis hyper-sensitive to the microtubule-disrupting drug oryzalin, implying SPI may mediate microtubule stability during trichome development. Accordingly, spi mutant has less-branched trichomes. Detailed live-cell imaging showed that the spatio-temporal microtubule organization during trichome morphogenesis is aberrant in spi mutants. Further genetic investigation indicated that SPI may cooperate with ZWICHEL (ZWI) to modulate microtubule dynamics during trichome morphogenesis. ZWI encodes a kinesin-like calmodulin-binding protein (KCBP), whose distribution is necessary for the proper microtubule organization in trichomes, and zwi mutants produce less-branched trichomes as well. Trichome branching is further inhibited in spi-3 zwi-101 double mutants compared to either of the single mutant. Moreover, we found SPI could co-localize with the MYTH4 domain of ZWI. Taken together, our results expand the role of SPI in regulating trichome morphogenesis and also reveal a molecular and genetic pathway in plant cell shape formation control. [ABSTRACT FROM AUTHOR]

The formation of a lytic immunological synapse (IS) is crucial for cytotoxic lymphocytes to accurately target and effectively eliminate malignant cells. While significant attention has been focused on the lymphocyte side of the IS, particularly its role as a secretory domain for lytic granules, the cancer cell side of the IS has remained relatively underexplored. Recent findings have revealed that cancer cells can rapidly polarize their actin cytoskeleton toward the IS upon interaction with natural killer (NK) cells, thereby evading NK cell-mediated cytotoxicity. In this Brief Research Report, we present preliminary findings suggesting that actin cytoskeleton remodeling at the cancer cell side of the IS is associated with the targeted secretion of small extracellular vesicles towards the interacting NK cell. We observed that multivesicular bodies (MVBs) preferentially accumulate in the synaptic region in cancer cells exhibiting synaptic accumulation of F-actin, compared to those lacking actin cytoskeleton remodeling. Extracellular immunofluorescence staining revealed increased surface exposure of CD63 at the cancer cell side of the IS, suggestive of the fusion of MVBs with the plasma membrane. This hypothesis was supported by a pH-sensitive probe demonstrating dynamic trafficking of CD63 to the extracellular region of the IS. Collectively, our data support the notion that cancer cells can engage in targeted secretion of extracellular vesicles in response to NK cell attack, underscoring the need for further research into the potential role of this process in facilitating cancer cell immune evasion. [ABSTRACT FROM AUTHOR]

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects millions globally, with increasing urban cases outside of Latin America. Treatment is based on two compounds, namely, benznidazole (BZ) and nifurtimox, but chronic cases pose several challenges. Targeting lysine acetylation, particularly bromodomain-containing proteins, shows promise as a novel antiparasitic target. Our research focuses on TcBDF3, a cytoplasmic protein, which is crucial for parasite differentiation that recognizes acetylated alpha-tubulin. In our previous study, A1B4 was identified as a high-affinity binder of TcBDF3, showing significant trypanocidal activity with low host toxicity in vitro. In this report, the binding of TcBDF3 to A1B4 was validated using differential scanning fluorescence, fluorescence polarization, and molecular modeling, confirming its specific interaction. Additionally, two new 1,3,4-oxadiazoles derived from A1B4 were identified, which exhibited improved trypanocide activity and cytotoxicity profiles. Furthermore, TcBDF3 was classified for the first time as an atypical divergent member of the bromodomain extraterminal family found in protists and plants. These results make TcBDF3 a unique target due to its localization and known functions not shared with higher eukaryotes, which holds promise for Chagas disease treatment. [ABSTRACT FROM AUTHOR]

Rho guanine nucleotide exchange factors (RhoGEFs) comprise a wide range of proteins with a common domain responsible for the activation of the Rho family of small GTPases and various domains in other regions. The evolutionary divergence of RhoGEFs enables actin cytoskeletal reorganization, leading to complex cellular responses in higher organisms. In this review, we address the involvement of RhoGEFs in the mechanical stress response of mammalian cells. The cellular mechanical stress response is essential for the proper and orderly regulation of cell populations, including the maintenance of homeostasis, tissue morphogenesis, and adaptation to the mechanical environment. In particular, this review focuses on the recent findings regarding the Dbl family of RhoGEFs involved in mechanical stress responses at the cell-cell and cell-substrate adhesion sites, and their molecular mechanisms underlying actin cytoskeleton remodeling and signal transduction. [ABSTRACT FROM AUTHOR]

Both the pollen tube and hyphae of filamentous pathogens penetrate the outer layer of the host and then grow within host tissues. Early epidermal responses are decisive for the outcome of these two-cell interaction processes. We identified a single cell type, the papilla in the stigma of Arabidospis, as a tool to conduct a comprehensive comparative analysis on how an epidermal cell responds to the invasion of an unwanted pathogen or a welcome pollen tube. We showed that Phytophtora parasitica , a root oomycete, effectively breaches the stigmatic cell wall and develops as a biotroph within the papilla cytoplasm. These invasive features resemble the behaviour exhibited by the pathogen within its natural host cell, but diverge from the manner in which the pollen tube progresses, being engulfed within the papilla cell wall. Quantitative analysis revealed that both invaders trigger reorganization of the stigmatic endomembrane system and the actin cytoskeleton. While some remodelling processes are shared between the two interactions, others appear more specific towards the respective invader. These findings underscore the remarkable ability of an epidermal cell to differentiate between two types of invaders, thereby enabling it to trigger the most suitable response during the onset of invasion. [ABSTRACT FROM AUTHOR]

AbstractCarbon nanomaterials have been widely applied for cutting edge therapeutic applications as they offer tunable physio-chemical properties with economic scale-up options. Nuclear delivery of cancer drugs has been of prime focus since it controls important cellular signaling functions leading to greater anti-cancer drug efficacies. Better cellular drug uptake per unit drug injection drastically reduces severe side-effects of cancer therapies. Similarly, carbon dots (CDs) uptaken by the nucleus can also be used to set-up cutting edge nano delivery systems. In an earlier paper, we showed the cellular uptake and plasma membrane impact of combustion generated yellow luminescing CDs produced by our group from fuel rich combustion reactors in a one-step tunable production. In this paper, we aim to specifically study the nucleus by establishing the uptake kinetics of these combustion-generated yellow luminescing CDs. At sub-lethal doses, after crossing the plasma membrane, they impact the actin and microtubule mesh, affecting cell adhesion and migration; enter nucleus by diffusion processes; modify the overall appearance of the nucleus in terms of morphology; and alter chromatin condensation. We thus establish how this one-step produced, cost and bulk production friendly carbon dots from fuel rich combustion flames can be innovatively repurposed as potential nano delivery agents in cancer cells. [ABSTRACT FROM AUTHOR]

During cell division, the microtubule cytoskeleton undergoes dramatic cell cycle-driven reorganizations of its architecture. Coordinated by changes in the phosphorylation patterns of a multitude of microtubule associated proteins, the mitotic spindle first self-assembles to capture the chromosomes and then reorganizes in anaphase as the chromosomes are segregated. A key protein for this reorganization is PRC1 which is differentially phosphorylated by the mitotic kinases CDK1 and PLK1. How the phosphorylation state of PRC1 orchestrates spindle reorganization is not understood mechanistically. Here, we reconstitute in vitro the transition between metaphase and anaphase-like microtubule architectures triggered by the changes in PRC1 phosphorylation. We find that whereas PLK1 regulates its own recruitment by PRC1, CDK1 controls the affinity of PRC1 for antiparallel microtubule binding. Dephosphorylation of CDK1-phosphorylated PRC1 is required and sufficient to trigger the reorganization of a minimal anaphase midzone in the presence of the midzone length controlling kinesin KIF4A. These results demonstrate how phosphorylation-controlled affinity changes regulate the architecture of active microtubule networks, providing new insight into the mechanistic underpinnings of the cell cycle-driven reorganization of the central spindle during mitosis. Lim et al. reconstitute in vitro the transition between metaphase and anaphase-like spindle midzone architectures and dissect the role of PRC1 phosphorylation by PLK and CDK1 in reorganizing the central spindle during mitosis. [ABSTRACT FROM AUTHOR]

Rho guanine nucleotide exchange factors (RhoGEFs) comprise a wide range of proteins with a common domain responsible for the activation of the Rho family of small GTPases and various domains in other regions. The evolutionary divergence of RhoGEFs enables actin cytoskeletal reorganization, leading to complex cellular responses in higher organisms. In this review, we address the involvement of RhoGEFs in the mechanical stress response of mammalian cells. The cellular mechanical stress response is essential for the proper and orderly regulation of cell populations, including the maintenance of homeostasis, tissue morphogenesis, and adaptation to the mechanical environment. In particular, this review focuses on the recent findings regarding the Dbl family of RhoGEFs involved in mechanical stress responses at the cell-cell and cell-substrate adhesion sites, and their molecular mechanisms underlying actin cytoskeleton remodeling and signal transduction. [ABSTRACT FROM AUTHOR]

Introduction: CD8+ cytotoxic T lymphocytes (CTLs) are highly effective in defending against viral infections and tumours. They are activated through the recognition of peptide--MHC-I complex by the T-cell receptor (TCR) and costimulation. This cognate interaction promotes the organisation of intimate cell-- cell connections that involve cytoskeleton rearrangement to enable effector function and clearance of the target cell. This is key for the asymmetric transport and mobilisation of lytic granules to the cell--cell contact, promoting directed secretion of lytic mediators such as granzymes and perforin. Mitochondria play a role in regulating CTL function by controlling processes such as calcium flux, providing the necessary energy through oxidative phosphorylation, and its own protein translation on 55S ribosomes. However, the effect of acute inhibition of cytosolic translation in the rapid response after TCR has not been studied in mature CTLs. Methods: Here, we investigated the importance of cytosolic protein synthesis in human CTLs after early TCR activation and CD28 co-stimulation for the dynamic reorganisation of the cytoskeleton, mitochondria, and lytic granules through short-term chemical inhibition of 80S ribosomes by cycloheximide and 80S and 55S by puromycin. Results: We observed that eukaryotic ribosome function is required to allow proper asymmetric reorganisation of the tubulin cytoskeleton and mitochondria and mTOR pathway activation early upon TCR activation in human primary CTLs. Discussion: Cytosolic protein translation is required to increase glucose metabolism and degranulation capacity upon TCR activation and thus to regulate the full effector function of human CTLs. [ABSTRACT FROM AUTHOR]

The actin cytoskeleton is a key determinant of cell structure and homeostasis. However, possible tissue-specific changes to actin dynamics during aging, notably brain aging, are not understood. Here, we show that there is an age-related increase in filamentous actin (F-actin) in Drosophila brains, which is counteracted by prolongevity interventions. Critically, decreasing F-actin levels in aging neurons prevents age-onset cognitive decline and extends organismal healthspan. Mechanistically, we show that autophagy, a recycling process required for neuronal homeostasis, is disabled upon actin dysregulation in the aged brain. Remarkably, disrupting actin polymerization in aged animals with cytoskeletal drugs restores brain autophagy to youthful levels and reverses cellular hallmarks of brain aging. Finally, reducing F-actin levels in aging neurons slows brain aging and promotes healthspan in an autophagy-dependent manner. Our data identify excess actin polymerization as a hallmark of brain aging, which can be targeted to reverse brain aging phenotypes and prolong healthspan. Changes to actin dynamics during brain aging are not well understood. Here, the authors report that there is an age-related increase in F-actin in Drosophila brain which disables autophagy within the tissue and limits the fly lifespan. [ABSTRACT FROM AUTHOR]

Epithelial ovarian carcinoma (EOC) is the most fatal among female reproductive system tumors. The immune tumor microenvironment and ubiquitin-proteasome pathway are closely related to the proliferation, invasion, and response to chemotherapy in EOC. However, their specific roles in EOC have not been fully elucidated. Therefore, we aimed to recognize potential prognostic markers and novel therapeutic targets for EOC. We constructed the ubiquitin-related signature risk model comprising HSP90AB1, FBXO9, SIGMAR1, STAT1, SH3KBP1, EPB41L2, DNAJB6, VPS18, PPM1G, AKAP12, FRK, and PYGB, specifically for patients with EOC. The high-risk model presented a worse prognosis, primarily associated with the B-cell receptor signaling pathway, ECM receptor interaction, focal adhesion, and actin cytoskeleton regulations. Analysis of the immune landscape revealed a higher abundance of B cells, M2 macrophages, neutrophil CD4 T cells, cancer-associated fibroblasts, macrophage neutrophils, and fibroblasts in the high-risk group. It also exhibited lower tumor mutation burden, mRNAsi, and EREG-mRNAsi and reduced sensitivity to other chemotherapy drugs, except dasatinib. These findings serve as a valuable indicator for personalized treatment strategies and clinical stratification in managing patients with EOC. Additionally, our study will serve as a foundation for future mechanistic research to explore the association between the ubiquitin-proteasome pathway and EOC. [ABSTRACT FROM AUTHOR]

Astrocytes are morphologically complex cells that serve essential roles. They are widely implicated in central nervous system (CNS) disorders, with changes in astrocyte morphology and gene expression accompanying disease. In the Sapap3 knockout (KO) mouse model of compulsive and anxiety-related behaviors related to obsessive-compulsive disorder (OCD), striatal astrocytes display reduced morphology and altered actin cytoskeleton and Gi-G-protein-coupled receptor (Gi-GPCR) signaling proteins. Here, we show that normalizing striatal astrocyte morphology, actin cytoskeleton, and essential homeostatic support functions by targeting the astrocyte Gi-GPCR pathway using chemogenetics corrected phenotypes in Sapap3 KO mice, including anxiety-related and compulsive behaviors. Our data portend an astrocytic pharmacological strategy for rescuing phenotypes in brain disorders that include compromised astrocyte morphology and tissue support. [Display omitted] • Astrocyte Gi-GPCR signaling corrects repetitive and anxiety behaviors in Sapap3 KO mice • Gi-GPCR signaling restores astrocyte morphology via the actin cytoskeleton • Astrocyte Gi-GPCR signaling rescues medium spiny neuron excitability • Improved phenotypes are accompanied with restored astrocyte homeostatic functions Soto et al. used multiple approaches to assess whether and how function is affected following astrocyte Gi-DREADD activation in a mouse model of compulsive and anxiety-related behaviors that are accompanied by astrocyte dysfunction. They found that Gi-DREADD activation restored decreased astrocyte morphology and homeostatic functions and led to the correction of compulsive and anxiety behaviors in obsessive-compulsive disorder (OCD)-related Sapap3 knockout mice. Soto et al. provide an astrocyte-specific pharmacological strategy for normalizing detrimental compulsive phenotypes related to OCD and potentially other disorders in which astrocyte morphology and homeostatic support are compromised. [ABSTRACT FROM AUTHOR]

Aim Methods Results Conclusion Whole‐exome sequencing (WES) studies have revealed that germline de novo variants (gDNVs) contribute to the genetic etiology of schizophrenia. However, the contribution of mosaic DNVs (mDNVs) to the risk of schizophrenia remains to be elucidated. In the present study, we systematically investigated the gDNVs and mDMVs that contribute to the genetic etiology of schizophrenia in a Japanese population.We performed deep WES (depth: 460×) of 73 affected offspring and WES (depth: 116×) of 134 parents from 67 families with schizophrenia. Prioritized rare nonsynonymous gDNV and mDNV candidates were validated using Sanger sequencing and ultra‐deep targeted amplicon sequencing (depth: 71,375×), respectively. Subsequently, we performed a Gene Ontology analysis of the gDNVs and mDNVs to obtain biological insights. Lastly, we selected DNVs in known risk genes for psychiatric and neurodevelopmental disorders.We identified 62 gDNVs and 98 mDNVs. The Gene Ontology analysis of mDNVs implicated actin filament and actin cytoskeleton as candidate biological pathways. There were eight DNVs in known risk genes: splice region gDNVs in AKAP11 and CUL1; a frameshift gDNV in SHANK1; a missense gDNV in SRCAP; missense mDNVs in CTNNB1, GRIN2A, and TSC2; and a nonsense mDNV in ZFHX4.Our results suggest the potential contributions of rare nonsynonymous gDNVs and mDNVs to the genetic etiology of schizophrenia. This is the first report of the mDNVs in schizophrenia trios, demonstrating their potential relevance to schizophrenia pathology. [ABSTRACT FROM AUTHOR]

The cytoskeleton is essential for spatial and temporal organisation of a wide range of cellular and tissue‐level processes, such as proliferation, signalling, cargo transport, migration, morphogenesis, and neuronal development. Cytoskeleton research aims to study these processes by imaging, or by locally manipulating, the dynamics and organisation of cytoskeletal proteins with high spatiotemporal resolution: which matches the capabilities of optical methods. To date, no photoresponsive microtubule‐stabilising tool has united all the features needed for a practical high‐precision reagent: a low potency and biochemically stable non‐illuminated state; then an efficient, rapid, and clean photoresponse that generates a high potency illuminated state; plus good solubility at suitable working concentrations; and efficient synthetic access. We now present CouEpo, a photocaged epothilone microtubule‐stabilising reagent that combines these needs. Its potency increases approximately 100‐fold upon irradiation by violet/blue light to reach low‐nanomolar values, allowing efficient photocontrol of microtubule dynamics in live cells, and even the generation of cellular asymmetries in microtubule architecture and cell dynamics. CouEpo is thus a high‐performance tool compound that can support high‐precision research into many microtubule‐associated processes, from biophysics to transport, cell motility, and neuronal physiology. [ABSTRACT FROM AUTHOR]

Metabolic memory (MM) is a major factor in the delayed wound healing observed in diabetic patients. While "Nocardia rubrum cell wall skeleton" (Nr-CWS) is utilized to enhance macrophage proliferation in immune diseases, its impact on MM wounds in diabetes is unclear. This study demonstrates that transient hyperglycemia leads to prolonged damage in vascular endothelial cells by decreasing METTL3 expression, leading to decreased RNA methylation and impaired cellular metabolism. Remarkably, Nr-CWS application increases METTL3 levels in these cells, facilitating the recovery of cell function. Further in vivo and in vitro analyses demonstrate that transient hyperglycemia-induced reduction in METTL3 hinders RNA methylation of the downstream gene Cds2, impacting mitochondrial function and energy metabolism and consequently reducing angiogenic capacity in endothelial cells. This impairment significantly influences diabetic wound healing. Our findings highlight the profound impact of transient hyperglycemia on wound healing, establishing METTL3 as a significant role in vascular complications of diabetes. This study not only elucidates the pathophysiological mechanisms behind MM in diabetic wounds but also suggests Nr-CWS as a potential therapeutic agent, offering a novel approach for treating diabetic wounds. METTL3 regulates RNA methylation and mitochondrial function in diabetic wound healing, highlighting Nocardia rubra cell wall skeleton as a potential therapeutic agent for improving vascular recovery in hyperglycemia-impaired wounds. [ABSTRACT FROM AUTHOR]

Human parathyroid hormone (1–34) (PTH) exhibits osteoanabolic and osteocatabolic effects, with shorter plasma exposure times favoring bone formation. Subcutaneous injection (SCI) is the conventional delivery route for PTH but faces low delivery efficiency due to limited passive diffusion and the obstruction of the vascular endothelial barrier, leading to prolonged drug exposure times and reduced osteoanabolic effects. In this work, a microcurrent delivery system (MDS) based on multimicrochannel microneedle arrays (MMAs) is proposed, achieving high efficiency and safety for PTH transdermal delivery. The internal microchannels of the MMAs are fabricated using high‐precision 3D printing technology, providing a concentrated and safe electric field that not only accelerates the movement of PTH but also reversibly increases vascular endothelial permeability by regulating the actin cytoskeleton and interendothelial junctions through Ca2+‐dependent cAMP signaling, ultimately promoting PTH absorption and shortening exposure times. The MDS enhances the osteoanabolic effect of PTH in an osteoporosis model by inhibiting osteoclast differentiation on the bone surface compared to SCI. Moreover, histopathological analysis of the skin and organs demonstrated the good safety of PTH delivered by MDS in vivo. In addition to PTH, the MDS shows broad prospects for the high‐efficiency transdermal delivery of macromolecular drugs. [ABSTRACT FROM AUTHOR]

The multicellular haploid stage of land plants develops from a single haploid cell produced by meiosis - the spore. Starting from a nonpolar state, these spores develop polarity, divide asymmetrically and establish the first axis of symmetry. Here, we show that the nucleus migrates from the cell centroid to the basal pole during polarisation of the Marchantia polymorpha spore cell. A microtubule organising centre on the leading edge of the nucleus initiates a microtubule array between the nuclear surface and the cortex at the basal pole. Simultaneously, cortical microtubules disappear from the apical hemisphere but persist in the basal hemisphere. This is accompanied by the formation a dense network of fine actin filaments between the nucleus and the basal pole cortex. Experimental depolymerisation of either microtubules or actin filaments disrupts cellular asymmetry. These data demonstrate that the cytoskeleton reorganises during spore polarisation and controls the directed migration of the nucleus to the basal pole. The presence of the nucleus at the basal pole provides the cellular asymmetry for the asymmetric cell division that establishes the apical-basal axis of the plant. [ABSTRACT FROM AUTHOR]

Summary: Plants delicately regulate endogenous auxin levels through the coordination of transport, biosynthesis, and inactivation, which is crucial for growth and development. While it is well‐established that the actin cytoskeleton can regulate auxin levels by affecting polar transport, its potential role in auxin biosynthesis has remained largely unexplored.Using LC–MS/MS‐based methods combined with fluorescent auxin marker detection, we observed a significant increase in root auxin levels upon deletion of the actin bundling proteins AtFIM4 and AtFIM5. Fluorescent observation, immunoblotting analysis, and biochemical approaches revealed that AtFIM4 and AtFIM5 affect the protein abundance of the key auxin synthesis enzyme YUC8 in roots.AtFIM4 and AtFIM5 regulate the auxin synthesis enzyme YUC8 at the protein level, with its degradation mediated by the 26S proteasome. This regulation modulates auxin synthesis and endogenous auxin levels in roots, consequently impacting root development. Based on these findings, we propose a molecular pathway centered on the 'actin cytoskeleton‐26S proteasome‐YUC8‐auxin' axis that controls auxin levels.Our findings shed light on a new pathway through which plants regulate auxin synthesis. Moreover, this study illuminates a newfound role of the actin cytoskeleton in regulating plant growth and development, particularly through its involvement in maintaining protein homeostasis via the 26S proteasome. [ABSTRACT FROM AUTHOR]

Platelets are critical mediators of hemostasis and thrombosis. Platelets circulate as discs in their resting form but change shape rapidly upon activation by vascular damage and/or soluble agonists such as thrombin. Platelet shape change is driven by a dynamic remodeling of the actin cytoskeleton. Actin filaments interact with the protein myosin, which is phosphorylated on the myosin light chain (MLC) upon platelet activation. Actin-myosin interactions trigger contraction of the actin cytoskeleton, which drives platelet spreading and contractile force generation. Filamin A (FLNA) is an actin cross-linking protein that stabilizes the attachment between subcortical actin filaments and the cell membrane. In addition, FLNA binds multiple proteins and serves as a critical intracellular signaling scaffold. Here, we used platelets from mice with a megakaryocyte/platelet-specific deletion of FLNA to investigate the role of FLNA in regulating platelet shape change. Relative to controls, FLNA-null platelets exhibited defects in stress fiber formation, contractile force generation, and MLC phosphorylation in response to thrombin stimulation. Blockade of Rho kinase (ROCK) and protein kinase C (PKC) with the inhibitors Y27632 and bisindolylmaleimide (BIM), respectively, also attenuated MLC phosphorylation; our data further indicate that ROCK and PKC promote MLC phosphorylation through independent pathways. Notably, the activity of both ROCK and PKC was diminished in the FLNA-deficient platelets. We conclude that FLNA regulates thrombin-induced MLC phosphorylation and platelet contraction, in a ROCK- and PKC-dependent manner. [ABSTRACT FROM AUTHOR]

Reelin, a secreted glycoprotein, plays a crucial role in guiding neocortical neuronal migration, dendritic outgrowth and arborization, and synaptic plasticity in the adult brain. Reelin primarily operates through the canonical lipoprotein receptors apolipoprotein E receptor 2 (Apoer2) and very low-density lipoprotein receptor (Vldlr). Reelin also engages with noncanonical receptors and unidentified coreceptors; however, the effects of which are less understood. Using high-throughput tandem mass tag (TMT) liquid chromatography tandem mass spectrometry (LC-MS/MS)-based proteomics and gene set enrichment analysis (GSEA), we identified both shared and unique intracellular pathways activated by Reelin through its canonical and noncanonical signaling in primary murine neurons of either sex during dendritic growth and arborization. We observed pathway cross talk related to regulation of cytoskeleton, neuron projection development, protein transport, and actin filament-based process. We also found enriched gene sets exclusively by the noncanonical Reelin pathway including protein translation, mRNA metabolic process, and ribonucleoprotein complex biogenesis suggesting Reelin fine-tunes neuronal structure through distinct signaling pathways. A key discovery is the identification of aldolase A, a glycolytic enzyme and actin-binding protein, as a novel effector of Reelin signaling. Reelin induced de novo translation and mobilization of aldolase A from the actin cytoskeleton. We demonstrated that aldolase A is necessary for Reelin-mediated dendrite growth and arborization in primary murine neurons and mouse brain cortical neurons. Interestingly, the function of aldolase A in dendrite development is independent of its known role in glycolysis. Altogether, our findings provide new insights into the Reelin-dependent signaling pathways and effector proteins that are crucial for dendritic development. [ABSTRACT FROM AUTHOR]

Escherichia coli K1 is the leading cause of neonatal gram-negative bacterial meningitis, but the pathogenesis of E coli K1 meningitis remains unclear. Blood-brain barrier (BBB) penetration is a crucial step in E coli meningitis development. Here, we uncovered the crucial role of CsiR, a GntR family regulator, in E coli K1 virulence. During infection, csiR expression was induced due to the derepression by Fur in the blood and human brain microvascular endothelial cells (HBMECs). CsiR positively regulated ilvB expression, which is associated with branched chain amino acid synthesis. Furthermore, we revealed that IlvB activated the FAK/PI3K pathway of HBMECs to induce actin cytoskeleton rearrangements, thereby promoting the bacterial invasion and penetration of the BBB. Overall, this study reveals a CsiR-mediated virulence regulation pathway in E coli K1, which may provide a useful target for the prevention or therapy of E coli meningitis. [ABSTRACT FROM AUTHOR]