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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.

While extracellular matrix (ECM) stress relaxation is increasingly appreciated to regulate stem cell fate commitment and other behaviors, much remains unknown about how cells process stress-relaxation cues in tissue-like three-dimensional (3D) geometries versus traditional 2D cell culture. Here, we develop an oligonucleotide-crosslinked hyaluronic acid-based ECM platform with tunable stress relaxation properties capable of use in either 2D or 3D. Strikingly, stress relaxation favors neural stem cell (NSC) neurogenesis in 3D but suppresses it in 2D. RNA sequencing and functional studies implicate the membrane-associated protein spectrin as a key 3D-specific transducer of stress-relaxation cues. Confining stress drives spectrins recruitment to the F-actin cytoskeleton, where it mechanically reinforces the cortex and potentiates mechanotransductive signaling. Increased spectrin expression is also accompanied by increased expression of the transcription factor EGR1, which we previously showed mediates NSC stiffness-dependent lineage commitment in 3D. Our work highlights spectrin as an important molecular sensor and transducer of 3D stress-relaxation cues.

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.

Synaptic plasticity is important for learning and memory formation; it describes the strengthening or weakening of connections between synapses. The postsynaptic part of excitatory synapses resides in dendritic spines, which are small protrusions on the dendrites. One of the key features of synaptic plasticity is its correlation with the size of these spines. A long-lasting synaptic strength increase [long-term potentiation (LTP)] is only possible through the reconfiguration of the actin spine cytoskeleton. Here, we develop an experimentally informed three-dimensional computational model in a moving boundary framework to investigate this reconfiguration. Our model describes the reactions between actin and actin-binding proteins leading to the cytoskeleton remodeling and their effect on the spine membrane shape to examine the spine enlargement upon LTP. Moreover, we find that the incorporation of perisynaptic elements enhances spine enlargement upon LTP, exhibiting the importance of accounting for these elements when studying structural LTP. Our model shows adaptation to repeated stimuli resulting from the interactions between spine proteins and mechanical forces.

Actin mediates insulin secretion in pancreatic β-cells through remodeling. Hampered by limited resolution, previous studies have offered an ambiguous depiction as depolymerization and repolymerization. We report the in situ structure of actin remodeling in INS-1E β-cells during glucose-stimulated insulin secretion at nanoscale resolution. After remodeling, the actin filament network at the cell periphery exhibits three marked differences: 12% of actin filaments reorient quasi-orthogonally to the ventral membrane; the filament network mainly remains as cell-stabilizing bundles but partially reconfigures into a less compact arrangement; actin filaments anchored to the ventral membrane reorganize from a netlike to a blooming architecture. Furthermore, the density of actin filaments and microtubules around insulin secretory granules decreases, while actin filaments and microtubules become more densely packed. The actin filament network after remodeling potentially precedes the transport and release of insulin secretory granules. These findings advance our understanding of actin remodeling and its role in glucose-stimulated insulin secretion.

Background: Liver sinusoidal endothelial cells (LSECs) have transcellular pores, called fenestrations, participating in the bidirectional transport between the vascular system and liver parenchyma. Fenestrated LSECs indicate a healthy phenotype of liver while loss of fenestrations (defenestration) in LSECs is associated with liver pathologies. Methods: We introduce a unique model of systemic inflammation triggered by the deletion of Mcpip1 in myeloid leukocytes (Mcpip1fl/flLysMCre) characterised by progressive alterations in LSEC phenotype. We implement multiparametric characterisation of LSECs by using novel real-time atomic force microscopy supported with scanning electron microscopy and quantitative fluorescence microscopy. In addition, we provide genetic profiling, searching for characteristic genes encoding proteins that might be connected with the structure of fenestrations. Results: We demonstrate that LSECs in Mcpip1fl/flLysMCre display two phases of defenestration: the early phase, with modest defenestration that was fully reversible using cytochalasin B and the late phase, with severe defenestration that is mostly irreversible. By thorough analysis of LSEC porosity, elastic modulus and actin abundance in Mcpip1fl/flLysMCre and in response to cytochalasin B, we demonstrate that proteins other than actin must be additionally responsible for inducing open fenestrations. We highlight several genes that were severely affected in the late but not in the early phase of LSEC defenestration shedding a light on complex structure of individual fenestrations. Conclusions: The presented model of LSEC derived from Mcpip1fl/flLysMCre provides a valuable reference for developing novel strategies for LSEC refenestration in the early and late phases of liver pathology. [ABSTRACT FROM AUTHOR]

Neurosteroids are undeniably regarded as neuroprotective mediators, regulating brain function by rapid non-genomic actions involving interference with microtubules. Conversely, hyperphosphorylated Tau is considered responsible for the onset of a plethora of neurodegenerative diseases, as it dissociates from microtubules, leading to their destabilization, thus impairing synaptic vesicle transport and neurotransmission. Consequently, we aimed to investigate the effects of neurosteroids, specifically allopregnanolone (Allo) and dehydroepiandrosterone (DHEA), on the levels of total and phosphorylated at Serine 404 Tau (p-Tau) in C57BL/6 mice brain slices. In total tissue extracts, we found that neurosteroids elevated both total and p-Tau levels without significantly altering the p-Tau/Tau ratio. In addition, the levels of several enzymes implicated in Tau phosphorylation did not display significant differences between conditions, suggesting that neurosteroids influence Tau distribution rather than its phosphorylation. Hence, we subsequently examined the mitochondria-enriched subcellular fraction where, again, both p-Tau and total Tau levels were increased in the presence of neurosteroids. These effects seem actin-dependent, as disrupting actin polymerization by cytochalasin B preserved Tau levels. Furthermore, co-incubation with high [Ca2+] and neurosteroids mitigated the effects of Ca2+ overload, pointing to cytoskeletal remodeling as a potential mechanism underlying neurosteroid-induced neuroprotection. [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 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]

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]

The editorial discusses the structure and function of immunological and redirecting artificial synapses and their clinical implications. It highlights the importance of the immunological synapse (IS) in facilitating intercellular communication for efficient T and B lymphocyte activation. The article also explores the role of microvilli in antigen sensing and effector functions, as well as the implications of tumor cell reorganization at the IS. Additionally, it delves into T cell redirecting strategies for cancer immunotherapy and the challenges associated with CAR-T therapy. [Extracted from the article]

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]

Mechanotransduction, the process of how cells sense and convert mechanical stimuli into biochemical response, is crucial in the migration of leukocytes or cancer cells through the endothelium during inflammation or metastasis. Migrating cells exert forces on the endothelium through cell surface adhesion molecules, such as platelet endothelial adhesion molecule PECAM-1, and this is essential for a successful transmigration. To study PECAM-1-mediated mechanotransduction, we applied PECAM-1-antibody-coated magnetic beads and exerted about 40 pN force on the endothelial monolayer. We show that force increases cell–ECM adhesion in the cell center and is accompanied by the opening of cell–cell junctions. Upon depletion of the MEK/ERK kinase, BRAF force increases cell–ECM adhesion both at the cell periphery and in the cell center, but this does not result in the opening of cell–cell junctions. Decreasing cell–ECM adhesion in BRAF-depleted cells through FAK inhibition results in the remodeling of cell–cell junctions. Force-induced increase in cell–ECM adhesion in the cell center correlates with the activation of the transcriptional cofactor Yes-associated protein (YAP). Furthermore, the induced activation of YAP through LATS inhibition prevents junctional remodeling in control cells. Thus, the activation of YAP might determine the strength of cell–cell junctions during PECAM-1-mediated mechanotransduction. [ABSTRACT FROM AUTHOR]

Dysregulation of Abelson interactor 1 (ABI1) is associated with various states of disease including developmental defects, pathogen infections, and cancer. ABI1 is an adaptor protein predominantly known to regulate actin cytoskeleton organization processes such as those involved in cell adhesion, migration, and shape determination. Linked to cytoskeleton via vasodilator-stimulated phosphoprotein (VASP), Wiskott-Aldrich syndrome protein family (WAVE), and neural-Wiskott-Aldrich syndrome protein (N-WASP)-associated protein complexes, ABI1 coordinates regulation of various cytoplasmic protein signaling complexes dysregulated in disease states. The roles of ABI1 beyond actin cytoskeleton regulation are much less understood. This comprehensive, protein-centric review describes molecular roles of ABI1 as an adaptor molecule in the context of its dysregulation and associated disease outcomes to better understand disease state-specific protein signaling and affected interconnected biological processes. [ABSTRACT FROM AUTHOR]

The LIM domain kinases (LIMKs) are important actin cytoskeleton regulators. These proteins, LIMK1 and LIMK2, are nodes downstream of Rho GTPases and are the key enzymes that phosphorylate cofilin/actin depolymerization factors to regulate filament severing. They therefore perform an essential role in cascades that control actin depolymerization. Signaling of the LIMKs is carefully regulated by numerous inter‐ and intra‐molecular mechanisms. In this review, we discuss recent findings that improve the understanding of LIM domain kinase regulation mechanisms. We also provide an up‐to‐date review of the role of the LIM domain kinases, their architectural features, how activity is impacted by other proteins, and the implications of these findings for human health and disease. [ABSTRACT FROM AUTHOR]

Bisphenol A (BPA) is widely exposed in populations worldwide and has negative effects on spermatogenesis both in animals and humans. The homeostasis of the actin cytoskeleton in the spermatogenic epithelium is crucial for spermatogenesis. Actin cytoskeleton destruction in the seminiferous epithelium is one of the important reasons for BPA‐induced spermatogenesis disorder. However, the underlying molecular mechanisms remain largely unexplored. Herein, we explored the role and mechanism of Rsad2, an interferon‐stimulated gene in BPA‐induced actin cytoskeleton disorder in mouse GC‐2 spermatocyte cell lines. After BPA exposure, the actin cytoskeleton was dramatically disrupted and the cell morphology was markedly altered accompanied by a significant increase in Rsad2 expression both in mRNA and protein levels in GC‐2 cells. Furthermore, the phalloidin intensities and cell morphology were restored obviously when interfering with the expression of Rsad2 in BPA‐treated GC‐2 cells. In addition, we observed a significant decrease in intracellular ATP levels after BPA treatment, while the ATP level was obviously upregulated when knocking down the expression of Rsad2 in BPA‐treated cells compared to cells treated with BPA alone. Moreover, Rsad2 relocated to mitochondria after BPA exposure in GC‐2 cells. BPA promoted Rsad2 expression by activating type I IFN‐signaling in GC‐2 cells. In summary, Rsad2 mediated BPA‐induced actin cytoskeletal disruption in GC‐2 cells, which provided data to reveal the mechanism of BPA‐induced male reproductive toxicity. BPA induces actin cytoskeleton disruption in the seminiferous epithelium. However, the underlying molecular mechanisms remain unclear. Herein, we found that Rsad2 was required for BPA‐induced cytoskeletal abnormality in GC‐2 cells. Mechanistically, BPA promoted Rsad2 expression by activating type I IFN‐signaling. Afterward, Rsad2 relocated to mitochondria, leading to a decrease in intracellular ATP levels and subsequently resulting in actin cytoskeletal disruption. Taken together, Rsad2 mediated BPA‐induced actin cytoskeleton disorder in GC‐2 cells. [ABSTRACT FROM AUTHOR]

A growth cone is a highly motile tip of an extending axon that is crucial for neural network formation. Three‐dimensional‐structured illumination microscopy, a type of super‐resolution light microscopy with a resolution that overcomes the optical diffraction limitation (ca. 200 nm) of conventional light microscopy, is well suited for studying the molecular dynamics of intracellular events. Using this technique, we discovered a novel type of filopodia distributed along the z‐axis ("z‐filopodia") within the growth cone. Z‐filopodia were typically oriented in the direction of axon growth, not attached to the substratum, protruded spontaneously without microtubule invasion, and had a lifetime that was considerably shorter than that of conventional filopodia. Z‐filopodia formation and dynamics were regulated by actin‐regulatory proteins, such as vasodilator‐stimulated phosphoprotein, fascin, and cofilin. Chromophore‐assisted laser inactivation of cofilin induced the rapid turnover of z‐filopodia. An axon guidance receptor, neuropilin‐1, was concentrated in z‐filopodia and was transported together with them, whereas its ligand, semaphorin‐3A, was selectively bound to them. Membrane domains associated with z‐filopodia were also specialized and resembled those of lipid rafts, and their behaviors were closely related to those of neuropilin‐1. The results suggest that z‐filopodia have unique turnover properties, and unlike xy‐filopodia, do not function as force‐generating structures for axon extension. [ABSTRACT FROM AUTHOR]

Simple Summary: Acute heat stress in animals impairs the intestinal barrier function, particularly tight junction (TJ) depression, which occurs in pigs owing to the lack of functional sweat glands for evaporative heat loss. However, the mechanisms by which heat stress affects TJ regulation are not fully understood. We investigated the effect of heat stress on the TJ and its interaction with potential genes using transcriptomic analysis of the porcine jejunum. We found that heat stress reduced the expression of TJ proteins and induced alterations in genes involved in the actin cytoskeleton and filament-binding pathways. We aimed to investigate the impact of heat stress (HS) on the expression of tight junction (TJ) proteins and the interaction between genes affecting intestinal barrier function using transcriptomics in the porcine jejunum. Twenty-four barrows (crossbred Yorkshire × Landrace × Duroc; average initial body weight, 56.71 ± 1.74 kg) were placed in different temperatures (normal temperature [NT]; HS) and reared for 56 days. At the end of the experiment, jejunal samples were collected from three pigs per treatment for transcriptome and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) analyses. We identified 43 differentially expressed genes, involving five Kyoto Encyclopedia of Genes and Genomes pathways, eight molecular functions, seven cellular components (CCs), and nine biological processes, using gene ontology enrichment analysis. Genes associated with the actin cytoskeleton, filament-binding pathways, and TJ proteins were selected and analyzed by RT-qPCR. Significant differences in relative mRNA expression showed that downregulated genes in the HS group included ZO1, CLDN1, OCLN, PCK1, and PCK2, whereas ACTG2, DES, MYL9, MYLK, TPM1, TPM2, CNN1, PDLIM3, and PCP4 were upregulated by HS (p < 0.05). These findings indicate that HS in growing-finishing pigs induces depression in gut integrity, which may be related to genes involved in the actin cytoskeleton and filaments of CC. [ABSTRACT FROM AUTHOR]

Extensive research has been conducted on the role of CXCR3 in immune responses and inflammation. However, the role of CXCR3 in the reproductive system, particularly in oocyte development, remains unknown. In this study, we present findings on the involvement of CXCR3 in the meiotic division process of mouse oocytes. We found CXCR3 was expressed consistently throughout the entire maturation process of mouse oocyte. Inhibition of CXCR3 impaired the asymmetric division of oocyte, while the injection of Cxcr3 mRNA was capable of restoring these defects. Further study showed that inhibition of CXCR3 perturbed spindle migration by affecting LIMK/cofilin pathway-mediated actin remodeling. Knockout of CXCR3 led to an upregulation of actin-binding protein and an increased ATP level in GV-stage oocytes, while maintaining normal actin dynamics during the process of meiosis. Additionally, we noticed the expression level of DYNLT1 is markedly elevated in CXCR3-null oocytes. DYNLT1 bound with the Arp2/3 complex, and knockdown of DYNLT1 in CXCR3-null oocytes impaired the organization of cytoplasmic actin, suggesting the regulatory role of DYNLT1 in actin organization, and the compensatory expression of DYNLT1 may contribute to maintain normal actin dynamics in CXCR3-knockout oocytes. In summary, our findings provide insights into the intricate network of actin dynamics associated with CXCR3 during oocyte meiosis. • CXCR3 is expressed consistently throughout the entire maturation process of mouse oocyte. • Inhibition of CXCR3 impaired the asymmetric division of oocyte. • Inhibition of CXCR3 perturbed spindle migration by affecting LIMK/cofilin pathway-mediated actin remodeling. • The compensatory increase in DYNLT1 contributes to the maintenance of normal actin dynamics in CXCR3-knockout oocytes. [ABSTRACT FROM AUTHOR]

Shortcomings in cell-based therapies for patients with diabetes have been revealed to be, in part, a result of an improper extracellular matrix (ECM) environment. In vivo, pancreatic islets are emersed in a diverse ECM that provides physical support and is crucial for healthy function. β1-Integrin receptors have been determined to be responsible for modulation of beneficial interactions with ECM proteins influencing beta-cell development, proliferation, maturation, and function. β1-Integrin signaling has been demonstrated to augment insulin secretion by impacting the actin cytoskeleton via activation of focal adhesion kinase and downstream signaling pathways. In other secretory cells, evidence of a bidirectional relationship between integrins and exocytotic machinery has been demonstrated, and, thus, this relationship could be present in pancreatic beta cells. In this review, we will discuss the role of ECM–β1-integrin interplay with exocytotic proteins in controlling pancreatic beta-cell insulin secretion through their dynamic and unique signaling pathway. [ABSTRACT FROM AUTHOR]

Plant organelles predominantly rely on the actin cytoskeleton and the myosin motors for long-distance trafficking, while using microtubules and the kinesin motors mostly for short-range movement. The distribution and motility of organelles in the plant cell are fundamentally important to robust plant growth and defense. Chloroplasts, mitochondria, and peroxisomes are essential organelles in plants that function independently and coordinately during energy metabolism and other key metabolic processes. In response to developmental and environmental stimuli, these energy organelles modulate their metabolism, morphology, abundance, distribution and motility in the cell to meet the need of the plant. Consistent with their metabolic links in processes like photorespiration and fatty acid mobilization is the frequently observed inter-organellar physical interaction, sometimes through organelle membranous protrusions. The development of various organelle-specific fluorescent protein tags has allowed the simultaneous visualization of organelle movement in living plant cells by confocal microscopy. These energy organelles display an array of morphology and movement patterns and redistribute within the cell in response to changes such as varying light conditions, temperature fluctuations, ROS-inducible treatments, and during pollen tube development and immune response, independently or in association with one another. Although there are more reports on the mechanism of chloroplast movement than that of peroxisomes and mitochondria, our knowledge of how and why these three energy organelles move and distribute in the plant cell is still scarce at the functional and mechanistic level. It is critical to identify factors that control organelle motility coupled with plant growth, development, and stress response.

Abstract Background Liver sinusoidal endothelial cells (LSECs) have transcellular pores, called fenestrations, participating in the bidirectional transport between the vascular system and liver parenchyma. Fenestrated LSECs indicate a healthy phenotype of liver while loss of fenestrations (defenestration) in LSECs is associated with liver pathologies. Methods We introduce a unique model of systemic inflammation triggered by the deletion of Mcpip1 in myeloid leukocytes (Mcpip1fl/flLysMCre) characterised by progressive alterations in LSEC phenotype. We implement multiparametric characterisation of LSECs by using novel real-time atomic force microscopy supported with scanning electron microscopy and quantitative fluorescence microscopy. In addition, we provide genetic profiling, searching for characteristic genes encoding proteins that might be connected with the structure of fenestrations. Results We demonstrate that LSECs in Mcpip1fl/flLysMCre display two phases of defenestration: the early phase, with modest defenestration that was fully reversible using cytochalasin B and the late phase, with severe defenestration that is mostly irreversible. By thorough analysis of LSEC porosity, elastic modulus and actin abundance in Mcpip1fl/flLysMCre and in response to cytochalasin B, we demonstrate that proteins other than actin must be additionally responsible for inducing open fenestrations. We highlight several genes that were severely affected in the late but not in the early phase of LSEC defenestration shedding a light on complex structure of individual fenestrations. Conclusions The presented model of LSEC derived from Mcpip1fl/flLysMCre provides a valuable reference for developing novel strategies for LSEC refenestration in the early and late phases of liver pathology.

Abstract Dysregulation of Abelson interactor 1 (ABI1) is associated with various states of disease including developmental defects, pathogen infections, and cancer. ABI1 is an adaptor protein predominantly known to regulate actin cytoskeleton organization processes such as those involved in cell adhesion, migration, and shape determination. Linked to cytoskeleton via vasodilator-stimulated phosphoprotein (VASP), Wiskott-Aldrich syndrome protein family (WAVE), and neural-Wiskott-Aldrich syndrome protein (N-WASP)-associated protein complexes, ABI1 coordinates regulation of various cytoplasmic protein signaling complexes dysregulated in disease states. The roles of ABI1 beyond actin cytoskeleton regulation are much less understood. This comprehensive, protein-centric review describes molecular roles of ABI1 as an adaptor molecule in the context of its dysregulation and associated disease outcomes to better understand disease state-specific protein signaling and affected interconnected biological processes.

Neutrophils are the most abundant leukocyte in humans and provide a critical early line of defense as part of our innate immune system. We perform a comprehensive, genome-wide assessment of the molecular factors critical to proliferation, differentiation, and cell migration in a neutrophil-like cell line. Through the development of multiple migration screen strategies, we specifically probe directed (chemotaxis), undirected (chemokinesis), and 3D amoeboid cell migration in these fast-moving cells. We identify a role for mTORC1 signaling in cell differentiation, which influences neutrophil abundance, survival, and migratory behavior. Across our individual migration screens, we identify genes involved in adhesion-dependent and adhesion-independent cell migration, protein trafficking, and regulation of the actomyosin cytoskeleton. This genome-wide screening strategy, therefore, provides an invaluable approach to the study of neutrophils and provides a resource that will inform future studies of cell migration in these and other rapidly migrating cells.

Key message: ARID-HMG DNA binding protein, AtHMGB15, regulates pollen development and pollen germination in Arabidopsis. Previous studies have shown that ARID-HMG DNA binding protein, AtHMGB15 regulate pollen development and pollen germination in Arabidopsis. Here, we performed transcriptome and cytological studies to understand the role of AtHMGB15 in regulating pollen wall morphology and the pollen tube germination rate. Our result showed abnormal vacuolization in the tapetal cells during anther maturation and prolonged PCD in AtHMGB15 loss-of-function mutant. The tapetum has the ability to perform both secretory and biosynthetic activities critical for pollen maturation and pollen viability. Interestingly, expression of PCD executer genes CEP1, MC9 and RNS3 were significant down-regulation of in athmgb15-4. The growth of pollen tubes is regulated by the actin cytoskeleton dynamics. To address the defect in pollen tube growth of athmgb15, we monitored the actin network in growing pollen tubes of wildtype and athmgb15-4 using Rhodamine-phalloidin fluorescence. Our results indicate a highly fragmented actin distribution in athmgb15-4 pollen tubes with a lesser number of long actin fibers and significantly low f-actin concentration at the apex. q-RTPCR further indicates significant downy-regulation of actin regulatory proteins VLN2 and PRF4. Collectively, our results suggest that AtHMGB15 being a nuclear architectural protein orchestrates high-order chromatin organization to promote the transcription of genes responsible for pollen development and pollen germination. [ABSTRACT FROM AUTHOR]

The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho‐associated coiled‐coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adult mice. Tamoxifen (TAM)‐inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1flox/flox and/or ROCK2flox/flox;Ubc‐CreERT2) were generated and administered a 5‐day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double‐floxed control mice. On Day 3 post‐TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post‐TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)‐cadherin, β‐catenin, and p120‐catenin at cell–cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE‐cadherin/β‐catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell–cell adhesion.

In nephrotic syndrome, the podocyte filtration structures are damaged in a process called foot process effacement. This is mediated by the actin cytoskeleton; however, which actins are involved and how they interact with other filtration components, like the basement membrane, remains poorly understood. Here, we used the well-established Drosophila pericardial nephrocyte—the equivalent of podocytes in flies—knockdown models (RNAi) to study the interplay of the actin cytoskeleton (Act5C, Act57B, Act42A, and Act87E), alpha- and beta-integrin (basement membrane), and the slit diaphragm (Sns and Pyd). Knockdown of an actin gene led to variations of formation of actin stress fibers, the internalization of Sns, and a disrupted slit diaphragm cortical pattern. Notably, deficiency of Act5C, which resulted in complete absence of nephrocytes, could be partially mitigated by overexpressing Act42A or Act87E, suggesting at least partial functional redundancy. Integrin localized near the actin cytoskeleton as well as slit diaphragm components, but when the nephrocyte cytoskeleton or slit diaphragm was disrupted, this switched to colocalization, both at the surface and internalized in aggregates. Altogether, the data show that the interdependence of the slit diaphragm, actin cytoskeleton, and integrins is key to the structure and function of the Drosophila nephrocyte. [ABSTRACT FROM AUTHOR]

Macroautophagy/autophagy is a catabolic process crucial for degrading cytosolic components and damaged organelles to maintain cellular homeostasis, enabling cells to survive in extreme extracellular environments. ENAH/MENA, a member of the Ena/VASP protein family, functions as a highly efficient actin elongation factor. In this study, our objective was to explore the role of ENAH in the autophagy process. Initially, we demonstrated that depleting ENAH in cancer cells inhibits autophagosome formation. Subsequently, we observed ENAH's colocalization with MAP1LC3/LC3 during tumor cell starvation, dependent on actin cytoskeleton polymerization and the interaction between ENAH and BECN1 (beclin 1). Additionally, mammalian ATG9A formed a ring-like structure around ENAH-LC3 puncta during starvation, relying on actin cytoskeleton polymerization. Furthermore, ENAH's EVH1 and EVH2 domains were found to be indispensable for its colocalization with LC3 and BECN1, while the PRD domain played a crucial role in the formation of the ATG9A ring. Finally, our study revealed ENAH-led actin comet tails in autophagosome trafficking. In conclusion, our findings provide initial insights into the regulatory role of the mammalian actin elongation factor ENAH in autophagy. Abbreviations: 3-MA 3-methyladenine; ABPs actin-binding proteins; ATG autophagy related; ATG9A autophagy related 9A; Baf A1 bafilomycin A1; CM complete medium; CytERM endoplasmic reticulum signal-anchor membrane protein; Cyto D cytochalasin D; EBSS Earl's balanced salt solution; ENAH/MENA ENAH actin regulator; EVH1 Ena/VASP homology 1 domain; EVH2 Ena/VASP homology 2 domain; GAPDH glyceraldehyde-3-phosphate dehydrogenase; Lat B latrunculin B; LC3-I unlipidated form of LC3; LC3-II phosphatidylethanolamine-conjugated form of LC3; MAP1LC3/LC3 microtubule associated protein 1 light chain 3; mEGFP monomeric enhanced green fluorescent protein; mTagBFP2 monomeric Tag blue fluorescent protein 2; OSER organized smooth endoplasmic reticulum; PRD proline-rich domain; PtdIns3K class III phosphatidylinositol 3-kinase; WM wortmannin. [ABSTRACT FROM AUTHOR]

The actin cytoskeleton regulates the integrity and repair of epithelial barriers by mediating the assembly of tight junctions (TJs), and adherens junctions (AJs), and driving epithelial wound healing. Actin filaments undergo a constant turnover guided by numerous actin-binding proteins, however, the roles of actin filament dynamics in regulating intestinal epithelial barrier integrity and repair remain poorly understood. Coactosin-like protein 1 (COTL1) is a member of the ADF/ cofilin homology domain protein superfamily that binds and stabilizes actin filaments. COTL1 is essential for neuronal and cancer cell migration, however, its functions in epithelia remain unknown. The goal of this study is to investigate the roles of COTL1 in regulating the structure, permeability, and repair of the epithelial barrier in human intestinal epithelial cells (IEC). COTL1 was found to be enriched at apical junctions in polarized IEC monolayers in vitro. The knockdown of COTL1 in IEC significantly increased paracellular permeability, impaired the steady state TJ and AJ integrity, and attenuated junctional reassembly in a calcium-switch model. Consistently, downregulation of COTL1 expression in Drosophila melanogaster increased gut permeability. Loss of COTL1 attenuated collective IEC migration and decreased cell-matrix attachment. The observed junctional abnormalities in COTL1-depleted IEC were accompanied by the impaired assembly of the cortical actomyosin cytoskeleton. Overexpression of either wild-type COTL1 or its actin-binding deficient mutant tightened the paracellular barrier and activated junction-associated myosin II. Furthermore, the actin-uncoupled COTL1 mutant inhibited epithelial migration and matrix attachment. These findings highlight COTL1 as a novel regulator of the intestinal epithelial barrier integrity and repair. [ABSTRACT FROM AUTHOR]

When stressed, cells need to adapt their proteome to maintain protein homeostasis. This requires increased proteasome assembly. Increased proteasome assembly is dependent on increased production of proteasome assembly chaperones. In Saccharomyces cerevisiae, inhibition of the growth-promoting kinase complex TORC1 causes increased proteasome assembly chaperone translation, including that of Adc17. This is dependent upon activation of the mitogen-activated protein kinase (MAPK) Mpk1 and relocalisation of assembly chaperone mRNA to patches of dense actin. We show here that TORC1 inhibition alters cell wall properties to induce these changes by activating the cell wall integrity pathway through the Wsc1, Wsc3 and Wsc4 sensor proteins. We demonstrate that, in isolation, these signals are insufficient to drive protein expression. We identify that the TORC1-activated S6 kinase Sch9 must be inhibited as well. This work expands our knowledge on the signalling pathways that regulate proteasome assembly chaperone production. [ABSTRACT FROM AUTHOR]

Inborn errors of immunity (IEI) are a group of diseases in humans that typically present as increased susceptibility to infections, autoimmunity, hyperinflammation, allergy, and in some cases malignancy. Among newly identified genes linked to IEIs include 3 independent reports of 9 individuals from 7 independent kindreds with severe primary immunodeficiency disease (PID) and autoimmunity due to loss-offunction mutations in the NCKAP1L gene encoding Hematopoietic protein 1 (HEM1). HEM1 is a hematopoietic cell specific component of the WASp family verprolin homologous (WAVE) regulatory complex (WRC), which acts downstream of multiple immune receptors to stimulate actin nucleation and polymerization of filamentous actin (F-actin). The polymerization and branching of F-actin is critical for creating force-generating cytoskeletal structures which drive most active cellular processes including migration, adhesion, immune synapse formation, and phagocytosis. Branched actin networks at the cell cortex have also been implicated in acting as a barrier to regulate inappropriate vesicle (e.g. cytokine) secretion and spontaneous antigen receptor crosslinking. Given the importance of the actin cytoskeleton in most or all hematopoietic cells, it is not surprising that HEM1 deficient children present with a complex clinical picture that involves overlapping features of immunodeficiency and autoimmunity. In this review, we will provide an overview of what is known about the molecular and cellular functions of HEM1 and the WRC in immune and other cells. We will describe the common clinicopathological features and immunophenotypes of HEM1 deficiency in humans and provide detailed comparative descriptions of what has been learned about Hem1 disruption using constitutive and immune cell-specific mouse knockout models. Finally, we discuss future perspectives and important areas for investigation regarding HEM1 and the WRC. [ABSTRACT FROM AUTHOR]

Isoprenyl cysteine carboxyl methyltransferase (ICMT) catalyzes the last step of the prenylation pathway. Previously, we found that high ICMT levels enhance tumorigenesis in vivo and that its expression is repressed by the p53 tumor suppressor. Based on evidence suggesting that some ICMT substrates affect invasive traits, we wondered if this enzyme may promote metastasis. In this work, we found that ICMT overexpression enhanced lung metastasis in vivo. Accordingly, ICMT overexpression also promoted cellular functions associated with aggressive phenotypes such as migration and invasion in vitro. Considering that some ICMT substrates are involved in the regulation of actin cytoskeleton, we hypothesized that actin-rich structures, associated with invasion and metastasis, may be affected. Our findings revealed that ICMT enhanced the formation of invadopodia. Additionally, by analyzing cancer patient databases, we found that ICMT is overexpressed in several tumor types. Furthermore, the concurrent expression of ICMT and CTTN , which encodes a crucial component of invadopodia, showed a significant correlation with clinical outcome. In summary, our work identifies ICMT overexpression as a relevant alteration in human cancer that promotes the development of metastatic tumors. • ICMT overexpression promotes metastasis in vivo. • ICMT enhances migration, invasion and invadopodia formation in cancer cells. • The ICMT gene is overexpressed in several human cancers. • High expression of ICMT and CTTN correlate with por clinical outcome in lung cancer. [ABSTRACT FROM AUTHOR]

The plant cell wall provides a strong yet flexible barrier to protect cells from the external environment. Modifications of the cell wall, either during development or under stress conditions, can induce cell wall integrity responses and ultimately lead to alterations in gene expression, hormone production, and cell wall composition. These changes in cell wall composition presumably require remodelling of the secretory pathway to facilitate synthesis and secretion of cell wall components and cell wall synthesis/remodelling enzymes from the Golgi apparatus. Here, we used a combination of live-cell confocal imaging and transmission electron microscopy to examine the short-term and constitutive impact of isoxaben, which reduces cellulose biosynthesis, and Driselase, a cocktail of cell-wall-degrading fungal enzymes, on cellular processes during cell wall integrity responses in Arabidopsis. We show that both treatments altered organelle morphology and triggered rebalancing of the secretory pathway to promote secretion while reducing endocytic trafficking. The actin cytoskeleton was less dynamic following cell wall modification, and organelle movement was reduced. These results demonstrate active remodelling of the endomembrane system and actin cytoskeleton following changes to the cell wall. [ABSTRACT FROM AUTHOR]

The eukaryotic cytoskeleton is a complex scaffold consisting of actin filaments, intermediate filaments, and microtubules. Although fungi and plants lack intermediate filaments, their dynamic structural network of actin filaments and microtubules regulates cell shape, division, polarity, and vesicular trafficking. However, the specialized functions of the cytoskeleton during plant–fungus interactions remain elusive. Recent reports demonstrate that the plant cytoskeleton responds to signal cues and pathogen invasion through remodeling, thereby coordinating immune receptor trafficking, membrane microdomain formation, aggregation of organelles, and transport of defense compounds. Emerging evidence also suggests that cytoskeleton remodeling further regulates host immunity by triggering salicylic acid signaling, reactive oxygen species generation, and pathogenesis-related gene expression. During host invasion, fungi undergo systematic cytoskeleton remodeling, which is crucial for successful host penetration and colonization. Furthermore, phytohormones act as an essential regulator of plant cytoskeleton dynamics and are frequently targeted by fungal effectors to disrupt the host's growth–defense balance. This review discusses recent advances in the understanding of cytoskeleton dynamics during plant–fungus interactions and provides novel insights into the relationship between phytohormones and cytoskeleton remodeling upon pathogen attack. We also highlight the importance of fungal cytoskeleton rearrangements during host colonization and suggest directions for future investigations in this field. [ABSTRACT FROM AUTHOR]

Contractile actomyosin bundles play crucial roles in various physiological processes, including cell migration, morphogenesis, and muscle contraction. The intricate assembly of actomyosin bundles involves the precise alignment and fusion of myosin II filaments, yet the underlying mechanisms and factors involved in these processes remain elusive. Our study reveals that LUZP1 plays a central role in orchestrating the maturation of thick actomyosin bundles. Loss of LUZP1 caused abnormal cell morphogenesis, migration, and the ability to exert forces on the environment. Importantly, knockout of LUZP1 results in significant defects in the concatenation and persistent association of myosin II filaments, severely impairing the assembly of myosin II stacks. The disruption of these processes in LUZP1 knockout cells provides mechanistic insights into the defective assembly of thick ventral stress fibers and the associated cellular contractility abnormalities. Overall, these results significantly contribute to our understanding of the molecular mechanism involved in actomyosin bundle formation and highlight the essential role of LUZP1 in this process. [ABSTRACT FROM AUTHOR]

The Ras homology (Rho) family of GTPases serves various functions, including promotion of cell migration, adhesion, and transcription, through activation of effector molecule targets. One such pair of effectors, the Rho‐associated coiled‐coil kinases (ROCK1 and ROCK2), induce reorganization of actin cytoskeleton and focal adhesion through substrate phosphorylation. Studies on ROCK knockout mice have confirmed that ROCK proteins are essential for embryonic development, but their physiological functions in adult mice remain unknown. In this study, we aimed to examine the roles of ROCK1 and ROCK2 proteins in normal adult mice. Tamoxifen (TAM)‐inducible ROCK1 and ROCK2 single and double knockout mice (ROCK1flox/flox and/or ROCK2flox/flox;Ubc‐CreERT2) were generated and administered a 5‐day course of TAM. No deaths occurred in either of the single knockout strains, whereas all of the ROCK1/ROCK2 double conditional knockout mice (DcKO) had died by Day 11 following the TAM course. DcKO mice exhibited increased lung tissue vascular permeability, thickening of alveolar walls, and a decrease in percutaneous oxygen saturation compared with noninducible ROCK1/ROCK2 double‐floxed control mice. On Day 3 post‐TAM, there was a decrease in phalloidin staining in the lungs in DcKO mice. On Day 5 post‐TAM, immunohistochemical analysis also revealed reduced staining for vascular endothelial (VE)‐cadherin, β‐catenin, and p120‐catenin at cell–cell contact sites in vascular endothelial cells in DcKO mice. Additionally, VE‐cadherin/β‐catenin complexes were decreased in DcKO mice, indicating that ROCK proteins play a crucial role in maintaining lung function by regulating cell–cell adhesion. [ABSTRACT FROM AUTHOR]

Membrane tension is thought to be a long-range integrator of cell physiology. Membrane tension has been proposed to enable cell polarity during migration through front-back coordination and long-range protrusion competition. These roles necessitate effective tension transmission across the cell. However, conflicting observations have left the field divided as to whether cell membranes support or resist tension propagation. This discrepancy likely originates from the use of exogenous forces that may not accurately mimic endogenous forces. We overcome this complication by leveraging optogenetics to directly control localized actin-based protrusions or actomyosin contractions while simultaneously monitoring the propagation of membrane tension using dual-trap optical tweezers. Surprisingly, actin-driven protrusions and actomyosin contractions both elicit rapid global membrane tension propagation, whereas forces applied to cell membranes alone do not. We present a simple unifying mechanical model in which mechanical forces that engage the actin cortex drive rapid, robust membrane tension propagation through long-range membrane flows.

Post-translational modifications of beta-amyloid (Aβ) play an important role in the pathogenesis of Alzheimer’s disease (AD). Aβ modifications such as Ser8 phosphorylation (pS8-Aβ42) and Asp7 isomerization (iso-Aβ42) can significantly alter the properties of Aβ and have been detected in vivo. One of the reasons for the different pathogenicity of Aβ isoforms may be the activation of different signaling cascades leading to changes in the mechanical properties of cells. In this paper, we used correlative scanning ion-conductance microscopy (SICM) and Pt-nanoelectrodes to compare the effects of Aβ isoforms on the Young’s modulus of SH-SY5Y cells and the level of ROS. It was found that unmodified Aβ42 resulted in the largest increase in cell Young’s modulus of all isoforms after 4 h of incubation, while pS8-Aβ42 induced the greatest increase in stiffness and ROS levels after 24 h of incubation. Analysis of signaling proteins involved in the regulation of the actin cytoskeleton showed that Aβ42, pS8-Aβ42 and iso-Aβ42 have different effects on cofilin, GSK3β, LIMK, ERK and p38. This indicates that post-translational modifications of Aβ modulate its effect on neuronal cells through the activation of various signaling cascades, which affects the mechanical properties of cells.

Post-traumatic stress disorder (PTSD) is characterized by hypermnesia of the trauma and a persistent fear response. The molecular mechanisms underlying the retention of traumatic memories remain largely unknown, which hinders the development of more effective treatments. Utilizing auditory fear conditioning, we demonstrate that a redox-dependent dynamic pathway for dendritic spine morphogenesis in the basolateral amygdala (BLA) is crucial for traumatic memory retention. Exposure to a fear-induced event markedly increased the reduction of oxidized filamentous actin (F-actin) and decreased the expression of the molecule interacting with CasL 1 (MICAL1), a methionine-oxidizing enzyme that directly oxidizes and depolymerizes F-actin, leading to cytoskeletal dynamic abnormalities in the BLA, which impairs dendritic spine morphogenesis and contributes to the persistence of fearful memories. Following fear conditioning, overexpression of MICAL1 in the BLA inhibited freezing behavior during fear memory retrieval via reactivating cytokinesis, whereas overexpression of methionine sulfoxide reductase B 1, a key enzyme that reduces oxidized F-actin monomer, increased freezing behavior during retrieval. Notably, intra-BLA injection of semaphorin 3A, an endogenous activator of MICAL1, rapidly disrupted fear memory within a short time window after conditioning. Collectively, our results indicate that redox modulation of actin cytoskeleton in the BLA is functionally linked to fear memory retention and PTSD-like memory.

We analyzed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates microtubule-organizing center (MTOC) and multivesicular bodies (MVB) polarization and exosome secretion at an immune synapse (IS) model in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which were restored by FMNL1βWT expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and exosome secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in PKCδ-interfered clones, indicating that S1086 FMNL1β phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the central region of the immune synapse (cIS), which is necessary for MTOC/MVB polarization. FMNL1βWT and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at the cIS. Thus, actin cytoskeleton reorganization at the IS underlies the effects of all these FMNL1β variants on polarized secretory traffic. FMNL1 was found in the IS made by primary T lymphocytes, both in T cell receptor (TCR) and chimeric antigen receptor (CAR)-evoked synapses. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.

ABSTRACT Phagocytosis of Mycobacterium tuberculosis (Mtb) followed by its integration into the matured lysosome is critical in the host defense against tuberculosis. How Mtb escapes this immune attack remains elusive. In this study, we unveiled a novel regulatory mechanism by which SIRT7 regulates cytoskeletal remodeling by modulating RAC1 activation. We discovered that SIRT7 expression was significantly reduced in CD14+ monocytes of TB patients. Mtb infection diminished SIRT7 expression by macrophages at both the mRNA and protein levels. SIRT7 deficiency impaired actin cytoskeleton-dependent macrophage phagocytosis, LC3II expression, and bactericidal activity. In a murine tuberculosis model, SIRT7 deficiency detrimentally impacted host resistance to Mtb, while Sirt7 overexpression significantly increased the host defense against Mtb, as determined by bacterial burden and inflammatory–histopathological damage in the lung. Mechanistically, we demonstrated that SIRT7 limits Mtb infection by directly interacting with and activating RAC1, through which cytoskeletal remodeling is modulated. Therefore, we concluded that SIRT7, in its role regulating cytoskeletal remodeling through RAC1, is critical for host responses during Mtb infection and proposes a potential target for tuberculosis treatment.IMPORTANCETuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a significant global health issue. Critical to macrophages’ defense against Mtb is phagocytosis, governed by the actin cytoskeleton. Previous research has revealed that Mtb manipulates and disrupts the host’s actin network, though the specific mechanisms have been elusive. Our study identifies a pivotal role for SIRT7 in this context: Mtb infection leads to reduced SIRT7 expression, which, in turn, diminishes RAC1 activation and consequently impairs actin-dependent phagocytosis. The significance of our research is that SIRT7 directly engages with and activates Rac Family Small GTPase 1 (RAC1), thus promoting effective phagocytosis and the elimination of Mtb. This insight into the dynamic between host and pathogen in TB not only broadens our understanding but also opens new avenues for therapeutic development.

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.

Arpin was discovered as an inhibitor of the Arp2/3 complex localized at the lamellipodial tip of fibroblasts, where it regulated migration steering. Recently, we showed that arpin stabilizes the epithelial barrier in an Arp2/3-dependent manner. However, the expression and functions of arpin in endothelial cells (EC) have not yet been described. Arpin mRNA and protein are expressed in EC and downregulated by pro-inflammatory cytokines. Arpin depletion in Human Umbilical Vein Endothelial Cells causes the formation of actomyosin stress fibers leading to increased permeability in an Arp2/3-independent manner. Instead, inhibitors of ROCK1 and ZIPK, kinases involved in the generation of stress fibers, normalize the loss-of-arpin effects on actin filaments and permeability. Arpin-deficient mice are viable but show a characteristic vascular phenotype in the lung including edema, microhemorrhage, and vascular congestion, increased F-actin levels, and vascular permeability. Our data show that, apart from being an Arp2/3 inhibitor, arpin is also a regulator of actomyosin contractility and endothelial barrier integrity.

Fluorescence microscopy is an important tool for cell biology and cancer research. Present-day approach of implementing advanced optical microscopy methods combined with immunofluorescence labelling of specific proteins in cells is now able to deliver optical super-resolution up to ∼25 nm. Here we perform super-resolved imaging using standard immunostaining protocol combined with easy stochastic optical reconstruction microscopy (easySTORM) to observe structural differences of two cytoskeleton elements, actin and tubulin in three different cell types namely human bone marrow-derived mesenchymal stem cells (MSCs), human glioblastoma (U87MG) and breast cancer (MDAMB-231) cells. The average width of the actin bundle obtained from STORM images of stem cells is observed to be larger than the same for U87MG and MDAMB-231 cells. No significant difference is however noticed in the width of the tubulin within the same cells. We also study the functional effect on the 2D migration potential of MDAMB-231 cells silenced for NICD1 and β-catenin. Although similar migration speed is observed for cells with the above two conditions compared to their control cells, easySTORM images show that widths of the actin in MDAMB-231 cells in β-catenin silenced is significantly lower than the same in control cells. Such minute differences however are not observable in widefield images. The outcome of our easySTORM investigation should benefit the researchers carrying out detailed investigations of the cellular structure and potential therapeutic applications.

To better understand the genetic basis of heart disease, we identified a variant in the Flightless-I homolog (FLII) gene that generates a R1243H missense change and predisposes to cardiac remodeling across multiple previous human genome-wide association studies (GWAS). Since this gene is of unknown function in the mammalian heart we generated gain- and loss-of-function genetically altered mice, as well as knock-in mice with the syntenic R1245H amino acid substitution, which showed that Flii protein binds the sarcomeric actin thin filament and influences its length. Deletion of Flii from the heart, or mice with the R1245H amino acid substitution, show cardiomyopathy due to shortening of the actin thin filaments. Mechanistically, Flii is a known actin binding protein that we show associates with tropomodulin-1 (TMOD1) to regulate sarcomere thin filament length. Indeed, overexpression of leiomodin-2 in the heart, which lengthens the actin-containing thin filaments, partially rescued disease due to heart-specific deletion of Flii. Collectively, the identified FLII human variant likely increases cardiomyopathy risk through an alteration in sarcomere structure and associated contractile dynamics, like other sarcomere gene-based familial cardiomyopathies.

Although sex differences in learning behaviors are well documented, sexual dimorphism in the synaptic processes of encoding is only recently appreciated. Studies in male rodents have built upon the discovery of long-term potentiation (LTP), and acceptance of this activity-dependent increase in synaptic strength as a mechanism of encoding, to identify synaptic receptors and signaling activities that coordinate the activity-dependent remodeling of the subsynaptic actin cytoskeleton that is critical for enduring potentiation and memory. These molecular substrates together with other features of LTP, as characterized in males, have provided an explanation for a range of memory phenomena including multiple stages of consolidation, the efficacy of spaced training, and the location of engrams at the level of individual synapses. In the present report, we summarize these findings and describe more recent results from our laboratories showing that in females the same actin regulatory mechanisms are required for hippocampal LTP and memory but, in females only, the engagement of both modulatory receptors such as TrkB and synaptic signaling intermediaries including Src and ERK1/2 requires neuron-derived estrogen and signaling through membrane-associated estrogen receptor α (ERα). Moreover, in association with the additional ERα involvement, females exhibit a higher threshold for hippocampal LTP and spatial learning. We propose that the distinct LTP threshold in females contributes to as yet unappreciated sex differences in information processing and features of learning and memory.

The photoreceptor outer segment (OS) is the phototransductive organelle in the vertebrate retina. OS tips are regularly ingested and degraded by the adjacent retinal pigment epithelium (RPE), offsetting the addition of new disk membrane at the base of the OS. This catabolic role of the RPE is essential for photoreceptor health, with defects in ingestion or degradation underlying different forms of retinal degeneration and blindness. Although proteins required for OS tip ingestion have been identified, spatiotemporal analysis of the ingestion process in live RPE cells is lacking; hence, the literature reflects no common understanding of the cellular mechanisms that affect ingestion. We imaged live RPE cells from mice (both sexes) to elucidate the ingestion events in real time. Our imaging revealed roles for f-actin dynamics and specific dynamic localizations of two BAR (Bin-Amphiphysin-Rvs) proteins, FBP17 and AMPH1-BAR, in shaping the RPE apical membrane as it surrounds the OS tip. Completion of ingestion was observed to occur by scission of the OS tip from the remainder of the OS, with a transient concentration of f-actin forming around the site of imminent scission. Actin dynamics were also required for regulating the size of the ingested OS tip, and the time course of the overall ingestion process. The size of the ingested tip is consistent with the term phagocytosis. However, phagocytosis usually refers to engulfment of an entire particle or cell, whereas our observations of OS tip scission indicate a process that is more specifically described as trogocytosis, in which one cell nibbles another cell.SIGNIFICANCE STATEMENT The ingestion of the photoreceptor outer segment (OS) tips by the retinal pigment epithelium (RPE) is a dynamic cellular process that has fascinated scientists for 60 years. Yet its molecular mechanisms had not been addressed in living cells. We developed a live-cell imaging approach to investigate OS tip ingestion, and focused on the dynamic participation of actin filaments and membrane-shaping BAR proteins. We observed scission of OS tips for the first time, and were able to monitor local changes in protein concentration preceding, during, and following scission. Our approach revealed that actin filaments were concentrated at the site of OS scission and were required for regulating the size of the ingested OS tip and the time course of the ingestion process.

Astrocytes and neurons extensively interact in the brain. Identifying astrocyte and neuron proteomes is essential for elucidating the protein networks that dictate their respective contributions to physiology and disease. Here we used cell-specific and subcompartment-specific proximity-dependent biotinylation1 to study the proteomes of striatal astrocytes and neurons in vivo. We evaluated cytosolic and plasma membrane compartments for astrocytes and neurons to discover how these cells differ at the protein level in their signalling machinery. We also assessed subcellular compartments of astrocytes, including end feet and fine processes, to reveal their subproteomes and the molecular basis of essential astrocyte signalling and homeostatic functions. Notably, SAPAP3 (encoded by Dlgap3), which is associated with obsessive-compulsive disorder (OCD) and repetitive behaviours2-8, was detected at high levels in striatal astrocytes and was enriched within specific astrocyte subcompartments where it regulated actin cytoskeleton organization. Furthermore, genetic rescue experiments combined with behavioural analyses and molecular assessments in a mouse model of OCD4 lacking SAPAP3 revealed distinct contributions of astrocytic and neuronal SAPAP3 to repetitive and anxiety-related OCD-like phenotypes. Our data define how astrocytes and neurons differ at the protein level and in their major signalling pathways. Moreover, they reveal how astrocyte subproteomes vary between physiological subcompartments and how both astrocyte and neuronal SAPAP3 mechanisms contribute to OCD phenotypes in mice. Our data indicate that therapeutic strategies that target both astrocytes and neurons may be useful to explore in OCD and potentially other brain disorders.