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Depletion interactions are thought to significantly contribute to the organization of intracellular structures in the crowded cytosol. The strength of depletion interactions depends on physical parameters such as the depletant number density and the depletant size ratio. Cells are known to dynamically regulate these two parameters by varying the copy number of proteins of a wide distribution of sizes. However, mammalian cells are also known to keep the total protein mass density remarkably constant, to within 0.5% throughout the cell cycle. We thus ask how the strength of depletion interactions varies when the total depletant mass is held fixed, a.k.a. fixed-mass depletion. We answer this question via scaling arguments, as well as by studying depletion effects on networks of reconstituted semiflexible actin in silico and in vitro. We examine the maximum strength of the depletion interaction potential U∗ as a function of q, the size ratio between the depletant and the matter being depleted. We uncover a scaling relation U∗ ∼ qζ for two cases: fixed volume fraction φ and fixed mass density ρ. For fixed volume fraction, we report ζ < 0. For the fixed mass density case, we report ζ > 0, which suggests that the depletion interaction strength increases as the depletant size ratio is increased. To test this prediction, we prepared our filament networks at fixed mass concentrations with varying sizes of the depletant molecule poly(ethylene glycol) (PEG). We characterize the depletion interaction strength in our simulations via the mesh size. In experiments, we observe two distinct actin network morphologies, which we call weakly bundled and strongly bundled. We identify a mass concentration where different PEG depletant sizes lead to weakly bundled or strongly bundled morphologies. For these conditions, we find that the mesh size and intra-bundle spacing between filaments across the different morphologies do not show significant differences, while the dynamic light scattering relaxation time and storage modulus between the two states do show significant differences. Our results demonstrate the ability to tune actin network morphology and mechanics by controlling depletant size and give insights into depletion interaction mechanisms under the fixed-depletant-mass constraint relevant to living cells. [ABSTRACT FROM AUTHOR]

Inherited mutations in human beta-cardiac myosin (M2β) can lead to severe forms of heart failure. The E525K mutation in M2β is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2β subfragment 1 (S1) we found that E525K enhances (threefold) the maximum steady-state actin-activated ATPase activity (k cat) and decreases (eightfold) the actin concentration at which ATPase is one-half maximal (K ATPase). We also found a twofold to fourfold increase in the actin-activated power stroke and phosphate release rate constants at 30 μM actin, which overall enhanced the duty ratio threefold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2β S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis.

In plant vegetative tissues, cell division employs a mitotic microtubule array called the preprophase band (PPB) that marks the cortical division site. This transient cytoskeletal array imprints the spatial information to be read by the cytokinetic phragmoplast at later stages of mitotic cell division. In Arabidopsis thaliana, we discovered that the PPB recruited the Myosin XI motor MYA1/Myo11F to the cortical division site, where it joined microtubule-associated proteins and motors to form a ring of prominent cytoskeletal assemblies that received the expanding phragmoplast. Such a myosin localization pattern at the cortical division site was dependent on the POK1/2 Kinesin-12 motors. This regulatory function of MYA1/Myo11F in phragmoplast guidance was dependent on intact actin filaments. The discovery of these cytoskeletal motor assemblies pinpoints a mechanism underlying how two dynamic cytoskeletal networks work in concert to govern PPB-dependent division plane orientation in flowering plants.

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.

Stroke, trauma, and neurodegenerative disorders cause loss of neurites (axons and dendrites) in addition to neuronal death. Neurite loss may result directly from a primary insult, secondary to parental neuron death, or secondary to a post-injury inflammatory response. Here, we use lipopolysaccharide and the alarmin S100β to selectively evaluate neurite loss caused by the inflammatory response. Activation of microglia and infiltrating macrophages by these stimuli causes neurite loss that far exceeds neuronal death, both in vitro and in vivo. Neurite loss is accompanied by the formation of cofilactin rods and aggregates (CARs), which are polymers of cofilin-1 and actin induced by oxidative stress and other factors. Mice deficient in either cofilin-1 or the superoxide-generating enzyme NADPH oxidase-2 show reduced CAR formation, neurite loss, and motor impairment. The findings identify a mechanism by which inflammation leads to neurite loss via CAR formation and highlight the relevance of neurite loss to functional impairment.

The failure of intracellular zinc accumulation is a key process in prostate carcinogenesis. Although prostate cancer cells can accumulate zinc after long-term exposure, chronic zinc oversupply may accelerate prostate carcinogenesis or chemoresistance. Because cancer progression is associated with energetically demanding cytoskeletal rearrangements, we investigated the effect of long-term zinc presence on biophysical parameters, ATP production, and EMT characteristics of two prostate cancer cell lines (PC-3, 22Rv1). Prolonged exposure to zinc increased ATP production, spare respiratory capacity, and induced a response in PC-3 cells, characterized by remodeling of vimentin and a shift of cell dry mass density and caveolin-1 to the perinuclear region. This zinc-induced remodeling correlated with a greater tendency to maintain actin architecture despite inhibition of actin polymerization by cytochalasin. Zinc partially restored epithelial characteristics in PC-3 cells by decreasing vimentin expression and increasing E-cadherin. Nevertheless, the expression of E-cadherin remained lower than that observed in predominantly oxidative, low-invasive 22Rv1 cells. Following long-term zinc exposure, we observed an increase in cell stiffness associated with an increased refractive index in the perinuclear region and an increased mitochondrial content. The findings of the computational simulations indicate that the mechanical response cannot be attributed exclusively to alterations in cytoskeletal composition. This observation suggests the potential involvement of an additional, as yet unidentified, mechanical contributor. These findings indicate that long-term zinc exposure alters a group of cellular parameters towards an invasive phenotype, including an increase in mitochondrial number, ATP production, and cytochalasin resistance. Ultimately, these alterations are manifested in the biomechanical properties of the cells. [ABSTRACT FROM AUTHOR]

Summary The effect of dry aging process (15 and 30 days) on some quality parameters, protein profile and formation of protein oxidation products of beef was investigated. The maximum weight and trim loss were calculated to be 47.07% and 23.06%, respectively. The meat dry‐aged for 30 days had higher dry matter, pH and protein content, but lower L* and a* values than those of the unaged meats. There was a significant decrease in actin protein band density in the meat dry‐aged for 30 days and a new protein band of 32 kDa appeared in the myofibrillar protein fraction. The dry aging process caused an insignificant increase in the total carbonyl content (P > 0.05). However, the γ‐glutamic semialdehyde (GGS), α‐aminoadipic semialdehyde (AAS) and total GGS and AAS contents were higher in the dry‐aged meats for 30 days. Total GGS and AAS contents ranged from 15.36% to 31.87% of the total carbonyl content. It was found that there was no significant difference between control group (unaged) meats and the meat dry‐aged for 15 days in terms of protein oxidation products. [ABSTRACT FROM AUTHOR]

The contractile apparatus, stress fiber (SF), is connected to the cell adhesion machinery, focal adhesion (FA), at the termini of SF. The SF-FA complex is essential for various mechanical activities of cells, including cell adhesion to the extracellular matrix (ECM), ECM rigidity sensing, and cell migration. This mini-review highlights the importance of SF mechanics in these cellular activities. Actin-crosslinking proteins solidify SFs by attenuating myosindriven flows of actin and myosin filaments within the SF. In the solidified SFs, viscous slippage between actin filaments in SFs and between the filaments and the surrounding cytosol is reduced, leading to efficient transmission of myosin-generated contractile force along the SFs. Hence, SF solidification via actin crosslinking ensures exertion of a large force to FAs, enabling FA maturation, ECM rigidity sensing and cell migration. We further discuss intracellular mechanisms for tuning crosslinker-modulated SF mechanics and the potential relationship between the aberrance of SF mechanics and pathology including cancer. [ABSTRACT FROM AUTHOR]

AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity. [ABSTRACT FROM AUTHOR]

The primary cilium is a small organelle protruding from the cell surface that receives signals from the extracellular milieu. Although dozens of studies have reported that several genetic factors can impair the structure of primary cilia, evidence for environmental stimuli affecting primary cilia structures is limited. Here, we investigated an extracellular stress that affected primary cilia morphology and its underlying mechanisms. Hyperosmotic shock induced reversible shortening and disassembly of the primary cilia of murine intramedullary collecting duct cells. The shortening of primary cilia caused by hyperosmotic shock followed delocalization of the pericentriolar material (PCM). Excessive microtubule and F-actin formation in the cytoplasm coincided with the hyperosmotic shockinduced changes to primary cilia and the PCM. Treatment with a microtubule-disrupting agent, nocodazole, partially prevented the hyperosmotic shock-induced disassembly of primary cilia and almost completely prevented delocalization of the PCM. An actin polymerization inhibitor, latrunculin A, also partially prevented the hyperosmotic shock-induced shortening and disassembly of primary cilia and almost completely prevented delocalization of the PCM. We demonstrate that hyperosmotic shock induces reversible morphological changes in primary cilia and the PCM in a manner dependent on excessive formation of microtubule and F-actin. [ABSTRACT FROM AUTHOR]

Synaptopodin 2-like protein (SYNPO2L) is localized in the sarcomere of cardiomyocytes and is involved in heart morphogenesis. However, the molecular function of SYNPO2L in the heart is not fully understood. We investigated the interaction of SYNPO2L with sarcomeric α-actinin and actin filaments in cultured mouse cardiomyocytes. Immunofluorescence studies showed that SYNPO2L colocalized with α-actinin and actin filaments at the Z-discs of the sarcomere. Recombinant SYNPO2La or SYNPO2Lb caused a bundling of the actin filaments in the absence of α-actinin and enhanced the α-actinin-dependent formation of actin bundles. In addition, high-speed atomic force microscopy revealed that SYNPO2La directly bound to α-actinin via its globular ends. The interaction between α-actinin and SYNPO2La fixed the movements of the two proteins on the actin filaments. These results strongly suggest that SYNPO2L cooperates with α-actinin during actin bundle formation to facilitate sarcomere formation and maintenance. [ABSTRACT FROM AUTHOR]

The omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified in 2021 as a variant with heavy amino acid mutations in the spike protein, which is targeted by most vaccines, compared to previous variants. Amino acid substitutions in the spike proteins may alter their affinity for host viral receptors and the host interactome. Here, we found that the receptor-binding domain (RBD) of the omicron variant of SARS-CoV-2 exhibited an increased affinity for human angiotensin-converting enzyme 2, a viral cell receptor, compared to the prototype RBD. Moreover, we identified β- and γ-actin as omicron-specific binding partners of RBD. Protein complex predictions revealed that many omicron-specific amino acid substitutions affected the affinity between RBD of the omicron variant and actin. Our findings indicate that proteins localized to different cellular compartments exhibit strong binding to the omicron RBD. [ABSTRACT FROM AUTHOR]

[Display omitted] • 1. AFM-based biophysical measurements are performed on a neural cell. • 2. Exposure to high-dose carbon dots affects cellular mechanics and morphology. • 3. Actin filaments are disorganized in the cells after exposure to high-dose carbon dots. • 4. Biological studies validate the cytotoxic effects disclosed by AFM. • 5. AFM is a powerful biophysical tool for the study of cell-nanomaterials interactions. Understanding the cytotoxicity of fluorescent carbon dots (CDs) is crucial for their applications, and various biochemical assays have been used to study the effects of CDs on cells. Knowledge on the effects of CDs from a biophysical perspective is integral to the recognition of their cytotoxicity, however the related information is very limited. Here, we report that atomic force microscopy (AFM) can be used as an effective tool for studying the effects of CDs on cells from the biophysical perspective. We achieve this by integrating AFM-based nanomechanics with AFM-based imaging. We demonstrate the performance of this method by measuring the influence of CDs on living human neuroblastoma (SH-SY5Y) cells at the single-cell level. We find that high-dose CDs can mechanically induce elevated normalized hysteresis (energy dissipation during the cell deformation) and structurally impair actin skeleton. The nanomechanical change highly correlates with the alteration of actin filaments, indicating that CDs-induced changes in SH-SY5Y cells are revealed in-depth from the AFM-based biophysical aspect. We validate the reliability of the biophysical observations using conventional biological methods including cell viability test, fluorescent microscopy, and western blot assay. Our work contributes new and significant information on the cytotoxicity of CDs from the biophysical perspective. [ABSTRACT FROM AUTHOR]

ABSTRACT It has been proposed that cortical fine actin filaments are needed for the morphogenesis of pavement cells (PCs). However, the precise role and regulation mechanisms of actin filaments in PC morphogenesis are not well understood. Here, we found that Arabidopsis thaliana ACTIN DEPOLYMERIZING FACTOR9 (ADF9) is required for the morphogenesis of PC, which is negatively regulated by the R2R3 MYELOBLASTOSIS (MYB) transcription factor MYB52. In adf9 mutants, the lobe number of cotyledon PCs was significantly reduced, while the average lobe length did not differ significantly compared to that of wild type (Col‐0), except for the variations in cell area and circularity, whereas the PC shapes in ADF9 overexpression seedlings showed different results. ADF9 decorated actin filaments, and colocalized with plasma membrane. The extent of filament bundling and actin filament bundling activity in adf9 mutant decreased. In addition, MYB52 directly targeted the promoter of ADF9 and negatively regulated its expression. The myb52‐2 mutant showed increased lobe number and cell area, reduced cell circularity of PCs, and the PC phenotypes were suppressed when ADF9 was knocked out. Taken together, our data demonstrate that actin filaments play an important role in the morphogenesis of PC and reveal a transcriptional mechanism underlying MYB52 regulation of ADF9‐mediated actin filament bundling in PC morphogenesis. [ABSTRACT FROM AUTHOR]

Endosomal membrane trafficking is mediated by specific protein coats and formation of actin-rich membrane domains. The Retromer complex coordinates with sorting nexin (SNX) cargo adaptors including SNX27, and the SNX27-Retromer assembly interacts with the Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex which nucleates actin filaments establishing the endosomal recycling domain. Crystal structures, modeling, biochemical, and cellular validation reveal how the FAM21 subunit of WASH interacts with both Retromer and SNX27. FAM21 binds the FERM domain of SNX27 using acidic-Asp-Leu-Phe (aDLF) motifs similar to those found in the SNX1 and SNX2 subunits of the ESCPE-1 complex. Overlapping FAM21 repeats and a specific Pro-Leu containing motif bind three distinct sites on Retromer involving both the VPS35 and VPS29 subunits. Mutation of the major VPS35-binding site does not prevent cargo recycling; however, it partially reduces endosomal WASH association indicating that a network of redundant interactions promote endosomal activity of the WASH complex. These studies establish the molecular basis for how SNX27-Retromer is coupled to the WASH complex via overlapping and multiplexed motif-based interactions required for the dynamic assembly of endosomal membrane recycling domains. [ABSTRACT FROM AUTHOR]

Many cytoskeletal networks consist of individual filaments that are organized into elaborate higher-order structures. While it is appreciated that the size and architecture of these networks are critical for their biological functions, much of the work investigating control over their assembly has focused on mechanisms that regulate the turnover of individual filaments through size-dependent feedback. Here, we propose a very different, feedback-independent mechanism to explain how yeast cells control the length of their actin cables. Our findings, supported by quantitative cell imaging and mathematical modeling, indicate that actin cable length control is an emergent property that arises from the cross-linked and bundled organization of the filaments within the cable. Using this model, we further dissect the mechanisms that allow cables to grow longer in larger cells and propose that cell length-dependent tuning of formin activity allows cells to scale cable length with cell length. This mechanism is a significant departure from prior models of cytoskeletal filament length control and presents a different paradigm to consider how cells control the size, shape, and dynamics of higher-order cytoskeletal structures. [ABSTRACT FROM AUTHOR]

Myelin is an insulator that forms around axons that enhance the conduction velocity of nerve fibers. Oligodendrocytes dramatically change cell morphology to produce myelin throughout the central nervous system (CNS). Cytoskeletal alterations are critical for the morphogenesis of oligodendrocytes, and actin is involved in cell differentiation and myelin wrapping via polymerization and depolymerization, respectively. Various protein members of the myosin superfamily are known to be major binding partners of actin filaments and have been intensively researched because of their involvement in various cellular functions, including differentiation, cell movement, membrane trafficking, organelle transport, signal transduction, and morphogenesis. Some members of the myosin superfamily have been found to play important roles in the differentiation of oligodendrocytes and in CNS myelination. Interestingly, each member of the myosin superfamily expressed in oligodendrocyte lineage cells also shows specific spatial and temporal expression patterns and different distributions. In this review, we summarize previous findings related to the myosin superfamily and discuss how these molecules contribute to myelin formation and regeneration by oligodendrocytes. [ABSTRACT FROM AUTHOR]

The monomer-binding protein profilin 1 (PFN1) plays a crucial role in actin polymerization. However, mutations in PFN1 are also linked to hereditary amyotrophic lateral sclerosis, resulting in a broad range of cellular pathologies which cannot be explained by its primary function as a cytosolic actin assembly factor. This implies that there are important, undiscovered roles for PFN1 in cellular physiology. Here we screened knockout cells for novel phenotypes associated with PFN1 loss of function and discovered that mitophagy was significantly upregulated. Indeed, despite successful autophagosome formation, fusion with the lysosome, and activation of additional mitochondrial quality control pathways, PFN1 knockout cells accumulate depolarized, dysmorphic mitochondria with altered metabolic properties. Surprisingly, we also discovered that PFN1 is present inside mitochondria and provide evidence that mitochondrial defects associated with PFN1 loss are not caused by reduced actin polymerization in the cytosol. These findings suggest a previously unrecognized role for PFN1 in maintaining mitochondrial integrity and highlight new pathogenic mechanisms that can result from PFN1 dysregulation. Synopsis: The actin monomer binding protein profilin 1 (PFN1) localizes inside mitochondria and has important roles in maintaining mitochondrial homeostasis. Loss of PFN1 increases mitophagy and the production of mitochondrial-derived vesicles. Depolarized, dysmorphic mitochondria with altered metabolic properties accumulate in PFN1 knockout cells. PFN1 knockout mitochondria phenotypes are not caused by a loss of cytoplasmic actin assembly. PFN1 localizes inside mitochondria. The actin monomer binding protein profilin 1 (PFN1) localizes inside mitochondria and has important roles in maintaining mitochondrial homeostasis. [ABSTRACT FROM AUTHOR]

Cell migration through complex 3D environments relies on the interplay between actin and microtubules. A new study shows that, when cells pass through narrow constrictions, CLASP-dependent microtubule stabilisation at the cell rear controls actomyosin contractility to enable nuclear translocation and preserve cell integrity. [ABSTRACT FROM AUTHOR]

Force generation and motility by actomyosin in nonmuscle cells are spatially regulated by (40 tropomyosin (Tpm) isoforms. The means by which Tpms are targeted to specific cellular regions and the mechanisms that result in differential activity of myosin paralogs are unknown. We show that Tpm3.1 and Tpm1.7 inhibit Myosin-IC (Myo1C), with Tpm1.7 more effectively reducing the number of gliding filaments than Tpm3.1. Strikingly, cosedimentation and fluorescence microscopy assays revealed that Tpm3.1 is displaced from actin by Myo1C and not by myosin-II. In contrast, Tpm1.7 is only weakly displaced by Myo1C. Unlike other characterized myosins, Myo1C motility is inhibited by Tpm when the Tpm-actin filament is activated by myosin-II. These results point to a mechanism for the exclusion of myosin-I paralogs from cellular Tpm-decorated actin filaments that are activated by other myosins. Additionally, our results suggest a potential mechanism for myosin-induced Tpm sorting in cells. [ABSTRACT FROM AUTHOR]

Actin is present in the cytoplasm and nucleus of every eukaryotic cell. In the cytoplasm, framework and motor functions of actin are associated with its ability to polymerize to form F-actin. In the nucleus, globular actin plays a significant functional role. For a globular protein, actin has a uniquely large number of proteins with which it interacts. Bioinformatics analysis of the actin interactome showed that only a part of actin-binding proteins are both cytoplasmic and nuclear. There are proteins that interact only with cytoplasmic, or only with nuclear actin. The first pool includes proteins associated with the formation, regulation, and functioning of the actin cytoskeleton predominate, while nuclear actin-binding proteins are involved in the majority of key nuclear processes, from regulation of transcription to DNA damage response. Bioinformatics analysis of the structure of actin-binding proteins showed that these are mainly intrinsically disordered proteins, many of which are part of membrane-less organelles. Interestingly, although the number of intrinsically disordered actin-binding proteins in the nucleus is greater than in the cytoplasm, the drivers for the formation of the membrane-less organelles in the cytoplasm are significantly (four times) greater than in the nucleus. [ABSTRACT FROM AUTHOR]

Eukaryotic cells have been evolving for billions of years, giving rise to wildly diverse cell forms and functions. Despite their variability, all eukaryotic cells share key hallmarks, including membrane-bound organelles, heavily regulated cytoskeletal networks and complex signaling cascades. Because the actin cytoskeleton interfaces with each of these features, understanding how it evolved and diversified across eukaryotic phyla is essential to understanding the evolution and diversification of eukaryotic cells themselves. Here, we discuss what we know about the origin and diversity of actin networks in terms of their compositions, structures and regulation, and how actin evolution contributes to the diversity of eukaryotic form and function. [ABSTRACT FROM AUTHOR]

The behavior and presence of actin-regulating proteins are characteristic of various clinical diseases. Changes in these proteins significantly impact the cytoskeletal and regenerative processes underlying pathological changes. Pituitary adenylate cyclase-activating polypeptide (PACAP), a cytoprotective neuropeptide abundant in the nervous system and endocrine organs, plays a key role in neuron differentiation and migration by influencing actin. This study aims to elucidate the role of PACAP as an actin-regulating polypeptide, its effect on actin filament formation, and the underlying regulatory mechanisms. We examined PACAP27, PACAP38, and PACAP6-38, measuring their binding to actin monomers via fluorescence spectroscopy and steady-state anisotropy. Functional polymerization tests were used to track changes in fluorescent intensity over time. Unlike PACAP27, PACAP38 and PACAP6-38 significantly reduced the fluorescence emission of Alexa488-labeled actin monomers and increased their anisotropy, showing nearly identical dissociation equilibrium constants. PACAP27 showed weak binding to globular actin (G-actin), while PACAP38 and PACAP6-38 exhibited robust interactions. PACAP27 did not affect actin polymerization, but PACAP38 and PACAP6-38 accelerated actin incorporation kinetics. Fluorescence quenching experiments confirmed structural changes upon PACAP binding; however, all studied PACAP fragments exhibited the same effect. Our findings indicate that PACAP38 and PACAP6-38 strongly bind to G-actin and significantly influence actin polymerization. Further studies are needed to fully understand the biological significance of these interactions. [ABSTRACT FROM AUTHOR]

Cells are capable of sensing and responding to the extracellular mechanical microenvironment via the actin skeleton. In vivo, tissues are frequently subject to mechanical forces, such as the rapid and significant shear flow encountered by vascular endothelial cells. However, the investigations about the transient response of intracellular actin networks under these intense external mechanical forces, their intrinsic mechanisms, and potential implications are very limited. Here, we observe that when cells are subject to the shear flow, an actin ring structure could be rapidly assembled at the periphery of the nucleus. To gain insights into the mechanism underlying this perinuclear actin ring assembly, we develop a computational model of actin dynamics. We demonstrate that this perinuclear actin ring assembly is triggered by the depolymerization of cortical actin, Arp2/3-dependent actin filament polymerization, and myosin-mediated actin network contraction. Furthermore, we discover that the compressive stress generated by the perinuclear actin ring could lead to a reduction in the nuclear spreading area, an increase in the nuclear height, and a decrease in the nuclear volume. The present model thus explains the mechanism of the perinuclear actin ring assembly under external mechanical forces and suggests that the spontaneous contraction of this actin structure can significantly impact nuclear morphology. [ABSTRACT FROM AUTHOR]

SUMMARY Being a bona fide actin bundler, Arabidopsis villin5 (VLN5) plays a crucial role in regulating actin stability and organization within pollen tubes. Despite its significance, the precise mechanism through which VLN5 bundles actin filaments has remained elusive. Through meticulous deletion analysis, we have unveiled that the link between gelsolin repeat 6 (G6) and the headpiece domain (VHP), rather than VHP itself, is indispensable for VLN5‐mediated actin bundling. Further refinement of this region has pinpointed a critical sequence spanning from Val763 to Ser823, essential for VLN5's actin‐bundling activity. Notably, the absence of Val763‐Ser823 in VLN5 results in decreased filamentous decoration within pollen tubes and a diminished ability to rescue actin bundling defects in vln2vln5 mutant pollen tubes compared to intact VLN5. Moreover, our findings highlight that the Val763‐Ser823 sequence harbors a binding site for F‐actin, suggesting that this linker‐based F‐actin binding site, in conjunction with the F‐actin binding site localized in G1–G6, enables a single VLN5 to concurrently bind to two adjacent actin filaments. Therefore, our study unveils a novel mechanism by which VLN5 bundles actin filaments. [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]

Cells exist in different phenotypes and can transition between them. A phenotype may be characterized by many different aspects. Here, we focus on the example of whether the cell is adhered or suspended and choose particular parameters related to the structure and mechanics of the actin cortex. The cortex is essential to cell mechanics, morphology, and function, such as for adhesion, migration, and division of animal cells. To predict and control cellular functions and prevent malfunctioning, it is necessary to understand the actin cortex. The structure of the cortex governs cell mechanics; however, the relationship between the architecture and mechanics of the cortex is not yet well enough understood to be able to predict one from the other. Therefore, we quantitatively measured structural and mechanical cortex parameters, including cortical thickness, cortex mesh size, actin bundling, and cortex stiffness. These measurements required developing a combination of measurement techniques in scanning electron, expansion, confocal, and atomic force microscopy. We found that the structure and mechanics of the cortex of cells in interphase are different depending on whether the cell is suspended or adhered. We deduced general correlations between structural and mechanical properties and show how these findings can be explained within the framework of semiflexible polymer network theory. We tested the model predictions by perturbing the properties of the actin within the cortex using compounds. Our work provides an important step toward predictions of cell mechanics from cortical structures and suggests how cortex remodeling between different phenotypes impacts the mechanical properties of cells. [ABSTRACT FROM AUTHOR]

Background: Cellular retinoic acid binding protein 1 (CRABP1) mediates rapid, non-canonical activity of retinoic acid (RA) by forming signalosomes via protein-protein interactions. Two signalosomes have been identified previously: CRABP1-MAPK and CRABP1-CaMKII. Crabp1 knockout (CKO) mice exhibited altered exosome profiles, but the mechanism of CRABP1 action was unclear. This study aimed to screen for and identify novel CRABP1 signalosomes that could modulate exosome secretion by using a combinatorial approach involving biochemical, bioinformatic and molecular studies. Methods: Immunoprecipitation coupled with mass spectrometry (IP-MS) identified candidate CRABP1-interacting proteins which were subsequently analyzed using GO Term Enrichment, Functional Annotation Clustering; and Pathway Analysis. Gene expression analysis of CKO samples revealed altered expression of genes related to exosome biogenesis and secretion. The effect of CRABP1 on exosome secretion was then experimentally validated using CKO mice and a Crabp1 knockdown P19 cell line. Results: IP-MS identified CRABP1-interacting targets. Bioinformatic analyses revealed significant association with actin cytoskeletal dynamics, kinases, and exosome secretion. The effect of CRABP1 on exosome secretion was experimentally validated by comparing circulating exosome numbers of CKO and wild type (WT) mice, and secreted exosomes from WT and siCRABP1-P19 cells. Pathway analysis identified kinase signaling and Arp2/3 complex as the major pathways where CRABP1-signalosomes modulate exosome secretion, which was validated in the P19 system. Conclusion: The combinatorial approach allowed efficient screening for and identification of novel CRABP1-signalosomes. The results uncovered a novel function of CRABP1 in modulating exosome secretion, and suggested that CRABP1 could play roles in modulating intercellular communication and signal propagation. [ABSTRACT FROM AUTHOR]

The molecular mechanisms driving the onset and metastasis of prostate cancer remain poorly understood. Actin, under the control of actin-binding proteins (ABPs), plays a crucial role in shaping the cellular cytoskeleton, which in turn supports the morphological alterations in normal cells, as well as the invasive spread of tumor cells. Previous research indicates that ABPs of various types serve distinct functions, and any disruptions in their activities could predispose individuals to prostate cancer. These ABPs are intricately implicated in the initiation and advancement of prostate cancer through a complex array of intracellular processes, such as severing, linking, nucleating, inducing branching, assembling, facilitating actin filament elongation, terminating elongation, and promoting actin molecule aggregation. As such, this review synthesizes existing literature on several ABPs linked to prostate cancer, including cofilin, filamin A, and fascin, with the aim of shedding light on the molecular mechanisms through which ABPs influence prostate cancer development and identifying potential therapeutic targets. Ultimately, this comprehensive examination seeks to contribute to the understanding and management of prostate diseases. [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]

Cofilin, an actin-severing protein, plays key roles in muscle sarcomere addition and maintenance. Our previous work found that Drosophila cofilin (DmCFL) knockdown in muscle causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, conducted an RNA-sequencing analysis that unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL muscle knockdown causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown resulted in mislocalization of GluRIIA class glutamate receptors in more deteriorated muscles and strongly impaired NMJ transmission strength. These findings expand our understanding of the roles of cofilin in muscle to include NMJ structural development and suggest that NMJ defects may contribute to the pathophysiology of nemaline myopathy. [ABSTRACT FROM AUTHOR]

Wounds that occur in adults form scars due to fibrosis, whereas those in embryos regenerate. If wound healing in embryos is mimicked in adults, scarring can be reduced. We found that mouse fetuses could regenerate tissues up to embryonic day (E) 13, but visible scars remained thereafter. This regeneration pattern requires actin cable formation at the epithelial wound margin via activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK). Here, we investigated whether the AMPK-activating effect of salicylate, an anti-inflammatory drug, promotes regenerative wound healing. Salicylate administration resulted in actin cable formation and complete wound regeneration in E14 fetuses, in which scarring should have normally occurred, and promoted contraction of the panniculus carnosus muscle, resulting in complete wound regeneration. In vitro, salicylate further induced actin remodeling in mouse epidermal keratinocytes in a manner dependent on cell and substrate target-specific AMPK activation and subsequent regulation of Rac1 signaling. Furthermore, salicylate promoted epithelialization, enhanced panniculus carnosus muscle contraction, and inhibited scar formation in adult mice. Administration of salicylates to wounds immediately after injury may be a novel method for preventing scarring by promoting a wound healing pattern similar to that of embryonic wounds. [ABSTRACT FROM AUTHOR]

Precise segregation of chromosomes during mitosis requires assembly of a bipolar mitotic spindle followed by correct attachment of microtubules to the kinetochores. This highly spatiotemporally organized process is controlled by various mitotic kinases and molecular motors. We have recently shown that Casein Kinase 1 (CK1) promotes timely progression through mitosis by phosphorylating FAM110A leading to its enrichment at spindle poles. However, the mechanism by which FAM110A exerts its function in mitosis is unknown. Using structure prediction and a set of deletion mutants, we mapped here the interaction of the N-and C-terminal domains of FAM110A with actin and tubulin, respectively. Next, we found that the FAM110A-Δ40-61 mutant deficient in actin binding failed to rescue defects in chromosomal alignment caused by depletion of endogenous FAM110A. Depletion of FAM110A impaired assembly of F-actin in the proximity of spindle poles and was rescued by expression of the wild-type FAM110A, but not the FAM110A-Δ40-61 mutant. Purified FAM110A promoted binding of F-actin to microtubules as well as bundling of actin filaments in vitro. Finally, we found that the inhibition of CK1 impaired spindle actin formation and delayed progression through mitosis. We propose that CK1 and FAM110A promote timely progression through mitosis by mediating the interaction between spindle microtubules and filamentous actin to ensure proper mitotic spindle formation. [ABSTRACT FROM AUTHOR]

Arf6 is a member of ADP‐ribosylation factor (Arf) family, which is widely implicated in the regulation of multiple physiological processes including endocytic recycling, cytoskeletal organization, and membrane trafficking during mitosis. In this study, we investigated the potential relationship between Arf6 and aging‐related oocyte quality, and its roles on organelle rearrangement and cytoskeleton dynamics in porcine oocytes. Arf6 expressed in porcine oocytes throughout meiotic maturation, and it decreased in aged oocytes. Disruption of Arf6 led to the failure of cumulus expansion and polar body extrusion. Further analysis indicated that Arf6 modulated ac‐tubulin for meiotic spindle organization and microtubule stability. Besides, Arf6 regulated cofilin phosphorylation and fascin for actin assembly, which further affected spindle migration, indicating the roles of Arf6 on cytoskeleton dynamics. Moreover, the lack of Arf6 activity caused the dysfunction of Golgi and ER for protein synthesis and signal transduction. Mitochondrial dysfunction was also observed in Arf6‐deficient porcine oocytes, which was supported by the increased ROS level and abnormal membrane potential. In conclusion, our results reported that insufficient Arf6 was related to aging‐induced oocyte quality decline through spindle organization, actin assembly, and organelle rearrangement in porcine oocytes. [ABSTRACT FROM AUTHOR]

Crosstalk between the actin and microtubule cytoskeletons is important for many cellular processes. Recent studies have shown that microtubules and F-actin can assemble to form a composite structure where F-actin occupies the microtubule lumen. Whether these cytoskeletal hybrids exist in physiological settings and how they are formed is unclear. Here, we show that the short-crossover Class I actin filament previously identified inside microtubules in human HAP1 cells is cofilin-bound F-actin. Lumenal F-actin can be reconstituted in vitro, but cofilin is not essential. Moreover, actin filaments with both cofilin-bound and canonical morphologies reside within human platelet microtubules under physiological conditions. We propose that stress placed upon the microtubule network during motor-driven microtubule looping and sliding may facilitate the incorporation of actin into microtubules. Using focused ion beam milling and cryoelectron tomography, Tsuji et al. found actin filaments, with both cofilin-bound and canonical morphologies, within the lumen of human platelet microtubules and reconstituted these structures in vitro. [ABSTRACT FROM AUTHOR]

Within a shared cytoplasm, filamentous actin (F-actin) plays numerous and critical roles across the cell body. Cells rely on actin-binding proteins (ABPs) to organize F-actin and to integrate its polymeric characteristics into diverse cellular processes. Yet, the multitude of ABPs that engage with and shape F-actin make studying a single ABP's influence on cellular activities a significant challenge. Moreover, without a means of manipulating actin-binding subcellularly, harnessing the F-actin cytoskeleton for synthetic biology purposes remains elusive. Here, we describe a suite of designed proteins, Controllable Actin-binding Switch Tools (CASTs), whose actin-binding behavior can be controlled with external stimuli. CASTs were developed that respond to different external inputs, providing options for turn-on kinetics and enabling orthogonality and multiplexing. Being genetically encoded, we show that CASTs can be inserted into native protein sequences to control F-actin association locally and engineered into structures to control cell and tissue shape and behavior. Numerous proteins interact with the actin cytoskeleton, convoluting their influence on cellular behaviour. Here the authors engineer actin-binding switch tools that can be activated with external stimuli to control the activity of just one protein. [ABSTRACT FROM AUTHOR]

An important question in cell biology is how cytoskeletal proteins evolved and drove the development of novel structures and functions. Here we address the origin of SPIRE actin nucleators. Mammalian SPIREs work with RAB GTPases, formin (FMN)-subgroup actin assembly proteins and class-5 myosin (MYO5) motors to transport organelles along actin filaments towards the cell membrane. However, the origin and extent of functional conservation of SPIRE among species is unknown. Our sequence searches show that SPIRE exist throughout holozoans (animals and their closest single-celled relatives), but not other eukaryotes. SPIRE from unicellular holozoans (choanoflagellate), interacts with RAB, FMN and MYO5 proteins, nucleates actin filaments and complements mammalian SPIRE function in organelle transport. Meanwhile SPIRE and MYO5 proteins colocalise to organelles in Salpingoeca rosetta choanoflagellates. Based on these observations we propose that SPIRE originated in unicellular ancestors of animals providing an actin-myosin driven exocytic transport mechanism that may have contributed to the evolution of complex multicellular animals. Genome analysis and molecular cell biology studies indicate that SPIRE actin nucleators originated in unicellular ancestors of animals, providing an actin-myosin driven exocytic transport mechanism that may have contributed to animal evolution. [ABSTRACT FROM AUTHOR]

Plakophilin 4 (PKP4) is a component of cell–cell junctions that regulates intercellular adhesion and Rho-signaling during cytokinesis with an unknown function during epidermal differentiation. Here we show that keratinocytes lacking PKP4 fail to develop a cortical actin ring, preventing adherens junction maturation and generation of tissue tension. Instead, PKP4-depleted cells display increased stress fibers. PKP4-dependent RhoA localization at AJs was required to activate a RhoA-ROCK2-MLCK-MLC2 axis and organize actin into a cortical ring. AJ-associated PKP4 provided a scaffold for the Rho activator ARHGEF2 and the RhoA effectors MLCK and MLC2, facilitating the spatio-temporal activation of RhoA signaling at cell junctions to allow cortical ring formation and actomyosin contraction. In contrast, association of PKP4 with the Rho suppressor ARHGAP23 reduced ARHGAP23 binding to RhoA which prevented RhoA activation in the cytoplasm and stress fiber formation. These data identify PKP4 as an AJ component that transduces mechanical signals into cytoskeletal organization. [ABSTRACT FROM AUTHOR]

Flow or collective movement is a frequently observed phenomenon for many cellular components including the cytoskeletal proteins actin and myosin. To study protein flow in living cells, we and others have previously used spatiotemporal image correlation spectroscopy (STICS) analysis on fluorescence microscopy image time series. Yet, in cells, multiple protein flows often occur simultaneously on different scales resulting in superimposed fluorescence intensity fluctuations that are challenging to separate using STICS. Here, we exploited the characteristic that distinct protein flows often occur at different spatial scales present in the image series to disentangle superimposed protein flow dynamics. We employed a newly developed and an established spatial filtering algorithm to alternatively accentuate or attenuate local image intensity heterogeneity across different spatial scales. Subsequently, we analysed the spatially filtered time series with STICS, allowing the quantification of two distinct superimposed flows within the image time series. As a proof of principle of our analysis approach, we used simulated fluorescence intensity fluctuations as well as time series of nonmuscle myosin II in endothelial cells and actin‐based podosomes in dendritic cells and revealed simultaneously occurring contiguous and noncontiguous flow dynamics in each of these systems. Altogether, this work extends the application of STICS for the quantification of multiple protein flow dynamics in complex biological systems including the actomyosin cytoskeleton. [ABSTRACT FROM AUTHOR]

Sponges (phylum Porifera) were among the first metazoans on Earth, and represent a unique global source of highly structured and diverse biosilica that has been formed and tested over more than 800 million years of evolution. Poriferans are recognized as a unique archive of siliceous multiscaled skeletal constructs with superficial micro-ornamentation patterned by biopolymers. In the present study, spicules and skeletal frameworks of selected representatives of sponges in such classes as Demospongiae, Homoscleromorpha, and Hexactinellida were desilicified using 10% HF with the aim of isolating axial filaments, which resemble the shape and size of the original structures. These filaments were unambiguously identified in all specimens under study as F-actin, using the highly specific indicators iFluor™ 594-Phalloidin, iFluor™ 488-Phalloidin, and iFluor™ 350-Phalloidin. The identification of this kind of F-actins, termed for the first time as silactins, as specific pattern drivers in skeletal constructs of sponges opens the way to the fundamental understanding of their skeletogenesis. Examples illustrating the biomimetic potential of sophisticated poriferan biosilica patterned by silactins are presented and discussed. [ABSTRACT FROM AUTHOR]

Cytokinesis, the last step in cell division, separates daughter cells through mechanical force. This is often through the force produced by an actomyosin contractile ring. In fission yeast cells, the ring helps recruit a mechanosensitive ion channel, Pkd2, to the cleavage furrow, whose activation by membrane tension promotes calcium influx and daughter cell separation. However, it is unclear how the activities of Pkd2 may affect the actomyosin ring. Here, through both microscopic and genetic analyses of a hypomorphic pkd2 mutant, we examined the potential role of this essential gene in assembling the contractile ring. The pkd2-81KD mutation significantly increased the counts of the type II myosin heavy chain Myo2 (+18%), its regulatory light chain Rlc1 (+37%) and actin (+100%) molecules in the ring, compared to the wild type. Consistent with a regulatory role of Pkd2 in the ring assembly, we identified a strong negative genetic interaction between pkd2-81KD and the temperature-sensitive mutant myo2-E1. The pkd2-81KD myo2-E1 cells often failed to assemble a complete contractile ring. We conclude that Pkd2 modulates the recruitment of type II myosin and actin to the contractile ring, suggesting a novel calcium-dependent mechanism regulating the actin cytoskeletal structures during cytokinesis. [ABSTRACT FROM AUTHOR]

Septins, a family of GTP-binding proteins that assemble into higher order structures, interface with the membrane, actin filaments and microtubules, and are thus important regulators of cytoarchitecture. Septin 9 (SEPT9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short microtubule-associated protein (MAP)-like motif unique to SEPT9 isoform 1 (SEPT9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring SEPT9_i1. Although outside of the MAP-like motif, HNA mutations abrogate this association, identifying a putative regulatory domain. Removal of this domain from SEPT9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering themechanisms underlying septin-associated pathologies. [ABSTRACT FROM AUTHOR]

Neutrophil extracellular traps (NETs) have a dual role in the innate immune response to thermal injuries. NETs provide an early line of defence against infection. However, excessive NETosis can mediate the pathogenesis of immunothrombosis, disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) in sepsis. Recent studies suggest that high interleukin-8 (IL-8) levels in intensive care unit (ICU) patients significantly contribute to excessive NET generation. This study aimed to determine whether IL-8 also mediates NET generation in patients with severe thermal injuries. IL-8 levels were measured in serum samples from thermally injured patients with ≥15% of the total body surface area (TBSA) and healthy controls (HC). Ex vivo NET generation was also investigated by treating isolated neutrophils with serum from thermal injured patients or normal serum with and without IL-8 and anti-IL-8 antibodies. IL-8 levels were significantly increased compared to HC on days 3 and 5 (p < 0.05) following thermal injury. IL-8 levels were also significantly increased at day 5 in septic versus non-septic patients (p < 0.001). IL-8 levels were also increased in patients who developed sepsis compared to HC at days 3, 5 and 7 (p < 0.001), day 10 (p < 0.05) and days 12 and 14 (p < 0.01). Serum containing either low, medium or high levels of IL-8 was shown to induce ex vivo NETosis in an IL-8-dependent manner. Furthermore, the inhibition of DNase activity in serum increased the NET-inducing activity of IL-8 in vitro by preventing NET degradation. IL-8 is a major contributor to NET formation in severe thermal injury and is increased in patients who develop sepsis. We confirmed that DNase is an important regulator of NET degradation but also a potential confounder within assays that measure serum-induced ex vivo NETosis. [ABSTRACT FROM AUTHOR]

Although more difficult to detect than in the cytoplasm, it is now clear that actin polymerization occurs in the nucleus and that it plays a role in the specific processes of the nucleus such as transcription, replication, and DNA repair. A number of studies suggest that nuclear actin polymerization is promoting precise DNA repair by homologous recombination, which could potentially be of help for precise genome editing and gene therapy. This review summarizes the findings and describes the challenges and chances in the field. [ABSTRACT FROM AUTHOR]

Rabies virus (RABV) is highly lethal and triggers severe neurological symptoms. The neuropathogenic mechanism remains poorly understood. Ras-related C3 botulinum toxin substrate 1 (Rac1) is a Rho-GTPase that is involved in actin remodeling and has been reported to be closely associated with neuronal dysfunction. In this study, by means of a combination of pharmacological inhibitors, small interfering RNA, and specific dominant-negatives, we characterize the crucial roles of dynamic actin and the regulatory function of Rac1 in RABV infection, dominantly in the viral entry phase. The data show that the RABV phosphoprotein interacts with Rac1. RABV phosphoprotein suppress Rac1 activity and impedes downstream Pak1-Limk1-Cofilin1 signaling, leading to the disruption of F-actin-based structure formation. In early viral infection, the EGFR-Rac1-signaling pathway undergoes a biphasic change, which is first upregulated and subsequently downregulated, corresponding to the RABV entry-induced remodeling pattern of F-actin. Taken together, our findings demonstrate for the first time the role played by the Rac1 signaling pathway in RABV infection and may provide a clue for an explanation for the etiology of rabies neurological pathogenesis. IMPORTANCE Though neuronal dysfunction is predominant in fatal rabies, the detailed mechanism by which rabies virus (RABV) infection causes neurological symptoms remains in question. The actin cytoskeleton is involved in numerous viruses infection and plays a crucial role in maintaining neurological function. The cytoskeletal disruption is closely associated with abnormal nervous symptoms and induces neurogenic diseases. In this study, we show that RABV infection led to the rearrangement of the cytoskeleton as well as the biphasic kinetics of the Rac1 signal transduction. These results help elucidate the mechanism that causes the aberrant neuronal processes by RABV infection and may shed light on therapeutic development aimed at ameliorating neurological disorders. [ABSTRACT FROM AUTHOR]

Despite being considered a normal flora, Providencia alcalifaciens can cause diarrhea. In a previous study, strain 2939/90, obtained from a diarrheal patient, caused invasion and actin condensation in mammalian cells, and diarrhea in a rabbit model. Four TnphoA mutants of 2939/90 produced negligible invasion and actin condensation in mammalian cells. Now, the parent strain and the mutants have been sequenced to locate TnphoA insertion sites and determine the effect on virulence. A TnphoA insertion was detected in the type three secretion system (T3SS) locus on a large plasmid and not in a T3SS locus on the chromosome. In 52 genomes of P. alcalifaciens surveyed, the chromosomal T3SS locus was present in all strains, including both P. alcalifaciens genomic clades, which we classified as group A and group B. Plasmid T3SS was present in 21 of 52 genomes, mostly in group A genomes, which included isolates from an outbreak of hemorrhagic diarrhea in dogs. The TnphoA insertion only in the plasmid T3SS locus affected the invasion phenotype, suggested that this locus is critical for causation of diarrhea. We conclude that a subgroup of P. alcalifaciens that possesses this plasmid-mediated T3SS is an enteric pathogen that can cause diarrheal disease. [ABSTRACT FROM AUTHOR]

Formins are large, multidomain proteins that nucleate new actin filaments and accelerate elongation through a processive interaction with the barbed ends of filaments. Their actin assembly activity is generally attributed to their eponymous formin homology (FH) 1 and 2 domains; however, evidence is mounting that regions outside of the FH1FH2 stretch also tune actin assembly. Here, we explore the underlying contributions of the tail domain, which spans the sequence between the FH2 domain and the C terminus of formins. Tails vary in length from ∼0 to >200 residues and contain a number of recognizable motifs. The most common and well-studied motif is the ∼15-residue-long diaphanous autoregulatory domain. This domain mediates all or nothing regulation of actin assembly through an intramolecular interaction with the diaphanous inhibitory domain in the N-terminal half of the protein. Multiple reports demonstrate that the tail can enhance both nucleation and processivity. In this study, we provide a high-resolution view of the alternative splicing encompassing the tail in the formin homology domain (Fhod) family of formins during development. While four distinct tails are predicted, we found significant levels of only two of these. We characterized the biochemical effects of the different tails. Surprisingly, the two highly expressed Fhod-tails inhibit processive elongation and diminish nucleation, while a third supports activity. These findings demonstrate a new mechanism of modulating actin assembly by formins and support a model in which splice variants are specialized to build distinct actin structures during development.

One important ecological question regarding the use of plant resistance genes against fungal pathogens concerns whether and how such resistance genes may modify pathogenic or beneficial members of the plant-associated microbiota. We studied the impact of a plant resistance gene by analyzing the mycobiota associated with Brassica napus organs over two cropping seasons. Sampling dates coincided with key stages of the life cycle of the B. napus pathogen Leptosphaeria maculans. Leaf samples were collected at three time points in autumn and spring, and stem base samples were collected at two time points a few weeks before and at harvest. Stem residues, where L. maculans survives in the intercropping season and develops sexual reproduction, were also analyzed at four time points between the two cropping seasons. The sampling was performed on two plant genotypes, Darmor and Darmor-Rlm11, only differing by the effective resistance gene against L. maculans, Rlm11. Altogether, 419 samples were analyzed using two barcode: internal transcribed spacer (ITS) and Actin. The plant organ was shown to be the main mycobiota structuring factor, as clear-cut alternation of the species suggested that each plant organ represented a specific ecological niche. The cropping season and plant genotype also significantly influenced the community structure in lower proportions. The resistance gene contributed differently to the community structure depending on the year and the organ concerned. A significant but low impact of Rlm11 on other B. napus fungal pathogens was detected. The ITS and Actin barcodes showed similar results, but the species assignation was limited for the latter.

Wooden breast is one of the myopathies that affected broilers and caused economic loss, but the causes are still unknown. This experiment aimed to investigate the effect of a high percentage of soybean oil in inducing this abnormality. Thirty chicks were fed into basal ratio as a control group and basal ratio with soybean oil 5.20% as the treatment group for 35 days. The histopathological examination of the Pectoralis muscle of broiler rearing for 12 days was represented by abnormal myofiber architecture, edema, and increased endomysium formation at percentages 40% and