Hyaluronidase inhibits TGF-ß-mediated rat periodontal ligament fibroblast expression of collagen and myofibroblast markers: An in vitro exploration of periodontal tissue remodeling.

OBJECTIVE: To determine the effect of hyaluronic acid (HA) degradation by hyaluronidase (HYAL) in inhibiting collagen fiber production by rat periodontal ligament cells (rPDLCs). DESIGN: Primary rPDLCs were isolated from the euthanized rats and used for in vitro experiments. The appropriate HYAL concentration was determined through CCK-8 testing for cytotoxicity detection and Alizarin red staining for mineralization detection. RT-qPCR and western blot assays were conducted to assess the effect of HYAL, with or without TGF-ß, on generation of collagen fiber constituents and expression of actin alpha 2, smooth muscle (ACTA2) of rPDLCs. RESULTS: Neither cell proliferation nor mineralization were significantly affected by treatment with 4 U/mL HYAL. HYAL (4 U/mL) alone downregulated type I collagen fiber (Col1a1 and Col1a2) and Acta2 mRNA expression; however, ACTA2 and COL1 protein levels were only downregulated by HYAL treatment after TGF-ß induction. CONCLUSIONS: Treatment of rPDLCs with HYAL can inhibit TGF-ß-induced collagen matrix formation and myofibroblast transformation.

BIN1 regulates actin-membrane interactions during IRSp53-dependent filopodia formation.

Amphiphysin 2 (BIN1) is a membrane and actin remodeling protein mutated in congenital and adult centronuclear myopathies. Here, we report an unexpected function of this N-BAR domain protein BIN1 in filopodia formation. We demonstrated that BIN1 expression is necessary and sufficient to induce filopodia formation. BIN1 is present at the base of forming filopodia and all along filopodia, where it colocalizes with F-actin. We identify that BIN1-mediated filopodia formation requires IRSp53, which allows its localization at negatively-curved membrane topologies. Our results show that BIN1 bundles actin in vitro. Finally, we identify that BIN1 regulates the membrane-to-cortex architecture and functions as a molecular platform to recruit actin-binding proteins, dynamin and ezrin, to promote filopodia formation.

The concerted action of SEPT9 and EPLIN modulates the adhesion and migration of human fibroblasts.

Septins are cytoskeletal proteins that participate in cell adhesion, migration, and polarity establishment. The septin subunit SEPT9 directly interacts with the single LIM domain of epithelial protein lost in neoplasm (EPLIN), an actin-bundling protein. Using a human SEPT9 KO fibroblast cell line, we show that cell adhesion and migration are regulated by the interplay between both proteins. The low motility of SEPT9-depleted cells could be partly rescued by increased levels of EPLIN. The normal organization of actin-related filopodia and stress fibers was directly dependent on the expression level of SEPT9 and EPLIN. Increased levels of SEPT9 and EPLIN enhanced the size of focal adhesions in cell protrusions, correlating with stabilization of actin bundles. Conversely, decreased levels had the opposite effect. Our work thus establishes the interaction between SEPT9 and EPLIN as an important link between the septin and the actin cytoskeleton, influencing cell adhesion, motility, and migration.

CLIC4 Function in the Epithelial-Mesenchymal Transition of Epithelial Odontogenic Lesions.

BACKGROUND: Odontogenic lesions constitute a heterogeneous group of lesions. CLIC4 protein regulates different cellular processes, including epithelial-mesenchymal transition and fibroblast-myofibroblast transdifferentiation. This study analyzed CLIC4, E-cadherin, Vimentin, and α-SMA immunoexpression in epithelial odontogenic lesions that exhibit different biological behavior. METHODS: It analyzed the immunoexpression of CLIC4, E-cadherin, and Vimentin in the epithelial cells, as well as CLIC4 and α-SMA in the mesenchymal cells, of ameloblastoma (AM) (n = 16), odontogenic keratocyst (OKC) (n = 20), and adenomatoid odontogenic tumor (AOT) (n = 8). Immunoexpressions were categorized as score 0 (0% positive cells), 1 (< 25%), 2 (≥ 25% - < 50%), 3 (≥ 50% - < 75%), or 4 (≥ 75%). RESULTS: Cytoplasmic CLIC4 immunoexpression was higher in AM and AOT (p < 0.001) epithelial cells. Nuclear-cytoplasmic CLIC4 was higher in OKC's epithelial lining (p < 0.001). Membrane (p = 0.012) and membrane-cytoplasmic (p < 0.001) E-cadherin immunoexpression were higher in OKC, while cytoplasmic E-cadherin expression was higher in AM and AOT (p < 0.001). Vimentin immunoexpression was higher in AM and AOT (p < 0.001). Stromal CLIC4 was higher in AM and OKC (p = 0.008). Similarly, α-SMA immunoexpression was higher in AM and OKC (p = 0.037). Correlations in these proteins' immunoexpression were observed in AM and OKC (p < 0.05). CONCLUSIONS: CLIC4 seems to regulate the epithelial-mesenchymal transition, modifying E-cadherin and Vimentin expression. In mesenchymal cells, CLIC4 may play a role in fibroblast-myofibroblast transdifferentiation. CLIC4 may be associated with epithelial odontogenic lesions with aggressive biological behavior.

NLRP4E regulates actin cap formation through SRC and CDC42 during oocyte meiosis.

BACKGROUND: Members of the nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing (NLRP) family regulate various physiological and pathological processes. However, none have been shown to regulate actin cap formation or spindle translocation during the asymmetric division of oocyte meiosis I. NLRP4E has been reported as a candidate protein in female fertility, but its function is unknown. METHODS: Immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were employed to examine the localization and expression levels of NLRP4E and related proteins in mouse oocytes. small interfering RNA (siRNA) and antibody transfection were used to knock down NLRP4E and other proteins. Immunoprecipitation (IP)-mass spectrometry was used to identify the potential proteins interacting with NLRP4E. Coimmunoprecipitation (Co-IP) was used to verify the protein interactions. Wild type (WT) or mutant NLRP4E messenger RNA (mRNA) was injected into oocytes for rescue experiments. In vitro phosphorylation was employed to examine the activation of steroid receptor coactivator (SRC) by NLRP4E. RESULTS: NLRP4E was more predominant within oocytes compared with other NLRP4 members. NLRP4E knockdown significantly inhibited actin cap formation and spindle translocation toward the cap region, resulting in the failure of polar body extrusion at the end of meiosis I. Mechanistically, GRIN1, and GANO1 activated NLRP4E by phosphorylation at Ser429 and Thr430; p-NLRP4E is translocated and is accumulated in the actin cap region during spindle translocation. Next, we found that p-NLRP4E directly phosphorylated SRC at Tyr418, while p-SRC negatively regulated p-CDC42-S71, an inactive form of CDC42 that promotes actin cap formation and spindle translocation in the GTP-bound form. CONCLUSIONS: NLRP4E activated by GRIN1 and GANO1 regulates actin cap formation and spindle translocation toward the cap region through upregulation of p-SRC-Tyr418 and downregulation of p-CDC42-S71 during meiosis I.

The actin-binding protein CAP1 represses MRTF-SRF-dependent gene expression in mouse cerebral cortex.

Serum response factor (SRF) is an essential transcription factor for brain development and function. Here, we explored how an SRF cofactor, the actin monomer-sensing myocardin-related transcription factor MRTF, is regulated in mouse cortical neurons. We found that MRTF-dependent SRF activity in vitro and in vivo was repressed by cyclase-associated protein CAP1. Inactivation of the actin-binding protein CAP1 reduced the amount of actin monomers in the cytoplasm, which promoted nuclear MRTF translocation and MRTF-SRF activation. This function was independent of cofilin1 and actin-depolymerizing factor, and CAP1 loss of function in cortical neurons was not compensated by endogenous CAP2. Transcriptomic and proteomic analyses of cerebral cortex lysates from wild-type and Cap1 knockout mice supported the role of CAP1 in repressing MRTF-SRF-dependent signaling in vivo. Bioinformatic analysis identified likely MRTF-SRF target genes, which aligned with the transcriptomic and proteomic results. Together with our previous studies that implicated CAP1 in axonal growth cone function as well as the morphology and plasticity of excitatory synapses, our findings establish CAP1 as a crucial actin regulator in the brain relevant for formation of neuronal networks.

An interphase actin wave promotes mitochondrial content mixing and organelle homeostasis.

Across the cell cycle, mitochondrial dynamics are regulated by a cycling wave of actin polymerization/depolymerization. In metaphase, this wave induces actin comet tails on mitochondria that propel these organelles to drive spatial mixing, resulting in their equitable inheritance by daughter cells. In contrast, during interphase the cycling actin wave promotes localized mitochondrial fission. Here, we identify the F-actin nucleator/elongator FMNL1 as a positive regulator of the wave. FMNL1-depleted cells exhibit decreased mitochondrial polarization, decreased mitochondrial oxygen consumption, and increased production of reactive oxygen species. Accompanying these changes is a loss of hetero-fusion of wave-fragmented mitochondria. Thus, we propose that the interphase actin wave maintains mitochondrial homeostasis by promoting mitochondrial content mixing. Finally, we investigate the mechanistic basis for the observation that the wave drives mitochondrial motility in metaphase but mitochondrial fission in interphase. Our data indicate that when the force of actin polymerization is resisted by mitochondrial tethering to microtubules, as in interphase, fission results.

FMNL2 regulates actin for endoplasmic reticulum and mitochondria distribution in oocyte meiosis.

During mammalian oocyte meiosis, spindle migration and asymmetric cytokinesis are unique steps for the successful polar body extrusion. The asymmetry defects of oocytes will lead to the failure of fertilization and embryo implantation. In present study, we reported that an actin nucleating factor Formin-like 2 (FMNL2) played critical roles in the regulation of spindle migration and organelle distribution in mouse and porcine oocytes. Our results showed that FMNL2 mainly localized at the oocyte cortex and periphery of spindle. Depletion of FMNL2 led to the failure of polar body extrusion and large polar bodies in oocytes. Live-cell imaging revealed that the spindle failed to migrate to the oocyte cortex, which caused polar body formation defects, and this might be due to the decreased polymerization of cytoplasmic actin by FMNL2 depletion in the oocytes of both mice and pigs. Furthermore, mass spectrometry analysis indicated that FMNL2 was associated with mitochondria and endoplasmic reticulum (ER)-related proteins, and FMNL2 depletion disrupted the function and distribution of mitochondria and ER, showing with decreased mitochondrial membrane potential and the occurrence of ER stress. Microinjecting Fmnl2-EGFP mRNA into FMNL2-depleted oocytes significantly rescued these defects. Thus, our results indicate that FMNL2 is essential for the actin assembly, which further involves into meiotic spindle migration and ER/mitochondria functions in mammalian oocytes.

A step-by-step guide to fragmenting bundled actin filaments.

There has long been conflicting evidence as to how bundled actin filaments, found in cellular structures such as filopodia, are disassembled. In this issue, Chikireddy et al. (https://doi.org/10.1083/jcb.202312106) provide a detailed in vitro analysis of the steps involved in fragmentation of fascin-bundled actin filaments and propose a novel mechanism for severing two-filament bundles.

Assessment of the association of myofibroblasts and structural components of the extracellular matrix with histopathological parameters of actinic cheilitis and lower lip squamous cell carcinoma.

BACKGROUND: To evaluate the presence of myofibroblasts (MFs) in the development of lip carcinogenesis, through the correlation of clinical, histomorphometric and immunohistochemical parameters, in actinic cheilitis (ACs) and lower lip squamous cell carcinomas (LLSCCs). METHODS: Samples of ACs, LLSCCs, and control group (CG) were prepared by tissue microarray (TMA) for immunohistochemical TGF-ß, α-SMA, and Ki-67 and histochemical hematoxylin and eosin, picrosirius red, and verhoeff van gieson reactions. Clinical and microscopic data were associated using the Mann-Whitney, Kruskal-Wallis/Dunn, and Spearman correlation tests (SPSS, p < 0.05). RESULTS: ACs showed higher number of α-SMA+ MFs when compared to CG (p = 0.034), and these cells were associated with the vertical expansion of solar elastosis (SE) itself (p = 0.027). Areas of SE had lower deposits of collagen (p < 0.001), immunostaining for TGF-ß (p < 0.001), and higher density of elastic fibers (p < 0.05) when compared to areas without SE. A positive correlation was observed between high-risk epithelial dysplasia (ED) and the proximity of SE to the dysplastic epithelium (p = 0.027). LLSCCs showed a higher number of α-SMA+ MFs about CG (p = 0.034), as well as a reduction in the deposition of total collagen (p = 0.009) in relation to ACs and CG. There was also a negative correlation between the amount of α-SMA+ cells and the accumulation of total collagen (p = 0.041). Collagen and elastic density loss was higher in larger tumors (p = 0.045) with nodal invasion (p = 0.047). CONCLUSIONS: Our findings show the possible role of MFs, collagen fibers, and elastosis areas in the lip carcinogenesis process.

The amoeboid migration of monocytes in confining channels requires the local remodeling of the cortical actin cytoskeleton by cofilin-1.

Within the bloodstream, monocytes must traverse the microvasculature to prevent leukostasis, which is the entrapment of monocytes within the confines of the microvasculature. Using the model cell line, THP-1, and VCAM-1 coated channels to simulate the microvasculature surface, we demonstrate that monocytes predominantly adopt an amoeboid phenotype, which is characterized by the formation of blebs. As opposed to cortical actin flow in leader blebs, cell movement is correlated with myosin contraction at the cell rear. It was previously documented that cofilin-1 promotes cortical actin turnover at leader bleb necks in melanoma cells. In monocytes, our data suggest that cofilin-1 promotes the local upregulation of myosin contractility through actin cytoskeleton remodeling. In support of this concept, cofilin-1 is found to localize to a single cell edge. Moreover, the widespread upregulation of myosin contractility was found to inhibit migration. Thus, monocytes within the microvasculature may avoid entrapment by adopting an amoeboid mode of migration.

Prolonged depletion of profilin 1 or F-actin causes an adaptive response in microtubules.

In addition to its well-established role in actin assembly, profilin 1 (PFN1) has been shown to bind to tubulin and alter microtubule growth. However, whether PFN1's predominant control over microtubules in cells occurs through direct regulation of tubulin or indirectly through the polymerization of actin has yet to be determined. Here, we manipulated PFN1 expression, actin filament assembly, and actomyosin contractility and showed that reducing any of these parameters for extended periods of time caused an adaptive response in the microtubule cytoskeleton, with the effect being significantly more pronounced in neuronal processes. All the observed changes to microtubules were reversible if actomyosin was restored, arguing that PFN1's regulation of microtubules occurs principally through actin. Moreover, the cytoskeletal modifications resulting from PFN1 depletion in neuronal processes affected microtubule-based transport and mimicked phenotypes that are linked to neurodegenerative disease. This demonstrates how defects in actin can cause compensatory responses in other cytoskeleton components, which in turn significantly alter cellular function.

Dynamic microvilli sculpt bristles at nanometric scale.

Organisms generate shapes across size scales. Whereas patterning and morphogenesis of macroscopic tissues has been extensively studied, the principles underlying the formation of micrometric and submicrometric structures remain largely enigmatic. Individual cells of polychaete annelids, so-called chaetoblasts, are associated with the generation of chitinous bristles of highly stereotypic geometry. Here we show that bristle formation requires a chitin-producing enzyme specifically expressed in the chaetoblasts. Chaetoblasts exhibit dynamic cell surfaces with stereotypical patterns of actin-rich microvilli. These microvilli can be matched with internal and external structures of bristles reconstructed from serial block-face electron micrographs. Individual chitin teeth are deposited by microvilli in an extension-disassembly cycle resembling a biological 3D printer. Consistently, pharmacological interference with actin dynamics leads to defects in tooth formation. Our study reveals that both material and shape of bristles are encoded by the same cell, and that microvilli play a role in micro- to submicrometric sculpting of biomaterials.

Tail length and E525K dilated cardiomyopathy mutant alter human ß-cardiac myosin super-relaxed state.

Dilated cardiomyopathy (DCM) is a condition characterized by impaired cardiac function, due to myocardial hypo-contractility, and is associated with point mutations in ß-cardiac myosin, the molecular motor that powers cardiac contraction. Myocardial function can be modulated through sequestration of myosin motors into an auto-inhibited "super-relaxed" state (SRX), which may be further stabilized by a structural state known as the "interacting heads motif" (IHM). Here, we sought to determine whether hypo-contractility of DCM myocardium results from reduced function of individual myosin molecules or from decreased myosin availability to interact with actin due to increased IHM/SRX stabilization. We used an established DCM myosin mutation, E525K, and characterized the biochemical and mechanical activity of wild-type and mutant human ß-cardiac myosin constructs that differed in the length of their coiled-coil tail, which dictates their ability to form the IHM/SRX state. We found that short-tailed myosin constructs exhibited low IHM/SRX content, elevated actin-activated ATPase activity, and fast velocities in unloaded motility assays. Conversely, longer-tailed constructs exhibited higher IHM/SRX content and reduced actomyosin ATPase and velocity. Our modeling suggests that reduced velocities may be attributed to IHM/SRX-dependent sequestration of myosin heads. Interestingly, longer-tailed E525K mutants showed no apparent impact on velocity or actomyosin ATPase at low ionic strength but stabilized IHM/SRX state at higher ionic strength. Therefore, the hypo-contractility observed in DCM may be attributable to reduced myosin head availability caused by enhanced IHM/SRX stability in E525K mutants.

A Magnetic Pincher for the Dynamic Measurement of the Actin Cortex Thickness in Live Cells.

The actin cortex is an essential element of the cytoskeleton allowing cells to control and modify their shape. It is involved in cell division and migration. However, probing precisely the physical properties of the actin cortex has proved to be challenging: it is a thin and dynamic material, and its location in the cell-directly under the plasma membrane-makes it difficult to study with standard light microscopy and cell mechanics techniques. In this chapter, we present a novel protocol to probe dynamically the thickness of the cortex and its fluctuations using superparamagnetic microbeads in a uniform magnetic field. A bead ingested by the cell and another outside the cell attract each other due to dipolar forces. By tracking their position with nanometer precision, one can measure the thickness of the cortex pinched between two beads and monitor its evolution in time. We first present the set of elements necessary to realize this protocol: a magnetic field generator adapted to a specific imaging setup and the aforementioned superparamagnetic microbeads. Then we detail the different steps of a protocol that can be used on diverse cell types, adherent or not.

Lumen expansion is initially driven by apical actin polymerization followed by osmotic pressure in a human epiblast model.

Post-implantation, the pluripotent epiblast in a human embryo forms a central lumen, paving the way for gastrulation. Osmotic pressure gradients are considered the drivers of lumen expansion across development, but their role in human epiblasts is unknown. Here, we study lumenogenesis in a pluripotent-stem-cell-based epiblast model using engineered hydrogels. We find that leaky junctions prevent osmotic pressure gradients in early epiblasts and, instead, forces from apical actin polymerization drive lumen expansion. Once the lumen reaches a radius of ∼12 µm, tight junctions mature, and osmotic pressure gradients develop to drive further growth. Computational modeling indicates that apical actin polymerization into a stiff network mediates initial lumen expansion and predicts a transition to pressure-driven growth in larger epiblasts to avoid buckling. Human epiblasts show transcriptional signatures consistent with these mechanisms. Thus, actin polymerization drives lumen expansion in the human epiblast and may serve as a general mechanism of early lumenogenesis.

Actin pushes open a leaky lumen.

Using a human stem cell-based model to understand how the human epiblast forms at the very beginning of implantation, Indana et al.1 establish a role for pushing forces that are generated by apical actin polymerization and reveal a two-stage, biomechanics-driven lumen growth process underlying epiblast cavity morphogenesis.

Peritumoral stroma and systemic inflammatory response in cervical cancer.

OBJECTIVE: To compare cervical stroma in advanced cervical cancer with the control group; to compare, in the pre-treatment period, hemogram parameters in patients with advanced cervical cancer with the same parameters as the control group; and to verify if there is an association of stromal markers with prognostic factors in cervical cancer. MATERIALS AND METHODS: We prospectively evaluated 16 patients diagnosed with advanced invasive cervical cancer. A control group of 22 patients was used (uterine leiomyoma). Immunohistochemistry was performed to verify the stromal immunostaining of alpha-smooth muscle actin (SMA) and fibroblast activation protein alpha (FAP). Immunostainings and hemogram parameters were compared using Fisher's exact and Mann-Whitney Test, respectively. RESULTS: Strong FAP immunostaining was more frequent in patients with cervical cancer when compared with patients with leiomyoma (P = 0.0002). Regarding SMA, strong immunostaining was also found more in the group of cancer patients compared to the control group (P < 0.00001). The neutrophil-lymphocyte ratio (NLR) values were higher in the cancer patient group compared to the control group (P = 0.0019). There was no association of the parameters studied with prognostic factors. CONCLUSIONS: Strong FAP and SMA immunostaining was found more in patients with cervical cancer when compared to the control group. NLR values were also higher in cervical cancer.

Actin-nucleation promoting factor N-WASP influences alpha-synuclein condensates and pathology.

Abnormal intraneuronal accumulation of soluble and insoluble α-synuclein (α-Syn) is one of the main pathological hallmarks of synucleinopathies, such as Parkinson's disease (PD). It has been well documented that the reversible liquid-liquid phase separation of α-Syn can modulate synaptic vesicle condensates at the presynaptic terminals. However, α-Syn can also form liquid-like droplets that may convert into amyloid-enriched hydrogels or fibrillar polymorphs under stressful conditions. To advance our understanding on the mechanisms underlying α-Syn phase transition, we employed a series of unbiased proteomic analyses and found that actin and actin regulators are part of the α-Syn interactome. We focused on Neural Wiskott-Aldrich syndrome protein (N-WASP) because of its association with a rare early-onset familial form of PD. In cultured cells, we demonstrate that N-WASP undergoes phase separation and can be recruited to synapsin 1 liquid-like droplets, whereas it is excluded from α-Syn/synapsin 1 condensates. Consistently, we provide evidence that wsp-1/WASL loss of function alters the number and dynamics of α-Syn inclusions in the nematode Caenorhabditis elegans. Together, our findings indicate that N-WASP expression may create permissive conditions that promote α-Syn condensates and their potentially deleterious conversion into toxic species.

Differential Anti-Fibrotic and Remodeling Responses of Human Dermal Fibroblasts to Artemisia sp., Artemisinin, and Its Derivatives.

Fibrosis is a ubiquitous pathology, and prior studies have indicated that various artemisinin (ART) derivatives (including artesunate (AS), artemether (AM), and dihydroartemisinin (DHA)) can reduce fibrosis in vitro and in vivo. The medicinal plant Artemisia annua L. is the natural source of ART and is widely used, especially in underdeveloped countries, to treat a variety of diseases including malaria. A. afra contains no ART but is also antimalarial. Using human dermal fibroblasts (CRL-2097), we compared the effects of A. annua and A. afra tea infusions, ART, AS, AM, DHA, and a liver metabolite of ART, deoxyART (dART), on fibroblast viability and expression of key fibrotic marker genes after 1 and 4 days of treatment. AS, DHA, and Artemisia teas reduced fibroblast viability 4 d post-treatment in up to 80% of their respective controls. After 4 d of treatment, AS DHA and Artemisia teas downregulated ACTA2 up to 10 fold while ART had no significant effect, and AM increased viability by 10%. MMP1 and MMP3 were upregulated by AS, 17.5 and 32.6 fold, respectively, and by DHA, 8 and 51.8 fold, respectively. ART had no effect, but A. annua and A. afra teas increased MMP3 5 and 16-fold, respectively. Although A. afra tea increased COL3A1 5 fold, MMP1 decreased >7 fold with no change in either transcript by A. annua tea. Although A. annua contains ART, it had a significantly greater anti-fibrotic effect than ART alone but was less effective than A. afra. Immunofluorescent staining for smooth-muscle α-actin (α-SMA) correlated well with the transcriptional responses of drug-treated fibroblasts. Together, proliferation, qPCR, and immunofluorescence results show that treatment with ART, AS, DHA, and the two Artemisia teas yield differing responses, including those related to fibrosis, in human dermal fibroblasts, with evidence also of remodeling of fibrotic ECM.