Distribution Of Cytoskeleton Research Articles

The Immunosuppressive Drug LF15-0195 Acts Also on Glomerular Lesions, by a Change in Cytoskeleton Distribution in Podocyte

Introduction: Buffalo/Mna rats spontaneously develop nephrotic syndrome (NS) which recurs after renal transplantation. The immunosuppressive drug LF15-0195 can promote regression of the initial and post-transplantation nephropathy via induction of regulatory T cells. We investigate if this drug has an additional effect on the expression and localization of podocyte specific proteins. Methods: Buffalo/Mna kidney samples were collected before and after the occurrence of proteinuria, and after the remission of proteinuria induced by LF15-0195 treatment and compared by quantitative RT-PCR, Western blot, electron, and confocal microscopy to kidney samples of age-matched healthy rats. Cytoskeleton changes were assessed in culture by stress fibers induction by TNFα. Results: We observed, by electron microscopy, a restoration of foot process architecture in the LF15-0195-treated Buff/Mna kidneys, consistent with proteinuria remission. Nephrin, podocin, CD2AP, and α-actinin-4 mRNA levels remained low during the active disease in the Buff/Mna, in comparison with healthy rats which increase, while podocalyxin and synaptopodin transcripts were elevated before the occurrence of the disease but did not differ from healthy animals after. No difference in the mRNA and protein expression between the untreated and the LF15-0195-treated proteinuric Buff/Mna were seen for these 6 proteins. No changes were observed by confocal microscopy in the protein distribution at a cellular level, but a more homogenous distribution similar to healthy rats, was observed within the glomeruli of LF15-0195-treated rats. In addition, LF15-0195 could partially restore actin cytoskeleton of endothelial cells in TNFα-induced-cell stress experiment. Conclusion: The effect of LF15-0195 treatment appears to be mediated by 2 mechanisms: an immunomodulatory effect via regulatory T cells induction, described in our previous work and which can act on immune cell involved in the disease pathogenesis, and an effect on the restoration of podocyte cytoskeleton, independent of expression levels of the proteins involved in the slit diaphragm and podocyte function, showed in this article.

Development of Novel Antibacterial Ti-Nb-Ga Alloys with Low Stiffness for Medical Implant Applications.

With the rising demand for medical implants and the dominance of implant-associated failures including infections, extensive research has been prompted into the development of novel biomaterials that can offer desirable characteristics. This study develops and evaluates new titanium-based alloys containing gallium additions with the aim of offering beneficial antibacterial properties while having a reduced stiffness level to minimise the effect of stress shielding when in contact with bone. The focus is on the microstructure, mechanical properties, antimicrobial activity, and cytocompatibility to inform the suitability of the designed alloys as biometals. Novel Ti-33Nb-xGa alloys (x = 3, 5 wt%) were produced via casting followed by homogenisation treatment, where all results were compared to the currently employed alloy Ti-6Al-4V. Optical microscopy, scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) results depicted a single beta (β) phase microstructure in both Ga-containing alloys, where Ti-33Nb-5Ga was also dominated by dendritic alpha (α) phase grains in a β-phase matrix. EDS analysis indicated that the α-phase dendrites in Ti-33Nb-5Ga were enriched with titanium, while the β-phase was richer in niobium and gallium elements. Mechanical properties were measured using nanoindentation and microhardness methods, where the Young's modulus for Ti-33Nb-3Ga and Ti-33Nb-5Ga was found to be 75.4 ± 2.4 and 67.2 ± 1.6 GPa, respectively, a significant reduction of 37% and 44% with respect to Ti-6Al-4V. This reduction helps address the disproportionate Young's modulus between titanium implant components and cortical bone. Importantly, both alloys successfully achieved superior antimicrobial properties against Gram-negative P. aeruginosa and Gram-positive S. aureus bacteria. Antibacterial efficacy was noted at up to 90 ± 5% for the 3 wt% alloy and 95 ± 3% for the 5 wt% alloy. These findings signify a substantial enhancement of the antimicrobial performance when compared to Ti-6Al-4V which exhibited very small rates (up to 6.3 ± 1.5%). No cytotoxicity was observed in hGF cell lines over 24 h. Cell morphology and cytoskeleton distribution appeared to depict typical morphology with a prominent nucleus, elongated fibroblastic spindle-shaped morphology, and F-actin filamentous stress fibres in a well-defined structure of parallel bundles along the cellular axis. The developed alloys in this work have shown very promising results and are suggested to be further examined towards the use of orthopaedic implant components.

Atheroprotective WSS effects on human aortic endothelial cells in a new generation bioreactor

Abstract Funding Acknowledgements None. Background Luminal arterial endothelial cells are mainly aligned along the blood flow direction, whereas the in vitro endothelial cells (EC) showed heterogeneous distribution. In atherosclerosis the altered blood flow, acting on mechano-transduction, contributes to endothelial dysfunction and barrier disorganization. A low wall shear stress (WSS) has a pro-atherosclerotic effect, whilst elevated WSS is atheroprotective. Purposes Aim of this work is to study the primary human aortic EC cells (HAOEC) capability to acquire a distribution reminiscent of the luminal alignment under atheroprotective WSS using a prototypal new generation bioreactor. Methods We have developed a novel device compatible with culture inside standard sterile incubator, allowing to apply complex WSS patterns in controlled conditions. The bioreactor includes a sample housing and a parallel plate flow chamber and is controlled by a custom electronic interface actuating motor and pump. The cells were settled over PMMA supports and coating conditions were setup. Atheroprotective WSS patterns were tested. We compared HAOEC submitted to high flow (1.2 Pa for 24h) vs. ramps with different slopes (3h, 8h, 24h) followed by steady-state flow (1.2Pa for 24h, 48h). Viability and distribution of Acridine Orange-labelled HAOEC were assessed over bioreactor supports at both beginning and end of the experiments using fluorescence microscope. Hematoxylin/Eosin and Coomassie Blue staining were tested after the experiment to evaluate the shape and cell orientation (directionality FiJi plugin). Confocal microscopy on HAOEC labelled with Phalloidin–TRITC, anti-CD31/PECAM1-AlexaFluor488 and DAPI displayed the flow-related changes. Results HAOEC settled over naked sterile PMMA supports mainly detached under WSS, independently from the cell number. Coating with extracellular matrix components (i.e., human laminin, bovine fibronectin, chicken collagen type II, human collagen type I) showed the superiority of collagen type I to obtain homogeneously distributed, flow-resistant cell monolayers. In static condition, HAOEC on coated supports demonstrated ≥50% detachment within 24h, those submitted to a direct atheroprotective WSS of 1.2Pa almost completely detached. Consistently, to ramps with high slope (range 0.2-1,2Pa in 3h or 8h) followed by1.2Pa WSS corresponded 60%-90% cell detachment. Conversely, a 24h ramp (range 0.2-1,2Pa) followed by 24h or 48h of 1.2Pa WSS preserved the HAOEC monolayer adherence, and aligned cells were found. Microscopy comparative analysis of selected areas before/after WSS showed a partial spatial reorganization, with changes in the F-actin cytoskeleton and CD31 distribution. Conclusion We have optimized a protocol for studying HAOEC under atheroprotective WSS. In perspective further increasing the duration of the stimulus under constant WSS will achieve full alignment and provide a pseudo-physiologic model for multidirectional atherogenic WSS studies.

Focal adhesion and actin orientation regulated by cellular geometry determine stem cell differentiation via mechanotransduction

Tuning cell adhesion geometry can affect cytoskeleton organization and the distribution of cytoskeleton forces, which play critical roles in controlling cell functions. To elucidate the geometrical relationship with cytoskeleton force distribution, it is necessary to control cell morphology. In this study, a series of dextral vortex micropatterns were prepared to precisely control cell morphology for investigating the influence of the curvature degree of adhesion curves on intracellular force distribution and stem cell differentiation at a sub-cellular level. Peripherial actin filaments of micropatterned cells were assembled along the adhesion curves and showed different orientations, filament thicknesses and densities. Focal adhesion and cytoskeleton force distribution were dependent on the curvature degree. Intracellular force distribution was also regulated by adhesion curves. The cytoskeleton and force distribution affected the osteogenic differentiation of mesenchymal stem cells through a YAP/TAZ-mediated mechanotransduction process. Thus, regulation of cell adhesion curvature, especially at cytoskeletal filament level, is critical for cell function manipulation. Statement of significanceIn this study, a series of dextral micro-vortexes were prepared and used for the culture of human mesenchymal stem cells (hMSCs) to precisely control adhesive curvatures (0°, 30°, 60°, and 90°). The single MSCs on the micropatterns had the same size and shape but showed distinct focal adhesion (FA) and cytoskeleton orientations. Cellular nanomechanics were observed to be correlated with the curvature degrees, subsequently influencing nuclear morphological features. As a consequence, the localization of the mechanotransduction sensor and activator-YAP/TAZ was affected, influencing osteogenic differentiation. The results revealed the pivotal role of adhesive curvatures in the manipulation of stem cell differentiation via the machanotransduction process, which has rarely been investigated.

Membrane potential dynamics of C5a-stimulated neutrophil granulocytes

Neutrophil granulocytes play a crucial role in host defense against invading pathogens and in inflammatory diseases. The aim of this study was to elucidate membrane potential dynamics during the initial phase of neutrophil activation and its relation to migration and production of reactive oxygen species (ROS). We performed ROS production measurements of neutrophils from healthy C57BL/6J mice after TNFα-priming and/or C5a stimulation. The actin cytoskeleton was visualized with fluorescence microscopy. Furthermore, we combined migration assays and measurements of membrane potential dynamics after stimulating unprimed and/or TNFα-primed neutrophils with C5a. We show that C5a has a concentration-dependent effect on ROS production and chemokinetic migration. Chemokinetic migration and chemotaxis are impaired at C5a concentrations that induce ROS production. The actin cytoskeleton of unstimulated and of ROS-producing neutrophils is not distributed in a polarized way. Inhibition of the phagocytic NADPH oxidase NOX2 with diphenyleneiodonium (DPI) leads to a polarized distribution of the actin cytoskeleton and rescues chemokinetic migration of primed and C5a-stimulated neutrophils. Moreover, C5a evokes a pronounced depolarization of the cell membrane potential by 86.6 ± 4.2 mV starting from a resting membrane potential of -74.3 ± 0.7 mV. The C5a-induced depolarization occurs almost instantaneously (within less than one minute) in contrast to the more gradually developing depolarization induced by PMA (lag time of 3—4 min). This initial depolarization is accompanied by a decrease of the migration velocity. Collectively, our results show that stimulation with C5a evokes parallel changes in membrane potential dynamics, neutrophil ROS production and motility. Notably, the amplitude of membrane potential dynamics is comparable to that of excitable cells.

Morphological Changes of 3T3 Cells under Simulated Microgravity.

Cells are sensitive to changes in gravity, especially the cytoskeletal structures that determine cell morphology. The aim of this study was to assess the effects of simulated microgravity (SMG) on 3T3 cell morphology, as demonstrated by a characterization of the morphology of cells and nuclei, alterations of microfilaments and microtubules, and changes in cycle progression. 3T3 cells underwent induced SMG for 72 h with Gravite®, while the control group was under 1G. Fluorescent staining was applied to estimate the morphology of cells and nuclei and the cytoskeleton distribution of 3T3 cells. Cell cycle progression was assessed by using the cell cycle app of the Cytell microscope, and Western blot was conducted to determine the expression of the major structural proteins and main cell cycle regulators. The results show that SMG led to decreased nuclear intensity, nuclear area, and nuclear shape and increased cell diameter in 3T3 cells. The 3T3 cells in the SMG group appeared to have a flat form and diminished microvillus formation, while cells in the control group displayed an apical shape and abundant microvilli. The 3T3 cells under SMG exhibited microtubule distribution surrounding the nucleus, compared to the perinuclear accumulation in control cells. Irregular forms of the contractile ring and polar spindle were observed in 3T3 cells under SMG. The changes in cytoskeleton structure were caused by alterations in the expression of major cytoskeletal proteins, including β-actin and α-tubulin 3. Moreover, SMG induced 3T3 cells into the arrest phase by reducing main cell cycle related genes, which also affected the formation of cytoskeleton structures such as microfilaments and microtubules. These results reveal that SMG generated morphological changes in 3T3 cells by remodeling the cytoskeleton structure and downregulating major structural proteins and cell cycle regulators.

Is bladder outlet obstruction rat model to induce overactive bladder (OAB) has similarity to human OAB? Research on the events in smooth muscle, collagen, interstitial cell and telocyte distribution

BackgroundCellular and cytoskeletal events of overactive bladder (OAB) have not been sufficiently explored in human bladder due to different limitations. Bladder outlet obstruction (BOO) had been induced in different animal models with different methods to induce (OAB). Similarity of the animal models of BOO to the human OAB is postulated but has not been confirmed. The interstitial cells of Cajal (ICCs), and telocytes (TCs) are an important players in smooth muscles conductivity, they had not been well investigated in the previous BOO models. Objectives are to investigate the morphological pattern of cellular, cytoskeleton and telocytes distribution in BOO rat model and to match the events in two time periods and compare it to the findings in real-world human OAB.MethodsFemale Sprague-Dawley rats (Rattus norvegicus) were randomly divided into: sham (n = 10), BOO 6 W (n = 10), BOO 8 W (n = 10). Operative procedure to Induce BOO was done under anesthesia with intraperitoneal Ketamine administration. The Effect of induction of BOO was evaluated after 6 and 8 weeks. The rats were anesthetized, and the urinary bladder was removed, while the rat was unconscious under anaesthesia it was transferred to the inhalation anaesthesia cage for euthanasia, rats were sacrificed under light anesthesia using isoflurane. Care of animals, surgical procedure, and euthanasia adhered to Guide for the Care and Use of Laboratory Animals, and AVMA Guidelines for the Euthanasia of Animals. The retrieved bladder was processed for examination with histopathology, immunohistochemistry (IHC), and transmission electron microscopy (EM).ResultsHistological examination of the bladder shows thinner urothelium, condensation of collagen between muscle bundles. IHC with c-kit shows the excess distribution of ICCs between smooth muscle bundles. EM shows frequent distribution of TCs that were situated between collagen fibers. Finings in BOO 6 W group and BOO 8 W group were comparable.ConclusionThe animal model study demonstrated increased collagen/ smooth muscle ratio, high intensity of ICCs and presence of TCs. Findings show that a minimally invasive procedure to induce BOO in rats had resulted in an OAB that has morphological changes that were stable in 6 & 8 weeks. We demonstrated the distribution of TCs and ICCs in the rat animal model and defined them. The population of TCs in the BOO rat model is described for the first time, suggests that the TCs and ICCs may contribute to the pathophysiology of OAB. Similarity of animal model to human events OAB was demonstrated. These findings warrant further study to define the role of TCs in OAB.Clinical trial registryThe study does not require a clinical trial registration; it is an experimental animal study in basic science and does not include human subjects.

ECM1 promotes migration and invasion in endometriosis

Extracellular matrix protein 1 (ECM1) is a glycoprotein that may be a key player in tumorigenesis and tumor progression. However, knowledge regarding the role of ECM1 in endometriosis (EM) is still lacking. Microarray analyses were performed to compare the mRNA expression patterns between paired EU tissues and ectopic endometrial (EC) tissues (n = 4) from EM patients. ECM1 expression was significantly increased in the eutopic endometrial (EU) tissues than paired EC tissues of endometriotic patients and normal endometrial (NE) tissues of controls without EM. Blocking ECM1 with siRNA attenuated the migration and invasion of hEM15A cells and modified the distribution of the F-actin cytoskeleton. We conducted microarray analyses and bioinformatics analyses to investigate the differentially expressed genes (DEGs) and related pathways regulated by ECM1. A total of 161 DEGs between the siECM1 and the negative control (siNC) treatments were identified, consisting of 79 downregulated genes and 82 upregulated genes. Enriched DEGs were associated with 9 gene ontology (GO) terms. Moreover, a protein-protein interaction (PPI) network was constructed for the hub genes and modules. Radixin (RDX) was the second most downregulated gene in the siECM1 group compared with the siNC group. ECM1 knockdown significantly decreased the expression of RDX, RhoC, ROCK1, N-cadherin and β-catenin but not ROCK2. ECM1 showed high tissue-specific expression in EU tissues from EM patients, and may contribute to the migration, invasion and reorganization of the F-actin cytoskeleton in eutopic endometrial stromal cells via the RhoC/ROCK1 signaling pathway in EM.

Nanomechanical-based classification of prostate tumor using atomic force microscopy.

The loss of mechanical homeostasis between tumor cells and microenvironment is an important factor in tumor metastasis. In the process, mechanical forces affect cell proliferation, differentiation, migration and tissue development. Using high spatial resolution of Atomic force microscopy (AFM) technology, our study provides the direct measurement of the nanomechanical properties of prostate cancer clinical tissue specimens. AFM was used to determine the biomechanical properties of prostate tissue with different grade scores. K-means clustering method and fuzzy C-means were used to distinguish the cellular component in prostate tissue from non-cellular component based on their viscoelasticity. Futhermore, AFM measurements in vitro cells, including metastatic prostate cells (PC-3) and normal human prostate cells (PZ-HPV-7) were carried out. The Young's modulus was decreased in prostate cancer progression, and the elasticity of cellular component in prostate cancer tissue was smaller than that of normal prostate tissue. PC-3 cells were softer than PZ-HPV-7 cells. Further mechanism investigation showed that the difference in modulus between cancerous and normal prostate tissue may be associated with a greater actin cytoskeleton distribution inside the cancer cells. The results suggests that the nanomechanical properties can classify the prostate tumor, which could be used as an index for the identification and classification of cancer at cellular level.

Protocells Capable of Generating a Cytoskeleton-like Structure from intracellular Membrane-active Artificial Organelles.

The intricate nature of eukaryotic cells with differently viscous intracellular compartments provides (membrane-active) enzymes to trigger time- and concentration-dependent processes in the intra-/extracellular matrix. Herein, we capitalize on membrane-active artificial organelles (AOs) to develop fluidic and stable proteinaceous membrane-based protocells. AOs in protocells induce the self-assembly of oligopeptides into an artificial cytoskeleton that underline their influence on the structure and functionality of protocells. A series of microscopical tools is used to validate the intracellular assembly and distribution of cytoskeleton, the changing protocells morphology, and AOs inclusion within cytoskeletal growth. Thus, the dynamics, diffusion and viscosity of intracellular components in the presence of cytoskeleton are evaluated by fluorescence tools and enzymatic assay. Membrane-active alkaline phosphatase in polymersomes as AOs fulfills the requirements of biomimetic eukaryotic cells to trigger intracellular environment, mobility, viscosity, diffusion and enzymatic activity itself as well as high mechanical stability and high membrane fluidity of protocells. Thus membrane-active AOs in protocells thoroughly provide a variable reaction space in a changing intracellular environment and underline their regulatory role in the fabrication of complex protocell architectures and functions. This study demonstrates an important contribution to effective biomimicry of cell-like structures, shapes and functions.

Leucine-Based Pseudo-Proteins (LPPs) as Promising Biomaterials: A Study of Cell-Supporting Properties.

Scaffold-based systems have become essential in biomedical research, providing the possibility of building in vitro models that can better mimic tissue/organic physiology. A relatively new family of biomimetics-pseudo-proteins (PPs)-can therefore be considered especially promising in this context. Three different artificial leucine-based LPP films were tested in vitro as potential scaffolding materials. In vitro experiments were performed using two types of cells: primary mouse skin fibroblasts and a murine monocyte/macrophages cell line, RAW264.7. Cell adhesion and cell spreading were evaluated according to morphological parameters via scanning electron microscopy (SEM), and they were assessed according to actin cytoskeleton distribution, which was studied via confocal laser microscopy. Cell proliferation was evaluated via an MTT assay. Cell migration was studied using time-lapse microscopy. SEM images for both types of cells demonstrated prominent adhesion and perfect cell spreading on all three LPPs. Analyses of actin cytoskeleton organization revealed a high number of focal adhesions and prominent motility-associated structures. A certain stimulation of cell proliferation was detected in the cases of all three LPPs, and two of them promoted macrophage migration. Overall, our data suggest that the LPPs used in the study can be considered potential cell-friendly scaffolding materials.

Microtubule detyrosination drives symmetry breaking to polarize cells for directed cell migration

To initiate directed movement, cells must become polarized, establishing a protrusive leading edge and a contractile trailing edge. This symmetry-breaking process involves reorganization of cytoskeleton and asymmetric distribution of regulatory molecules. However, what triggers and maintains this asymmetry during cell migration remains largely elusive. Here, we established a micropatterning-based 1D motility assay to investigate the molecular basis of symmetry breaking required for directed cell migration. We show that microtubule (MT) detyrosination drives cell polarization by directing kinesin-1-based transport of the adenomatous polyposis coli (APC) protein to cortical sites. This is essential for the formation of cell's leading edge during 1D and 3D cell migration. These data, combined with biophysical modeling, unveil a key role for MT detyrosination in the generation of a positive feedback loop linking MT dynamics and kinesin-1-based transport. Thus, symmetry breaking during cell polarization relies on a feedback loop driven by MT detyrosination that supports directed cell migration.

Beta-arrestin-biased agonism promotes TRPC6-mediated calcium influx in human podocytes

Background: Angiotensin Receptor blockers (ARBs) are the first-line treatment for hypertension and other chronic kidney diseases and act by inhibiting signaling through the angiotensin 1 receptor (AT1R). AT1R activation initiates signaling through G protein-coupled receptor (GPCR) effector signaling and β-arrestins. Recently, a novel AT1R agonist TRV120027 (TRV) that selectively activates the β-arrestin cascade without impacting the GCPR pathway has become available. Therefore, we hypothesized that β-arrestin and G-protein signaling pathways via AT1R have distinct downstream effects in podocytes. Furthermore, we propose that β-arrestin signaling initiates TRPC6-mediated Ca 2+ entry. Methods: We used a conditionally immortalized human podocyte cell line to determine β-arrestin’s involvement in podocyte calcium signaling and cytoskeletal reorganization. Intracellular Ca 2+ influx (Fluo-4 AM) and NO response (DAF-FM) to Ang II or TRV applications were analyzed with confocal microscopy. The role of TRPC channels was tested by single-channel electrophysiology and confocal imaging using a variety of commercially available inhibitors (AC1903 for TRPC5 and GSK283350A, SAR7334 for TRPC6). Western blotting for apoptosis-associated proteins, TUNEL staining to detect DNA cleavage, and actin cytoskeleton rearrangements were performed in podocytes exposed to TRV or Ang II treatment. Results: Our experiments determined that TRV-mediated β-arrestin pathway activation in podocytes promotes rapid elevation of intracellular Ca 2+ in a dose-dependent manner (IC 50 =15μM, ligand-receptor binding fit modeled with Levenberg–Marquardt algorithm, adj. R 2 =0.93). Interestingly, the amplitude of β-arrestin-mediated Ca 2+ influx was four times higher than the response to similar Ang II concentrations (t-test, n≥30, p<0.05). Moreover, the TRV response was significantly increased under hyperglycemic stress conditions, and promoted rapid apoptosis in podocytes (one-way ANOVA, n≥21, p=0.003). The pharmacological blockade of TRPC6, but not TRPC5, significantly attenuated Ca 2+ influx in response to β-arrestin. In contrast to Ang II, TRV does not induce AT1R-mediated NO production, suggesting that β-arrestin activation only targets AT1R, and not AT2R. Single-channel analyses show rapid activation of TRPC activity in response to acute TRV application in podocytes (one-way ANOVA, n=8, p=0.004). Overall prolonged activation of the β-arrestin pathway in podocytes results in enhanced actin bundle thickness, abnormal actin cytoskeleton distribution, and increased apoptotic cell markers. Conclusions: TRV-mediated β-arrestin signaling in podocytes promotes high ionotropic TRPC6 channel-mediated Ca 2+ influx, cytoskeleton rearrangement, and apoptosis, possibly leading to severe defects in glomerular filtration barrier integrity and kidney health. Under these circumstances, the potential therapeutic application of TRV for hypertension treatment is controversial and requires further investigation. DK126720 (to OP), DK129227 (to AS and OP), and NIH/NCATS/SCTR UL1TR001450/SCTR 2214 (to OP), endowed funds from the SC SmartState Centers of Excellence (to OP), Veterans Affairs Support Veterans Affairs (Merit Award I01 BX000820 to JL), research grant from Dialysis Clinic, Inc (to JL). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role of cell division cycle 42 in root development of tooth and the involved mechanisms

Objective: To explore the effect of cell division cycle 42 (CDC42) on root development and its regulation on cell proliferation and migration in Hertwig's epithelial root sheath (HERS). Methods: Trace the spatiotemporal expression of CDC42 in root development process [postnatal day 5 (P5), P7, P14] through immunofluorescence staining. Nine eight-week-old C57BL/6J male mice were randomly divided into 3 groups using a simple random sample method (n=3 in each group). P3 tooth germ was cultured in air-liquid system for 1 day and then transplanted to renal capsule each to observe tooth root development. The control group implanted tooth germ only. The phosphate buffered saline (PBS) group implanted tooth germ and gel beads soaked with PBS, while the ML141 group implanted tooth germ and gel beads soaked with CDC42 inhibitor (ML141). Cdc42 in HERS cells was inhibited via lentivirus transfection. Cell counting kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay and scratch assay were performed. The distribution of Golgi apparatus (GM130) and cytoskeleton (F-actin) in migrated cells were mapped via immunofluorescence staining. Results: CDC42 was expressed in epithelial cells of HERS, polarized ameloblasts and odontoblasts, as well as adjacent dental papilla and dental follicle cells during tooth root development. The root length of the ML141 group [(0.61±0.09) mm] was substantially shorter than that of control group [(1.03±0.19) mm, P=0.007] and PBS group [(0.98±0.10) mm, P=0.021] according to the data of renal capsule transplantation. After lentiviral transfection, the relative expression of Cdc42 in knockdown group (0.31±0.33) was significantly lower than that in control group (1.05±0.08) (t=15.38, P<0.001), demonstrating the knockdown efficiency closed to 70%. Cell viabilities were significantly inhibited in knockdown group (0.87±0.04, 0.96±0.10, 0.59±0.06, respectively) compared with those in control group (1.09±0.13, 1.55±0.32, 1.10±0.09, respectively) after 3, 4 and 5 days (t=3.16, P=0.016; t=4.23, P=0.002; t=5.08, P<0.01), and the cell proliferation ability in knockdown group [(1.65±0.64)%] also decreased than that in the control group [(4.02±1.12)%](t=5.21, P<0.001). In addition, the cell migration rates after 24 and 48 h [(45.1±4.2)%, (56.4±8.3)%] in knockdown group were obviously lower than those in the control group [(63.8±7.4)%, (80.2±7.8)%] (t=3.78, P=0.019; t=3.62, P=0.023). After Cdc42 was knocked down, Golgi apparatus distributed along the nucleus while behaved oriented in the control group. Conclusions: CDC42 plays an important role in the regulation of root length during root development, which may mediate root elongation by affecting the migration and proliferation of HERS cells.

Podocyte Geranylgeranyl Transferase Type-I Is Essential for Maintenance of the Glomerular Filtration Barrier.

A tightly regulated actin cytoskeleton attained through balanced activity of RhoGTPases is crucial to maintaining podocyte function. However, how RhoGTPases are regulated by geranylgeranylation, a post-translational modification, has been unexplored. The authors found that loss of the geranylgeranylation enzyme geranylgeranyl transferase type-I (GGTase-I) in podocytes led to progressive albuminuria and foot process effacement in podocyte-specific GGTase-I knockout mice. In cultured podocytes, the absence of geranylgeranylation resulted in altered activity of its downstream substrates Rac1, RhoA, Cdc42, and Rap1, leading to alterations of β1-integrins and actin cytoskeleton structural changes. These findings highlight the importance of geranylgeranylation in the dynamic management of RhoGTPases and Rap1 to control podocyte function, providing new knowledge about podocyte biology and glomerular filtration barrier function. Impairment of the glomerular filtration barrier is in part attributed to podocyte foot process effacement (FPE), entailing disruption of the actin cytoskeleton and the slit diaphragm. Maintenance of the actin cytoskeleton, which contains a complex signaling network through its connections to slit diaphragm and focal adhesion proteins, is thus considered crucial to preserving podocyte structure and function. A dynamic yet tightly regulated cytoskeleton is attained through balanced activity of RhoGTPases. Most RhoGTPases are post-translationally modified by the enzyme geranylgeranyl transferase type-I (GGTase-I). Although geranylgeranylation has been shown to regulate activities of RhoGTPases and RasGTPase Rap1, its significance in podocytes is unknown. We used immunofluorescence to localize GGTase-I, which was expressed mainly by podocytes in the glomeruli. To define geranylgeranylation's role in podocytes, we generated podocyte-specific GGTase-I knockout mice. We used transmission electron microscopy to evaluate FPE and measurements of urinary albumin excretion to analyze filtration barrier function. Geranylgeranylation's effects on RhoGTPases and Rap1 function were studied in vitro by knockdown or inhibition of GGTase-I. We used immunocytochemistry to study structural modifications of the actin cytoskeleton and β1 integrins. Depletion of GGTase-I in podocytes in vivo resulted in FPE and concomitant early-onset progressive albuminuria. A reduction of GGTase-I activity in cultured podocytes disrupted RhoGTPase balance by markedly increasing activity of RhoA, Rac1, and Cdc42 together with Rap1, resulting in dysregulation of the actin cytoskeleton and altered distribution of β1 integrins. These findings indicate that geranylgeranylation is of crucial importance for the maintenance of the delicate equilibrium of RhoGTPases and Rap1 in podocytes and consequently for the maintenance of glomerular integrity and function.

CDT of Clostridioides difficile Induces MLC-Dependent Intestinal Barrier Dysfunction in HT-29/B6 Epithelial Cell Monolayers.

Clostridioides&amp;nbsp;difficile binary toxin (CDT) defines the hypervirulence of strains in nosocomial antibiotic-induced colitis with the highest mortality. The objective of our study was to investigate the impact of CDT on the intestinal epithelial barrier and to enlighten the underlying molecular mechanisms. Functional measurements of epithelial barrier function by macromolecular permeability and electrophysiology were performed in human intestinal HT-29/B6 cell monolayers. Molecular analysis of the spatial distribution of tight junction protein and cytoskeleton was performed by super-resolution STED microscopy. Sublethal concentrations of CDT-induced barrier dysfunction with decreased TER and increased permeability for 332 Da fluorescein and 4 kDa FITC-dextran. The molecular correlate to the functional barrier defect by CDT was found to be a tight junction protein subcellular redistribution with tricellulin, occludin, and claudin-4 off the tight junction domain. This redistribution was shown to be MLCK-dependent. CDT compromised epithelial barrier function in a human intestinal colonic cell model, even in sublethal concentrations, pointing to barrier dysfunction in the intestine and leak flux induction as a diarrheal mechanism. However, this cannot be attributed to the appearance of apoptosis and necrosis, but rather to an opening of the paracellular leak pathway as the result of epithelial tight junction alterations.

Confocal Microscopy Studies of F-Actin Cytoskeleton Distribution and Dynamics Using Fluorescent LifeAct Constructs in Bovine Adrenal Chromaffin Cells.

Cultured bovine chromaffin cells have been characterized as a successful model to study changes in the cytoskeleton during the secretory process. In this sense, the distribution and dynamics of the F-actin cytoskeleton can be studied by confocal microscopy using appropriate molecular tools such as LifeAct, a peptide that stains the structures of F-actin. In this work, we describe some methodological protocols making possible to study, under controlled stimulus conditions, the local dynamic changes of F-actin in the cortical zone and also to detect the simultaneous displacements of chromaffin granules and organelles in active zones.

Coronin 2B Regulates Neuronal Migration via Rac1-Dependent Multipolar-Bipolar Transition.

In the developing cortex, excitatory neurons migrate along the radial fibers to their final destinations and build up synaptic connection with each other to form functional circuitry. The shaping of neuronal morphologies by actin cytoskeleton dynamics is crucial for neuronal migration. However, it is largely unknown how the distribution and assembly of the F-actin cytoskeleton are coordinated. In the present study, we found that an actin regulatory protein, coronin 2B, is indispensable for the transition from a multipolar to bipolar morphology during neuronal migration in ICR mice of either sex. Loss of coronin 2B led to heterotopic accumulation of migrating neurons in the intermediate zone along with reduced dendritic complexity and aberrant neuronal activity in the cortical plate. This was accompanied by increased seizure susceptibility, suggesting the malfunction of cortical development in coronin 2B-deficient brains. Coronin 2B knockdown disrupted the distribution of the F-actin cytoskeleton at the leading processes, while the migration defect in coronin 2B-deficient neurons was partially rescued by overexpression of Rac1 and its downstream actin-severing protein, cofilin. Our results collectively reveal the physiological function of coronin 2B during neuronal migration whereby it maintains the proper distribution of activated Rac1 and the F-actin cytoskeleton.SIGNIFICANCE STATEMENT Deficits in neuronal migration during cortical development result in various neurodevelopmental disorders (e.g., focal cortical dysplasia, periventricular heterotopia, epilepsy, etc.). Most signaling pathways that control neuronal migration process converge to regulate actin cytoskeleton dynamics. Therefore, it is important to understand how actin dynamics is coordinated in the critical processes of neuronal migration. Herein, we report that coronin 2B is a key protein that regulates neuronal migration through its ability to control the distribution of the actin cytoskeleton and its regulatory signaling protein Rac1 during the multipolar-bipolar transition in the intermediate zone, providing insights into the molecular machinery that drives the migration process of newborn neurons.

Modulation of DRG neurons response to semaphorin 3A via substrate stiffness.

Semaphorin 3A (Sema3a) is a chemotropic protein that acts as a neuronal guidance cue and plays a major role in dorsal root ganglion (DRG) sensory neurons projection during embryo development. The present study evaluated the impact of stiffness in the repulsive response of DRG neurons to Sema3a when cultured over substrates of variable stiffness. Stiffness modified DRG neurons morphology and regulated their response to Sema3a, reducing the collapse of growth cones when they were cultured on softer substrates. Sema3a receptors expression was also regulated by stiffness, neuropilin-1 was overexpressed and plexin A4 mRNA was downregulated in stiffer substrates. Cytoskeleton distribution was also modified by stiffness. In softer substrates, βIII-tubulin and actin co-localized up to the leading edge of the growth cones, and as the substrate became stiffer, βIII-tubulin was confined to the transition and peripheral domains of the growth cone. Moreover, a decrease in the α-actinin adaptor protein was also observed in softer substrates. Our results show that substrate stiffness plays an important role in regulating the collapse response to Sema3a and that the modulation of cytoskeleton distribution and Sema3a receptors expression are related to the differential collapse responses of the growth cones.

Intercellular adhesion molecule-1-targeted near-infrared photoimmunotherapy of triple-negative breast cancer.

Triple‐negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and conventional chemotherapy and molecular‐targeted therapies show limited efficacy. Near‐infrared photoimmunotherapy (NIR‐PIT) is a new anticancer treatment that selectively damages the cell membrane of cancer cells based on NIR light‐induced photochemical reactions of the antibody (Ab)‐photoabsorber (IRDye700Dx) conjugate and the cell membrane. TNBC is known to express several adhesion molecules on the cell surface providing a potential new target for therapy. Here, we investigated the therapeutic efficacy of intercellular adhesion molecule‐1 (ICAM‐1)‐targeted NIR‐PIT using xenograft mouse models subcutaneously inoculated with two human ICAM‐1‐expressing TNBC cell lines, MDAMB468‐luc and MDAMB231 cells. In vitro ICAM‐1‐targeted NIR‐PIT damaged both cell types in a NIR light dose‐dependent manner. In vivo ICAM‐1‐targeted NIR‐PIT in both models showed early histological signs of cancer cell damage, such as cytoplasmic vacuolation. Even among the cancer cells that appeared to be morphologically intact within 2 h post treatment, abnormal distribution of the actin cytoskeleton and a significant decrease in Ki‐67 positivity were observed, indicating widespread cellular injury reflected in cytoplasmic degeneration. Such damage to cancer cells by NIR‐PIT significantly inhibited subsequent tumor growth and improved survival. This study suggests that ICAM‐1‐targeted NIR‐PIT could have potential clinical application in the treatment of TNBC.