Organization Of Actin Filaments Research Articles

The unique organization of filamentous actin in the medullary canal of the medulla oblongata.

In the central canal, F-actin is predominantly localized in the apical region, forming a ring-like structure around the circumference of the lumen. However, an exception is found in the medulla oblongata, where the apical F-actin becomes interrupted in the ventral aspect of the canal. To clarify the precise localization of F-actin, the fluorescence signals for F-actin were converted to the peroxidase/DAB reaction products in this study by a phalloidin-based ultrastructural technique, which demonstrated that F-actin is located mainly in the microvilli and terminal webs in the ependymocytes. It is because the ventrally oriented ependymocytes do not possess well-developed microvilli or terminal web that led to a discontinuous labeling of F-actin in the medullary canal. Since spinal motions can change the shape and size of the central canal, we next examined the cytoskeletons in the medullary canal in both rats and monkeys, because these two kinds of animals show different kinematics at the atlanto-occipital articulation. Our results first demonstrated that the apical F-actin in the medullary canal is differently organized in the animals with different head-neck kinemics, which suggests that the mechanic stretching of spinal motions is capable of inducing F-actin reorganization and the subsequent cell-shape changes in the central canal.

ADF10 shapes the overall organization of apical actin filaments by promoting their turnover and ordering in pollen tubes.

Here, we show that Arabidopsis ADF10 plays an important role in shaping the overall organization of apical actin filaments by promoting their turnover and ordering. ADF10 severs and depolymerizes actin filaments in vitro and is distributed throughout the entire pollen tube. In adf10 mutants, severing and monomer dissociation events for apical actin filaments are reduced, and the apical actin structure extends further toward the tube base than in wild-type tubes. In particular, the percentage of apical actin filaments that form large angles to the tube growth axis is much higher in adf10 pollen tubes, and the actin filaments are more randomly distributed, implying that ADF10 promotes their ordering. Consistent with the role of apical actin filaments in physically restricting the movement of vesicles, the region in which apical vesicles accumulate is enlarged at the tip of adf10 pollen tubes. Both tipward and backward movements of small vesicles are altered within the growth domain of adf10 pollen tubes. Thus, our study suggests that ADF10 shapes the organization of apical actin filaments to regulate vesicle trafficking and pollen tube growth.

Melatonin-Induced Protective Effects on Cardiomyocytes Against Reperfusion Injury Partly Through Modulation of IP3R and SERCA2a Via Activation of ERK1.

BackgroundMelatonin is a neuroendocrine hormone synthesized primarily by the pineal gland that is indicated to effectively prevent myocardial reperfusion injury. It is unclear whether melatonin protects cardiac function from reperfusion injury by modulating intracellular calcium homeostasis.ObjectiveDemonstrate that melatonin protect against myocardial reperfusion injury through modulating IP3R and SERCA2a to maintain calcium homeostasis via activation of ERK1 in cardiomyocytes.MethodsIn vitro experiments were performed using H9C2 cells undergoing simulative hypoxia/reoxygenation (H/R) induction. Expression level of ERK1, IP3R and SERCA2a were assessed by Western Blots. Cardiomyocytes apoptosis was detected by TUNEL. Phalloidin-staining was used to assess alteration of actin filament organization of cardiomyocytes. Fura-2 /AM was used to measure intracellular Ca2+ concentration. Performing in vivo experiments, myocardial expression of IP3R and SERCA2a were detected by immunofluorescence staining using myocardial ischemia/ reperfusion (I/R) model in rats.ResultsIn vitro results showed that melatonin induces ERK1 activation in cardiomyocytes against H/R which was inhibited by PD98059 (ERK1 inhibitor). The results showed melatonin inhibit apoptosis of cardiomyocytes and improve actin filament organization in cardiomyocytes against H/R, because both could be reversed by PD98059. Melatonin was showed to reduce calcium overload, further to inhibit IP3R expression and promote SERCA2a expression via ERK1 pathway in cardiomyocytes against H/R. Melatonin induced lower IP3R and higher SERCA2a expression in myocardium that were reversed by PD98059.Conclusionmelatonin-induced cardioprotection against reperfusion injury is at least partly through modulation of IP3R and SERCA2a to maintain intracellular calcium homeostasis via activation of ERK1.

Drebrin and Spermatogenesis.

Drebrin is a family of actin-binding proteins with two known members called drebrin A and E. Apart from the ability to stabilize F-actin microfilaments via their actin-binding domains near the N-terminus, drebrin also regulates multiple cellular functions due to its unique ability to recruit multiple binding partners to a specific cellular domain, such as the seminiferous epithelium during the epithelial cycle of spermatogenesis. Recent studies have illustrated the role of drebrin E in the testis during spermatogenesis in particular via its ability to recruit branched actin polymerization protein known as actin-related protein 3 (Arp3), illustrating its involvement in modifying the organization of actin microfilaments at the ectoplasmic specialization (ES) which includes the testis-specific anchoring junction at the Sertoli-spermatid (apical ES) interface and at the Sertoli cell-cell (basal ES) interface. These data are carefully evaluated in light of other recent findings herein regarding the role of drebrin in actin filament organization at the ES. We also provide the hypothetical model regarding its involvement in germ cell transport during the epithelial cycle in the seminiferous epithelium to support spermatogenesis.

Simulated Microgravity Promotes Angiogenesis through RhoA-Dependent Rearrangement of the Actin Cytoskeleton

Background/aims: Microgravity leads to hydrodynamic alterations in the cardiovascular system and is associated with increased angiogenesis, an important aspect of endothelial cell behavior to initiate new vessel growth. Given the critical role of Rho GTPase-dependent cytoskeleton rearrangement in cell migration, small GTPase RhoA might play a potential role in microgravity-induced angiogenesis. Methods: We examined the organization of actin filaments by FITC-conjugated phalloidin staining, as well as the expression and activity of RhoA by quantitative PCR and Western blot, in human umbilical vein endothelial cells (HUVECs) under normal gravity and simulated microgravity. Effect of simulated microgravity on the wound closure and tube formation in HUVECs, and their dependence on RhoA, were also analyzed by cell migration and tube formation assays. Results: We show that in HUVECs actin filaments are disorganized and RhoA activity is reduced by simulated microgravity. Blocking RhoA activity either by C3 transferase Rho inhibitor or siRNA knockdown mimicked the effect of simulated microgravity on inducing actin filament disassembly, followed by enhanced wound closure and tube formation in HUVECs, which closely resembled effects seen on microgravity-treated cells. In contrast, overexpressing RhoA in microgravity-treated HUVECs restored the actin filaments, and decreased wound closure and tube formation abilities. Conclusion: These results suggest that RhoA inactivation is involved in the actin rearrangement-associated angiogenic responses in HUVECs during simulated microgravity.

Nano-scale actin-network characterization of fibroblast cells lacking functional Arp2/3 complex.

Arp2/3 complex is thought to be the primary protrusive force generator in cell migration by controlling the assembly and turnover of the branched filament network that pushes the leading edge of moving cells forward. However, mouse fibroblasts without functional Arp2/3 complex migrate at rates similar to wild-type cells, contradicting this paradigm. We show by correlative fluorescence and large-scale cryo-tomography studies combined with automated actin-network analysis that the absence of functional Arp2/3 complex has profound effects on the nano-scale architecture of actin networks. Our quantitative analysis at the single-filament level revealed that cells lacking functional Arp2/3 complex fail to regulate location-dependent fine-tuning of actin filament growth and organization that is distinct from its role in the formation and regulation of dendritic actin networks.

Complex Cell Physiology on Topographically and Chemically Designed Material Surfaces

A crucial factor for ingrowth of permanent implants in the bone is the rapid cellular acceptance. The topographical features often follow mechanical aspects for implant stability. But several of these implants fail due to insufficient cell adhesion. Cells are able to perceive the physico-chemical properties of their surrounding and to pass these signals into the cell to modulate their adhesion structures, growth or production of extracellular matrix. However, the complex cell physiology at the material interface is not yet fully understood, particular on stochastically structured topographies resulting from industrial production. We could find out that corundum blasted titanium hampered the organization of actin filaments inside the cells, clustered adhesion components, e. g. beta-1 integrins and tensin, and the cells bridged the valleys which reduces cell-substrate contacts. These morphological changes strongly diminished the mineralization of osteoblasts. To shed light on cause and effect we reduced the physical complexity of the material surface by introduction of regular micro-structures (pillars, grooves) using deep reactive ion etching. Now it was more obvious what cells are doing on sharp edged topographies ‒ the actin filaments of our cells were clustered around the pillars. As a result the intracellular calcium signaling and the protein synthesis were impaired. Our recent findings indicated an attempted phagocytosis of the micro-pillars by osteoblasts. Therefore we conclude that implants used in orthopedic surgery should avoid any sharp-edged topographical features that could induce phagocytosis by the surrounding cells, which is an unnecessarily energy consuming process.

Challenge Integrity: The Cell-Penetrating Peptide BP100 Interferes with the Auxin-Actin Oscillator.

Actin filaments are essential for the integrity of the cell membrane. In addition to this structural role, actin can modulate signaling by altering polar auxin flow. On the other hand, the organization of actin filaments is modulated by auxin constituting a self-referring signaling hub. Although the function of this auxin–actin oscillator is not clear, there is evidence for a functional link with stress signaling activated by the NADPH oxidase Respiratory burst oxidase Homolog (RboH). In the current work, we used the cell-penetrating peptide BP100 to induce a mild and transient perturbation of membrane integrity. We followed the response of actin to the BP100 uptake in a green fluorescent protein (GFP)-tagged actin marker line of tobacco Bright Yellow 2 (BY-2) cells by spinning disc confocal microscopy. We observed that BP100 enters in a stepwise manner and reduces the extent of actin remodeling. This actin ‘freezing’ can be rescued by the natural auxin IAA, and mimicked by the auxin-efflux inhibitor 1-napthylphthalamic acid (NPA). We further tested the role of the membrane-localized NADPH oxidase RboH using the specific inhibitor diphenyl iodonium (DPI), and found that DPI acts antagonistically to BP100, although DPI alone can induce a similar actin ‘freezing’ as well. We propose a working model, where the mild violation of membrane integrity by BP100 stimulates RboH, and the resulting elevated levels of reactive oxygen species interfere with actin dynamicity. The mitigating effect of auxin is explained by competition of auxin- and RboH-triggered signaling for superoxide anions. This self-referring auxin–actin–RboH hub might be essential for integrity sensing.

Flexible nanopillars to regulate cell adhesion and movement

Flexible polymer nanopillar substrates were used to systematically demonstrate cell alignment and migration guided by the directional formation of focal adhesions. The polymer nanopillar substrates were constructed to various height specifications to provide an extensive variation of flexibility; a rectangular arrangement created spatial confinement between adjacent nanopillars, providing less spacing in the horizontal and vertical directions. Three polymer nanopillar substrates with the diameter of 400 nm and the heights of 400, 800, and 1200 nm were fabricated. Super-resolution localization imaging and protein pair–distance analysis of vinculin proteins revealed that Chinese hamster ovary (CHO) cells formed mature focal adhesions on 1200 nm high nanopillar substrates by bending adjacent nanopillars to link dot-like adhesions. The spacing confinement of the adjacent nanopillars enhanced the orthogonal directionality of the formation tendency of the mature focal adhesions. The directional formation of the mature focal adhesions also facilitated the organization of actin filaments in the horizontal and vertical directions. Moreover, 78% of the CHO cells were aligned in these two directions, in conformity with the flexibility and nanotopographical cues of the nanopillars. Biased cell migration was observed on the 1200 nm high nanopillar substrates.

Formin DAAM1 Organizes Actin Filaments in the Cytoplasmic Nodal Actin Network.

A nodal cytoplasmic actin network underlies actin cytoplasm cohesion in the absence of stress fibers. We previously described such a network that forms upon Latrunculin A (LatA) treatment, in which formin DAAM1 was localized at these nodes. Knock down of DAAM1 reduced the mobility of actin nodes but the nodes remained. Here we have investigated DAAM1 containing nodes after LatA washout. DAAM1 was found to be distributed between the cytoplasm and the plasma membrane. The membrane binding likely occurs through an interaction with lipid rafts, but is not required for F-actin assembly. Interesting the forced interaction of DAAM1 with plasma membrane through a rapamycin-dependent linkage, enhanced F-actin assembly at the cell membrane (compared to the cytoplasm) after the LatA washout. However, immediately after addition of both rapamycin and LatA, the cytoplasmic actin nodes formed transiently, before DAAM1 moved to the membrane. This was consistent with the idea that DAAM1 was initially anchored to cytoplasmic actin nodes. Further, photoactivatable tracking of DAAM1 showed DAAM1 was immobilized at these actin nodes. Thus, we suggest that DAAM1 organizes actin filaments into a nodal complex, and such nodal complexes seed actin network recovery after actin depolymerization.

Cortical flow aligns actin filaments to form a furrow.

Cytokinesis in eukaryotic cells is often accompanied by actomyosin cortical flow. Over 30 years ago, Borisy and White proposed that cortical flow converging upon the cell equator compresses the actomyosin network to mechanically align actin filaments. However, actin filaments also align via search-and-capture, and to what extent compression by flow or active alignment drive furrow formation remains unclear. Here, we quantify the dynamical organization of actin filaments at the onset of ring assembly in the C. elegans zygote, and provide a framework for determining emergent actomyosin material parameters by the use of active nematic gel theory. We characterize flow-alignment coupling, and verify at a quantitative level that compression by flow drives ring formation. Finally, we find that active alignment enhances but is not required for ring formation. Our work characterizes the physical mechanisms of actomyosin ring formation and highlights the role of flow as a central organizer of actomyosin network architecture.

Immunohistochemical and electronmicroscopic features of mesenchymal-to-epithelial transition in human developing, postnatal and nephrotic podocytes.

Differentiation of human podocytes starts with mesenchymal-to-epithelial transition (MET) of the metanephric mesenchyme into the S-shaped nephrons. During further development, differentiating podocytes regain mesenchyme-like cell characteristics by epithelial-to-mesenchymal transition (EMT), leading to formation of the terminally differentiated, non-dividing cell. Both MET and EMT processes involve changes in content and organization of cytoskeletal and actin filaments, accompanied by the increased glomerular vascularization. Here, we analyze and compare normal human developing, postnatal and nephrotic podocytes and glomeruli, using immunohistochemical and double immunofluorescent methods for detection of markers of cytoskeletal filaments (nestin, cytokeratin 10-CK10, vimentin and α-SMA), vasculogenesis (CD31 and VEGF) and podocyte function (receptor for advanced glycation end products, RAGE). In addition, electron microscopy is used to detect ultrastructural changes of the podocytes. Early metanephric cup mesenchyme expresses all investigated markers except α-SMA, which characterizes only surface mesenchymal cells. In differentiating podocytes and cells of Bowman's capsule (parietal podocytes) nestin decreases, vimentin increases, while CK10 gradually disappears. Increase in α-SMA is associated with blood vessels development, appearance of podocyte pedicles and slit diaphragm and loss of intercellular connections (zonulae adherentes). Increase in CD31 characterizes vascular glomerular tufts development, while decrease in RAGE expression accompanies normal podocyte differentiation. In congenital nephrotic syndrome of the Finnish type, dedifferentiated podocytes display changes in cytoskeletal filaments and depletion of podocyte pedicles, while glomerular vascular supply is diminished. Our data also suggest high potential of metanephric mesenchyme and parietal podocytes in possible regeneration of the damaged podocytes.

Fascin- and α-Actinin-Bundled Networks Contain Intrinsic Structural Features that Drive Protein Sorting

Cells assemble and maintain functionally distinct actin cytoskeleton networks with various actin filament organizations and dynamics through the coordinated action of different sets of actin-binding proteins. The biochemical and functional properties of diverse actin-binding proteins, both alone and in combination, have been increasingly well studied. Conversely, how different sets of actin-binding proteins properly sort to distinct actin filament networks in the first place is not nearly as well understood. Actin-binding protein sorting is critical for the self-organization of diverse dynamic actin cytoskeleton networks within a common cytoplasm. Using invitro reconstitution techniques including biomimetic assays and single-molecule multi-color total internal reflection fluorescence microscopy, we discovered that sorting of the prominent actin-bundling proteins fascin and α-actinin to distinct networks is an intrinsic behavior, free of complicated cellular signaling cascades. When mixed, fascin and α-actinin mutually exclude each other by promoting their own recruitment and inhibiting recruitment of the other, resulting in the formation of distinct fascin- or α-actinin-bundled domains. Subdiffraction-resolution light microscopy and negative-staining electron microscopy revealed that fascin domains are densely packed, whereas α-actinin domains consist of widely spaced parallel actin filaments. Importantly, other actin-binding proteins such as fimbrin and espin show high specificity between these two bundle types within the same reaction. Here we directly observe that fascin and α-actinin intrinsically segregate to discrete bundled domains that are specifically recognized by other actin-binding proteins.

In vitro cultures of ectodermal monolayers from the model sea anemone Nematostella vectensis.

We report here a novel approach for the extraction, isolation and culturing of intact ectodermal tissue layers from a model marine invertebrate, the sea anemone Nematostella vectensis. A methodology is described in which a brief exposure of the animal to the mucolytic agent N-acetyl-L-cysteine (NAC) solution triggers the dislodging of the ectodermis from its underlying basement membrane and mesoglea. These extracted fragments of cell sheets adherent to culture-dish substrates, initially form 2D monolayers that are transformed within 24h post-isolation into 3D structures. These ectodermal tissues were sustained in vitro for several months, retaining their 3D structure while continuously releasing cells into the surrounding media. Cultures were then used for cell type characterizations and, additionally, the underlying organization of actin filaments in the 3D structures are demonstrated. Incorporation of BrdU and immunohistochemical labeling using p-histone H3 primary antibody were performed to compare mitotic activities of ectodermal cells originating from intact and from in vivo regenerating animals. Results revealed no change in mitotic activities at 2h after bisection and a 1.67-, 1.71- and 3.74-fold increase over 24, 48 and 72h of regeneration, respectively, depicting a significant correlation coefficient (p < 0.05; R 2 = 0.74). A significant difference was found only between the control and 3-day regenerations (p = 0.016). Cell proliferation was demonstrated in the 3D ectodermis after 6 culturing days. Moreover, monolayers that were subjected to Ca++/Mg++ free medium for the first 2h after isolation and then replaced by standard medium, showed, at 6days of culturing, profuse appearance of positive p-histone H3-labeled nuclei in the 3D tissues. Cytochalasin administered throughout the culturing period abolished all p-histone H3 labeling. This study thus depicts novel in vitro tissue culturing of ectodermal layers from a model marine invertebrate, demonstrating the ease with which experiments can be performed and cellular and molecular pathways can be revealed, thus opening studies on 2D tissue organizations and morphogenesis as well as the roles of cellular components in the formation of tissues in this organism.

Influence of cold on organization of actin filaments of different types of root cells in Arabidopsis thaliana

The effect of the low temperature (+4°C) on the organization of actin filaments (microfilaments) of cells from different growth zones has been studied in the roots of Arabidopsis thaliana (L.). It was found that cold treatment inhibited the growth of the primary root and changed its morphology, causing a formation of large number of deformed (ectopic) root hairs in differentiation zone. The temporal relationship between the disorientation and the organization of actin filaments and the detected changes of growth and morphology of roots after cold treatment was shown. It has been found that actin filaments of root hairs, meristematic cells, cells of elongation zone, and epidermal cells of all root zones of A. thaliana are the most sensitive to the cold.

Actin filament-microtubule interactions in axon initiation and branching.

Neurons begin life as spherical cells. A major hallmark of neuronal development is the formation of elongating processes from the cell body which subsequently differentiate into dendrites and the axon. The formation and later development of neuronal processes is achieved through the concerted organization of actin filaments and microtubules. Here, we review the literature regarding recent advances in the understanding of cytoskeletal interactions in neurons focusing on the initiation of processes from neuronal cell bodies and the collateral branching of axons. The complex crosstalk between cytoskeletal elements is mediated by a cohort of proteins that either bind both cytoskeletal systems or allow one to regulate the other. Recent studies have highlighted the importance of microtubule plus-tip proteins in the regulation of the dynamics and organization of actin filaments, while also providing a mechanism for the subcellular capture and guidance of microtubule tips by actin filaments. Although the understanding of cytoskeletal crosstalk and interactions in neuronal morphogenesis has advanced significantly in recent years the appreciation of the neuron as an integrated cytoskeletal system remains a frontier.

Therapeutic innovation: Inflammatory-reactive astrocytes as targets of inflammation

This study aimed to test pharmaceutical compounds targeting astrocytes showing inflammatory dysregulation. The primary rat brain cultures were treated with different batches of serum with or without microglia added to make the cells inflammatory-reactive. Lipopolysaccharide (LPS) and tryptase were used as inflammatory inducers. Expression levels of Toll-like receptor 4 (TLR4), Na+/K+-ATPase, and matrix metalloprotease-13 (MMP-13), as well as actin filament organization, pro-inflammatory cytokines, and intracellular Ca2+ release, were evaluated. LPS combined with tryptase upregulated TLR4 expression, whereas Na+/K+-ATPase expression was downregulated, ATP-evoked Ca2+ transients were increased, actin filaments were reorganized and ring structures instead of stress fibers were observed. Other aims of the study were to prevent astrocytes from becoming inflammatory-reactive and to restore inflammatory dysregulated cellular changes. A combination of the μ-opioid antagonist (−)-naloxone in ultra-low concentrations, the non-addictive μ-opioid agonist (−)-linalool, and the anti-epileptic agent levetiracetam was examined. The results indicated that this drug cocktail prevented the LPS- and tryptase-induced inflammatory dysregulation. The drug cocktail could also restore the LPS- and tryptase-treated cells back to a normal physiological level in terms of the analyzed parameters.

Quantitative Evaluation of Stomatal Cytoskeletal Patterns during the Activation of Immune Signaling in Arabidopsis thaliana.

Historically viewed as primarily functioning in the regulation of gas and water vapor exchange, it is now evident that stomata serve an important role in plant immunity. Indeed, in addition to classically defined functions related to cell architecture and movement, the actin cytoskeleton has emerged as a central component of the plant immune system, underpinning not only processes related to cell shape and movement, but also receptor activation and signaling. Using high resolution quantitative imaging techniques, the temporal and spatial changes in the actin microfilament array during diurnal cycling of stomatal guard cells has revealed a highly orchestrated transition from random arrays to ordered bundled filaments. While recent studies have demonstrated that plant stomata close in response to pathogen infection, an evaluation of stimulus-induced changes in actin cytoskeletal dynamics during immune activation in the guard cell, as well as the relationship of these changes to the function of the actin cytoskeleton and stomatal aperture, remains undefined. In the current study, we employed quantitative cell imaging and hierarchical clustering analyses to define the response of the guard cell actin cytoskeleton to pathogen infection and the elicitation of immune signaling. Using this approach, we demonstrate that stomatal-localized actin filaments respond rapidly, and specifically, to both bacterial phytopathogens and purified pathogen elicitors. Notably, we demonstrate that higher order temporal and spatial changes in the filament array show distinct patterns of organization during immune activation, and that changes in the naïve diurnal oscillations of guard cell actin filaments are perturbed by pathogens, and that these changes parallel pathogen-induced stomatal gating. The data presented herein demonstrate the application of a highly tractable and quantifiable method to assign transitions in actin filament organization to the activation of immune signaling in plants.

The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites

Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics.

Simulated Microgravity Disrupts Cytoskeleton Organization and Increases Apoptosis of Rat Neural Crest Stem Cells Via Upregulating CXCR4 Expression and RhoA-ROCK1-p38 MAPK-p53 Signaling.

Neural crest stem cells (NCSCs) are a population of multipotent stem cells that are distributed broadly in many tissues and organs and are capable of differentiating into a variety of cell types that are dispersed throughout three germ layers. We are interested in studying the effects of simulated microgravity on the survival and self-renewal of NCSCs. NCSCs extracted from the hair follicle bulge region of the rat whisker pad were cultured in vitro, respectively, in a 2D adherent environment and a 3D suspension environment using the rotatory cell culture system (RCCS) to simulate microgravity. We found that rat NCSCs (rNCSCs) cultured in the RCCS for 24 h showed disrupted organization of filamentous actin, increased globular actin level, formation of plasma membrane blebbing and neurite-like artifact, as well as decreased levels of cortactin and vimentin. Interestingly, ∼70% of RCCS-cultured rNCSCs co-expressed cleaved (active) caspase-3 and neuronal markers microtubule-associated protein 2 (MAP2) and Tuj1 instead of NCSC markers, suggesting stress-induced formation of neurite-like artifact in rNCSCs. In addition, rNCSCs showed increased C-X-C chemokine receptor 4 (CXCR4) expression, RhoA GTPase activation, Rho-associated kinase 1 (ROCK1) and p38 mitogen-activated protein kinase (MAPK) phosphorylation, and p53 expression in the nucleus. Incubation of rNCSCs with the Gα protein inhibitor pertussis toxin or CXCR4 siRNA during RCCS-culturing prevented cytoskeleton disorganization and plasma membrane blebbing, and it suppressed apoptosis of rNCSCs. Taken together, we demonstrate for the first time that simulated microgravity disrupts cytoskeleton organization and increases apoptosis of rNCSCs via upregulating CXCR4 expression and the RhoA-ROCK1-p38 MAPK-p53 signaling pathway.