Imbalance between Actin Isoforms Contributes to Tumour Progression in Taxol-Resistant Triple-Negative Breast Cancer Cells.

The widespread occurrence of breast cancer and its propensity to develop drug resistance highlight the need for a comprehensive understanding of the molecular mechanisms involved. This study investigates the intricate pathways associated with secondary resistance to taxol in triple-negative breast cancer (TNBC) cells, with a particular focus on the changes observed in the cytoplasmic actin isoforms. By studying a taxol-resistant TNBC cell line, we revealed a shift between actin isoforms towards γ-actin predominance, accompanied by increased motility and invasive properties. This was associated with altered tubulin isotype expression and reorganisation of the microtubule system. In addition, we have shown that taxol-resistant TNBC cells underwent epithelial-to-mesenchymal transition (EMT), as evidenced by Twist1-mediated downregulation of E-cadherin expression and increased nuclear translocation of ß-catenin. The RNA profiling analysis revealed that taxol-resistant cells exhibited significantly increased positive regulation of cell migration, hormone response, cell-substrate adhesion, and actin filament-based processes compared with naïve TNBC cells. Notably, taxol-resistant cells exhibited a reduced proliferation rate, which was associated with an increased invasiveness in vitro and in vivo, revealing a complex interplay between proliferative and metastatic potential. This study suggests that prolonged exposure to taxol and acquisition of taxol resistance may lead to pro-metastatic changes in the TNBC cell line.

Actin-Dependent Mechanism of Tumor Progression Induced by a Dysfunction of p53 Tumor Suppressor.

Cancer cell aggressiveness, marked by actin cytoskeleton reconfiguration critical for metastasis, may result from an imbalanced ratio favoring γ-actin. Dysfunctional p53 emerges as a key regulator of invasiveness and migration in various cancer cells, both in vitro and in vivo. P53 inactivation (via mutants R175H, R248W, R273H, or TP53 repression) significantly enhanced the migration, invasion, and proliferation of human lung adenocarcinoma A549 cells in vitro and in vivo, facilitating intrapulmonary xenograft metastasis in athymic mice. Conversely, wild-type TP53 (TP53 WT) overexpression in p53-deficient non-small- cell lung cancer (NSCLC) H1299 cells substantially reduced proliferation and migration in vitro, effectively curbing orthotopic tumorigenicity and impeding in vivo metastasis. These alterations in cell motility were closely associated with actin cytoskeleton restructuring, favoring γ-actin, and coincided with ERK1/2-mediated signaling activation, unveiling an innovative regulatory mechanism in malignancy progression. Cancer cell aggressiveness, driven by actin cytoskeleton reorganization and a shift towards γ-actin predominance, may be regulated by p53 dysfunction, thereby providing novel insight into tumor progression mechanisms.

Cingulin and paracingulin tether myosins-2 to junctions to mechanoregulate the plasma membrane.

The mechanisms that regulate the spatial sorting of nonmuscle myosins-2 (NM2) isoforms and couple them mechanically to the plasma membrane are unclear. Here we show that the cytoplasmic junctional proteins cingulin (CGN) and paracingulin (CGNL1) interact directly with NM2s through their C-terminal coiled-coil sequences. CGN binds strongly to NM2B, and CGNL1 to NM2A and NM2B. Knockout (KO), exogenous expression, and rescue experiments with WT and mutant proteins show that the NM2-binding region of CGN is required for the junctional accumulation of NM2B, ZO-1, ZO-3, and phalloidin-labeled actin filaments, and for the maintenance of tight junction membrane tortuosity and apical membrane stiffness. CGNL1 expression promotes the junctional accumulation of both NM2A and NM2B and its KO results in myosin-dependent fragmentation of adherens junction complexes. These results reveal a mechanism for the junctional localization of NM2A and NM2B and indicate that, by binding to NM2s, CGN and CGNL1 mechanically couple the actomyosin cytoskeleton to junctional protein complexes to mechanoregulate the plasma membrane.

The Role of Aurora B Kinase in Normal and Cancer Cells.

Aurora kinases are essential players in mammalian cell division. These kinases are involved in the regulation of spindle dynamics, microtubule-kinetochore interactions, and chromosome condensation and orientation during mitosis. At least three members of the Aurora family - Aurora kinases A, B, and C - have been identified in mammals. Aurora B is essential for maintaining genomic stability and normal cell division. Mutations and dysregulation of this kinase are implicated in tumor initiation and progression. In this review, we discuss the functions of Aurora B, the relationship between increased Aurora B activity and carcinogenesis, and the prospects for the use of Aurora B kinase inhibitors in antitumor therapy.

Significance of NOTCH1 Expression in the Progression of Human Lung and Colorectal Cancers.

Lung and colorectal cancers are the most common types of cancer characterized by a poor prognosis and a high mortality rate. Mutations in the genes encoding components of the main intra- and extracellular signaling pathways, in particular the NOTCH1 gene (Notch1, a member of the Notch family of receptors), play one of the key roles in progression of these malignancies. Notch signaling is involved in maintaining homeostasis of the intestinal epithelium and structural and functional lung elements. Therefore, it is not surprising that the constitutive activity and hyperactivity of Notch signaling due to somatic mutations in genes coding for the products directly involved into its activation, could lead to the progression of these cancer types. The aim of our study was to investigate how the NOTCH1 downregulation via RNA interference (RNAi) affects the phenotype, characteristics, and Notch-dependent signaling of human A549 lung and HCT116 colorectal carcinoma cells. Several small harpin RNAs (shRNAs) were selected using the bioinformatic analysis and tested for their ability to suppress the NOTCH1 expression. The most efficient one was used to produce the A549 and HCT116 cells with NOTCH1 knockdown. The obtained cell lines demonstrated decreased proliferation rates, reduced colony-forming capacity under adhesive conditions, and decreased migration activity in a Boyden chamber. The NOTCH1 knockdown also significantly decreased expression of some Notch signaling target genes potentially involved in the acquisition and maintenance of more invasive and malignant cell phenotype. In vivo experiments in immunodeficient athymic female Balb/c nu/nu mice confirmed the results obtained in vitro: the NOTCH1 inhibition decreased the growth rates of the subcutaneous xenografts formed by A549 and HCT116 tumor cells. Therefore, downregulation of the gene encoding the Notch1 receptor potentially reduces malignant characteristics of human lung and colorectal carcinoma cells.

The Cytoplasmic Actins in the Regulation of Endothelial Cell Function.

The primary function of the endothelial cells (EC) lining the inner surface of all vessels is to regulate permeability of vascular walls and to control exchange between circulating blood and tissue fluids of organs. The EC actin cytoskeleton plays a crucial role in maintaining endothelial barrier function. Actin cytoskeleton reorganization result in EC contraction and provides a structural basis for the increase in vascular permeability, which is typical for many diseases. Actin cytoskeleton in non-muscle cells presented two actin isoforms: non-muscle ß-cytoplasmic and γ-cytoplasmic actins (ß-actins and γ-actins), which are encoded by ACTB and ACTG1 genes, respectively. They are ubiquitously expressed in the different cells in vivo and in vitro and the ß/γ-actin ratio depends on the cell type. Both cytoplasmic actins are essential for cell survival, but they perform various functions in the interphase and cell division and play different roles in neoplastic transformation. In this review, we briefly summarize the research results of recent years and consider the features of the cytoplasmic actins: The spatial organization in close connection with their functional activity in different cell types by focusing on endothelial cells.

Impaired Expression of Cytoplasmic Actins Leads to Chromosomal Instability of MDA-MB-231 Basal-Like Mammary Gland Cancer Cell Line.

We have shown previously that two cytoplasmic actin isoforms play different roles in neoplastic cell transformation. Namely, ß-cytoplasmic actin acts as a tumor suppressor, whereas γ-cytoplasmic actin enhances malignant features of tumor cells. The distinct participation of each cytoplasmic actin in the cell cycle driving was also observed. The goal of this study was to describe the diverse roles of cytoplasmic actins in the progression of chromosomal instability of MDA-MB-231 basal-like human carcinoma cell line. We performed traditional methods of chromosome visualization, as well as 3D-IF microscopy and western blotting for CENP-A detection/quantification, to investigate chromosome morphology. Downregulation of cytoplasmic actin isoforms alters the phenotype and karyotype of MDA-MB-231 breast cancer cells. Moreover, ß-actin depletion leads to the progression of chromosomal instability with endoreduplication and aneuploidy increase. On the contrary, γ-actin downregulation results not only in reduced percentage of mitotic carcinoma cells, but leads to chromosome stability, reduced polyploidy, and aneuploidy.

Inhibition of FGF2-Mediated Signaling in GIST-Promising Approach for Overcoming Resistance to Imatinib.

Inhibition of KIT-signaling is a major molecular target for gastrointestinal stromal tumor (GIST) therapy, and imatinib mesylate (IM) is known as the most effective first-line treatment option for patients with advanced, unresectable, and/or metastatic GISTs. We show here for the first time that the inhibition of KIT-signaling in GISTs induces profound changes in the cellular secretome, leading to the release of multiple chemokines, including FGF-2. IM increased migration, invasion, and colony formation of IM-resistant GISTs in an FGF2-dependent manner, whereas the use of blocking anti-FGF2 antibodies or BGJ398, a selective FGFR inhibitor, abolished these effects, thus suggesting that the activation of FGF2-mediated signaling could serve as a compensatory mechanism of KIT-signaling inhibited in GISTs. Conversely, FGF-2 rescued the growth of IM-naive GISTs treated by IM and protected them from IM-induced apoptosis, consistent with the possible involvement of FGF-2 in tumor response to IM-based therapy. Indeed, increased FGF-2 levels in serum and tumor specimens were found in IM-treated mice bearing IM-resistant GIST xenografts, whereas BGJ398 used in combination with IM effectively inhibited their growth. Similarly, increased FGF-2 expression in tumor specimens from IM-treated patients revealed the activation of FGF2-signaling in GISTs in vivo. Collectively, the continuation of IM-based therapy for IM-resistant GISTs might facilitate disease progression by promoting the malignant behavior of tumors in an FGF2-dependent manner. This provides a rationale to evaluate the effectiveness of the inhibitors of FGF-signaling for IM-resistant GISTs.

Drug Targeting the Actin Cytoskeleton Potentiates the Cytotoxicity of Low Dose Vincristine by Abrogating Actin-Mediated Repair of Spindle Defects.

Antimicrotubule vinca alkaloids are widely used in the clinic but their toxicity is often dose limiting. Strategies that enhance their effectiveness at lower doses are needed. We show that combining vinca alkaloids with compounds that target a specific population of actin filaments containing the cancer-associated tropomyosin Tpm3.1 result in synergy against a broad range of tumor cell types. We discovered that low concentrations of vincristine alone induce supernumerary microtubule asters that form transient multi-polar spindles in early mitosis. Over time these asters can be reconstructed into functional bipolar spindles resulting in cell division and survival. These microtubule asters are organized by the nuclear mitotic apparatus protein (NuMA)-dynein-dynactin complex without involvement of centrosomes. However, anti-Tpm3.1 compounds at nontoxic concentrations inhibit this rescue mechanism resulting in delayed onset of anaphase, formation of multi-polar spindles, and apoptosis during mitosis. These findings indicate that drug targeting actin filaments containing Tpm3.1 potentiates the anticancer activity of low-dose vincristine treatment. IMPLICATIONS: Simultaneously inhibiting Tpm3.1-containing actin filaments and microtubules is a promising strategy to potentiate the anticancer activity of low-dose vincristine.

Divergent impact of actin isoforms on cell cycle regulation.

We have shown that cytoplasmic actin isoforms play different roles in neoplastic cell transformation. ß-Cytoplasmic actin acts as a tumor suppressor, affecting epithelial differentiation, cell growth, cell invasion and tumor growth of colon and lung carcinoma cells. In contrast, γ-cytoplasmic actin enhances malignant features of tumor cells whose actin network regulation is carried out via the γ-actin isoform. The goal of this study was to describe the role of cytoplasmic actins in cell cycle regulation of breast cancer cell lines MCF-7 and MDA-MB-231. The distinct roles of each cytoplasmic actin in the cell cycle driving were observed. ß-Actin as well as γ-actin down-regulation inhibited proliferation of breast cancer cells, but only down-regulation of ß-actin induced a significant decrease in diploid cell population and accumulation of tetraploid cells. Down-regulation of ß-actin stimulated cyclin A2, B1 and D3 expression, whereas down-regulation of γ-actin reduced expression of these cyclins in both cell lines. Moreover, cyclin B1 and γ-actin were co-localized in mitotic control and ß-actin-deficient cells. In mitotic MCF-7 cells down-regulation of ß-actin caused an enrichment of prophase/metaphase population compared with control. γ-Actin down-regulation induced telophase enrichment. ERK1/2 and γ-actin co-localization and possible selective binding were revealed in MCF7 cells. ß-Actin down-regulation induced ERK1/2 activation, while γ-actin down-regulation led to reduction of p-ERK1/2. A direct interaction of ERK1/2 with γ-actin and cyclin A2 in the same protein complex was also discovered. We suggest that γ-actin down-regulation leads to decrease of cyclin A2 level, inhibits ERK1/2 signaling and deceleration of breast cancer cells proliferation.

Mitochondria-targeted antioxidant SkQ1 suppresses fibrosarcoma and rhabdomyosarcoma tumour cell growth.

Mitochondria are important regulators of tumour growth and progression due to their specific role in cancer metabolism and modulation of apoptotic pathways. In this paper we describe that mitochondria-targeted antioxidant SkQ1 designed as a conjugate of decyl-triphenylphosphonium cation (TPP+) with plastoquinone, suppressed the growth of fibrosarcoma HT1080 and rhabdomyosarcoma RD tumour cells in culture and tumour growth of RD in xenograft nude mouse model. Under the same conditions, no detrimental effect of SkQ1 on cell growth of primary human subcutaneous fibroblasts was observed. The tumour growth suppression was shown to be a result of the antioxidant action of low nanomolar concentrations of SkQ1. We have revealed significant prolongation of mitosis induced by SkQ1 in both tumour cell cultures. Prolonged mitosis and apoptosis could be responsible for growth suppression after SkQ1 treatment in RD cells. Growth suppression in HT1080 cells was accompanied by the delay of telophase and cytokinesis, followed by multinuclear cells formation. The effects of SkQ1 on the cell cycle were proved to be at least partially mediated by inactivation of Aurora family kinases. ABBREVIATIONS: TPP+: Triphenylphosphonium cation; ROS: Reactive oxygen species; mtROS: Mitochondrial reactive oxygen species; NAC: N-acetyl-L-cysteine; DCFH-DA: Dichlorodihydrofluorescein diacetate; APC: Anaphase promoting complex; ABPs: Actin-binding proteins; DMEM: Dulbecco's modified Eagle media; SDS: sodium dodecyl sulfate; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.

Identification of Cancer-Targeted Tropomyosin Inhibitors and Their Synergy with Microtubule Drugs.

Actin filaments, with their associated tropomyosin polymers, and microtubules are dynamic cytoskeletal systems regulating numerous cell functions. While antimicrotubule drugs are well-established, antiactin drugs have been more elusive. We previously targeted actin in cancer cells by inhibiting the function of a tropomyosin isoform enriched in cancer cells, Tpm3.1, using a first-in-class compound, TR100. Here, we screened over 200 other antitropomyosin analogues for anticancer and on-target activity using a series of in vitro cell-based and biochemical assays. ATM-3507 was selected as the new lead based on its ability to disable Tpm3.1-containing filaments, its cytotoxicity potency, and more favorable drug-like characteristics. We tested ATM-3507 and TR100 alone and in combination with antimicrotubule agents against neuroblastoma models in vitro and in vivo Both ATM-3507 and TR100 showed a high degree of synergy in vitro with vinca alkaloid and taxane antimicrotubule agents. In vivo, combination-treated animals bearing human neuroblastoma xenografts treated with antitropomyosin combined with vincristine showed minimal weight loss, a significant and profound regression of tumor growth and improved survival compared with control and either drug alone. Antitropomyosin combined with vincristine resulted in G2-M phase arrest, disruption of mitotic spindle formation, and cellular apoptosis. Our data suggest that small molecules targeting the actin cytoskeleton via tropomyosin sensitize cancer cells to antimicrotubule agents and are tolerated together in vivo This combination warrants further study. Mol Cancer Ther; 16(8); 1555-65. ©2017 AACR.

Depletion of mitochondrial reactive oxygen species downregulates epithelial-to-mesenchymal transition in cervical cancer cells.

In the course of cancer progression, epithelial cells often acquire morphological and functional characteristics of mesenchymal cells, a process known as epithelial-to-mesenchymal transition (EMT). EMT provides epithelial cells with migratory, invasive, and stem cell capabilities. Reactive oxygen species produced by mitochondria (mtROS) could be of special importance for pro-tumorigenic signaling and EMT.In our study, we used mitochondria-targeted antioxidant SkQ1 to lower the mtROS level and analyze their role in the regulation of the actin cytoskeleton, adhesion junctions, and signaling pathways critical for tumorigenesis of cervical carcinomas. A decrease in mtROS was found to induce formation of ß-cytoplasmic actin stress fibers and circumferential rings in cervical cancer SiHa and Ca-Ski cells. It was accompanied by an upregulation of E-cadherin in SiHa cells and a downregulation of N-cadherin in Ca-Ski cells. In SiHa cells, an increase in E-cadherin expression was accompanied by a reduction of Snail, E-cadherin negative regulator. A stimulation of mtROS by epidermal growth factor (EGF) caused a Snail upregulation in SiHa cells that could be downregulated by SkQ1. SkQ1 caused a decrease in activation of extracellular-signal-regulated kinases 1 and 2 (ERK1/2) in SiHa and Ca-Ski. EGF produced an opposite effect. Incubation with SkQ1 suppressed EGF-induced p-ERK1/2 upregulation in SiHa, but not in Ca-Ski cells. Thus, we showed that scavenging of mtROS by SkQ1 initiated reversal of EMT and suppressed proliferation of cervical cancer cells.

Interaction of microtubules with the actin cytoskeleton via cross-talk of EB1-containing +TIPs and γ-actin in epithelial cells.

Actin microfilaments and microtubules are both highly dynamic cytoskeleton components implicated in a wide range of intracellular processes as well as cell-cell and cell-substrate interactions. The interactions of actin filaments with the microtubule system play an important role in the assembly and maintenance of 3D cell structure. Here we demonstrate that cytoplasmic actins are differentially distributed in relation to the microtubule system. LSM, 3D-SIM, proximity ligation assay (PLA) and co-immunoprecipitation methods applied in combination with selective depletion of ß- or γ-cytoplasmic actins revealed a selective interaction between microtubules and γ-, but not ß-cytoplasmic actin via the microtubule +TIPs protein EB1. EB1-positive comet distribution analysis and quantification have shown more effective microtubule growth in the absence of ß-actin. Our data represent the first demonstration that microtubule +TIPs protein EB1 interacts mainly with γ-cytoplasmic actin in epithelial cells.

Stable incorporation of α-smooth muscle actin into stress fibers is dependent on specific tropomyosin isoforms.

α-Smooth Muscle Actin (α-SMA), a widely characterized cytoskeletal protein, represents the hallmark of myofibroblast differentiation. Transforming growth factorß1 (TGFß1) stimulates α-SMA expression and incorporation into stress fibers, thus providing an increased myofibroblast contractile force that participates in tissue remodeling. We have addressed the molecular mechanism by which α-SMA is stably incorporated into stress fibers in human myofibroblasts following exposure to TGFß1. The unique N-terminal sequence AcEEED, which is critical for α-SMA incorporation into stress fibers, was used to screen for AcEEED binding proteins. Tropomyosins were identified as candidate binding proteins. We find that after TGFß1 treatment elevated levels of the Tpm1.6/7 isoforms, and to a lesser extent Tpm2.1, precede the increase in α-SMA. RNA interference experiments demonstrate that α-SMA fails to stably incorporate into stress fibers of TGFß1 treated fibroblasts depleted of Tpm1.6/7, but not other tropomyosins. This does not appear to be due to exclusive interactions between α-SMA and just the Tpm1.6/7 isoforms. We propose that an additional AcEEED binding factor may be required to generate α-SMA filaments containing just Tpm1.6/7 which result in stable incorporation of the resulting filaments into stress fibers.

Tumor promotion by γ and suppression by ß non-muscle actin isoforms.

Here we have shown that ß-cytoplasmic actin acts as a tumor suppressor, inhibiting cell growth and invasion in vitro and tumor growth in vivo. In contrast, γ-cytoplasmic actin increases the oncogenic potential via ERK1/2, p34-Arc, WAVE2, cofilin1, PP1 and other regulatory proteins. There is a positive feedback loop between γ-actin expression and ERK1/2 activation. We conclude that non-muscle actin isoforms should not be considered as merely housekeeping proteins and the ß/γ-actins ratio can be used as an oncogenic marker at least for lung and colon carcinomas. Agents that increase ß- and/or decrease γ-actin expression may be useful for anticancer therapy.

Loss of γ-cytoplasmic actin triggers myofibroblast transition of human epithelial cells.

Transdifferentiation of epithelial cells into mesenchymal cells and myofibroblasts plays an important role in tumor progression and tissue fibrosis. Such epithelial plasticity is accompanied by dramatic reorganizations of the actin cytoskeleton, although mechanisms underlying cytoskeletal effects on epithelial transdifferentiation remain poorly understood. In the present study, we observed that selective siRNA-mediated knockdown of γ-cytoplasmic actin (γ-CYA), but not ß-cytoplasmic actin, induced epithelial-to-myofibroblast transition (EMyT) of different epithelial cells. The EMyT manifested by increased expression of α-smooth muscle actin and other contractile proteins, along with inhibition of genes responsible for cell proliferation. Induction of EMyT in γ-CYA-depleted cells depended on activation of serum response factor and its cofactors, myocardial-related transcriptional factors A and B. Loss of γ-CYA stimulated formin-mediated actin polymerization and activation of Rho GTPase, which appear to be essential for EMyT induction. Our findings demonstrate a previously unanticipated, unique role of γ-CYA in regulating epithelial phenotype and suppression of EMyT that may be essential for cell differentiation and tissue fibrosis.

Smooth muscle actin isoforms: a tug of war between contraction and compliance.

In higher vertebrates, smooth muscle (SM) contains two tissue-specific actin isoforms: α-SMA and γ-SMA, which predominate in vascular and visceral SM, respectively. Whether α-SMA has been extensively studied and recognized for its contractile activity in SM and SM-like cells such as myofibroblasts, myoepithelial and myoid cells, the distribution and role of γ-SMA remained largely unknown. We developed a new specific monoclonal antibody against γ-SMA and confirmed that γ-SMA predominates in the visceral system and is minor in the vascular system, although more expressed in highly compliant veins than in stiff arteries. Contrary to α-SMA, γ-SMA is absent from myofibroblasts in vitro, and in fibrotic diseases in vivo. We raised the hypothesis that, whereas α-SMA is responsible for the "contractile" activity, γ-SMA would be involved in the "compliance" of SM and SM-like cells. Several models support this hypothesis, namely veins vs. arteries and the physiological modifications occurring in the uterus and mammary glands during pregnancy and lactation. Our results suggest that, in addition to enteric smooth muscles, γ-SMA is expressed in all the tissues submitted to an important dilation including veins, gravid uterus, and lactating mammary glands. The hypothesis of two complementary mechanical roles for the two SMA isoforms is sustained by their different intracellular distributions and by functional assays.

In search of novel highly active mitochondria-targeted antioxidants: thymoquinone and its cationic derivatives.

Since the times of the Bible, an extract of black cumin seeds was used as a medicine to treat many human pathologies. Thymoquinone (2-demethylplastoquinone derivative) was identified as an active antioxidant component of this extract. Recently, it was shown that conjugates of plastoquinone and penetrating cations are potent mitochondria-targeted antioxidants effective in treating a large number of age-related pathologies. This review summarizes new data on the antioxidant and some other properties of membrane-penetrating cationic compounds where 2-demethylplastoquinone substitutes for plastoquinone. It was found that such a substitution significantly increases a window between anti- and prooxidant concentrations of the conjugates. Like the original plastoquinone derivatives, the novel compounds are easily reduced by the respiratory chain, penetrate through model and natural membranes, specifically accumulate in mitochondria in an electrophoretic fashion, and strongly inhibit H2O2-induced apoptosis at pico- and nanomolar concentrations in cell cultures. At present, cationic demethylplastoquinone derivatives appear to be the most promising mitochondria-targeted drugs of the quinone series.

Cooperation between ß- and γ-cytoplasmic actins in the mechanical regulation of endothelial microparticle formation.

Elevated endothelial microparticle (MP) levels are observed in numerous diseases, increasingly supporting roles as effectors and valuable markers of vascular dysfunction. While a contractile role for the actin cytoskeleton has been implicated in vesiculation, i.e., MP production, the precise interactions and mechanisms of its constituents, ß- and γ-cytoplasmic actins, is unknown. Human cerebral microvascular endothelial cells were stimulated with known agonists, and vesiculation development was monitored by scanning electron microscopy (SEM) and flow cytometry. These data in combination provide new insight into the kinetics, patterns of vesiculating cell recruitment, and degrees of response specific to stimuli. Reorganization of ß- and γ-actins, F-actin, vinculin, and talin accompanied significant MP release. ß-Actin redistribution into basal stress fibers following stimulation was associated with increased apically situated actin-rich particulate structures, which in turn directly correlated with electron-lucent membrane protrusions observed by SEM. Y-27632 Rho-kinase inhibition abolished basal ß-actin fiber formation, minimizing apically associated actin-rich structures, significantly reducing membrane protrusions and MP release to near basal levels. Cytoskeletal protein expression and distribution varied between MPs and mother cells, as determined by Western blot. These data strongly suggest that ß-actin plays an active facilitative role in agonist-induced protuberance formation, through mechanical interactions with newly described actin-rich structures.