Cytochemical dopamine visualization as a method for estimation of globular actin content in cytosol of living cells

The possibility to reveal globular (G-) actin in cell cytosole by means of microscopy has been studied. The applicability of this method was in particular evaluated for diagnostics of malignant cells, whose main pathocytological feature is an anomalously high content of G-actin in cytosol. The cells of a common origin but with different states of cytosolic actin were analyzed by means of cytochemical reaction for biogenic amines using Falck-Hillarp method after 40-h incubation of the cells in dopamine-containing cultivation medium. Mouse embryo cell line BALB/3T3, clone A31 with differentiated actin cytoskeleton were used as a control cell line. The same cells infected with pathogenic virus SV-40 (cell line 3T3B-SV40) exhibited a malignant phenotype; their cytosol mainly consisted of G-actin. Manifold increase in fluorescence intensity of cytosol and karyoplasms, the loci with the highest G-actin concentration, was revealed in malignant cells in comparison with their healthy prototype. Thus, it was shown that G-actin of malignant cells is a diagnostic target for dopamine, which, as it was earlier shown, penetrates into the cytosol, polymerizes G-actin, incorporating into the filaments as integral component in the 100:1 ratio, and thus fluorescently labels G-actin due to conversion into isoquinoline by reaction with formaldehyde. Besides, dopamine exhibited a strong cytotoxicity that considerably reduced the viability of malignant cells. The data suggest that the content of Gactin in cytosol of living cells can be quantitatively estimated by fluorescence intensity of cytosol following incubation of the cells in dopamine-containing medium.     

Cytoskeletal regulation of the cellular function by dopamine

The interaction of dopamine with model membranes, isolated G-actin, and living cells, such as Mauthner neurons and fibroblast-like BHK-21 cells has been studied. It was found that in vitro dopamine passes through the phospholipid membrane and directly polymerizes G-actin due to incorporation into threads as their integral part. In in vivo conditions, it penetrates inside the cell and induces the appearance of a network of actin filaments in loci rich in globular actin. The data suggest that there exists a mechanism of dopamine interaction with living cells, which is based on direct polymerization of cytosolic G-actin as its cellular target. The reorganization of the actin cytoskeleton leads to changes in the morphofunctional status of cells.     

超短脉冲技术测量癌细胞荧光光谱与荧光寿命

研究用于癌症诊断与治疗的光敏剂血卟啉（hematoporphyrin derivative,HPD）的超快光动力学过程,采用超短脉冲激光光谱技术和皮秒时间相关单光子计数系统,测量经血卟啉培养的活体癌细胞与正常细胞的荧光光谱、荧光寿命特性及荧光峰值强度随时间变化曲线,并测量单一细胞内部不同位置的荧光寿命特性,观测到：癌细胞样品在645 nm处具有特有的光谱谱峰;癌细胞样品荧光寿命的快成分约150 ps慢成分约1200 ps,而正常细胞样品快成分约300 ps慢成分约2500 ps;癌细胞样品的荧光峰值强度经12小时衰减约10%,而正常细胞样品衰减约55%;在细胞内部荧光寿命300 ps的快成分十分显著,且中心部位血卟啉浓度最高.癌细胞与正常细胞的荧光光谱、荧光寿命特性及荧光峰值强度随时间变化曲线相差十分明显,反映了癌细胞与正常细胞对血卟啉亲和特性有显著的差异,测量结果确认了荧光光谱技术诊断与治疗癌症的可行性,并对发展超短脉冲激光光谱技术早期诊断与治疗癌症具有重要的指导意义和临床应用价值.     

Calreticulin attenuated microwave radiation-induced human microvascular endothelial cell injury through promoting actin acetylation and polymerization

Recent work reveals that actin acetylation modification has been linked to different normal and disease processes and the effects associated with metabolic and environmental stressors. Herein, we highlight the effects of calreticulin on actin acetylation and cell injury induced by microwave radiation in human microvascular endothelial cell (HMEC). HMEC injury was induced by high-power microwave of different power density (10, 30, 60, 100 mW/cm², for 6 min) with or without exogenous recombinant calreticulin. The cell injury was assessed by lactate dehydrogenase (LDH) activity and Cell Counting Kit-8 in culture medium, migration ability, intercellular junction, and cytoskeleton staining in HMEC. Western blotting analysis was used to detected calreticulin expression in cytosol and nucleus and acetylation of globular actin (G-actin). We found that HMEC injury was induced by microwave radiation in a dose-dependent manner. Pretreatment HMEC with calreticulin suppressed microwave radiation-induced LDH leakage and increased cell viability and improved microwave radiation-induced decrease in migration, intercellular junction, and cytoskeleton. Meanwhile, pretreatment HMEC with exogenous calreticulin upregulated the histone acetyltransferase activity and the acetylation level of G-actin and increased the fibrous actin (F-actin)/G-actin ratio. We conclude that exogenous calreticulin protects HMEC against microwave radiation-induced injury through promoting actin acetylation and polymerization.     

Tolrestat对高糖所致肾小球系膜细胞肌动蛋白去组装的影响

研究了醛糖还原酶抑制剂Tolrestat对高浓度葡萄糖(HG)所致肾小球系膜细胞(MC)肌动蛋白(actin)组装的影响。结果证明,与正常浓度葡萄糖(NG)相比,在HG培养的MC,F-actin失去束状外观呈不规则网状,显示F-actin部分去组装;F-actin荧光强度降低,G-actin荧光强度升高和F-/G-actin荧光强度比值下降。Tolrestat加入培养后,明显防止HG引起的F-actin去组装及F-和G-actin荧光强度的变化。提示多元醇通路激活在HG引起的MCactin去组装改变中起一定作用。     

An unexplained sequestration of latrunculin A is required in neutrophils for inhibition of actin polymerization

Latrunculin A (LatA) is a toxic natural product that causes disruption of the actin cytoskeleton in many eukaryotic cells at submicromolar concentrations. LatA has been found to bind G-actin with a dissociation constant of 0.2 microM, and more recently to bind profilin-G-actin and, weakly, thymosin beta4-G-actin. A number of investigators have used LatA as a G-actin sequestering agent. Thus, we studied neutrophil chemotaxis and its requisite conversion of G-actin to F-actin, supported by an extensive pool of G-actin, mainly bound to thymosin beta4. Calculations suggest that the affinity of LatA is insufficient to cause significant sequestration of this pool, and the pool's buffering action should protect neutrophils from depletion of productive G-actin species by submicromolar LatA. Nonetheless, we found that both chemoattractant stimulated migration and F-actin polymerization in neutrophils were inhibited by LatA at these concentrations. The latter effect was accompanied by sequestration of LatA and showed a cell density dependence that was consistent with G-actin sequestration. The apparent contradiction between the calculations and the experimental observations could be reconciled by assuming the presence of an accessory species, of unknown normal function, which forms a high affinity ternary complex with LatA and G-actin, thus causing the cells to concentrate LatA. Other models that could not be ruled out also invoke new actions of LatA, suggesting caution in the interpretation of its effects on cells.     

Characterization of endogenous fluorescence in nonsmall lung cancerous cells: A comparison with nonmalignant lung normal cells

Monitoring fluorescence properties of endogenous fluorophores such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in normal and cancerous cells provide substantial information noninvasively on biochemical and biophysical aspects of metabolic dysfunction of cancerous cells. Time‐resolved spectral profiles and fluorescence lifetime images of NADH and FAD were obtained in human lung nonsmall carcinomas (H661 and A549) and normal lung cells (MRC‐5). Both fluorophores show the fast and slowly decaying emission components upon pulsed excitation, and fluorescence spectra of NADH and FAD show blue‐ and red‐shifts, respectively, during their decay. All identified lifetime components of NADH and FAD were found to be shorter in cancerous cells than in normal cells, no matter how they were measured under different extra‐cellular conditions (cells suspended in cuvette and cells attached on glass substrate), indicating that the changes in metabolism likely altered the subcellular milieu and potentially also affected the interaction of NADH and FAD with enzymes to which these cofactors were bound. The intensity ratio of NADH and FAD of cancerous cells was also shown to be larger than that of normal cells.     

Ultraviolet-induced autofluorescence characterization of normal and tumoral esophageal epithelium cells with quantitation of NAD(P)H.

Cellular autofluorescence was characterized in normal human esophageal cells and in malignant esophageal epithelial cells. The study was performed under excitation at 351 nm where the cell fluorescence is mainly due to the reduced pyridine nucleotides (NAD(P)H) with a very small contribution from the oxidized flavins (FMN, FAD) or lipopigments. The autofluorescence emission of squamous cell carcinoma, adenocarcinoma on Barrett's mucosa and normal cells was characterized by microspectrofluorimetry on monolayers and by spectrofluorimetry on cell suspensions. The relative contribution of each fluorophore to the fluorescence emission of the different cell types was evaluated by a curve-fitting analysis. A statistically highly significant difference was observed between the average intensity of the raw spectra of the different cell types. Tumoral cells had a fluorescence intensity approximately twice as high as that of normal cells. The results of the NAD(P)H quantitation analyzed by microspectrofluorimetry on single living cells and spectrofluorimetry on cell suspensions were consistent with those obtained by biochemical cycling assays, showing that the amount of intracellular NAD(P)H is higher in tumoral cells than in normal cells. Bound NAD(P)H concentration was found to be quite stable whatever the cell type while the amount of free NAD(P)H showed a very important increase in tumoral cells.     

Comparisons of Endothelial Cell G- and F-Actin Distribution in Situ and in Vitro

Numerous studies have described the F-actin cytoskeleton; however, little information relevant to C-actin is available. The actin pools of bovine aortic endothelial cells were examined using in situ and in vitro conditions and fluorescent probes for G-(deoxyribonuclease I.0.3 μM) or F-actin (phalloidin, 0.2 μM). Cells in situ displayed a diffuse G-actin distribution, while F-actin was concentrated in the cell periphery and in fine stress fibers that traversed some cells. Cells of subconfluent or just confluent cultures demonstrated intense fluorescence, with many F-actin stress fibers. Postconfluent cultures resembled the condition in situ; peripheral F-actin was prominent, traversing actin stress fibers were greatly reduced and fluorescent intensity was diminished. Postconfluency had little influence on G-actin. with only an enhancement in the intensity of G-actin punctate fluorescence. When post-confluent cultures were incubated with cytochalasin D (15 min; 10^--⁴ M), F-actin networks were disrupted and actin punctate and diffuse fluorescence increased. G-actin fluorescence was not altered by the incubation. Although its unstructured nature may account for the minor changes observed, the stability of the G-actin pool in the presence of notable F-actin modulations suggested that filamentous actin was the key constituent involved in these actin cytoskeletal alterations. A separate finding illustrated that the concomitant use of actin probes with image enhancement and fluorescent microscopy could reveal simultaneously the G- and F-actin pools within the same cell.     

Peptide Pheromone Plantaricin A Produced by Lactobacillus plantarum Permeabilizes Liver and Kidney Cells

Certain antimicrobial peptides from multicellular animals kill a variety of tumor cells at concentrations not affecting normal eukaryotic cells. Recently, it was reported that also plantaricin A (PlnA), which is a peptide pheromone with strain-specific antibacterial activity produced by Lactobacillus plantarum, permeabilizes cancerous rat pituitary cells (GH₄ cells), whereas normal rat anterior pituitary cells are resistant to the peptide. To examine whether the preferential permeabilization of cancerous cells is a general feature of PlnA, we studied its effect on primary cultures of cells from rat liver (hepatocytes, endothelial, and Kupffer cells) and rat kidney cortex, as well as two epithelial cell lines of primate kidney origin (Vero cells from green monkey and human Caki-2 cells). The Vero cell line is derived from normal cells, whereas the Caki-2 cell line is derived from a cancerous tumor. The membrane effects were studied by patch clamp recordings and microfluorometric (fura-2) monitoring of the cytosolic concentrations of Ca²⁺ ([Ca²⁺]_i) and fluorophore. In all the tested cell types except Kupffer cells, exposure to 10–100 μM PlnA induced a nearly instant permeabilization of the membrane, indicated by the following criteria: increased membrane conductance, membrane depolarization, increased [Ca²⁺]_i, and diffusional loss of fluorophore from the cytosol. At a concentration of 5 μM, PlnA had no effect on any of the cell types. The Kupffer cells were permeabilized by 500 μM PlnA. We conclude that the permeabilizing effect of PlnA is not restricted to cancerous cells.     

Combined effect of dopamine and MPP+ on membrane permeability in mitochondria and cell viability in PC12 cells

The present study examined the combined effect of dopamine and 1-methyl-4-phenylpyridinium (MPP(+)) on the membrane permeability in isolated brain mitochondria and on cell viability in PC12 cells. MPP(+) increased effect of dopamine against the swelling, membrane potential, and Ca(2+) transport in isolated mitochondria, which was not inhibited by the addition of antioxidant enzymes (SOD and catalase). Dopamine or MPP(+) caused the decrease in transmembrane potential, increase in reactive oxygen species, depletion of GSH, and cell death in PC12 cells. Antioxidant enzymes reduced each effect of dopamine and MPP(+) against PC12 cells. Co-addition of dopamine and MPP(+) caused the decrease in the transmembrane potential and increase in the formation of reactive oxygen species in PC12 cells, in which they showed an additive effect. Dopamine plus MPP(+)-induced the depletion of GSH and cell death in PC12 cells were not decreased by the addition of antioxidant enzymes, rutin, diethylstilbestrol, and ascorbate. Melanin caused a cell viability loss in PC12 cells. The N-acetylcysteine, N-phenylthiourea, and 5-hydroxyindole decreased the cell death and the formation of dopamine quinone and melanin induced by co-addition of dopamine and MPP(+), whereas deprenyl and chlorgyline did not show an inhibitory effect. The results suggest that co-addition of dopamine and MPP(+) shows an enhancing effect on the change in mitochondrial membrane permeability and cell death, which may be accomplished by toxic quinone and melanin derived from the MPP(+)-stimulated dopamine oxidation.     

Identification of actin as a 15-deoxy-Delta12,14-prostaglandin J2 target in neuroblastoma cells: mass spectrometric, computational, and functional approaches to investigate the effect on cytoskeletal derangement

A proteomic approach was used to identify 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) protein targets in human neuroblastoma SH-SY5Y cells. By using biotinylated 15d-PGJ2, beta-actin was found as the major adducted protein; at least 12 proteins were also identified as minor biotin-positive spots, falling in different functional classes, including glycolytic enzymes (enolase and lactate dehydrogenase), redox enzymes (biliverdin reductase), and a eukaryotic regulatory protein (14-3-3gamma). 15d-PGJ2 induced marked morphological changes in the actin filament network and in particular promoted F-actin depolymerization as confirmed by Western blot analysis. By using a mass spectrometric approach, we found that 15d-PGJ2 reacts with isolated G-actin in a 1:1 stoichiometric ratio and selectively binds the Cys374 site through a Michael adduction mechanism. Computational studies showed that the covalent binding of 15d-PGJ2 induces a significant unfolding of actin structure and in particular that 15d-PGJ2 distorts the actin subdomains 2 and 4, which define the nucleotide binding sites impeding the nucleotide exchange. The functional effect of 15d-PGJ2 on G-actin was studied by polymerization measurement: in the presence of 15d-PGJ2, a lower amount of F-actin forms, as followed by the increase in pyrenyl-actin fluorescence intensity, as the major effect of increasing 15d-PGJ2 concentrations occurs on the maximum extent of actin polymerization, whereas it is negligible on the initial rate of reaction. In summary, the results here reported give an insight into the role of 15d-PGJ2 as a cytotoxic compound in neuronal cell dysfunction. Actin is the main protein cellular target of 15d-PGJ2, which specifically binds through a Michael adduction to Cys374, leading to a protein conformational change that can explain the disruption of the actin cytoskeleton, F-actin depolymerization, and impairment of G-actin polymerization.     

钙调蛋白结合蛋白通过调节细胞骨架稳定性影响肿瘤细胞运动能力

轻链钙调蛋白结合蛋白（light-chain Caldesmon,l-CaD）是一种重要的肌动蛋白结合蛋白,普遍存在于众多非肌肉细胞中。体外研究证明,l-CaD能通过与肌动蛋白的结合起到促进原肌动蛋白（G-actin）聚合、稳定肌动蛋白纤维（F-actin）结构的作用。在磷酸化作用下,l-CaD能从肌动蛋白纤维上脱离并促进肌动蛋白纤维的解聚。该研究拟考察l-CaD在细胞内对细胞肌动蛋白骨架的调节作用,阐明l-CaD对细胞运动能力的影响,作者将天然低表达l-CaD的人源性乳腺癌细胞MCF-7作为细胞模型,在MCF-7胞内以基因转染的方式高表达外源野生型l-CaD及其磷酸化突变株A1234-CaD（不可磷酸化CaD）、D1234-CaD（完全磷酸化CaD）。首先,通过激光共聚焦扫描,探讨了l-CaD对细胞骨架重排的调节;其次,通过细胞迁移transwell阵列,检测了l-CaD对细胞迁移能力的影响;最后,在单细胞层次上测定了细胞基底牵张力、胰酶刺激下的细胞基底脱附能力,并进一步检测了l-CaD对细胞迁移子过程中细胞伸张、收缩的影响。研究结果显示,l-CaD在胞内对细胞骨架的形成有显著的调控作用。非磷酸化l-CaD主要富集在细胞骨架上,增强了细胞骨架的强度,导致细胞基底牵张力以及对胰酶的耐受性增强,但对细胞的迁移能力有显著的抑制作用;磷酸化l-CaD跟细胞骨架结合能力很弱,对细胞的运动能力没有显著影响。通过磷酸化,l-CaD起到了一个“蛋白开关”的作用,通过控制细胞骨架的解聚、重排来调节细胞的运动能力。     

Rat mesencephalic neuronal cells cultured for different periods as a model of dopamine transporter ontogenesis

Ventral mesencephalic neurons contained only low-affinity and sodium-independent binding sites of [³H]WIN 35,428 (marker of dopamine transporter) during the first 10d in primary cultures. These sites were present in cytosol, and they are not very probably related to dopamine transporter. After 12 d in culture, membrane-bound, high-affinity, and sodium-dependent [³H]WIN 35,428 binding sites were detected. In membranes prepared from cells 14 d in culture, cocaine displaced [³H]WIN 35,428 binding with similar potency to that in striatal membranes of adult rat brain. The high-affinity [³H]WIN 35,428 binding sites in mesencephalic neuronal cell cultures are very probably related to dopamine transporter. The development of high-affinity [³H]WIN 35,428 binding sites in neurons cultured for different time periods could be a useful model of dopamine transporter ontogenesis.     

Laser-guidance based cell detection for identifying malignant cancerous cells without any fluorescent markers

Laser guidance technique employs the optical forces generated from a focused Gaussian laser beam incident on a biological cell to trap and guide the cell along the laser propagation direction. The optical force, which determines the guidance speed, is dependent on the cellular characteristics of the cell being guided, such as size, shape, composition and morphology. Different cell populations or subpopulations can be detected without any fluorescent markers by measuring their guidance speeds. We found that cell guidance speeds were sensitive enough to monitor the subtle changes during the progression of mouse fibroblast cells from normal to cancerous phenotype. The results also demonstrated that this technique can effectively distinguish mouse mammary cancerous cells with different metastatic competence. Laser guidance technique can be used as a label-free cell detection method for basic cell biological investigation and cancer diagnosis.     

Immunofluorescent patterns of clathrin and dopamine beta-hydroxylase in chromaffin cells in culture

Summary Bovine chromaffin cells maintained in culture for eight days were loaded with [³H]noradrenaline and then stimulated by a depolarizing concentration (56 mM) of K⁺. Control and stimulated cells were fixed in 3.7% formaldehyde, treated with acetone or Triton X-100, and then exposed to antibodies raised against dopamine beta-hydroxylase (a secretory granule marker) and clathrin, and purified by affinity chromatography. The cellular distribution of the correspondent antigens was investigated by indirect immunofluorescence. Cells treated with anti-dopamine beta-hydroxylase exhibited a granular pattern of fluorescence in the cytosol of the cell body, neurites, and terminal cones. Chromaffin cells exposed to anti-clathrin also showed a punctate pattern of fluorescence staining. However, in this case, the fluorescent dots were smaller than those observed with anti-dopamine beta-hydroxylase, and they were differently distributed. The speckled anti-clathrin fluorescence was preferentially condensed in the juxtanuclear region of the cell bodies, suggesting the possibility that clathrin was concentrated at the level of the Golgi apparatus.The stimulation of cultured chromaffin cells by 10 pulses of 56 mM K⁺ produced 91±2% (n = 5) depletion in the [³H]noradrenaline cell content and a concomitant displacement of the dopamine beta-hydroxylase fluorescence to the periphery of the cells. Four days after cell stimulation the dopamine beta-hydroxylase fluorescence was similar to that observed in control cells. Under the same conditions of stimulation, the distribution of the clathrin fluorescence was unaltered suggesting either that K⁺induced stimulation of the chromaffin cells does not change the cellular distribution of clathrin, or that the changes in the distribution of clathrin are of such low magnitude that they escape detection by fluorescence microscopy.     

神经生长因子对PC12细胞脂多糖损伤的保护作用及其机制

目的：细胞水平研究神经生长因子（NGF）对大鼠嗜铬细胞瘤细胞株（PC12细胞）脂多糖（LPS）损伤后的保护作用以及核转录因子（NF-κB）的活性影响,探讨药物作用机制。方法：PC12细胞常规培养后,建立LPS损伤模型,随后MTT观察不同浓度的LPS对PC12细胞损伤及NGF对LPS损伤的保护作用,同时用倒置显微镜和荧光显微镜下观察细胞状态,最后RT-PCR检测NF-κB的含量。结果：①PC12细胞LPS损伤有浓度梯度,随着LPS浓度的增加,PC12细胞的存活率不断下降;LPS损伤的同时加入不同浓度NGF,LPS损伤均有明显的改善。②显微镜观察显示PC12细胞形态学上的改变,表明NGF对LPS损伤有保护作用。③RT-PCR结果显示,LPS损伤细胞的NF-κB的相对表达量明显高于正常对照细胞,而药物治疗组的NF-κB表达量则接近于正常细胞。结论：目前,神经生长因子在脑内炎症后的细胞修复作用报道甚少,而本实验研究神经生长因子对PC12细胞LPS损伤起到保护作用,尤其是损伤后再修复作用,且其作用机制可能与NF-κB信号通路的调控有关。     

Crosslinking of isolated cytoskeletal proteins with hemoglobin: a possible damage inflicted to the red cell membrane

Crosslinking of isolated red cell membrane cytoskeletal proteins and hemoglobin mediated by H2O2 was studied. The products of spectrin and hemoglobin interaction were demonstrated electrophoretically to be high-molecular-weight polypeptides crosslinked by nondisulfide covalent bonds. The molecular weight of the protein bands correlated with various combinations of spectrin and hemoglobin chains and the relative amount of the different products was dependent on the molar ratio of the interacting proteins. Free hemin caused spectrin crosslinking as well, but globin in the absence of hemin was inactive. Since the H2O2-mediated reaction resulted in reduction of the spectrin tryptophan fluorescence, the latter was used to monitor the reaction progress under various conditions. Both oxyhemoglobin and methemoglobin were found to be most efficient, whereas cyanmethemoglobin and hemichrome were relatively inactive. Analysis of the data implied that tryptophan oxidation as well as spectrin conformational changes follow an iron-induced crosslinking of the interacting proteins. Actin, the second major protein in the red cell cytoskeleton, behaved similarly to spectrin. The intrinsic fluorescence intensity of both G- and F-actin was decreased upon addition of H2O2 to the mixture of hemoglobin and each of the actin forms. SDS-polyacrylamide gel electrophoresis revealed that G-actin crosslinked one or two hemoglobin chains. F-actin-hemoglobin interaction induced by H2O2 produced very high aggregates that could not penetrate the gel. It is suggested that crosslinking of cytoskeletal proteins in red cells containing membrane-associated hemoglobin provides a rationale for the loss of membrane flexibility.     

Actin-filled nuclear invaginations indicate degree of cell de-differentiation

For years the existence of nuclear actin has been heavily debated, but recent data have clearly demonstrated that actin, as well as actin-binding proteins (ABPs), are located in the nucleus. We examined live EGFP-actin-expressing cells using confocal microscopy and saw the presence of structures strongly resembling actin filaments in the nuclei of MDA-MB-231 human mammary epithelial tumor cells. Many nuclei had more than one of these filamentous structures, some of which appeared to cross the entire nucleus. Extensive analysis, including fluorescence recovery after photobleaching (FRAP), showed that all EGFP-actin in the nucleus is monomeric (G-actin) rather than filamentous (F-actin) and that the apparent filaments seen in the nucleus are invaginations of cytoplasmic monomeric actin. Immunolocalization of nuclear pore complex proteins shows that similar invaginations are seen in cells that are not overexpressing EGFP-actin. To determine whether there is a correlation between increased levels of invagination in the cell nuclei and the state of de-differentiation of the cell, we examined a variety of cell types, including live Xenopus embryonic cells. Cells that were highly de-differentiated, or cancerous, had an increased incidence of invagination, while cells that were differentiated had few nuclear invaginations. The nuclei of embryonic cells that were not yet differentiated underwent multiple shape changes throughout interphase, and demonstrated numerous transient invaginations of varying sizes and shapes. Although the function of these actin-filled invaginations remains speculative, their presence correlates with cells that have increased levels of nuclear activity.