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Cytoplasm
Kinds of Cytoplasm Selected AbstractsExpression of HuR in Merkel cell carcinoma and in normal skinJOURNAL OF CUTANEOUS PATHOLOGY, Issue 1 2008Virve Koljonen Background:, HuR is a ubiquitously expressed member of the Elav/Hu family of mRNA-binding proteins, and its cytoplasmic expression has been recognised to participate in carcinogenesis. The aims of this study were to explore the expression pattern of HuR in primary Merkel cell carcinoma (MCC), lymph node metastases and non-neoplastic skin. Methods:, Twenty-two primary MCC samples and five lymph node metastases were evaluated for HuR expression by immunohistochemistry. The data were compared with clinical parameters. Results:, Nuclear and cytoplasmic HuR-staining patterns were observed. Nuclear immunoreactivity was observed in 91% of the primary tumors and in 80% of the lymph node metastases. Cytoplasm was positive in 27% of the primary tumors and in 60% of the lymph node metastases. No cytoplasmic HuR immunoreactivity was detected in non-neoplastic skin. However, moderate to strong nuclear staining was found in normal epidermis and in the epithelium of hair follicles and sebaceous glands. Expression of HuR in MCC did not associate with clinicopathological parameters. Conclusions:, Primary MCCs and their lymph node metastases as well as non-neoplastic skin show nuclear expression of HuR protein. In contrast to non-neoplastic skin, a subset of MCC tumors show cytoplasmic HuR staining, which may contribute to carcinogenesis in MCC. [source] Efficient Delivery of Bioactive Antibodies into the Cytoplasm of Living Cells by Charge-Conversional Polyion Complex Micelles,ANGEWANDTE CHEMIE, Issue 14 2010Yan Lee Dr. Nichts wie rein! Immunglobulin,G (IgG) kann nach Ladungsumkehr und Behandlung mit einem kationischen Blockcopolymer in Form von Polyionkomplex(PIC)-Micellen in das Plasma lebender Zellen eingebracht werden (siehe Bild). In der Zelle erlangt IgG in Abhängigkeit vom pH-Wert seine biologische Wirkung zurück, sodass das Wachstum humaner Hepatomzellen durch IgG-Bindung an intrazelluläre Zielmoleküle gesteuert werden kann. [source] Development of the pons in human fetusesCONGENITAL ANOMALIES, Issue 2 2007Toshihisa Hatta ABSTRACT Morphometric and histological studies of the pons were performed by light microscopy in 28 cases of externally normal human fetuses ranging from 90 to 246 mm in crown-rump length (CRL) and from 13 to 28 weeks of gestation. The brainstems of fetuses were embedded in celloidin or paraffin, and transverse sections were prepared. The pons was divided into two regions at the most ventral margin of the medial lemniscus at the level of the motor trigeminal nucleus. The relationships between the total dorsoventral length, ventral length, and dorsal length of the pons versus CRL and gestational ages were calculated, and empiric formulas were fitted. It was found that the ventral portion increased in size more rapidly than the dorsal portion. The proportion of the ventral portion in the total dorsoventral length was constitutively higher than that of the dorsal portion in the present range of CRL. In the pontine nuclei, from 235 mm in the CRL, some large cells with rich cytoplasm, pale nuclei, and a distinct nucleolus appeared on the dorsal side of the pyramidal tract. According to Weigert stained preparations, the first myelinated fibers in each motor root of the trigeminal, abducent, and facial nerves were recognized at 130,140 mm in CRL and the medial lemniscus at 230,235 mm. [source] Mineralocorticoid Receptor Is Overexpressed in Cardiomyocytes of Patients With Congestive Heart FailureCONGESTIVE HEART FAILURE, Issue 1 2005Masahiro Yoshida MD Mineralocorticoid receptors (MRs) have been identified in the human cardiovascular tissues. We determined MR expression in the failing heart to clarify the mechanism of action of aldosterone antagonist in the treatment of congestive heart failure. MR protein and MR mRNA content were detected by immunohistochemical staining and in situ hybridization in the cardiac tissues. Immunohistochemical staining of the receptor, as well as in situ hybridization of MR mRNA, was dense in cardiomyocytes of the failing left ventricle as compared with the controls. The staining ratio of the cytoplasm to the interstitium showed that MRs were located mainly in the cytoplasm. The cytoplasm to the interstitium in the failing left ventricle was 1.53±0.13, which was significantly higher than that of the controls 1.25±0.19 (p<0.05). These findings suggest that the efficacy of aldosterone antagonists in treating congestive heart failure may be in part through blocking the MRs, which are upregulated in the failing heart. [source] Ageing influences myonuclear domain size differently in fast and slow skeletal muscle of ratsACTA PHYSIOLOGICA, Issue 1 2009N. E. Brooks Abstract Aim:, In multinucleated skeletal muscle, a myonuclear domain is the region of cytoplasm governed by one nucleus, and myofibres are mosaics of overlapping myonuclear domains. Association of ageing and myonuclear domain is important in the understanding of sarcopenia and with prevention or combating age-related muscle declines. This study examined the effects of age, fibre type and muscle on nucleo-cytoplasmic (N/C) relationships as reflecting myonuclear domain size. Methods:, The N/C was compared in fibre types of soleus and plantaris muscles from young (n = 6) and ageing (n = 8) male Fisher 344 rats. Results:, There were no significant differences in fibre type composition or cross-sectional area of the soleus across ages. The old soleus had significantly more myonuclei, resulting in a significantly smaller myonuclear domain size. The plantaris muscle showed a higher percentage of slow fibres in old compared with young fibres. There were no differences in the number of myonuclei or in myonuclear domain size between young and older animals. Conclusion:, We found muscle-specific differences in the effects of ageing on myonuclear domain, possibly as a result of reduced efficiency of the myonuclei in the slow muscles. [source] Cytoplasmic fatty acid-binding protein facilitates fatty acid utilization by skeletal muscleACTA PHYSIOLOGICA, Issue 4 2003J. F. C. Glatz Abstract The intracellular transport of long-chain fatty acids in muscle cells is facilitated to a great extent by heart-type cytoplasmic fatty acid-binding protein (H-FABP). By virtue of the marked affinity of this 14.5-kDa protein for fatty acids, H-FABP dramatically increases their concentration in the aqueous cytoplasm by non-covalent binding, thereby facilitating both the transition of fatty acids from membranes to the aqueous space and their diffusional transport from membranes (e.g. sarcolemma) to other cellular compartments (e.g. mitochondria). Striking features are the relative abundance of H-FABP in muscle, especially in oxidative muscle fibres, and the modulation of the muscular H-FABP content in concert with the modulation of other proteins and enzymes involved in fatty acid handling and utilization. Newer studies with mice carrying a homozygous or heterozygous deletion of the H-FABP gene show that, in comparison with wild-type mice, hindlimb muscles from heterozygous animals have a markedly lowered (,66%) H-FABP content but unaltered palmitate uptake rate, while in hindlimb muscles from homozygous animals (no H-FABP present) palmitate uptake was reduced by 45%. These findings indicate that H-FABP is present in relative excess and plays a substantial, but merely permissive role in fatty acid uptake by skeletal muscles. [source] Mesothelioma Symposium 11.30,12.30 Tuesday 16 September 2003CYTOPATHOLOGY, Issue 2003Darrel Whitaker Dr The diagnosis of malignant mesothelioma on the cytology of serous effusions is a two-phase process. First is to determine that the effusion is malignant based on morphological features such as a highly cellular fluid with many large three dimensional cell aggregates, and/or the recognition of minor malignant criteria including prominent cell engulfment, uniformly present very prominent nucleoli, or the finding of very large (giant) cells. In cell block sections, strong positive staining with EMA often with cell membrane accentuation provides compelling support for a cytological diagnosis of malignancy. Second is to recognize that the malignant cells have a mesothelial phenotype and do not represent metastatic malignancy (usually adenocarcinoma). Criteria in support of mesothelioma include the lack of a ,two cell' population, that is one native (mesothelial) and one foreign (metastatic), cells with abundant dense staining cytoplasm, the presence of ,windows' where mesothelioma cells lie in close apposition and intracytoplasmic glycogen presenting either as small peripheral vacuoles on MGG stained smears or large yellow refractile crescents on Papanicolaou stained smears. In addition, mesothliomas often possess connective tissue stromal cores occurring as either well-formed collagen within papillary aggregates or lying free as pink (MGG) or light green (Pap) amorphous material in the background of the smear or in loose association with mesothelioma cells. Finally small orange staining squamous-like cells can occasionally be identified and sometimes this may be a very prominent finding and has resulted in the false impression of a squamous cell carcinoma. Almost certainly these cells represent apoptotic tumour cells. The connective tissue mucin hyaluronic acid may be found as a net-like pattern in the smear background or as large hard-edged magenta-stained vacuoles on MGG-stained smears. Cell block sections provide architectural information and it is usually possible to separate mesothelioma aggregates with their cuboidal cells, central nuclei and abundant dense cytoplasm arranged in solid, papillary or hollow clusters from those of adenocarcinoma with less dense, often foamy cytoplasm, often composed of columnar cells with elongated nuclei. Aggregate form in adenocarcinoma can be variable but true acini are a rare finding. These cell block sections provide an ideal medium for histochemistry (PAS with and without diastase digestion) and immunocytochemistry. By using a panel of antibodies (Calretinin and CK 5/6, BerEp4, CEA, B72.3) it is almost always possible to distinguish mesothelioma from metastatic adenocarcinoma. Calretinin and CK 5/6 positive staining and absent staining with BerEp4, CEA and B72.3 is considered diagnostic of mesothelioma. [source] Force propagation and force generation in cells,CYTOSKELETON, Issue 9 2010Oliver Jonas Abstract Determining how forces are produced by and propagated through the cytoskeleton (CSK) of the cell is of great interest as dynamic processes of the CSK are intimately correlated with many molecular signaling pathways. We are presenting a novel approach for integrating measurements on cell elasticity, transcellular force propagation, and cellular force generation to obtain a comprehensive description of dynamic and mechanical properties of the CSK under force loading. This approach uses a combination of scanning force microscopy (SFM) and Total Internal Reflection Fluorescence (TIRF) microscopy. We apply well-defined loading schemes onto the apical cell membrane of fibroblasts using the SFM and simultaneously use TIRF microscopy to image the topography of the basal cell membrane. The locally distinct changes of shape and depth of the cytoskeletal imprints onto the basal membrane are interpreted as results of force propagation through the cytoplasm. This observation provides evidence for the tensegrity model and demonstrates the usefulness of our approach that does not depend on potentially disturbing marker compounds. We confirm that the actin network greatly determines cell stiffness and represents the substrate that mediates force transduction through the cytoplasm of the cell. The latter is an essential feature of tensegrity. Most importantly, our new finding that, both intact actin and microtubule networks are required for enabling the cell to produce work, can only be understood within the framework of the tensegrity model. We also provide, for the first time, a direct measurement of the cell's mechanical power output under compression at two femtowatts. © 2010 Wiley-Liss, Inc. [source] Actin on DNA,An ancient and dynamic relationship,CYTOSKELETON, Issue 8 2010Kari-Pekka Skarp Abstract In the cytoplasm of eukaryotic cells the coordinated assembly of actin filaments drives essential cell biological processes, such as cell migration. The discovery of prokaryotic actin homologues, as well as the appreciation of the existence of nuclear actin, have expanded the scope by which the actin family is utilized in different cell types. In bacteria, actin has been implicated in DNA movement tasks, while the connection with the RNA polymerase machinery appears to exist in both prokaryotes and eukaryotes. Within the nucleus, actin has further been shown to play a role in chromatin remodeling and RNA processing, possibly acting to link these to transcription, thereby facilitating the gene expression process. The molecular mechanism by which actin exerts these newly discovered functions is still unclear, because while polymer formation seems to be required in bacteria, these species lack conventional actin-binding proteins to regulate the process. Furthermore, although the nucleus contains a plethora of actin-regulating factors, the polymerization status of actin within this compartment still remains unclear. General theme, however, seems to be actin's ability to interact with numerous binding partners. A common feature to the novel modes of actin utilization is the connection between actin and DNA, and here we aim to review the recent literature to explore how this connection is exploited in different contexts. [source] Actin-like protein 1 (ALP1) is a component of dynamic, high molecular weight complexes in Toxoplasma gondii,CYTOSKELETON, Issue 1 2010Jennifer L. Gordon Abstract Apicomplexan parasites, such as Toxoplasma gondii, rely on actin-based motility for cell invasion, yet conventional actin does not appear to be required for cell division in these parasites. Apicomplexans also contain a variety of actin-related proteins (Arps); however, most of these not directly orthologous to Arps in well-studied systems. We recently identified an apicomplexan-specific member of this family called Actin-Like Protein 1, (ALP1), which plays a role in the assembly of vesicular components recruited to the inner membrane complex (IMC) of daughter cells during cell division. In addition to its enrichment at daughter cell membranes, ALP1 is localized throughout the cytoplasm both diffusely distributed and concentrated in clusters that are detected by fluorescence microscopy, suggesting it forms complexes. Using quantitative optical imaging methods, including fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP), we demonstrated that ALP1 is a component of a large complex, and that it readily exchanges between diffusible and complex-bound forms. Sedimentation and density gradient analyses revealed that ALP1 is found in a freely soluble state as well as high molecular weight complexes. During cell division, ALP1 was dynamically associated with the IMC, suggesting it rapidly cycles between freely diffusible and complex forms during daughter cell assembly. © 2009 Wiley-Liss, Inc. [source] An in vitro model system for cytoskeletal confinementCYTOSKELETON, Issue 10 2009Sarah Köster Abstract The motility, shape, and functionality of the cell depend sensitively on cytoskeletal mechanics which in turn is governed by the properties of filamentous proteins - mainly actin, microtubules, and intermediate filaments. These biopolymers are confined in the dense cytoplasm and therefore experience strong geometric constraints on their equilibrium thermal fluctuations. To obtain a better understanding of the influence of confinement on cytoskeletal filaments we study the thermal fluctuations of individual actin filaments in a microfluidic in vitro system by fluorescence microscopy and determine the persistence length of the filaments by analyzing the radial distribution function. A unique feature of this method is that we obtain the persistence length without detailed knowledge of the complete contour of the filament which makes the technique applicable to a broad range of biological polymers, including those with a persistence length smaller than the optical resolution. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Centrioles to basal bodies in the spermiogenesis of Mastotermes darwiniensis (Insecta, Isoptera)CYTOSKELETON, Issue 5 2009Maria Giovanna Riparbelli Abstract In addition to their role in centrosome organization, the centrioles have another distinct function as basal bodies for the formation of cilia and flagella. Centriole duplication has been reported to require two alternate assembly pathways: template or de novo. Since spermiogenesis in the termite Mastotermes darwiniensis lead to the formation of multiflagellate sperm, this process represents a useful model system in which to follow basal body formation and flagella assembly. We present evidence of a possible de novo pathway for basal body formation in the differentiating germ cell. This cell also contains typical centrosomal proteins, such as centrosomin, pericentrin-like protein, ,-tubulin, that undergo redistribution as spermatid differentiation proceeds. The spermatid centrioles are long structures formed by nine doublet rather than triplet microtubules provided with short projections extending towards the surrounding cytoplasm and with links between doublets. The sperm basal bodies are aligned in parallel beneath the nucleus. They consist of long regions close to the nucleus showing nine doublets in a cartwheel array devoid of any projections; on the contrary, the short region close to the plasma membrane, where the sperm flagella emerge, is characterized by projections similar to those observed in the centrioles linking the basal body to the plasma membrane. It is hypothesized that this appearance is in connection with the centriole elongation and further with the flagellar axonemal organization. Microtubule doublets of sperm flagellar axonemes are provided with outer dynein arms, while inner arms are rarely visible. Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Assembling microtubules disintegrate the postsynaptic density in vitroCYTOSKELETON, Issue 1 2007Li-Ping Lo Abstract The postsynaptic density (PSD), a disk-shaped protein aggregation of several hundred nm in diameter, plays important roles in the signal transduction and molecular organization of the excitatory synapses in mammalian CNS. The PSD resides in the microfilament-enriched cytoplasm of dendritic spines where the transient appearance of microtubules has been reported. When PSD isolated from porcine brain was incubated with polymerizing ,,,-tubulins, its turbidity became greater than that of the original PSD, suggesting that the PSD's structure was altered upon incubating with assembling microtubules. By transmission electron microscopy, smaller PSD fragments and several novel structures, including holes and finger-like extensions, were found in the PSD after incubation with assembling microtubules, but not in the original PSD or in the PSD incubated with ,,,-tubulins pretreated with vincristine. The results suggest that the interactions with assembling microtubules may result in the formation of holes in the PSD, and the rupture of these holes subsequently leads to the formation of smaller PSD fragments. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Cryopreservable neutrophil surrogates: Granule-poor, motile cytoplasts from polymorphonuclear leukocytes home to inflammatory lesions in vivoCYTOSKELETON, Issue 5 2006Stephen E. Malawista Abstract Cytokineplasts (CKP) are anucleate, motile, granule-poor fragments induced from polymorphonuclear leukocytes on surfaces by the brief application of heat. Derived from the peripheral cytoplasm and membranes of PMN, they retain the sensing, transducing, and effector mechanisms necessary for chemotaxis and phagocytosis, and appear to represent a functional, self-purification of the motile apparatus. Unlike their parent PMN, CKP are cryopreservable. We have shown that they can adhere to endothelial cell monolayers, open interendothelial cell junctions, and migrate to the abluminal side when stimulated by a chemoattractant. Employing an animal model, we now show that, given intravenously, they can home to an inflammatory target lesion in vivo. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc. [source] Caenorhabditis elegans expresses three functional profilins in a tissue-specific mannerCYTOSKELETON, Issue 1 2006D. Polet Abstract Profilins are actin binding proteins, which also interact with polyphosphoinositides and proline-rich ligands. On the basis of the genome sequence, three diverse profilin homologues (PFN) are predicted to exist in Caenorhabditis elegans. We show that all three isoforms PFN-1, PFN-2, and PFN-3 are expressed in vivo and biochemical studies indicate they bind actin and influence actin dynamics in a similar manner. In addition, they bind poly(L -proline) and phosphatidylinositol 4,5-bisphosphate micelles. PFN-1 is essential whereas PFN-2 and PFN-3 are nonessential. Immunostainings revealed different expression patterns for the profilin isoforms. In embryos, PFN-1 localizes in the cytoplasm and to the cell,cell contacts at the early stages, and in the nerve ring during later stages. During late embryogenesis, expression of PFN-3 was specifically detected in body wall muscle cells. In adult worms, PFN-1 is expressed in the neurons, the vulva, and the somatic gonad, PFN-2 in the intestinal wall, the spermatheca, and the pharynx, and PFN-3 localizes in a striking dot-like fashion in body wall muscle. Thus the model organism Caenorhabditis elegans expresses three profilin isoforms and is the first invertebrate animal with tissue-specific profilin expression. Cell Motil. Cytoskeleton, 2006.© 2005 Wiley-Liss, Inc. [source] Preparing to move: Assembly of the MSP amoeboid motility apparatus during spermiogenesis in AscarisCYTOSKELETON, Issue 4 2005Maria Antonia Rodriguez Abstract We exploited the rapid, inducible conversion of non-motile Ascaris spermatids into crawling spermatozoa to examine the pattern of assembly of the MSP motility apparatus that powers sperm locomotion. In live sperm, the first detectable motile activity is the extension of spikes and, later, blebs from the cell surface. However, examination of cells by EM revealed that the formation of surface protrusions is preceded by assembly of MSP filament tails on the membranous organelles in the peripheral cytoplasm. These organelle-associated filament meshworks assemble within 30 sec after induction of spermiogenesis and persist until the membranous organelles are sequestered into the cell body when the lamellipod extends. The filopodia-like spikes, which are packed with bundles of filaments, extend and retract rapidly but last only a few seconds before giving way to, or converting into, blebs. Coalescence of these blebs, each supported by a dense mesh of filaments, often initiates lamellipod extension, which culminates in the formation of the robust, dynamic MSP fiber complexes that generate sperm motility. The same membrane phosphoprotein that orchestrates assembly of the fiber complexes at the leading edge of the lamellipod of mature sperm is also found at all sites of filament assembly during spermiogenesis. The orderly progression of steps that leads to construction of a functional motility apparatus illustrates the precise spatio-temporal control of MSP filament assembly in the developing cell and highlights the remarkable similarity in organization and plasticity shared by the MSP cytoskeleton and the actin filament arrays in conventional crawling cells. Cell Motil. Cytoskeleton 60:191,199, 2005 © 2005 Wiley-Liss, Inc. [source] The ,II isotype of tubulin is present in the cell nuclei of a variety of cancersCYTOSKELETON, Issue 2 2004I-Tien Yeh Abstract Tubulin, the subunit protein of microtubules, has generally been thought to be exclusively a cytoplasmic protein in higher eukaryotes. We have previously shown that cultured rat kidney mesangial cells contain the ,II isotype of tubulin in their nuclei in the form of an ,,II dimer [Walss et al., 1999: Cell Motil. Cytoskeleton 42:274,284, 1999]. More recently, we examined a variety of cancerous and non-cancerous cell lines and found ,II in the nuclei of all of the former and only a few of the latter (Walss-Bass et al., 2002: Cell Tissue Res. 308:215,223]. In order to determine if ,II -tubulin occurs in the nuclei of actual cancers as well as in cancer cell lines, we used the immunoperoxidase method to look for nuclear ,II in a variety of tumors excised from 201 patients. We found that 75% of these tumors contain ,II in their nuclei. Distribution of nuclear ,II was highly dependent on the type of cancer, with 100% of the colon and prostate cancers, but only 19% of the skin tumors, having nuclear ,II. Nuclear ,II was particularly marked in tumors of epithelial origin, of which 83% showed nuclear ,II, in contrast to 54% in tumors of non-epithelial origin. In many cases, ,II staining occurred very strongly in the nuclei and not in the cytoplasm; in other cases, ,II was present in both. In many cases, particularly metastases, otherwise normal cells adjacent to the tumor also showed nuclear ,II, suggesting that cancer cells may influence nearby cells to synthesize ,II and localize it to their nuclei. Our results have implications for the diagnosis, biology, and chemotherapy of cancer. Cell Motil. Cytoskeleton 57:96,106, 2004. © 2004 Wiley-Liss, Inc. [source] Plant profilin isovariants are distinctly regulated in vegetative and reproductive tissuesCYTOSKELETON, Issue 1 2002Muthugapatti K. Kandasamy Abstract Profilin is a low-molecular weight, actin monomer-binding protein that regulates the organization of actin cytoskeleton in eukaryotes, including higher plants. Unlike the simple human or yeast systems, the model plant Arabidopsis has an ancient and highly divergent multi-gene family encoding five distinct profilin isovariants. Here we compare and characterize the regulation of these profilins in different organs and during microspore development using isovariant-specific monoclonal antibodies. We show that PRF1, PRF2, and PRF3 are constitutive, being strongly expressed in all vegetative tissues at various stages of development. These profilin isovariants are also predominant in ovules and microspores at the early stages of microsporogenesis. In contrast, PRF4 and PRF5 are late pollen-specific and are not detectable in other cell types of the plant body including microspores and root hairs. Immunocytochemical studies at the subcellular level reveal that both the constitutive and pollen-specific profilins are abundant in the cytoplasm. In vegetative cell types, such as root apical cells, profilins showed localization to nuclei in addition to the cytoplasmic staining. The functional diversity of profilin isovariants is discussed in light of their spatio-temporal regulation during vegetative development, pollen maturation, and pollen tube growth. Cell Motil. Cytoskeleton 52:22,32, 2002. © 2002 Wiley-Liss, Inc. [source] Quantification of Shigella IcsA required for bacterial actin polymerizationCYTOSKELETON, Issue 4 2002Juana Magdalena Abstract Shigella move through the cytoplasm of host cells by active polymerization of host actin to form an "actin tail." Actin tail assembly is mediated by the Shigella protein IcsA. The process of Shigella actin assembly has been studied extensively using IcsA-expressing Escherichia coli in cytoplasmic extracts of Xenopus eggs. However, for reasons that have been unclear, wild type Shigella does not assemble actin in these extracts. We show that the defect in actin assembly in Xenopus extracts by Shigella can be rescued by increasing IcsA expression by approximately 3-fold. We calculate that the number of IcsA molecules required on an individual bacterium to assemble actin filaments in extracts is approximately 1,500,2,100 molecules, and the number of IcsA molecules required to assemble an actin tail is approximately 4,000 molecules. The majority of wild type Shigella do not express these levels of IcsA when grown in vitro. However, in infected host cells, IcsA expression is increased 3.2-fold, such that the number of IcsA molecules on a significant percentage of intracellular wild type Shigella would exceed that required for actin assembly in extracts. Thus, the number of IcsA molecules estimated from our studies in extracts as being required on an individual bacterium to assemble actin filaments or an actin tail is a reasonable prediction of the numbers required for these functions in Shigella -infected cells. Cell Motil. Cytoskeleton 51:187,196, 2002. © 2002 Wiley-Liss, Inc. [source] Regulation of focal adhesion targeting and inhibitory functions of the FAK related protein FRNK using a novel estrogen receptor "switch"CYTOSKELETON, Issue 2 2002Karen H. Martin Abstract Focal adhesion kinase (FAK) is a regulator of numerous adhesion-dependent processes including cell migration, cell proliferation, and cell survival. The C-terminal domain of FAK, FAK-related nonkinase (FRNK), is autonomously expressed and functions as an inhibitor of FAK signaling. Previous attempts to use FRNK as a tool to dissect FAK signaling have been limited because of an inability to temporally regulate the inhibitory functions of FRNK. In this report, we describe and characterize a conditionally targeted form of FRNK that was created by fusing the hormone-binding domain of the estrogen receptor (ER*) to the C-terminus of FRNK. In the absence of added hormone, FRNK-ER* was diffusely distributed throughout the cytoplasm of cells. Upon addition of hormone, the cytoplasmic pool of FRNK-ER* was rapidly redistributed to focal adhesions. We demonstrate that cells expressing FRNK-ER* show a hormone-dependent decrease in FAK tyrosine phosphorylation and cell migration. Furthermore, when cells expressing of FRNK-ER* were treated with hormone, the cells responded with a dramatic change in cell morphology, suggesting a role for FAK in the regulation of the adhesive properties of focal adhesions. Cell Motil. Cytoskeleton 51:76,88, 2002. © 2002 Wiley-Liss, Inc. [source] Vascular smooth muscle cell phenotypic modulation in culture is associated with reorganisation of contractile and cytoskeletal proteinsCYTOSKELETON, Issue 3 2001Nathalie F. Worth Abstract Smooth muscle cells (SMC) exhibit a functional plasticity, modulating from the mature phenotype in which the primary function is contraction, to a less differentiated state with increased capacities for motility, protein synthesis, and proliferation. The present study determined, using Western analysis, double-label immunofluorescence and confocal microscopy, whether changes in phenotypic expression of rabbit aortic SMC in culture could be correlated with alterations in expression and distribution of structural proteins. "Contractile" state SMC (days 1 and 3 of primary culture) showed distinct sorting of proteins into subcellular domains, consistent with the theory that the SMC structural machinery is compartmentalised within the cell. Proteins specialised for contraction (,-SM actin, SM-MHC, and calponin) were highly expressed in these cells and concentrated in the upper central region of the cell. Vimentin was confined to the body of the cell, providing support for the contractile apparatus but not co-localising with it. In line with its role in cell attachment and motility, ,-NM actin was localised to the cell periphery and basal cortex. The dense body protein ,-actinin was concentrated at the cell periphery, possibly stabilising both contractile and motile apparatus. Vinculin-containing focal adhesions were well developed, indicating the cells' strong adhesion to substrate. In "synthetic" state SMC (passages 2,3 of culture), there was decreased expression of contractile and adhesion (vinculin) proteins with a concomitant increase in cytoskeletal proteins (,-non-muscle [NM] actin and vimentin). These quantitative changes in structural proteins were associated with dramatic changes in their distribution. The distinct compartmentalisation of structural proteins observed in "contractile" state SMC was no longer obvious, with proteins more evenly distributed throughout the cytoplasm to accommodate altered cell function. Thus, SMC phenotypic modulation involves not only quantitative changes in contractile and cytoskeletal proteins, but also reorganisation of these proteins. Since the cytoskeleton acts as a spatial regulator of intracellular signalling, reorganisation of the cytoskeleton may lead to realignment of signalling molecules, which, in turn, may mediate the changes in function associated with SMC phenotypic modulation. Cell Motil. Cytoskeleton 49:130,145, 2001. © 2001 Wiley-Liss, Inc. [source] Calyculin-A, an inhibitor for protein phosphatases, induces cortical contraction in unfertilized sea urchin eggsCYTOSKELETON, Issue 4 2001Yukako Asano Abstract When an unfertilized sea urchin egg was exposed to calyculin-A (CL-A), an inhibitor of protein phosphatases, for a short period and then lysed, the cortex contracted to exclude cytoplasm and became a cup-shaped mass. We call the contracted cortex "actin cup" since actin filaments were major structural components. Electron microscopic observation revealed that the cup consisted of inner electron-dense layer, middle microfilamentous layer, and outermost granular region. Microfilaments were heavily accumulated in the inner electron-dense layer. The middle layer also contained numerous microfilaments, which were determined to be actin filaments by myosin S1 decoration, and they were aligned so that their barbed ends directed toward the outermost region. Myosin II, Arp2, Arp3, and spectrin were concentrated in the actin cup. Immuno-electron microscopy revealed that myosin II was localized to the electron-dense layer. We further found that the cortical tension of the egg increased just after application of CL-A and reached maximum within 10 min. Cytochalasin B or butanedione monoxime blocked the contraction, which suggested that both actin filaments and myosin ATPase activity were required for the contraction. Myosin regulatory light chain (MRLC) in the actin cup was shown to be phosphorylated at the activation sites Ser-19 and Thr-18, by immunoblotting with anti-phosphoepitope antibodies. The phosphorylation of MRLC was also confirmed by a 32P in vivo labeling experiment. The CL-A-induced cortical contraction may be a good model system for studying the mechanism of cytokinesis. Cell Motil. Cytoskeleton 48:245,261, 2001. © 2001 Wiley-Liss, Inc. [source] Programmed cell death of the ovarian nurse cells during oogenesis of the silkmoth Bombyx moriDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 7 2006Vicky E. Mpakou In the present study, we describe the features of programmed cell death of the ovarian nurse cells occurring during vitellogenesis of the silkmoth Bombyx mori. At developmental stage 5, the nurse cells occupy one-half of the follicular volume and obtain a rather spherical shape, while the nurse cell nuclei appear large and elongated, forming impressive projections. At the following stage, stage 6, the nurse cells decrease in size and their shape becomes elliptic. The nuclei remain elongated, being also characterized by large lobes. The lobes of the ramified nurse cell nuclei seem to retain the nucleus in the center of the cell during the dumping of the nurse cell cytoplasm into the growing oocyte. At stage 7, membrane enclosed vacuoles can be easily detected into the nurse cells cytoplasm. Ultrastructural analysis and fluorescent microscopy using mono-dansyl-cadaverine staining of these vacuoles also reveal that they represent autolysosomes. Caspase activity is detected during stage 7, as it is demonstrated by using the Red-VAD-FMK staining reagent. At developmental stages 8 and 9, the nurse cells exhibit chromatin condensation, DNA fragmentation and caspase activity. Finally, during the following stage 10, the nuclear remnants are assembled into apoptotic vesicles, which, after being phagocytosed, are observed in the cytoplasm of adjacent follicle cells. We propose that apoptosis and autophagy operate synergistically during vitellogenesis of B. mori, in order to achieve an efficient and rapid clearance of the degenerated nurse cell cluster. [source] Identification of asymmetrically localized transcripts along the animal,vegetal axis of the Xenopus eggDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 8 2005Kensuke Kataoka In many organisms, proper embryo development depends on the asymmetrical distribution of mRNA in the cytoplasm of the egg. Here we report comprehensive screening of RNA localized in the animal or vegetal hemisphere of the Xenopus egg. Macroarrays including over 40 000 independent embryonic cDNA clones, representing at least 17 000 unigenes, were differentially hybridized with labeled probes synthesized from the mRNA of animal or vegetal blastomeres. After two rounds of screening, we identified 33 clones of transcripts that may be preferentially distributed in the vegetal region of the early stage embryo, but transcripts localized in the animal region were not found. To assess the array results, we performed northern blot and quantitative real-time reverse transcription,polymerase chain reaction analysis. As a result, 21 transcripts of the 33 were confirmed to be localized in the vegetal region of the early stage embryo. Whole-mount in situ hybridization analysis revealed that 11 transcripts, including 7 previously reported genes, were localized in the vegetal hemisphere of the egg. These 11 transcripts were categorized into three groups according to their expression patterns in the egg. The first group, which contained four transcripts, showed uniform expression in the vegetal hemisphere, similar to VegT. The second group, which contained three transcripts, showed gradual expression from the vegetal pole to the equator, similar to Vg1. The last group, which contained three transcripts, was expressed at the germ plasm, similar to Xdazl. One transcript, Xwnt11, showed both the second and the third expression patterns. [source] Midblastula transition (MBT) of the cell cycles in the yolk and pigment granule-free translucent blastomeres obtained from centrifuged Xenopus embryosDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 5 2005Yasuhiro Iwao We obtained translucent blastomeres free of yolk and pigment granules from Xenopus embryos which had been centrifuged at the beginning of the 8-cell stage with cellular integrity. They divided synchronously regardless of their cell size until they had decreased to 37.5 µm in radius; those smaller than this critical size, however, divided asynchronously with cell cycle times inversely proportional to the square of the cell radius after midblastula transition (MBT). The length of the S phase was determined as the time during which nuclear DNA fluorescence increased in Hoechst-stained blastomeres. When the cell cycle time exceeded 45 min, S and M phases were lengthened; when the cell cycle times exceeded 70 min, the G2 phase appeared; and after cell cycle times became longer than 150 min, the G1 phase appeared. Lengths of G1, S and M phases increased linearly with increasing cell cycle time. Enhanced green fluorescent protein (EGFP)-tagged proliferating cell nuclear antigen (PCNA) expressed in the blastomeres appeared in the S phase nucleus, but suddenly dispersed into the cytoplasm at the M phase. The system developed in this study is useful for examining the cell cycle behavior of the cell cycle-regulating molecules in living Xenopus blastomeres by fluorescence microscopy in real time. [source] Developmental delay and unstable state of the testes in the rdw rat with congenital hypothyroidismDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 4 2004Yasuhiro Sakai From the present study of the rdw rat, it is clear that the thyroid hormone is essential for the development and maintenance of the testes. In previous studies, the thyroid hormone has few serious effects on the testes except during the neonatal stage when the thyroid hormone receptor is mainly present. However, there is little knowledge concerning the prolonged effect of thyroid hormone deficiency throughout the rat's life span. In the present study, a morphological analysis was performed on the testes of rdw rats with congenital hypothyroidism. The rdw testes required a longer time to develop into the normal adult structure. Moreover, the developed, normal structure began to degenerate after full maturation. Specific characteristics of the rdw testes include: (i) a prolonged proliferation of Sertoli cells during postnatal development; (ii) a developmental delay in the appearance of spermatocytes and spermatid; (iii) direct contact with each other for both spermatocytes and spermatids, without Sertoli cell cytoplasm completely intervening between adjacent germ cells; (iv) subsequent apoptosis of germ cells after maturation; (v) reduction in the height of the seminiferous epithelium; and (vi) lower testosterone levels in the rdw rats, especially during old age. Thus, we conclude that the thyroid hormone plays an important role in developing and maintaining normal function of testes. [source] Hypotonic buffer induces meiosis and formation of anucleate cytoplasmic islands in the egg of the two-spotted cricket Gryllus bimaculatusDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2003Isao Sarashina In insects, egg activation is known to occur in vivo and independently of fertilization, but its mechanisms are poorly understood. To gain understanding of these mechanisms, an attempt was made to activate the egg of Gryllus bimaculatus in vitro. It was found that meiosis resumed and was completed in unfertilized eggs treated with hypotonic buffer. Early developmental processes in activated, unfertilized eggs were investigated and compared with those in fertilized eggs. Mitosis did not progress, resulting in formation of anucleate cytoplasmic islands (pseudoenergids). Development in the activated, unfertilized eggs stopped at this stage and both yolk subdivision and cellularization did not occur. To elucidate the role of the nucleus in the developmental process to the syncytial stage in fertilized eggs, eggs were treated with aphidicolin to inhibit DNA polymerization. It was found that pseudoenergids also formed in these aphidicolin-treated fertilized eggs. These results demonstrate that pseudoenergids can increase in number independently of nuclei, suggesting that the cytoplasm rather than the nucleus plays the primary role in development to the syncytial stage in G. bimaculatus. [source] Sequential activation of Notch family receptors during mouse spermatogenesisDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 1 2003Shintaro Mori The expression pattern of Notch family receptors during mouse spermatogenesis was examined by immunohistochemistry. The entire cytoplasm of spermatogonia, spermatocytes and spermatids showed staining with antibodies against extracellular domains of Notch1, 2 and 4. In contrast, the nuclei of spermatogonia showed staining with an antibody against the intracellular domain of Notch3, and the nuclei of spermatocytes and spermatids showed staining with antibodies against the intracellular domains of Notch1 and 4. During regeneration of spermatogonia in busulfan-treated mice, the nuclei of all proliferating cells showed staining for the intracellular domain of Notch3. Western blot analysis showed that the molecular weights of the intracellular domains of Notch1 and 3 localizing in the nuclear fraction were smaller than those in the cytoplasmic fraction. This was consistent with the theory that the intracellular domain of Notch was cleaved in the cytoplasm and translocated to the nucleus. These results suggest that different Notch signals are sequentially activated during mouse spermatogenesis and control the proliferation and differentiation of spermatogenic stem cells. [source] Development and fine structure of the yolk nucleus of previtellogenic oocytes in the medaka Oryzias latipesDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2000Hirokuni Kobayashi The development and fine structure of yolk nuclei in the cytoplasm of previtellogenic oocytes were examined by electron microscopy during several stages of oogenesis in the medaka, Oryzias latipes. Shortly after oogenesis starts, oocytes 20,30 ,m in diameter have much electron-dense (basophilic) cytoplasm, within which a continuous or discontinuous, irregular ring-shaped lower electron-dense area of flocculent appearance (LF) begins to emerge around the nucleus. The yolk nucleus is first recognized within an LF area as a few fragments of dense granular thread measuring 20,25 nm in width. The threads consist of two rows of very dense granules resembling ribosomes or ribonucleoprotein (RNP)-like particles in size and electron density. These thread-like fragments gradually increase in number and length until they assemble into a compact, spherical mass of complicated networks. Analysis of serial sections suggests that the yolk nucleus is a complicated mass of numerous, small deformed vacuoles composed of a single lamella with double layers of ribosomes or RNP-like granules, rather than a mass of granular threads. When oocytes develop to greater than 100 ,m in diameter, the yolk nucleus begins to fragment before dispersing throughout the surrounding cytoplasm, concomitantly with the disappearance of LF areas. At this stage of oogenesis, a restricted region of the granulosa cell layer adjacent to the yolk nucleus becomes somewhat columnar in morphology, fixing the vegetal pole region of the oocyte. [source] Spatio-temporal expression of Xenopus vasa homolog, XVLG1, in oocytes and embryos: The presence of XVLG1 RNA in somatic cells as well as germline cellsDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2000Kohji Ikenishi The expression of Xenopus vasa homolog or XVLG1 was examined in oocytes and embryos by whole-mount in situ hybridization and reverse transcription,polymerase chain reaction (RT-PCR). To confirm the results in embryos, both methods were also applied to explants of germ plasm-bearing cells (GPBC) from 32-cell embryos and to those of partial embryos deprived of GPBC. By hybridization, XVLG1 ribonucleic acid (RNA) was shown to be present throughout the cytoplasm in oocytes at stages I,III, except for the mitochondrial cloud. It was barely recognizable in a portion of germline cells of embryos at specific stages, notwithstanding that XVLG1 protein was present in those cells almost throughout their life-span. A weak signal for the RNA was detectable in some of the presumptive primordial germ cells (pPGC, descendants of GPBC from the gastrula stage onward) from the late gastrula (stage 12) to the hatching tadpole stage (stage 33/34), and in some of the PGC at stages 49,50. The results for pPGC were confirmed by the hybridization of explants of GPBC at equivalent stages in control embryos. In contrast, XVLG1 RNA was detected in certain somatic cells of embryos until stage 46. These observations were supported in part by the results of RT-PCR for embryos and explants. The possible role of the product of XVLG1 was reconsidered given its presence in both germline and somatic cells. [source] | ||||||||||||||||||||||||||||||||||||||||||||||