[Show abstract][Hide abstract] ABSTRACT: A deficit of exogenous arginine affects growth and viability of numerous cancer cells. Although arginine deprivation-based strategy is currently undergoing clinical trials, molecular mechanisms of tumor cells’ response to arginine deprivation are not yet elucidated. We have examined effects of arginine starvation on cell motility, adhesion and invasiveness as well as on actin cytoskeleton organization of human glioblastoma cells. We observed for the first time that arginine, but not lysine, starvation affected cell morphology, significantly inhibited their motility and invasiveness, and impaired adhesion. No effects on glia cells were observed. Also, arginine deprivation in glioblastoma evoked specific changes in actin assembly, decreased β-actin filament content, and affected its N-terminal arginylation. We suggest that alterations in organization of β-actin resulted from a decrease of its arginylation could be responsible for the observed effects of arginine deprivation on cell invasiveness and migration. Our data indicate that arginine deprivation-based treatment strategies could inhibit, at least transiently, the invasion process of highly malignant brain tumors and may have a potential for combination therapy to extend overall patient survival.
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[Show abstract][Hide abstract] ABSTRACT: Myosin VI (MVI) is the only known myosin walking towards minus end of actin filaments and is believed to play distinct role(s) than other myosins. We addressed a role of this unique motor in secretory PC12 cells, derived from rat adrenal medulla pheochromocytoma using cell lines with reduced MVI synthesis (produced by means of siRNA). Decrease of MVI expression caused severe changes in cell size and morphology, and profound defects in actin cytoskeleton organization and Golgi structure. Also, significant inhibition of cell migration as well as cell proliferation was observed. Flow cytometric analysis revealed that MVI-deficient cells were arrested in G0/G1 phase of the cell cycle but did not undergo increased senescence as compared with control cells. Also, neither polyploidy nor aneuploidy were detected. Surprisingly, no significant effect on noradrenaline secretion was observed. These data indicate that in PC12 cells MVI is involved in cell migration and proliferation but is not crucial for stimulation-dependent catecholamine release.
Journal of Muscle Research and Cell Motility 11/2011; 32(4-5):291-302. DOI:10.1007/s10974-011-9279-0 · 1.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inhibition of Rho-associated protein kinase (ROCK) activity in glioma C6 cells induces changes in actin cytoskeleton organization and cell morphology similar to those observed in other types of cells with inhibited RhoA/ROCK signaling pathway. We show that phosphorylation of myosin light chains (MLC) induced by P2Y₂ receptor stimulation in cells with blocked ROCK correlates in time with actin cytoskeleton reorganization, F-actin redistribution and stress fibers assembly followed by recovery of normal cell morphology. Presented results indicate that myosin light-chain kinase (MLCK) is responsible for the observed phosphorylation of MLC. We also found that the changes induced by P2Y₂ stimulation in actin cytoskeleton dynamics and morphology of cells with inhibited ROCK, but not in the level of phosphorylated MLC, depend on the presence of calcium in the cell environment.
[Show abstract][Hide abstract] ABSTRACT: Recently, we found a 130-kDa myosin VI immunoanalog in amoeba, which bound to actin in an ATP-sensitive manner and in migrating amoebae colocalized to filamentous actin and dynamin II-containing vesicular structures. To further characterize this protein, we assessed its involvement in amoeba pinocytosis and phagocytosis. Confocal immunofluorescence microscopy and electron microscopy of immunogold-stained cells revealed that, in pinocytotic and phagocytotic amoebae, the myosin VI immunoanalog was visible throughout the cells, including pinocytotic channels and pinocytotic vesicles as well as phagosomes and emerging phagocytic cups. Blocking endogenous protein with anti-porcine myosin VI antibody (introduced into cells by means of microinjection) caused severe defects in pinocytosis and phagocytosis. In comparison with control cells, the treated amoebae formed ~75% less pinocytotic channels and phagocytosed ~65% less Tetrahymena cells. These data indicate that the myosin VI immunoanalog has an important role in pinocytosis and phagocytosis in Amoeba proteus (Pal.).
[Show abstract][Hide abstract] ABSTRACT: Amoeba proteus and smaller by an order of magnitude (and evolutionary younger) Acanthamoeba castellanii have been for many years model cells for studies of amoeboidal (crawling) type of movement, characteristic also for some of metazoan cells such as fibroblasts, granulocytes and macrophages. Amoeboidal migration is indispensable of organization and dynamics of actin-based cytoskeleton. While there is a number of data on molecular mechanisms of motility of A. castellanii, there is very little known about bases of migration of A. proteus. Noteworthy, a large A. proteus (length approximately 600 microm) have been from over a century an object for studies on biology and physiology of cellular migration. This review describes the current knowledge on molecular aspects of force generation required for migration of these two amoebae and attempts to compare the functioning and regulation of actin cytoskeleton in these free-living unicellular species.
[Show abstract][Hide abstract] ABSTRACT: Objective Pituitary adenomas occur rarely in childhood and adolescence. Pituitary adenoma predisposition (PAP) has been recently associated with germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene. The aim of the study was to examine the proportion of germline AIP mutations in apparently sporadic paediatric pituitary adenomas.Design Genomic DNA was analysed for mutations in the AIP gene, by PCR amplification and direct sequencing.Patients A population-based cohort consisting of 36 apparently sporadic paediatric pituitary adenoma patients, referred to two medical centres in Italy, was included in the study. Patients were either less than 18 years at diagnosis, or showed clinical evidence of adenoma development before the age of 18 years.Results A heterozygous in-frame deletion Y248del (c.742_744delTAC) was identified in one GH-secreting adenoma patient. Loss of heterozygosity (LOH) analysis of tumour DNA revealed the loss of the wild-type allele. First degree relatives carrying the mutation were clinically unaffected.Conclusions While mutations were absent in non-GH-secreting adenoma patients, germline AIP mutations can be found in children and adolescents with GH-secreting tumours, even in the absence of family history. The present study reports the AIP mutation analysis results on patients of a single ethnic origin. Clearly, further studies are needed to improve our knowledge on the role of AIP in paediatric pituitary adenomas.
[Show abstract][Hide abstract] ABSTRACT: Pituitary adenomas are common neoplasms of the anterior pituitary gland. Germ-line mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene cause pituitary adenoma predisposition (PAP), a recent discovery based on genetic studies in Northern Finland. In this population, a founder mutation explained a significant proportion of all acromegaly cases. Typically, PAP patients were of a young age at diagnosis but did not display a strong family history of pituitary adenomas. To evaluate the role of AIP in pituitary adenoma susceptibility in other populations and to gain insight into patient selection for molecular screening of the condition, we investigated the possible contribution of AIP mutations in pituitary tumorigenesis in patients from Europe and the United States. A total of 460 patients were investigated by AIP sequencing: young acromegaly patients, unselected acromegaly patients, unselected pituitary adenoma patients, and endocrine neoplasia-predisposition patients who were negative for MEN1 mutations. Nine AIP mutations were identified. Because many of the patients displayed no family history of pituitary adenomas, detection of the condition appears challenging. Feasibility of AIP immunohistochemistry (IHC) as a prescreening tool was tested in 50 adenomas: 12 AIP mutation-positive versus 38 mutation-negative pituitary tumors. AIP IHC staining levels proved to be a useful predictor of AIP status, with 75% sensitivity and 95% specificity for germ-line mutations. AIP contributes to PAP in all studied populations. AIP IHC, followed by genetic counseling and possible AIP mutation analysis in IHC-negative cases, a procedure similar to the diagnostics of the Lynch syndrome, appears feasible in identification of PAP.
Proceedings of the National Academy of Sciences 04/2007; 104(10):4101-5. DOI:10.1073/pnas.0700004104 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We studied the distribution of the endogenous Arp2/3 complex in Amoeba proteus and visualised the ratio of filamentous (F-actin) to total actin in living cells. The presented results show that in the highly motile Amoeba proteus, Arp2/3 complex-dependent actin polymerisation is involved in the formation of the branching network of the contractile layer, adhesive structures, and perinuclear cytoskeleton. The aggregation of the Arp2/3 complex in the cortical network, with the exception of the uroid and advancing fronts, and the spatial orientation of microfilaments at the leading edge suggest that actin polymerisation in this area is not sufficient to provide the driving force for membrane displacement. The examined proteins were enriched in the pinocytotic pseudopodia and the perinuclear cytoskeleton in pinocytotic amoebae. In migrating amoebae, the course of changes in F-actin concentration corresponded with the distribution of tension in the cell cortex. The maximum level of F-actin in migrating amoebae was observed in the middle-posterior region and in the front of retracting pseudopodia. Arp2/3 complex-dependent actin polymerisation did not seem to influence F-actin concentration. The strongly condensed state of the microfilament system could be attributed to strong isometric contraction of the cortical layer accompanied by its retraction from distal cell regions. Isotonic contraction was limited to the uroid.
[Show abstract][Hide abstract] ABSTRACT: The organization of Amoeba proteus cortical layer is highly associated with cofilin-like protein activity. This protein is involved in actin dynamics in the middle-anterior region of migrating cells, but does not take place in processes of the cortical network disorganization that occurred in the uroid. Cofilin homologue and actin co-localized at the leading edge, in the cortical and perinuclear cytoskeleton, in the area of cellular adhesion and in streaming endoplasm. Actin dynamics induced by cofilin-like protein are important for normal morphology and motility of A. proteus.
[Show abstract][Hide abstract] ABSTRACT: Using scanning electron microscopy, Amoeba proteus cells migrating on the glass have been shown to develop dense coats of minipodia, which are discrete microprotrusions up to 8 microm long and approximately 0.5 microm across. They cover the middle-anterior area of the ventral cell surface, i.e. the region previously determined as the zone of most efficient adhesion of an amoeba to its substratum. Minipodia are sparse underneath the frontal zone and lacking from the tail region. In amoebae that adhere to the glass without moving, have just started moving, or show unstable motor polarity, minipodia are grouped in rosette contacts, cauliflower-like papillae composed of supporting platforms with crowns of minipodia emerging from them. Both structures abound with cytoskeletal F-actin, as shown by staining with fluorescein-conjugated phalloidin. Amoebae experimentally prevented from adhering to the substratum neither develop discrete minipodia nor rosette contacts.
Cell Biology International 02/2001; 25(12):1279-83. DOI:10.1006/cbir.2001.0806 · 1.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Amoeba proteus cells, strains A and C t , with well pronounced motor polarity (the polytactic and orthotactic forms) develop dense coats of discrete minipodia which anchor them to the glass substratum. The scanning electron microscopy demonstrates that minipodia are surface microprotrusions about 0.5 µm thick and up to 8 µm long, covering mainly the middle-anterior area of the ventral cell surface. They are few at the frontal zone and absent at the tail region. It means that the dense felt of discrete minipodia is located in the same region which has been earlier described as the zone of most efficient adhesion of a directionally moving amoeba. The cells without stable motor polarity and those which adhere to the glass without moving, or just start locomotion, lack areas covered by discrete minipodia; instead, minipodia are grouped in rosette contacts, which have the form of papillae composed of supporting platforms with crones of minipodia projected from them. The cells detached from the substratum by simple experimental procedures: radiate heterotactic forms produced by mechanical shocks, anucleate fragments obtained by microdissection, and amoebae in course of cation-induced pinocytosis, neither have separate minipodia nor rosette contacts. In contrast, during phagocytosis amoebae strongly adhere and produce dense sheaths of discrete minipodia extending along the whole surface up to the tail, except the naked front enclosing the phagosome. It was demonstrated by staining with the fluorescein-conjugated phalloidin that both types of adhesive structures: the discrete minipodia as well as the rosette contacts are very rich in the cytoskeletal F-actin.
[Show abstract][Hide abstract] ABSTRACT: Scanning electron microscopy of Amoeba proteus cells, fractured by micromanipulation after fixation and drying, reveals cable-like strands intertwining around the surface of the nucleus. Some strands leave the nucleus, cross the fluid endoplasm and reach the ectoplasmic cortical gel built of F-actin network. These strands contain large F-actin bundles, and are non-permanent structures. They cont rol the nucleus drift forwards with the endoplasmic flow, by anchoring it periodically to the ectoplasmic cylinder.
[Show abstract][Hide abstract] ABSTRACT: The abundance and species composition of diatoms adhering to the loricae of four agglutinated Tintinnina species, Laackmanniella naviculaefera, Codonellopsis gaussi Cd. balechi and Tintinnopsis lobiancoi, were determined. Diatoms from the Fragilariopsis group, F. cylindrus and F. pseudonana, dominated on tintinnid loricae from the Antarctic waters, whilst Thalassiosira spp. were predominant on loricae from the Baltic Sea. Although tintinnids utilized diatoms in the environment, it is not a rule that they use only these which are dominant. Our results suggest that certain diatoms are actively selected and agglutinated by particular tintinnid species.
Journal of Plankton Research 10/1996; 18(10):1931-1940. DOI:10.1093/plankt/18.10.1931 · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In water samples collected from the middle of Admiralty Bay (King George Island 62°08'S 58°25'W) between February 1990 and January 1991, 17 Tintinnina species were noted. Total Tintinnina numbers in summer were very high (up to 5000 cells m−3), but species diversity was low, consisting mainly of Cymatocylis affnislconvallaria , forma convallaria . During austral winter, cell numbers were very low, but species composition was diverse. Cymatocylis affnislconvallaria , forma affinis, and C.affmis/convallaria , forma convallaria , the polymorphic forms of one species C.affinis/convallaria , appear to be interchangeable during the year. The transition from one form to the other occurs in the spring and autumn. The typical Tintinnina polymorphism can be attributed to the prevailing environmental conditions.
Journal of Plankton Research 01/1994; DOI:10.1093/plankt/16.1.1 · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Using the interference-contrast videomicroscopy the speed of cytoplasmic streaming was measured during the sequence of division stages in thigmotactically settled specimens of Paramecium bursaria. The speed of cytoplasmic flow gradually decreased during the first stages of binary fission and movement became indistinguishable at stage D(3). Almost at the same time cytoplasm started to move in the opposite direction, pushing or pulling the dividing micronucleus into the prospective posterior daughter cell and eventually stopped at stage D(5)-D(6). Further cell division events proceeded without detectable movement of cytoplasmic components. Cytoplasmic streaming in the normal interphase route was gradually restored in daughter cells about 30-40 min after cell separation. During the whole period of binary fission phagocytosis was arrested. Transportation and participation in the positioning of prospective micronuclei in daughter cells seems to be the main function of cytoplasmic streaming activity in cell division of Paramecium bursaria. The possible relationship between the stages of cytoskeleton transitions and the kinetics of cytoplasmic streaming associated with cell divison is discussed.
European Journal of Protistology 11/1991; 27(4):352-6. DOI:10.1016/S0932-4739(11)80251-8 · 2.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Discocilia are observed in the marine ciliate Cymatocylis convallaria fixed with formalin, osmium in sublimate, and glutaraldehyde. They are restricted to the oral ciliature, while the somatic cilia have a normal, cylindrical profile. The swelling of the ciliary shaft can be located near or at the ciliary tip. In the bleb, located below the ciliary tip, the axoneme completely loses contact with the membrane. In the swelling at the tip, the axoneme is detached from the membrane unilaterally, forming a loop. Suggestions concerning the role of discocilia are presented.
Cell Biology International Reports 06/1991; 15(6):485-491. DOI:10.1016/0309-1651(91)90117-2
[Show abstract][Hide abstract] ABSTRACT: A sudden increase of temperature of surrounding medium, even in the sublethal range, induced in Amoeba proteus (strain War) and Amoeba borockensis the synthesis of a new class of proteins, which might be classified as the heat shock proteins (hsp). The new pattern of protein synthesis, determined by incorporation of (35)S-methionine, was detectable within 30 min after beginning of the exposure of the cells to the elevated temperature. Amoeba proteus (strain War) and Amoeba borokensis differ distinctly in thermosensitivity, however, no distinct difference in the rate of the heat shock proteins' synthesis between the studied species was found.
European Journal of Protistology 12/1988; 24(1):64-8. DOI:10.1016/S0932-4739(88)80010-5 · 2.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The morphology of pinocytosing Amoeba proteus induces by two monovalent cations: Na + and K + were examined at different calcium concentration. It was demonstrated that pinocytotic response of amoeba (number, size and shape of pinocytotic pseudopodia) was related to the amount of Ca 2+ accumulated on the cell surface.