[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. · 1.36 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.).
Biochemistry and Cell Biology 01/2009; 86(6):509-19. · 2.92 Impact Factor
[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. · 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: 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: Before the furrow formation the non-polar general contraction resulting in cell de-adhesion and spherulation, and the following relaxation leading to re-adhesion, flattening and spreading, are both necessary prerequisites of a successful cytokinesis in A. proteus. The bipartition begins by sudden generation of two divergent cytoplasmic streamings at the late anaphase, always before the formation of furrow. All these facts fit better with the polar relaxation model than with the equatorial contraction model of initiating the fission of amoeba. After formation of the furrow the contractile ring gradually constricts the cytoplasmic connection between daughter cells. Endopasm flow in the connection bridge is no more bipolar but irregularly reversing; it compensates hydrostatic pressure differences between daughter cells. The final break of the connection is explained by its stretching because of the disparate locomotor activities on both sides of the furrow and owing to the cytoskeleton disassembly inside the connecting bridge.
[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.