Involvement of myosin VI immunoanalog in pinocytosis and phagocytosis in Amoeba proteus

2008 ◽  
Vol 86 (6) ◽  
pp. 509-519 ◽  
Author(s):  
Magdalena Sobczak ◽  
Anna Wasik ◽  
Wanda Kłopocka ◽  
Maria Jolanta Rędowicz
Keyword(s):  
Electron Microscopy ◽  
Immunofluorescence Microscopy ◽  
Amoeba Proteus ◽  
Myosin Vi ◽  
Filamentous Actin ◽  
Confocal Immunofluorescence Microscopy ◽  
Endogenous Protein ◽  
Confocal Immunofluorescence ◽  
Pinocytotic Vesicles ◽  
Phagocytic Cups

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.).

Basket-shaped structures formed by F-actin in the nuclei of elongating cells of Nicotiana tabacum

1993 ◽  
Vol 71 (5) ◽  
pp. 725-731 ◽  
Author(s):  
Harry M. P. Kengen ◽  
Ton van Amstel ◽  
Bart Knuiman
Keyword(s):  
Nicotiana Tabacum ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Filamentous Actin ◽  
Suspension Cells ◽  
Confocal Immunofluorescence Microscopy ◽  
Cytoplasmic Actin ◽  
Extraction Buffer ◽  
Confocal Immunofluorescence

Phalloidin labelled with trimethyl rhodamine isothiocyanate in extraction buffer was used to stain actin in suspension cells of tobacco. Confocal immunofluorescence microscopy indicated the presence of filamentous actin containing structures in interphase nuclei of elongating cells of Nicotiana tabacum ‘Bright Yellow 2 Go’. The results were not affected by the omission of DMSO from the extraction medium. These structures, called actin baskets, were present in about 20% of the cells after induced elongation and varied in size, shape, and number per nucleus. The cytoplasmic actin array remained intact. It is proposed that the baskets have a transient character and are related to differentiation. Key words: confocal laser scanning microscopy, elongation, F-actin, nucleus, protoplasts, TRITC–phalloidin.

scholarly journals Human Rhinovirus Type 2 Is Internalized by Clathrin-Mediated Endocytosis

2003 ◽  
Vol 77 (9) ◽  
pp. 5360-5369 ◽  
Author(s):  
Luc Snyers ◽  
Hannes Zwickl ◽  
Dieter Blaas
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Immunofluorescence Microscopy ◽  
Dominant Negative ◽  
Human Rhinovirus ◽  
Coated Pits ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Transient Overexpression

ABSTRACT Using several approaches, we investigated the importance of clathrin-mediated endocytosis in the uptake of human rhinovirus serotype 2 (HRV2). By means of confocal immunofluorescence microscopy, we show that K+ depletion strongly reduces HRV2 internalization. Viral uptake was also substantially reduced by extraction of cholesterol from the plasma membrane with methyl-β-cyclodextrin, which can inhibit clathrin-mediated endocytosis. In accordance with these data, overexpression of dynamin K44A in HeLa cells prevented HRV2 internalization, as judged by confocal immunofluorescence microscopy, and strongly reduced infection. We also demonstrate that HRV2 bound to the surface of HeLa cells is localized in coated pits but not in caveolae. Finally, transient overexpression of the specific dominant-negative inhibitors of clathrin-mediated endocytosis, the SH3 domain of amphiphysin and the C-terminal domain of AP180, potently inhibited internalization of HRV2. Taken together, these results indicate that HRV2 uses clathrin-mediated endocytosis to infect cells.

scholarly journals Rab11 regulates recycling through the pericentriolar recycling endosome.

1996 ◽  
Vol 135 (4) ◽  
pp. 913-924 ◽  
Author(s):  
O Ullrich ◽  
S Reinsch ◽  
S Urbé ◽  
M Zerial ◽  
R G Parton
Keyword(s):  
Immunofluorescence Microscopy ◽  
Bound State ◽  
Small Gtpases ◽  
Recycling Endosome ◽  
Functional Studies ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Recycling Pathway ◽  
And Function ◽  
Perinuclear Area

Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic. In the present study, we examined the distribution and function of rab11. Rab11 was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells. Expression of rab11 mutants that are preferentially in the GTP- or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rab11-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rab11-GDP caused a dispersion of the transferrin labeling. Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rab11 proteins. However, recycling from the later recycling endosome was inhibited in cells overexpressing the rab11-GDP mutant. Rab5, which regulates early endocytic trafficking, acted before rab11 in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11-positive recycling endosome. These results suggest a novel role for rab11 in controlling traffic through the recycling endosome.

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