Animals ; Fluorescence ; Fluorescence Recovery After Photobleaching ; Fluorescence Resonance Energy Transfer ; Fluorometry ; Green Fluorescent Proteins ; Humans ; Immunohistochemistry

BACKGROUND: Intermediate filaments as well as microtubule and microfilament are major components of cytoskeleton of human cells. Melanocytes have vimentin intermediate filament, which have not been well investigated as other cytoskeletons, especially in their function. OBJECTIVE: The purpose of this study was to observe the motile characteristics of vimentin intermediate filament in living B16 melanoma cells. METHODS: The motile properties of vimentin intermediate filament have been studied in living B16 melanoma cells using green fluorescent protein(GFP). cDNA expressing GFP-vimentin fusion protein was cloned and transfected into living B16 melanoma cells. Living cells were observed under fluorescent microscope and confocal microscope. Time-lapse images were collected and analysed. RESULTS: GFP-vimentin is incorporated into the endogenous vimentin networks. Time-lapse observations of vimentin fibrils demonstrate that they are constantly changing their configurations. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Short filamentous structures('squiggle') are also seen actively translocating. Melanosomes also are changing their position back-and-pro constantly. They are co-localized very well with kinesin molecules in B16 melanoma cells. CONCLUSION: The vimentin intermediate filament and melanosomes in B16 melanoma cells have very active movement, which seem to have close relation with kinesin motor proteins.
Actin Cytoskeleton ; Clone Cells ; Cytoskeleton ; DNA, Complementary ; Fluorescence ; Fluorescence Recovery After Photobleaching ; Humans ; Intermediate Filaments* ; Kinesin ; Melanocytes ; Melanoma, Experimental* ; Melanosomes ; Microtubules ; Vimentin*

The cytoplasmic polyadenylation element (CPE)-binding protein (CPEB) binds to CPE containing mRNAs on their 3' untranslated regions (3'UTRs). This RNA binding protein comes out many important tasks, especially in learning and memory, by modifying the translational efficiency of target mRNAs via poly (A) tailing. Overexpressed CPEB has been reported to induce the formation of stress granules (SGs), a sort of RNA granule in mammalian cell lines. RNA granule is considered to be a potentially important factor in learning and memory. However, there is no study about RNA granule in Aplysia. To examine whether an Aplysia CPEB, ApCPEB1, forms RNA granules, we overexpressed ApCPEB1-EGFP in Aplysia sensory neurons. Consistent with the localization of mammalian CPEB, overexpressed ApCPEB1 formed granular structures, and was colocalized with RNAs and another RNA binding protein, ApCPEB, showing that ApCPEB1 positive granules are RNA-protein complexes. In addition, ApCPEB1 has a high turnover rate in RNA granules which were mobile structures. Thus, our results indicate that overexpressed ApCPEB1 is incorporated into RNA granule which is a dynamic structure in Aplysia sensory neuron. We propose that ApCPEB1 granule might modulate translation, as other RNA granules do, and furthermore, influence memory.
Animals ; Aplysia/genetics/*metabolism ; Fluorescence Recovery After Photobleaching ; RNA/genetics/metabolism ; Sensory Receptor Cells/*metabolism ; mRNA Cleavage and Polyadenylation Factors/genetics/metabolism/*physiology

OBJECTIVESTo explore intervention with electromagnetic noise for co-suppression effect on gap-junctional intercellular communication (GJIC) induced or strengthened by low intensity magnetic field with carcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA).

METHODSFibroblast cells from NIH 3T3 mice were exposed to extremely low intensity magnetic field (MF) 0.2 mT, 0.2 mT + TPA or/and electromagnetic noise with the same intensity of MF for 24 h, and GJIC was determined using fluorescence recovery analysis after photobleaching (FRAP) with a laser-scanning confocal microscope (Leica, Germany).

RESULTSGJIC function could be co-suppressed by MF of 0.2 mT with TPA, with fluorescence recovery of (23 +/- 11)%, lower than that in the control group [(46 +/- 19)%] and in the group with TPA only [(34 +/- 17) %] (P < 0.01), indicating 0.2 mT MF plus TPA could co-inhibit GJIC (P < 0.01). Superposition of 0.2 mT noise MF could get a fluorescence recovery of (35 +/- 19)% and significantly antagonize its co-suppression by TPA.

CONCLUSIONElectromagnetic noise of 0.2 mT could block the intensifying effect of power frequency magnetic field on TPA-induced GJIC inhibition.

Animals ; Cell Communication ; drug effects ; physiology ; radiation effects ; Cell Line ; Electromagnetic Fields ; adverse effects ; Fluorescence Recovery After Photobleaching ; Gap Junctions ; drug effects ; physiology ; radiation effects ; Mice ; NIH 3T3 Cells ; Noise ; adverse effects ; Tetradecanoylphorbol Acetate ; pharmacology

OBJECTIVETo explore the effect of millimeter wave (MW) at low power density on gap junctional intercellular communication (GJIC) in human keratinocytes (HaCaTs).

METHODSFluorescence recovery after photobleaching (FRAP) technique was employed to determine effect of 30.16 GHz MW exposure at 1.0 and 3.5 mW/cm(2) on GJIC with laser confocal scanning microscope.

RESULTSFRAP analysis revealed that 12-O-tetradecanoylphorbol-13-acetate (TPA) at a dose of 5 microg/L could inhibit GJIC in HaCaTs. Fluorescence recovery rate fell from (55 +/- 17)% in the controls to (34 +/- 13)% after photobleaching, with a very significant difference (P < 0.001). Exposure to MW alone for one hour at either 1.0 mW/cm(2) or 3.5 mW/cm(2) did not affect GJIC, with fluorescence recovery rates of (52 +/- 16)% and (50 +/- 17)%, respectively. GJIC suppression induced by TPA was weakened by MW combined with 5 microg/L TPA treatment for one hour, which could be partially recovered by exposure to 1.0 mW/cm(2) MW with fluorescence recovery rate of (47 +/- 16)%, P < 0.01, and fully recovered by exposure to 3.5 mW/cm(2) MW with fluorescence recovery rate of (50 +/- 16)%, P < 0.001, with a very significant difference.

CONCLUSIONSGJIC suppression induced by TPA could be eliminated or diminished by exposure to millimeter wave in HaCaTs.

Cell Communication ; drug effects ; radiation effects ; Cell Line ; Fluorescence Recovery After Photobleaching ; methods ; Gap Junctions ; drug effects ; physiology ; radiation effects ; Humans ; Keratinocytes ; cytology ; physiology ; Microwaves ; adverse effects ; Tetradecanoylphorbol Acetate ; pharmacology