scholarly journals Detection of single microtubules in living cells: particle transport can occur in both directions along the same microtubule.

1984 ◽  
Vol 99 (5) ◽  
pp. 1785-1793 ◽  
Author(s):  
J H Hayden ◽  
R D Allen
Keyword(s):  
Electron Microscopy ◽  
Particle Transport ◽  
Corneal Stroma ◽  
Differential Interference Contrast Microscopy ◽  
Whole Mount ◽  
Contrast Microscopy ◽  
Linear Elements ◽  
Interference Contrast Microscopy ◽  
Cytoplasmic Structures ◽  
Cellular Components

Video-enhanced contrast/differential interference-contrast microscopy was used in conjunction with whole mount electron microscopy to study particle transport along linear elements in fibroblasts. Keratocytes from the corneal stroma of Rana pipiens were grown on gold indicator grids and examined with video microscopy. Video records were taken of the linear elements and associated particle transport until lysis and/or fixation of the cells was completed. The preparations were then processed for whole mount electron microscopy. By combining these two methods, we demonstrated that linear elements detected in the living cell could be identified as single microtubules, and that filaments as small as 10 nm could be detected in lysed and fixed cells. The visibility of different cytoplasmic structures changed after lysis with many more cellular components becoming visible. Microtubules became more difficult to detect after lysis while bundles of microfilaments became more prominent. All particle translocations were observed to take place along linear elements composed of one or more microtubules. Furthermore, particles were observed to translocate in one or both directions on the same microtubule.

Protein phosphatases independently regulate vesicle movement and microtubule subpopulations in hepatocytes

1996 ◽  
Vol 271 (3) ◽  
pp. C929-C943 ◽  
Author(s):  
S. F. Hamm-Alvarez ◽  
X. Wei ◽  
N. Berndt ◽  
M. Runnegar
Keyword(s):  
Immunofluorescence Microscopy ◽  
Maximum Dose ◽  
Protein Phosphatases ◽  
Differential Interference Contrast Microscopy ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Microscopy Analysis ◽  
High Doses ◽  
Vesicle Movement ◽  
Cellular Components

To investigate the regulation of microtubule (MT)-based vesicle transport and the interphase MT array in hepatocytes, we have used okadaic acid (OKA) and microcystin (MCYST), two toxins that inhibit serine-threonine protein phosphatases (PP) 1 and 2A, to alter cellular phosphorylation. Video-enhanced differential interference contrast microscopy analysis revealed that both toxins inhibited the frequency, velocity, and run length of MT-dependent vesicle movements dose dependently between 50 and 500 nM. At our maximum dose of 500 nM, both toxins significantly decreased PP2A activity (OKA to 45 +/- 12% and MCYST to 57 +/- 2%), whereas PP1 was inhibited only by MCYST. Because no additional effects on vesicle movements were caused by MCYST over the changes caused by OKA, these data implicate PP2A in the regulation of MT-dependent vesicle movement. To understand whether the changes in parameters of vesicle movements were due to changes in the MT array, the effects of these toxins on MT distribution were examined by immunofluorescence microscopy. Although lower doses of OKA produced no effects, treatment with 500 nM OKA altered MT organization and also caused fragmentation and loss of acetylated (stable) MTs. In contrast, MCYST concentrations up to 500 nM elicited no changes in MT organization in general or in the acetylated (stable) array. From these findings we conclude that inhibition of MT-dependent vesicle movement by the PP inhibitors, MCYST and OKA, in hepatocytes cannot result from changes or disruption in the MT array. Because OKA (an inhibitor of PP2A only in our system) at high doses caused loss of stable MTs, whereas MCYST (an inhibitor of both PP1 and PP2A) did not, we conclude that the control of the preservation of the stable MT array in hepatocytes is complex. Stable MTs require active PP2A for maintenance, but the disruption of the array through inhibition of PP2A can be prevented if PP1 is also inhibited, suggesting that the relative degree of phosphorylation of multiple cellular components is the determinant of MT stability.

scholarly journals Studies on the motility of the foraminifera. II. The dynamic microtubular cytoskeleton of the reticulopodial network of Allogromia laticollaris.

1983 ◽  
Vol 97 (6) ◽  
pp. 1668-1676 ◽  
Author(s):  
J L Travis ◽  
J F Kenealy ◽  
R D Allen
Keyword(s):  
Polarized Light ◽  
Differential Interference Contrast ◽  
Differential Interference Contrast Microscopy ◽  
Transmission Electron ◽  
Microtubular Cytoskeleton ◽  
Whole Mount ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Birefringent Fibrils ◽  
Positively Charged

Lamellipodia have been induced to form within the reticulopodial networks of Allogromia laticollaris by being plated on positively charged substrata. Video-enhanced, polarized light, and differential interference contrast microscopy have demonstrated the presence of positively birefringent fibrils within these lamellipodia. The fibrils correspond to the microtubules and bundles of microtubules observed in whole-mount transmission electron micrographs of lamellipodia. Microtubular fibrils exhibit two types of movements within the lamellipodia: lateral and axial translocations. Lateral movements are often accompanied by reversible lateral associations between adjacent fibrils within a lamellipodium. This lateral association-dissociation of adjacent fibrils has been termed 'zipping' and 'unzipping'. Axial translocations are bidirectional. The axial movements of the microtubular fibrils can result in the extension of filopodia by pushing against the plasma membrane of the lamellipodia. Shortening, or complete withdrawal, of such filopodia is accomplished by the reversal of the direction of the axial movement. The bidirectional streaming characteristic of the reticulopodial networks also occurs within the lamellipodia. In these flattened regions the streaming is clearly seen to occur exclusively in association with the intracellular fibrils. Transport of both organelles and bulk hyaline cytoplasm occurs bidirectionally along the fibrils.

Correlative light microscopy, electron microscopy, and cytochemistry of cytoskeletal structures in motile fibroblasts

1991 ◽  
Vol 49 ◽  
pp. 168-169
Author(s):  
Julian P. Heath ◽  
Donna Turner ◽  
Bruce F. Holifield
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Differential Interference Contrast Microscopy ◽  
Dynamic Interactions ◽  
Fine Grained ◽  
Motile Cells ◽  
Contrast Microscopy ◽  
Carbon Coated ◽  
Interference Contrast Microscopy ◽  
Triton X

Contrast-enhanced video differential interference contrast microscopy (VDICM) is revealing new details about the dynamics of F-actin assemblies in motile cells. We are using correlative single cell light and electron microscopy and immunocytochemistry to understand the dynamic interactions of the actin cytoskeleton in lamellipodia and the leading lamella.Fibroblasts were cultured on carbon-coated glass #1 coverslips and examined on a Zeiss Axiophot. Cells were perfused with 1% glutaraldehyde (GA) in PIPES buffer and fixed for 15 min., quenched with 1 mg/ml borohydride/PBS and extracted with 0.1% Triton X-100 in PBS for 1 min. Cell postions were marked with a diamond objective, and the cells were incubated for 30 min. with 0.6 uM rhodamine-conjugated phalloidin to stain for F-actin. Light and fluorescence micrographs were taken on fine-grained film. After photography, cells were further fixed in 2.5% GA, postfixed in 1% osmium, dehydrated in ethanol and embedded in Spurrs resin. The glass was removed by scoring with a diamond pencil followed by immersion in liquid N2. Cells were relocated in the blocks by phase-contrast microscopy. Thin (80 nm) sections were examined in Philips EM 410 at 60 kV on a dicentric goniometric stage.

The translocation of mitochondria along insect ovarian microtubules from isolated nutritive tubes: a simple reactivated model

1987 ◽  
Vol 88 (5) ◽  
pp. 641-648
Author(s):  
H. Stebbings ◽  
C. Hunt
Keyword(s):  
Electron Microscopy ◽  
Energy Metabolism ◽  
Differential Interference Contrast ◽  
Differential Interference Contrast Microscopy ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy

Nutritive tubes, the microtubule-based translocation channels that link the trophic tissue to the developing oocytes in the ovaries of hemipteran insects, have been isolated and examined using video-enhanced differential interference contrast microscopy. When viewed in this way the nutritive tubes are seen to fray into linear strands, which, on the addition of exogenous ATP, support the translocation of particles along their lengths. The movement is also seen with GTP but not AMP-PNP. It is not affected by the addition of inhibitors of dynein or of energy metabolism. Electron microscopy shows the strands to consist of bundles of parallel microtubules of different sizes and the moving particles to be mitochondria. Comparisons are drawn between the movement of mitochondria along isolated insect ovarian microtubules and the reported translocation of vesicles along microtubules from squid axoplasm. The simplicity of the insect system is emphasized. The fact that it can be isolated easily and characterized biochemically makes it potentially valuable for investigating microtubule-based translocation.

The application of video-enhanced constrast/differential interference constrast microscopy in conjunction with whole mount electron microscopy to the study of organelle transport

1988 ◽  
Vol 46 ◽  
pp. 66-67
Author(s):  
J. H. Hayden
Keyword(s):  
Electron Microscopy ◽  
Rana Pipiens ◽  
Silver Film ◽  
Immunofluorescence Microscopy ◽  
Organelle Transport ◽  
Linear Element ◽  
Whole Mount ◽  
Carbon Coated ◽  
Linear Elements ◽  
Dic Microscopy

In a previous study, Allen video-enhanced constrast/differential interference constrast (AVEC-DIC) microscopy was used in conjunction with immunofluorescence microscopy to demonstrate that organelles and vesicle move in either direction along linear elements composed of microtubules. However, this study was limited in that the number of microtubules making up a linear element could not be determined. To overcome this limitation, we have used AVEC-DIC microscopy in conjunction with whole mount electron microscopy.Keratocytes from Rana pipiens were grown on glass coverslips as described elsewhere. Gold London Finder grids were Formvar- and carbon coated, and sterilized by exposure to ultraviolet light. It is important to select a Formvar film that gives a grey reflection when it is floated on water. A silver film is too thick and will detract from the image in the light microscope.

scholarly journals Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis

2003 ◽  
Vol 160 (5) ◽  
pp. 671-683 ◽  
Author(s):  
Alexey Khodjakov ◽  
Lily Copenagle ◽  
Michael B. Gordon ◽  
Duane A. Compton ◽  
Tarun M. Kapoor
Keyword(s):  
Three Dimensional ◽  
Differential Interference Contrast ◽  
Differential Interference Contrast Microscopy ◽  
Spindle Formation ◽  
Capture Mechanism ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Kinesin Eg5 ◽  
Mitotic Kinesin Eg5 ◽  
Dependent Mechanism

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living α-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient chromosome-free bipolar array whose orientation specified the axis along which chromosomes segregated. We propose that the capture and incorporation of preformed K-fibers complements the microtubule plus-end capture mechanism and contributes to spindle formation in vertebrates.

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