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 Oscillating mitotic newt lung cell kinetochores are, on average, under tension and rarely push

1996 ◽  
Vol 109 (12) ◽  
pp. 2823-2831 ◽  
Author(s):  
J.C. Waters ◽  
R.V. Skibbens ◽  
E.D. Salmon
Keyword(s):  
Cell Cycle Progression ◽  
Immunofluorescence Microscopy ◽  
Differential Interference Contrast Microscopy ◽  
Cycle Progression ◽  
Spindle Poles ◽  
Constant Tension ◽  
Contrast Microscopy ◽  
Interference Contrast Microscopy ◽  
Regulate Cell Cycle Progression ◽  
Rest Length

Experimentally introduced tension on kinetochores and their centromeres has been shown to stabilize kinetochore attachment to microtubules, modify kinetochore directional instability, and regulate cell-cycle progression into anaphase. In mitosis, kinetochore tension and the stretch of centromere chromatin are produced by the movement of sister kinetochores toward opposite poles and astral ejection forces on the chromosome arms. However, newt lung cell kinetochores oscillate between poleward and away from the pole motility states throughout mitosis, indicating kinetochores are not under constant tension. To test whether kinetochores are under net tension while they are oscillating, and how often they are under compression and pushing into the chromosome, we measured the distance between sister kinetochores in newt lung cells using both video-enhanced differential interference contrast microscopy (VE-DIC) and immunofluorescence microscopy. We found that for chromosomes in which sister kinetochores are attached to opposite spindle poles, centromeres are, on average, stretched (2.2 microns in living cells and 1.8 microns in fixed cells) with respect to the inter-kinetochore ‘rest’ length (1.1 microns in living and fixed cells). For chromosomes in which only one kinetochore is attached to the spindle, the centromere chromatin associated with the tethered kinetochore is, on average, stretched to approximately half of the average inter-kinetochore distance measured for chromosomes in which both kinetochores are attached. We conclude that while newt lung cell kinetochores oscillate between states of P and AP movement, they are under tension approximately 90% of the time and under compression less than 6% of the time.

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.

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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