Fluorescence cytometry of microtubules and nuclear DNA during cell‐cycle and reverse‐transformation

Abstract

Synchronized CHO-K1 cells and their dibutyryl c-AMP treated counterparts have been characterized by means of static and flow fluorescence cytometry at the level of nuclear DNA and cytoplasmic microtubules. In order to confirm earlier findings on synchronized population, Carnoy fixed and hydrolyzed, several new findings are here reported at the level of single intact cell. The fluorescence intensity of DAPI-stained glutaraldehyde fixed 2C cells correlates well with the average absorbance of the corresponding Feulgen-stained cells, thereby appearing also to be a measure of chromatin condensation during the G1 phase. In the early part of G1, the drastic alteration in anti-beta tubulin immunostaining is shown to parallel microtubule depolymerization induced by calcium or colcemide. The known 1-2 h lengthening of the G1 period after reverse-transformation appears to correlate with a similar delay in the abrupt chromatin decondensation. The above results are discussed in terms of the role of microtubules and nuclear morphometry (and their coupling) in the control of cell cycle progression of transformed vs. fibroblast-like cells.

Protein bioelectronics: a review of what we do and do not know

“…These techniques include measurements of their topography, as well as spectroscopic, and ET properties. 28,[173][174][175][176][177][178][179] Atomic force microscopy (see section 3.6.2) and scanning tunneling microscopy (STM) have been employed to investigate morphological properties, ETp, and redox activity of individual metalloproteins, chemisorbed on gold substrates. 2,32,49,85,149,[180][181][182][183][184][185][186] The arrangement and orientation of the proteins on a gold substrate, and their structural and dynamic properties, have been simulated using molecular dynamics.…”

Relationship between chromatin compactness and dye uptake for in situ chromatin stained with DAPI

“…Recently, new instruments based on laser scanning cytometry (20) demonstrated an ability to analyze intensitometric and morphometric parameters that makes possible the estimation of differences in chromatin structure. It is well‐known that the integrated fluorescence intensity of nuclei stained with DNA‐specific dyes is related not only to the total DNA content but also to the chromatin‐DNA structure (6, 21, 22). In fact, only a portion of the double helix is accessible to the dye when DNA is organized in a chromatin fiber, and this portion changes depending on differences in fiber condensation (23).…”

Effect of fixatives on calf thymocytes chromatin as analyzed by 3D high-resolution fluorescence microscopy

“…The quantitative analysis of the processed images allowed us to reconstruct the topological distribution of DNA inside the nucleus and to correlate the data with the results obtained by differential scanning calorimetry on similar samples. o 1996 Wiley-Liss, I~C .The effects of common fixatives at the level of chromatin-DNA distribution and organization have been extensively studied in recent years (6,7,17,23,31,32,36,37) because for the most part, structural analyses of in situ chromatin is usually carried out on fixed and sectioned cells. Several recent reports noted numerous details and hypotheses on chromatin organization inside the eukaryotic nucleus: the existence of different structural domains has been reported both for interphase chromatin and for mitotic chromosomes (7,9,20,22,24,28,30).…”