Myosin subfragment binding for the localization of actin-like microfilaments in cultured cells. A light and electron microscope study.

Abstract

Fluorescein-labeled heavy meromyosin subfragment-1 (F-S-l) has been purified by ion exchange chromatography and characterized in terms of its ability to bind specifically to actin. F-S-1 activates the Mg+ § triphosphatase activity of rabbit skeletal muscle actin and decorates actin as shown by negative stains and thin sections of rabbit actin and rat embryo cell microfilament bundles, respectively. Binding of F-S-1 to cellular structures is prevented by pyrophosphate and by competition with excess unlabeled S-1.The F-S-1 is used in light microscope studies to determine the distribution of actin-containing structures in interphase and mitotic rat embryo and rat kangaroo cells. Interphase cells display the familiar pattern of fluorescent stress fibers. Chromosome-to-pole fibers are fluorescent in mitotic cells.The glycerol extraction procedures employed provide an opportunity to examine cells prepared in an identical manner by light and electron microscopy. The latter technique reveals that actin-like microfilaments are identifiable in spindles of glycerinated cells before and after addition of S-1 or HMM. In some cases, microfilaments appear to be closely associated with spindle microtubules. Comparison of the light and electron microscope results aids in the evaluation of the fluorescent myosin fragment technique and provides further evidence for possible structural and functional roles of actin in the mitotic apparatus.KEY WORDS microfilaments HMM subfragment-1 cultured cells actin mitotic spindle fluorescence microscopy Determination of the distribution and organization of actin-like microfilaments in nonmuscle cells is an important approach to discerning the mechanisms of cellular motility (see references 40 and 41 for reviews). Microfilaments are identified in cells fixed and thin-sectioned for electron microscopy by their size (-6 nm diameter) and ability to bind the myosin fragments heavy meromyosin (HMM) and HMM subfragment-1 (S-l), resulting in "arrowhead" configurations or decorated complexes (26,27,18). This reaction is considered to be specific for actin, as HMM and S-1 do not decorate other fibrous structures in the cell (27,19,40 also been employed to study the overall distribution of structures thought to contain actin (5). However, the supramolecular organization of aco tin cannot be determined by these techniques because of the limits of resolution of the light microscope (22). Only a few attempts have been made to correlate directly information from light and electron microscope studies. For instance, the distribution of phase-dense stress fibers (5) and birefringent fibers seen with polarized light microscopy (17) has been shown to correspond to that of microfilament bundles in several cultured animal cells (20,22).Fluorescein-labeled HMM (F-HMM), S-1 (F-S-1), and antiactin antibody in indirect immunofluorescence studies have been shown to stain stress fibers in extensively spread cells which correspond to microfilament bundles (45,47,22,29,20). We have shown that comparison of light an...

Differential staining of actin in metaphase spindles with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin and fluorescent DNase: is actin involved in chromosomal movement?

“…Evidence against cytoplasmic actin contamination of the spindle comes from experiments in which glutaraldehyde fixation, which should prevent gross transfer ofactin, was used to fix specimens. These experiments also reported the presence ofactin in mitotic spindles (18)(19)(20). Further complications arise from the report of actin filaments in close proximity to microtubules (18).…”

“…These experiments also reported the presence ofactin in mitotic spindles (18)(19)(20). Further complications arise from the report of actin filaments in close proximity to microtubules (18). This observation suggests that actin filaments in the spindle might be obscured by microtubules, thus accounting for the apparent lack of spindle actin filaments as reported (1,21,22).…”

“…After fixation and extraction, the cells were incubated with 200 A.l of 150 nM NBD-Ph for 20 min at 220C, Fl-HMM at [10][11][12][13][14][15][16][17][18][19][20] ,ug/ml for 40 min at 370C, or Fl-DNase or Rh-DNase at 40 (1981) -44% (Rh-DNase) or 70% (Fl-DNase) of the activity of the unlabeled starting DNase I. When measured in the presence of equimolar actin, the DNase activities of Rh-DNase, Fl-DNase, and unlabeled DNase decreased to 39%, 60%, and 26% of the respective uninhibited rates.…”

Calcium-dependent regulator protein: Localization in mitotic apparatus of eukaryotic cells

“…However, evidence for the presence of actin or myosin in the mitotic apparatus is controversial. Actin inimunofluorescence (21) or heavy meromyosin decoration (19,20,22) of actin in the mitotic apparatus has been demonstrated only after lysis and extraction of cells with either glycerol (19,20,22) or Triton X-100 (21) prior to fixation. Extraction of the cells before fixing may allow artifactual redistribution of actin to the spindle.…”

“…1). Beginning in early prophase of mitosis, the cytoskeleton of cells became disorganized, as reflected by actin (17) and tubulin immunofluorescence (15 (18) and actin (19)(20)(21)(22) in the mitotic apparatus. In fact, the specific localization of actin in the half-spindles is similar to the pattern described for CDR in the present study.…”