Nuclear and cytoplasmic actin in dinoflagellates

Abstract

Experiments using monoclonal and polyclonal anti-actin antibodies allowed us to demonstrate the presence of F- or G-actin in original protists, dinoflagellates, either by biochemistry, immunofluorescence and in TEM. SDS-PAGE electrophoresis and immunoblottings made either from total or nuclear protein extracts revealed the presence of a 44-kDa band reacting with monoclonal anti-actin antibody in two species, Prorocentrum micans and Crypthecodinium cohnii, and thus demonstrated the presence of actin in nuclear and cytoplasmic fractions. After squash preparation of P micans cells, actin was identified within the nucleus and in some regions of the cytoplasm by immunofluorescence microscopy. Labelling of both the nucleolus and the centrosome region was evident together with amorphous nucleoplasmic material surrounding the chromosomes. The use of cryosections of intact P micans and C cohnii cells for immunofluorescence along with staining with DAPI to delineate the chromosomes themselves, yielded finer resolution of the intranuclear network labelling pattern and allowed us to complete our observations, in particular on the cytoplasmic labelling. In P micans, in addition to the centrosome region, the cytoplasmic channels passing through the nucleus in dividing cells are labelled. In C cohnii, the cortex, the centrosome region, the cytoplasmic channels, the region surrounding the nucleus, the filaments linking it to the cortex and the cleavage furrow are also labelled. In the nucleus of the two species, there is a prominent "weft' of fine actin filaments in the nucleoplasm forming a matrix of varying density around the persistent chromosomes. This actin matrix, of unknown function, is most conspicuous at the end of the S-phase of the cell cycle. Fluorescent derivatives of phalloidin, used as diagnostic cytochemical probes for polymeric actin (F-actin), gave similar results. Positive TEM immunolabelling of intranuclear actin confirms its presence in the nucleoplasm, in the nucleolus where the preribosomal region is labelled while C cohnii chromosomes are unlabelled and the P micans chromosomes very slightly. In the cytoplasm, lips of the cleavage furrow and kinetosome regions are labelled as well as the centrosome region. The possible functions of this protein located in several compartments of dinoflagellate cells are discussed.

Dynamic changes in microtubule organization during division of the primitive dinoflagellate Oxyrrhis marina

“…The presence of an actin band at furrowing cell surface reported in this paper confirms the presence of actin previously described in other dinoflagellates (Soyer-Gobillard et al, 1996). The cytokinesis is likely to be mediated by the contraction of this band.…”

Understanding Interactions between Cellular Matrices and Metal Complexes: Methods To Improve Silver Nanodot‐Specific Staining

“…The N2/N1 ratio gradually increased from 1.0±0.2 to 2.2±0.4 over the course of cell cycle, in line with the activity of nucleoli . Moreover, the high signal‐to‐background ratio created by the 615‐emitter staining enabled us to distinguish each nucleolus in a nucleus and to measure the average area of nucleoli in the whole image using ImageJ software (Figure S5 in the Supporting Information) significantly better than had been possible using either the traditional TEM imaging method or the silver staining method . These results indicate that both the number of nucleoli per cell and the average area of a nucleolus increased from the G1 stage to the G2 stage (Figure C and D), suggesting a more active cellular activity…”

Topology of splicing and snRNP biogenesis in dinoflagellate nuclei

“…The existence of specific subnuclear compartments for transcription/splicing and snRNP biogenesis in dinoflagellates was demonstrated by immunofluorescence and EM studies. Transcription occurs on extrachromosomal DNA loops, protruding from the condensed chromosome cores (Sigee, 1984), which contain histone-like proteins (Sala Rovira et al, 1991), transcription factors (Guillebault et al, 2001), stretches of Z-DNA configuration associated with the transcribing polymerases (Soyer-Gobillard et al, 1990;Černá et al, 2004) and nuclear actin (Soyer-Gobillard et al, 1996), involved in chromatin remodelling and RNA transcription and splicing (Bettinger et al, 2004). [H 3 ]Uridine incorporation (Echeverría et al, 1993) and detection of Sm proteins and the 2,2,7-methyl-G cap of snRNAs (O.M.…”