Notwithstanding the controversial evidence for an extensive meshwork of filaments in the interchromatin space, it still behooves us to ask with an open mind whether the electron-translucent nucleoplasm is simply a concentrated sea of individual protein molecules or has, in addition, some formed elements.
In a study of IGCs labeled with a green fluorescent protein–mRNA splicing factor protein (Misteli et al., 1997
), real-time observations in living cells revealed that ~80% of the IGCs remain stationary. This might signify tethering to a putative nuclear matrix or to nascent pre-mRNAs extending from their transcription sites to a vicinal IGC. In addition, a small portion of the IGCs were observed to undergo short-range movements (Misteli et al., 1997
). Although the observed degree of movement of IGCs can, at present, be taken neither as supporting nor negating the nuclear matrix concept, these important in vivo observations prompt one to further ponder whether the interchromatin space is simply a concentrated protein solution or has some degree of preformed structure. A good place to start is nuclear actin.
Nonmuscle actin is ubiquitously present in eukaryotic cells and has been shown to equilibrate between nucleus and cytoplasm in amphibian oocytes (Clark and Merriam, 1977
). Evidence for the presence of actin in the nucleus of several other species and cell types has also been reported (Fukui, 1978
; Fukui and Katsumaru, 1979
; Krohne and Franke, 1980
; Osborne and Weber, 1980
; Gounon and Karsenti, 1981
; Welch and Suhan, 1985
; Milankov and DeBoni, 1993
; Amankwah and DeBoni, 1994
; Yan et al., 1997
; Wada et al., 1998
; Gonsior et al., 1999
). Moreover, a number of actin-binding nuclear proteins have been described (Ankenbauer et al., 1989
; Rimm and Pollard, 1989
; Nowak et al., 1997
; Cairns et al., 1998
; Harata et al., 1999
). The nuclear actin of Xenopus
oocytes exists as a gel within the intact nucleus under certain, gentle conditions of germinal vesicle preparation (Clark and Rosenbaum, 1979
; J.G. Gall, personal communication of unpublished results) and can be microsurgically extirpated by teasing away the nuclear envelope (Clark and Rosenbaum, 1979
). This suggests that one of the abundant nuclear proteins in living amphibian oocytes is on a delicate edge of polymerization.
Recently, monomeric β-actin in the nucleus has emerged in the context of studies of chromatin remodeling during gene transcription activation. A group of mammalian nuclear proteins termed BAFs has been described that are related to the well-characterized yeast SWI/SNF chromatin remodeling complex (Wang et al., 1996
). Biochemical characterization of the nuclear BAF complex from calf thymus and activated mouse lymphocytes revealed that its subunits include both monomeric β-actin as well as a novel actin-related protein (Zhao et al., 1998
). The association of actin with the nuclear BAF complex in vivo was confirmed in studies using a cell-permeant protein–protein cross-linking agent, and additional data indicated that the BAF complexes also bind to profilin and cofilin, further emphasizing the central role of actin-binding proteins, as well as actin, in this chromatin remodeling complex (Zhao et al., 1998
). These results bring to mind an earlier publication in which it was reported that actin antibodies inhibited transcription on lampbrush chromosomes when injected into amphibian oocyte nuclei (Scheer et al., 1984
). Whether actin is normally other than monomeric in the nucleus remains unclear. There may be gene transcription site–proximal actin, present as monomers or perhaps short filaments, possibly capped in a transcription-linked regulated mechanism or conceivably dynamically unstable. However, the described extensive, anastomizing nuclear matrix does not appear to be substantially composed of actin by either ultrastructural criteria or polypeptide composition (Pederson, 1998
Are there any other clues to structure in the nucleoplasmic ground substance, if not as multimicrometer-spanning scaffolds then perhaps at least as shorter-range elements? Here there are recent, encouraging clues.
Nup 153 and Tpr
are nuclear pore complex–associated proteins that are organized into filaments extending 100–350 nm into the nucleus (Cordes et al., 1993
; Zimowska et al., 1997
). Although these filaments do not extend sufficiently deeply or intersectionally into the nucleus to be candidates for the observed extensively anastomosing nuclear matrix, their suggested role in mRNA export (Bangs et al., 1998
) nonetheless presents an alternative element of nonchromatin nuclear structure that may facilitate a late step in gene readout, albeit confined to the outer nuclear perimeter.
A second and intriguing group of proteins for careful consideration as elements of internal nuclear structure are the nuclear lamins. These cousins of the cytoplasmic intermediate filaments were originally thought to exist solely as a fence–wire network underneath the nuclear envelope. But subsequent studies have revealed the presence of a population of internal nuclear lamins as well (Goldman et al., 1992
; Bridger et al., 1993
; Neri et al., 1999
; R. Goldman, personal communication of unpublished results; C. Hutchison, personal communication of unpublished results). Although the oligomerization–polymerization state of these intranuclear lamins is not known, their mobility measured by fluorescence recovery after photobleaching in living cells suggests that they may not be monomeric (Moir et al., 1998
). This is a very important subject for further investigation.
These studies of nuclear actin, Tpr
proteins, and nucleoplasmic lamins remind us that filament-forming protein families are present in cell nuclei. If short arrays of filaments were to nucleate around gene transcription and RNA processing sites, these local “gene expression matrices” might help tether the necessary transcription and RNA processing machinery and yet would not necessarily comprise a nucleus-filling, long-range filament system such as the one seen in extracted preparations called the nuclear matrix. Such local structure might be important as an organized framework for final transcript processing and active release of the finished RNA before a diffusion-based transport to the Nup 153/Tpr
and possibly other filament systems at the nuclear perimeter (Strambio-de-Castillia et al., 1999
; Politz and Pederson, 2000
Nothing in the foregoing considerations rules out the possibility that mRNA might move by diffusion and yet also transiently interact with some sort of structural elements in the interchromatin space. Although these two notions might seem somewhat contradictory, or even mutually exclusive, the issue comes down to the lifetimes of the postulated mRNA–structural element interaction (Politz and Pederson, 2000
). A recent electron microscopic tomography study of Chironomus
Balbiani ring mRNP particles in the nucleoplasm reveals that a portion of these RNPs is in contact with thin fibers (Miralles et al., 2000
), even though kinetic analysis of the movement of mRNP in this very same system (using living Chironomus
salivary gland cells) indicates that the particles overall display random movement that is compatible with diffusion (Daneholt, 1999
; Singh et al., 1999
). The thin nucleoplasmic fibers observed by Miralles et al. (2000)
are described by the authors as not resembling the extensive, nucleoplasm-filling meshwork observed in typical nuclear matrix preparations.