Various types of membranous and nonmembranous organelles have been described in eukaryotic cells, and their structures and functions have been analyzed in detail. However, there are likely to be many organelles that have not been identified or characterized. The centriolar satellite, electron-dense spherical granules ~70–100 nm in diameter, occurring around centrioles in most types of cells, is one such uncharacterized type of nonmembranous organelle. In this study, we identified PCM-1 as the first component of the centriolar satellite in Xenopus A6 and mouse Eph4 cells. Transfection experiments of a truncated form of XPCM-1 showed that multiple XPCM-1 molecules were incorporated into each granule, and our preliminary experiments showed that these molecules bind directly to each other to form dimers or oligomers, suggesting that PCM-1 is a kind of scaffold protein constituting the centriolar satellites. Fibrous granules also constitute an uncharacterized type of nonmembranous organelle. These granules were thought to appear in ciliated cells only during ciliologenesis, but we found that they were also distributed close to ciliary basal bodies in nonciliogenic phase. These granules also had the appearance of electron-dense spherical granules ~80–90 nm in diameter, and were indistinguishable morphologically from the centriolar satellites, although this resemblance has not been described previously. Interestingly, we found that these granules also contained PCM-1. Therefore, we propose here that centriolar satellites and fibrous granules can be regarded as the same novel nonmembranous organelles, defined by their specific component, PCM-1.
One of the most characteristic features of centriolar satellites (so probably also fibrous granules) is their ability to move along MTs; they moved along MTs toward their minus ends, i.e., toward centrosomes, in reconstituted asters in vitro in the presence of ATP. The effects of AMP-PNP, vanadate, and antidynein mAb suggested that dynein, but not kinesin, was involved in their movement. In good agreement, Balczon et al. 1999
recently reported that PCM-1 was coprecipitated with MTs from CHO cell extracts, and that immunodepletion with antidynein antibody, not antikinesin antibody, from CHO extracts significantly decreased the amount of coprecipitated PCM-1. Furthermore, PCM-1 was shown to directly bind to Huntingtin-associated protein 1 (HAP1) by yeast two hybrid analyses (Engelender et al. 1997
). Since HAP1 bound to the p150Glued
subunit of dynactin complex (Engelender et al. 1997
), HAP1 may function as a cross-linker between PCM-1, i.e., the centriolar satellites (and also fibrous granules), and the dynein/dynactin complexes.
The dynein-dependent, minus end-directed movement can explain the pericentriolar localization of centriolar satellites. In live A6 cells, GFP-tagged centriolar satellites moved not only toward centrosomes, but also toward the cell periphery. It is clear that the centripetal movement dominates as a whole, since centriolar satellites accumulated around centrosomes in live cells, but it remains unclear whether centriolar satellites also bear plus end-directed motors such as kinesins, or some fraction of MTs of A6 cells do not originate from centrosomes and these MTs are responsible for the centrifugal movement of centriolar satellites. The localization of fibrous granules close to ciliary basal bodies in ciliated epithelial cells in the nonciliogenic phase could also be explained in the same way. In simple epithelial cells, most of the minus ends of MTs are not anchored at the centrosome, but are scattered throughout their apical regions with MTs running parallel along the apico-basal axis (Bacallao et al. 1989
; Mogensen et al. 1989
). Therefore, if the fibrous granules are also associated with dynein, they would accumulate at the apical regions of epithelial cells.
The physiological functions of the centriolar satellites remain unclear. As shown in this study, however, their resemblance to the fibrous granules not only morphologically, but also in their molecular composition, suggested that they may play some roles in centriolar replication. Fibrous granules were reported to be associated with the replication of basal bodies (Sorokin 1968
; Anderson and Brenner 1971
). In good agreement, the level of PCM-1 was markedly elevated when ciliogenesis was induced in nasal epithelial cells ( and ). In addition to fibrous granules, larger electron-dense spherical structures called deuterosomes also emerged during centriolar replication in ciliogenic cells, from which multiple procentrioles grew (Sorokin 1968
), and previous electron microscopic observations suggested that fibrous granules were fused to form deuterosomes (Sorokin 1968
; Anderson and Brenner 1971
). However, this was not likely since PCM-1 was detected in fibrous granules, but not in deuterosomes ( and ). The experimentally induced ciliogenesis examined in this study will be an advantageous system to further analyze the relationship between PCM-1–containing granules and centriolar replication in future studies.
Previous studies on PCM-1 itself also suggested its possible association with centriolar replication. It is widely accepted that centriolar replication begins near the G1/S boundary, continues through S phase, and is completed during G2 phase (Robbins et al. 1968
; Brinkley 1985
; Vandré and Borisy 1989
). In good agreement, PCM-1 at centrosomes is released into the cytoplasm on the entry to M phase, and on the entry to interphase this molecule is reconcentrated at centrosomes (Rattner 1992
; Balczon et al. 1994
). PCM-1 mRNA levels increase through G1 and S phases, and became undetectable during G2 and M phases in CHO cells (Balczon et al. 1995
). Interestingly, PCM-1 mRNA levels remained elevated during multiple rounds of centrosome replication in CHO cells arrested at the G1/S boundary by hydroxyurea with a concomitant increase in number of centriolar satellites (see Figure 4 in Balczon et al. 1995
On the other hand, fibrous granules were also suggested to function as axonemal precursors (Steinman 1968
). Recent studies using Chlamydomonas
identified intraflagellar transport (IFT) particles as large preassembled precursors for various axonemal structures in cytoplasm that were concentrated around centrioles (Cole et al. 1998
; Rosenbaum et al. 1999
). However, it is not likely that fibrous granules are the counterparts of IFT particles; IFT particles are lollipop-shaped electron-dense granules, ~14–19 nm in diameter (see Figure 3 in Kozminski et al. 1993
), which is much smaller than fibrous granules. IFT particles were detected within flagella, while fibrous granules or PCM-1 was not observed within cilia. Furthermore, PCM-1 immunofluorescence was abundant in the apical cytoplasm of nonciliated epithelial cells, such as intestinal and gastric epithelial cells (Kubo, A., A. Yuba-Kubo, S. Tsukita, and N. Shiina, unpublished data). These findings are against the notion that fibrous granules function as axonemal precursors. Further identification of other components of fibrous granules/centriolar satellites will answer these questions more clearly.
In this study, we identified pericentriolar satellites and fibrous granules as PCM-1–containing novel nonmembranous organelles, which were accumulated around centrosomes and ciliary basal bodies, respectively, through their minus end-directed movement along MTs. These findings then suggested the possible association of these PCM-1–containing organelles with centriolar replication. Further detailed analyses of these organelles, as well as PCM-1 molecules, will lead to a better understanding of the molecular mechanism of centriologenesis in general.