Thymosin beta 4 may translocate from the cytoplasm in to the nucleus in HepG2 cells following serum starvation. An ultrastructural study

Abstract

Due to its actin-sequestering properties, thymosin beta-4 (Tβ4) is considered to play a significant role in the cellular metabolism. Several physiological properties of Tβ4 have been reported;, however, many questions concerning its cellular function remain to be ascertained. To better understand the role of this small peptide we have analyzed by means of transmission immunoelectron microscopy techniques the ultrastructural localization of Tβ4 in HepG2 cells. Samples of HepG2 cells were fixed in a mixture of 3% formaldehyde and 0.1% glutaraldehyde in 0.1 M cacodylate buffer and processed for standard electron microscopic techniques. The samples were dehydrated in a cold graded methanol series and embedded in LR gold resin. Ultrathin sections were labeled with rabbit antibodies to Tβ4, followed by gold-labeled goat anti-rabbit, stained with uranyl acetate and bismuth subnitrate, observed and photographed in a JEOL 100S transmission electron microscope. High-resolution electron microscopy showed that Tβ4 was mainly restricted to the cytoplasm of HepG2 growing in complete medium. A strong Tβ4 reactivity was detected in the perinuclear region of the cytoplasmic compartment where gold particles appeared strictly associated to the nuclear membrane. In the nucleus specific Tβ4 labeling was observed in the nucleolus. The above electron microscopic results confirm and extend previous observations at light microscopic level, highlighting the subcellular distribution of Tβ4 in both cytoplasmic and nuclear compartments of HepG2 cells. The meaning of Tβ4 presence in the nucleolus is not on the best of our knowledge clarified yet. It could account for the interaction of Tβ4 with nucleolar actin and according with this hypothesis, Tβ4 could contribute together with the other nucleolar acting binding proteins to modulate the transcription activity of the RNA polymerases.

Fig 1. (A-C) Electron micrographs of HepG2 cells growing in complete medium.

Specific Tβ4 reactivity is detected in the cytoplasmic compartment (cc), where gold particles are observed strictly associated to the endoplasmic reticulum (arrows). In the nuclearcompartment (nc) the nucleoplasm is devoid of labeling whereas the nucleolus (nu) shows evident labeling. (D) Portion of HepG2 cell growing for 48h in the absence of fetal bovine serum. Specific Tβ4 immunostaining is observed in both cytoplasm (cc) and nucleoplasm (nc). On the contrary, few gold particles decorate the nucleolus (nu). Bars = 0,5 μm

Fig 2. Tβ4 mRNA expression detected by RT-PCR reaction in HepG2 cells growing in normal serum and in starvation conditions.

The ratio value Tβ4/β actin detected at 24h in normal condition was assumed as equal to 1 (calibrator sample). In these conditions the Tβ4 expression patterns suggest a moderate Tβ4 mRNA increase expression rate from 24 to 48h in normal conditions and, a more high Tβ4 mRNA expression values, during starvation.(2 folds in difference at 48h). The vertical bars, reppresent the range of standard error (+-SE) of the mean.

Fig 3. Three dimensional (3D) reconstruction of Tβ4 cellular trafficking in HepG2 cells growing at different cell conditions.

3D reconstruction of the main steps of the hypothetical Tβ4 nuclear trafficking from nucleoplasm to nucleolus in HepG2 cells growing in complete medium(1A-4A). In HepG2 cells growing for 48h in the absence of fetal bovine serum, the starvation induced stress could affect the molecular mechanisms involved in the Tβ4 cellular trafficking, preventing the localization of Tβ4 in the nucleolus (1B-4B).