Polymerization of monomeric actin into filaments has pivotal roles in cell motility, growth, differentiation, and gene expression. Therefore, techniques of manipulating actin polymerization, including actin-binding chemicals, have been developed for understanding and regulating multiple biological functions. Here, we demonstrate that irradiation with terahertz (THz) waves is a novel method of modulating actin polymerization. When actin polymerization reaction is performed under irradiation with 0.46 THz waves generated by a Gyrotron, actin polymerization was observed to be activated by monitoring the fluorescence of pyrene actin fluorophores. We also observed the number of actin filaments under a fluorescence microscope using the polymerized actin probe SiR-actin. The number of actin filaments was increased by 3.5-fold after THz irradiation for 20 min. When the THz irradiation was applied to a steady-state actin solution, in which elongation and depolymerization of actin filaments were equilibrated, increased actin polymerization was observed, suggesting that the THz irradiation activates actin polymerization, at least in the elongation process. These results suggest that THz waves could be applied for manipulating biomolecules and cells.

The effect of terahertz (THz) radiation on deep tissues of human body has been considered negligible due to strong absorption by water molecules. However, we observed that the energy of THz pulses transmits a millimeter thick in the aqueous solution, possibly as a shockwave, and demolishes actin filaments. Collapse of actin filament induced by THz irradiation was also observed in the living cells under an aqueous medium. We also confirmed that the viability of the cell was not affected under the exposure of THz pulses. The potential of THz waves as an invasive method to alter protein structure in the living cells is demonstrated.

Although actin monomers polymerize into filaments in the cytoplasm, the form of actin in the nucleus remains elusive. We searched for the form and function of β-actin fused to nuclear localization signal and to enhanced yellow fluorescent protein (EN-actin). Our results reveal that EN-actin is either dispersed in the nucleoplasm (homogenous EN-actin) or forms bundled filaments in the nucleus (EN-actin filaments). Formation of such filaments was not connected with increased EN-actin levels. Among numerous actin-binding proteins tested, only cofilin is recruited to the EN-actin filaments. Overexpression of EN-actin causes increase in the nuclear levels of actin-related protein 3 (Arp3). Although Arp3, a member of actin nucleation complex Arp2/3, is responsible for EN-actin filament nucleation and bundling, the way cofilin affects nuclear EN-actin filaments dynamics is not clear. While cells with homogenous EN-actin maintained unaffected mitosis during which EN-actin re-localizes to the plasma membrane, generation of nuclear EN-actin filaments severely decreases cell proliferation and interferes with mitotic progress. The introduction of EN-actin manifests in two mitotic-inborn defects—formation of binucleic cells and generation of micronuclei—suggesting that cells suffer aberrant cytokinesis and/or impaired chromosomal segregation. In interphase, nuclear EN-actin filaments passed through chromatin region, but do not co-localize with either chromatin remodeling complexes or RNA polymerases I and II. Surprisingly presence of EN-actin filaments was connected with increase in the overall transcription levels in the S-phase by yet unknown mechanism. Taken together, EN-actin can form filaments in the nucleus which affect important cellular processes such as transcription and mitosis.

Biological phenomena induced by terahertz (THz) irradiation are described in recent reports, but underlying mechanisms, structural and dynamical change of specific molecules are still unclear. In this paper, we performed time-lapse morphological analysis of human cells and found that THz irradiation halts cell division at cytokinesis. At the end of cytokinesis, the contractile ring, which consists of filamentous actin (F-actin), needs to disappear; however, it remained for 1 hour under THz irradiation. Induction of the functional structures of F-actin was also observed in interphase cells. Similar phenomena were also observed under chemical treatment (jasplakinolide), indicating that THz irradiation assists actin polymerization. We previously reported that THz irradiation enhances the polymerization of purified actin in vitro; our current work shows that it increases cytoplasmic F-actin in vivo. Thus, we identified one of the key biomechanisms affected by THz waves.

Keratins represent important structural components of intermediate filament proteins. Their expression profiles are remarkably tissue-specific. Recent data have shown that keratins associate with many proteins including heat shock proteins (HSP). We recently identified cell-specific keratin and HSP expression. We aimed to gain further insight into the regulation of keratins by specific inhibition through knockdown of Hsp40 in human keratinocyte cells. Keratin-HSP interaction in HaCaT cell lysate was evaluated by immunoprecipitation followed by Western blotting. Immunofluorescence, was used to examine the co-localization of keratins and Hsp40. Hsp40 depletion led to an increase in the levels of keratin proteins (K5, K14, K10) and a decrease in keratin ubiquitination without influencing keratin gene expression. Our results demonstrate direct or indirectly association of Hsp40 and imply that expressed keratin proteins were regulated by Hsp40 depending on the ubiquitin-proteasome pathway in HaCaT. Furthermore, the K10 differentiation marker was increased by knockdown of Hsp40. The results presented in this study indicate that Hsp40 is related to the differentiation exchange of keratin pairs.