Kazunori Watanabe

The molecular mechanism of photochemical internalization of cell penetrating peptide-cargo-photosensitizer conjugates

In many drug delivery strategies, an inefficient transfer of macromolecules such as proteins and nucleic acids to the cytosol often occurs because of their endosomal entrapment. One of the methods to overcome this problem is photochemical internalization, which is achieved using a photosensitizer and light to facilitate the endosomal escape of the macromolecule. In this study, we examined the molecular mechanism of photochemical internalization of cell penetrating peptide-cargo (macromolecule)-photosensitizer conjugates. We measured the photophysical properties of eight dyes (photosensitizer candidates) and determined the respective endosomal escape efficiencies using these dyes. Correlation plots between these factors indicated that the photogenerated 1O2 molecules from photosensitizers were highly related to the endosomal escape efficiencies. The contribution of 1O2 was confirmed using 1O2 quenchers. In addition, time-lapse fluorescence imaging showed that the photoinduced endosomal escape occurred at a few seconds to a few minutes after irradiation (much longer than 1O2 lifetime), and that the pH increased in the endosome prior to the endosomal escape of the macromolecule.

Formation of tRNA granules in the nucleus of heat-induced human cells

The stress response, which can trigger various physiological phenomena, is important for living organisms. For instance, a number of stress-induced granules such as P-body and stress granule have been identified. These granules are formed in the cytoplasm under stress conditions and are associated with translational inhibition and mRNA decay. In the nucleus, there is a focus named nuclear stress body (nSB) that distinguishes these structures from cytoplasmic stress granules. Many splicing factors and long non-coding RNA species localize in nSBs as a result of stress. Indeed, tRNAs respond to several kinds of stress such as heat, oxidation or starvation. Although nuclear accumulation of tRNAs occurs in starved Saccharomyces cerevisiae, this phenomenon is not found in mammalian cells. We observed that initiator tRNA(Met) (Meti) is actively translocated into the nucleus of human cells under heat stress. During this study, we identified unique granules of Meti that overlapped with nSBs. Similarly, elongator tRNA(Met) was translocated into the nucleus and formed granules during heat stress. Formation of tRNA granules is closely related to the translocation ratio. Then, all tRNAs may form the specific granules.

Degradation of initiator tRNAMet by Xrn1/2 via its accumulation in the nucleus of heat-treated HeLa cells

Stress response mechanisms that modulate the dynamics of tRNA degradation and accumulation from the cytoplasm to the nucleus have been studied in yeast, the rat hepatoma and human cells. In the current study, we investigated tRNA degradation and accumulation in HeLa cells under various forms of stress. We found that initiator tRNAMet (tRNA(iMet)) was specifically degraded under heat stress. Two exonucleases, Xrn1 and Xrn2, are involved in the degradation of tRNA(iMet) in the cytoplasm and the nucleus, respectively. In addition to degradation, we observed accumulation of tRNA(iMet) in the nucleus. We also found that the mammalian target of rapamycin (mTOR), which regulates tRNA trafficking in yeast, is partially phosphorylated at Ser2448 in the presence of rapamycin and/or during heat stress. Our results suggest phosphorylation of mTOR may correlate with accumulation of tRNA(iMet) in heat-treated HeLa cells.

Three-dimensional CT image analysis of the digging system in the aardvark

We examined the bone movement in the forepaw and hind paw in the aardvark (Orycteropus afer) by using three-dimensional (3D)-computed tomography (CT) techniques and osteometrical methods to confirm the functional adaptation of the extremities as a digging system. The four metacarpal bones could be strongly bent from the distal carpal bones. The distal end of the second and third metacarpal bones possessed enlarged smooth articulation surfaces that allowed the proximal phalanx to bend at a sharp angle. However, the articulation surface was not well-developed in the distal end of the fourth and fifth metacarpal bones and the proximal phalanx could bend at smaller angle in these two lateral digits. The proximal phalanges sharply crook from the metatarsal in the first, second, third and fourth digits in the hind paw. We suggest that the medial two digits in the forepaw directly contribute to the crushing, when these proximal phalanges crook in the phase of power stroke. In contrast the lateral third and fourth digits may act as sweeper of the crushed soil. These suggestions regarding the different functional adaptation between medial two digits and lateral two digits are consistent with the anatomical data of the forearm musculature. In the hind paw, we suggest that the second, third and fourth digits are functionally similar and that the hind paw may not act as a crushing apparatus but as a running motor or soil-sweeper similarly using these main three digits.

Near-infrared light-directed RNAi using a photosensitive carrier molecule.

Controlled activation of small RNAs, such as small interfering RNA, in cells is very useful for various biological applications. Light is an effective inducer of controlled activation; in particular, near-infrared light is favorable because it can penetrate deeper into tissues than UV or visible light. In this study, near-infrared light control of RNA interference (RNAi) was demonstrated in mammalian cells using a photosensitive RNA carrier molecule, consisting of an RNA carrier protein and a fluorochrome. The photosensitive carrier molecule was identified from six candidates, each with a different fluorochrome. Using this carrier molecule, cytosolic RNA delivery and RNAi can be triggered by near-infrared light. Cytotoxicity was not observed after photoinduction of RNAi.

mTOR regulates the nucleoplasmic diffusion of Xrn2 under conditions of heat stress

Stress induces various responses, including translational suppression and tRNA degradation in mammals. Previously, we showed that heat stress induces degradation of initiator tRNAMet (iMet) through 5′–3′ exoribonuclease Xrn1 and Xrn2, respectively. In addition, we found that rapamycin inhibits the degradation of iMet under heat stress conditions. Here, we report that the mammalian target of rapamycin (mTOR) regulates the diffusion of Xrn2 from the nucleolus to the nucleoplasm, facilitating the degradation of iMet under conditions of heat stress. Our results suggest a mechanism of translational suppression through mTOR‐regulated iMet degradation in mammalian cells.

Photocontrolled Intracellular RNA Delivery Using Nanoparticles or Carrier–Photosensitizer Conjugates

Small interfering RNA (siRNA) and short hairpin RNA (shRNA) may potentially treat a wide variety of diseases through RNA interference-mediated silencing of specific genes. MicroRNAs (miRNAs) are endogenous regulatory RNA molecules that also modulate gene expression, and thus potential therapeutic approaches using miRNAs have attracted attention. For clinical application of these small RNAs, efficient and safe RNA delivery to target tissues and cells is necessary. Current challenges to RNA delivery are the penetration of negatively charged RNAs through the cell membrane and specific delivery of RNA into target cells. Photocontrolled intracellular RNA delivery is a promising strategy with high target specificity. This strategy includes photodependent endosomal escape of RNA or photodependent release of RNAs from carrier particles. In this chapter, photocontrolled intracellular RNA delivery methods employing gold or silver nanoparticles, upconversion nanoparticles, proteins, or polymers are discussed.

Phototriggered protein syntheses by using (7-diethylaminocoumarin-4-yl)methoxycarbonyl-caged aminoacyl tRNAs

The possibility of spatiotemporally photocontrolling translation holds considerable promise for studies on the biological roles of local translation in cells and tissues. Here we report caged aminoacyl-tRNAs (aa-tRNAs) synthesized using a (7-diethylaminocoumarin-4-yl)methoxycarbonyl (DEACM)-cage compound. DEACM-caged aa-tRNA does not spontaneously deacylate for at least 4u2009h in neutral aqueous solution, and does not bind to the elongation factor Tu. On irradiation at ∼405u2009nm at 125u2009mWu2009cm−2, DEACM-aa-tRNA is converted into active aa-tRNA with a half-life of 19u2009s. Notably, this rapid uncaging induced by visible light does not impair the translation system. Translation is photoinduced when DEACM-aa-tRNA carrying a CCCG or a CUA anticodon is uncaged in the presence of mRNAs harbouring a CGGG four-base codon or a UAG amber codon, respectively. Protein synthesis is phototriggered in several model systems, including an in vitro translation system, an agarose gel, in liposomes and in mammalian cells.

Photoinduced apoptosis using a peptide carrying a photosensitizer

A novel molecule, TatBim-Alexa, consisting of the HIV1 Tat cell-penetrating peptide, the Bim apoptosis-inducing peptide, and Alexa Fluor 546 was synthesized for photoinducion of apoptosis. The Alexa Fluor 546 was used as a photosensitizer and covalently attached at the C-terminus of TatBim peptide by the thiol-maleimide reaction. Photo-dependent cytosolic internalization of TatBim-Alexa and photo-dependent apoptosis using TatBim-Alexa were demonstrated in several kinds of mammalian cells including human cancer cell lines.

MicroRNA-664a-5p promotes neuronal differentiation of SH-SY5Y cells

MicroRNAs (miRNAs) belong to a class of small noncoding RNAs that play important roles in the translational regulation of gene expression. A number of miRNAs are known to act as key regulators of diverse processes such as neuronal differentiation. In this study, we have attempted to identify novel miRNAs related to neuronal differentiation via microarray analysis in the human neuronal differentiation model neuroblastoma SH‐SY5Y cells. We identified 15 up‐regulated and eight down‐regulated miRNAs in SH‐SY5Y cells treated with all‐trans retinoic acid to induce differentiation. We further showed that one of the up‐regulated miRNAs, miR‐664a‐5p, promoted neuronal differentiation of SH‐SY5Y cells. These findings enhance our understanding of the miRNAs involved in the process of neurogenesis and, in particular, highlight an important role of miR‐664a‐5p in SH‐SY5Y cell neuronal differentiation. Further studies will be required to confirm the function of miR‐664‐5p in neuronal development and disease and to identify its relevant target genes.