Melanin Distribution in Human Skin: Influence of Cytoskeletal,Polarity, and Centrosome-Related Machinery of Stratum basale Keratinocytes

Melanin granules cluster within supra-nuclear caps in basal keratinocytes (KCs) of the human epidermis, where they protect KC genomic DNA against ultraviolet radiation (UVR) damage. While much is known about melanogenesis in melanocytes (MCs) and a moderate amount about melanin transfer from MC to KC, we know little about the fate of melanin once inside KCs. We recently reported that melanin fate in progenitor KCs is regulated by rare asymmetric organelle movement during mitosis. Here, we explore the role of actin, microtubules, and centrosome-associated machinery in distributing melanin within KCs. Short-term cultures of human skin explants were treated with cytochalasin-B and nocodazole to target actin filaments and microtubules, respectively. Treatment effects on melanin distribution were assessed by the Warthin–Starry stain, on centrosome-associated proteins by immunofluorescence microscopy, and on co-localisation with melanin granules by brightfield microscopy. Cytochalasin-B treatment disassembled supra-nuclear melanin caps, while nocodazole treatment moved melanin from the apical to basal KC domain. Centrosome and centriolar satellite-associated proteins showed a high degree of co-localisation with melanin. Thus, once melanin granules are transferred to KCs, their preferred apical distribution appears to be facilitated by coordinated movement of centrosomes and centriolar satellites. This mechanism may control melanin’s strategic position within UVR-exposed KCs.     

An E3‐14.7K Peptide that Promotes Microtubules‐Mediated Transport of Plasmid DNA Increases Polyplexes Transfection Efficiency

Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non‐viral vectors. Here, the interaction between E3‐14.7K and FIP‐1 to favor migration of pDNA along microtubules is exploited. E3‐14.7K is an early protein of human adenoviruses that interacts via FIP‐1 (Fourteen.7K Interacting Protein 1) protein with the light‐chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79‐98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non‐directional movements in the cytoplasm. Remarkably, P79‐98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.     

Terpendole E and its Derivative Inhibit STLC‐ and GSK‐1‐Resistant Eg5

Terpendole E is first natural product found to inhibit mitotic kinesin Eg5, but its inhibitory mechanism remains to be revealed. Here, we report the effects of terpendole E and 11ketopaspaline (a new natural terpendole E analogue) on the Eg5–microtubule interaction and in several Eg5 mutants. 11‐Ketopaspaline is a shunt product from terpendole E, and it shows potent inhibitory activity against the microtubule‐stimulated ATPase activity of Eg5. Unlike other Eg5 inhibitors, such as S‐trityl‐L ‐cysteine (STLC) and GSK‐1, both terpendole E and 11‐ketopaspaline only partially inhibited Eg5–microtubule interaction. Furthermore, terpendole E and 11‐ketopaspaline inhibited several Eg5 mutants that are resistant to STLC (Eg5^D130A, Eg5^L214A) or GSK‐1 (Eg5^I299F, Eg5^A356T), but with the same extent of inhibition against wild‐type Eg5. Because Eg5^D130A and Eg5^L214A show cross‐resistance to most known Eg5 inhibitors, which bind the L5 loop, these results suggest that terpendole E and its analogues have a different binding site and/or inhibitory mechanism to those for L5 loop‐binding type Eg5 inhibitors.     

细胞骨架与辐射所致神经细胞迁移障碍研究进展

微管和微丝的动态变化是正常细胞迁移的主要动力。电离辐射引发机体组织和细胞的生物效应与细胞骨架的改变密切相关,辐射引起微丝解聚微管聚合障碍,使神经细胞无法进入迁移模式或迁移异常。本文综述了辐射所致细胞骨架改变对神经细胞迁移的影响。     

Quantitative Analysis of Tau-Microtubule Interaction Using FRET

The interaction between the microtubule associated protein, tau and the microtubules is investigated. A fluorescence resonance energy transfer (FRET) assay was used to determine the distance separating tau to the microtubule wall, as well as the binding parameters of the interaction. By using microtubules stabilized with Flutax-2 as donor and tau labeled with rhodamine as acceptor, a donor-to-acceptor distance of 54 ± 1 Å was found. A molecular model is proposed in which Flutax-2 is directly accessible to tau-rhodamine molecules for energy transfer. By titration, we calculated the stoichiometric dissociation constant to be equal to 1.0 ± 0.5 µM. The influence of the C-terminal tails of αβ-tubulin on the tau-microtubule interaction is presented once a procedure to form homogeneous solution of cleaved tubulin has been determined. The results indicate that the C-terminal tails of α- and β-tubulin by electrostatic effects and of recruitment seem to be involved in the binding mechanism of tau.     

Making Epothilones Fluoresce: Design,Synthesis, and Biological Characterization of a Fluorescent N12‐Aza‐Epothilone (Azathilone)

A green fluorescent 12‐aza‐epothilone (azathilone) derivative has been prepared through the attachment of the 4‐nitro‐2,1,3‐benzoxadiazole (NBD) fluorophore to the 12‐nitrogen atom of the azamacrolide core structure. While less potent than natural epothilones or different N12‐acylated azathilone derivatives, NBD‐azathilone ( 3 ) promotes tubulin assembly, inhibits cancer cell proliferation in vitro and arrests the cell cycle at the G2/M transition. Most significantly, the binding of 3 to cellular microtubules (MTs) could be directly visualized by confocal fluorescence microscopy. Based on competition binding experiments with laulimalide‐stabilized MTs in vitro, the N12‐Boc substituted azathilone 1 , Epo A, and NBD‐azathilone ( 3 ) all interact with the same tubulin‐binding site. Computational studies provided a structural model of the complexes between β‐tubulin and 1 or 3 , respectively, in which the NBD moiety of 3 or the BOC moiety of 1 directly and specifically contribute to MT binding. Collectively, these data demonstrate that the cellular effects of 3 and, by inference, also of other azathilones are the result of their interactions with the cellular MT network.