Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments.

We used electron tomography to examine microtubules (MTs) growing from pure tubulin in vitro as well as two classes of MTs growing in cells from six species. The tips of all these growing MTs display bent protofilaments (PFs) that curve away from the MT axis, in contrast with previously reported MTs growing in vitro whose tips are either blunt or sheetlike. Neither high pressure nor freezing is responsible for the PF curvatures we see. The curvatures of PFs on growing and shortening MTs are similar; all are most curved at their tips, suggesting that guanosine triphosphate-tubulin in solution is bent and must straighten to be incorporated into the MT wall. Variations in curvature suggest that PFs are flexible in their plane of bending but rigid to bending out of that plane. Modeling by Brownian dynamics suggests that PF straightening for MT growth can be achieved by thermal motions, providing a simple mechanism with which to understand tubulin polymerization.

Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential.

UVB irradiation induces harmful photochemical reactions, including formation of Cyclobutane Pyrimidine Dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2days. Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less CPD in those cells that were also treated with photoreactivating light, which is required for photolyase activity. The functional photolyase also diminished other UVB-mediated effects, including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA lesions. The pseudouridine-modified mRNA approach has a strong potential to discern cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient expression of proteins offers a number of opportunities for future application in medicine.

The Potorous CPD photolyase rescues a cryptochrome-deficient mammalian circadian clock.

Despite the sequence and structural conservation between cryptochromes and photolyases, members of the cryptochrome/photolyase (flavo)protein family, their functions are divergent. Whereas photolyases are DNA repair enzymes that use visible light to lesion-specifically remove UV-induced DNA damage, cryptochromes act as photoreceptors and circadian clock proteins. To address the functional diversity of cryptochromes and photolyases, we investigated the effect of ectopically expressed Arabidopsis thaliana (6-4)PP photolyase and Potorous tridactylus CPD-photolyase (close and distant relatives of mammalian cryptochromes, respectively), on the performance of the mammalian cryptochromes in the mammalian circadian clock. Using photolyase transgenic mice, we show that Potorous CPD-photolyase affects the clock by shortening the period of behavioral rhythms. Furthermore, constitutively expressed CPD-photolyase is shown to reduce the amplitude of circadian oscillations in cultured cells and to inhibit CLOCK/BMAL1 driven transcription by interacting with CLOCK. Importantly, we show that Potorous CPD-photolyase can restore the molecular oscillator in the liver of (clock-deficient) Cry1/Cry2 double knockout mice. These data demonstrate that a photolyase can act as a true cryptochrome. These findings shed new light on the importance of the core structure of mammalian cryptochromes in relation to its function in the circadian clock and contribute to our further understanding of the evolution of the cryptochrome/photolyase protein family.

Monitoring reproduction in the critically endangered marsupial, Gilbert's potoroo (Potorous gilbertii): preliminary analysis of faecal oestradiol-17beta, cortisol and progestagens.

Gilbert's potoroo (Potorous gilbertii) was rediscovered in 1994 after having been presumed extinct for 120 years. Estimates indicate fewer than 40 individuals remain at Two Peoples Bay Nature Reserve on the south coast of Western Australia although a translocated population of approximately 20 animals has recently been established on nearby Bald Island. A captive breeding facility has been established adjacent to the mainland population but few young have been produced (8 since 1995). Faecal levels of oestradiol-17beta (E(2)) were monitored over a 2-year period in an effort to determine cyclic reproductive activity, and faecal cortisol levels were also monitored to gauge whether chronic stress may be a factor limiting breeding in captivity. Faecal steroids were monitored in six captive females, and four captive male potoroos, and four wild females. The only captive births recorded after 1998 were one in August 1999 and one in February 2001, both to the same female. Peaks in E(2) concentration, up to 10 ng g(-1) of dried faecal mass were measured and results to date suggest the main breeding period to be November-December based on elevated E(2) levels at this time. Clear patterns of reproductive activity in the captive females, however, were not evident. Analysis of epithelial cell counts from urinogenital swabs and faecal E(2) and progestagen (PM) levels from a single female kept at the Perth Zoo, suggest that Gilbert's potoroo has an oestrous cycle of approximately 39 days. Faecal cortisol levels in captive females were significantly lower than those in wild-caught individuals and thus there is no indication that elevated cortisol levels per se inhibited reproduction in captive females.

Lamellipodial contractions during crawling and spreading.

Most eukaryotic cells can crawl over surfaces. In general, this motility requires three distinct actions: polymerization at the leading edge, adhesion to the substrate, and retraction at the rear. Recent experiments with mouse embryonic fibroblasts showed that during spreading and crawling the lamellipodium undergoes periodic contractions that are substrate-dependent. Here I show that a simple model incorporating stick-slip adhesion and a simplified mechanism for the generation of contractile forces is sufficient to explain periodic lamellipodial contractions. This model also explains why treatment of cells with latrunculin modifies the period of these contractions. In addition, by coupling a diffusing chemical species that can bind actin, such as myosin light-chain kinase, with the contractile model leads to periodic rows and waves in the chemical species, similar to what is observed in experiments. This model provides a novel and simple explanation for the generation of contractile waves during cell spreading and crawling that is only dependent on stick-slip adhesion and the generation of contractile force and suggests new experiments to test this mechanism.