Ankyrin repeats are an amino-acid motif believed to function in protein recognition; they are present in tandem copies in diverse proteins in nearly all phyla 1 . Ankyrin repeats contain antiparallel a-helices that can stack to form a superhelical spiral 2 . Visual inspection of the extrapolated structure of 24 ankyrin-R repeats 2 indicates the possibility of spring-like behaviour of the putative superhelix. Moreover, stacks of 17-29 ankyrin repeats in the cytoplasmic domains of transient receptor potential (TRP) channels have been identified as candidates for a spring that gates mechanoreceptors in hair cells as well as in Drosophila bristles 3-5 . Here we report that tandem ankyrin repeats exhibit tertiary-structure-based elasticity and behave as a linear and fully reversible spring in single-molecule measurements by atomic force microscopy. We also observe an unexpected ability of unfolded repeats to generate force during refolding, and report the first direct measurement of the refolding force of a protein domain. Thus, we show that one of the most common aminoacid motifs has spring properties that could be important in mechanotransduction and in the design of nanodevices.The atomic structure of 12 ankyrin-R repeats suggests that ankyrin stacks composed of n $ 24 repeats should form a full superhelical turn with putative spring properties 2,3 . We used an atomic force microscope (AFM) to identify individual stacks of 24 ankyrin-B repeats ( Supplementary Fig. S2) and found that they do indeed have a hook-like shape 2 with the molecules' end-to-end distance closely matching the ,12 nm determined for the extrapolated structure 2 (Fig. 1a). Thus, the AFM images strongly suggest that the engineered protein, bearing at its terminus a glutathione S-transferase (GST) module, is correctly folded and does not aggregate. These conclusions are further supported by circular dichroism and hydrodynamic measurements (Supplementary Table 1 and Supplementary Fig. S1).For elasticity measurements, heptahistidine-tagged polypeptides containing 24 ankyrin-B repeats with or without GST, or 12 repeats with GST, were immobilized on a glass surface bearing the metal chelate N-nitrilotriacetic acid (NTA) 6,7 (Fig. 1a). Molecules were stretched vertically, in solution, by the AFM cantilever, and their length and tension were measured with subnanometre and ,10 pN precision [8][9][10] . Most trials revealed complex force-extension profiles with irregularly spaced force peaks typical of multiple molecules ( Supplementary Fig. S4a). However, ,5% of the force-extension curves had simple and consistent features that, we argue, represent LETTERSFigure 1 | Atomic force microscopy measurements reveal the linear elasticity of ankyrin-B repeats. a, The extrapolated structure of 24 ankyrin-R repeats 2 and a diagram of the elasticity measurement on a His-tagged ankyrin fragment bound to NTA (red handles) and stretched with the AFM cantilever. b-e, Force-extension curves of individual ankyrins: 24 repeats with GST (b-d); 24 repeats with no GST (e)....

Postnatal/adult neural stem cells (NSCs) within the rodent subventricular/subependymal zone (SVZ/SEZ) generate Doublecortin (DCX)+ neuroblasts that migrate and integrate into olfactory bulb circuitry1,2. Continuous production of neuroblasts is controlled by SVZ microenvironmental niche3,4. It is generally believed that enhancing neurogenic activities of endogenous NSCs may provide needed therapeutic options for disease states and after brain injury. However, SVZ NSCs can also differentiate into astrocytes. It remains unclear if there are conditions that favor astrogenesis over neurogenesis in the SVZ niche, and if astrocytes produced there exhibit different properties from others in the brain. We have uncovered that SVZ-generated astrocytes express high levels of Thrombospondin-4 (Thbs4)5,6, a secreted homopentameric glycoprotein, in contrast to cortical astrocytes which are Thbs4low. We found that localized photothrombotic/ischemic cortical injury initiates a marked increase in Thbs4hi astrocyte production from the postnatal SVZ niche. Tamoxifen-inducible nestin-CreERtm4 lineage-tracing demonstrated that it is these SVZ-generated Thbs4hi astrocytes, and not DCX+ neuroblasts, that home-in on the injured cortex. This robust post-injury astrogenic response required SVZ Notch activation, modulated by Thbs4 via direct Notch1 receptor binding and endocytosis to activate downstream signals, including increased Nfia transcription factor expression important for glia production7. Consequently, Thbs4KO/KO animals showed severe defects in cortical injury-induced SVZ astrogenesis, instead producing cells expressing DCX from SVZ to the injury sites. These alterations in cellular responses resulted in abnormal glial scar formation after injury, and significantly increased microvascular hemorrhage into the brain parenchyma of Thbs4KO/KO animals. Taken together, these findings have significant implications for post-injury applications of endogenous and transplanted NSCs in the therapeutic setting, as well as disease states where Thbs family members play important roles8,9.

SUMMARY The rodent subventricular/subependymal zone (SVZ/SEZ) houses neural stem cells (NSCs) that generate olfactory bulb interneurons. It is unclear how the SVZ environment sustains neuronal production into adulthood. We discovered that the adapter molecule Ankyrin-3 (Ank3) is specifically upregulated in radial glia destined to become SVZ ependymal niche cells, but not in NSCs, and is required for SVZ assembly through progenitor lateral adhesion. Furthermore, we found that Ank3 expression is controlled by Foxj1, a transcriptional regulator of multicilia formation, and genetic deletion of this pathway led to complete loss of SVZ niche structure. In its absence, radial glia continued to transition into postnatal NSCs. However, inducible ependymal deletion of Foxj1-Ank3 after SVZ niche assembly resulted in dramatic depletion of neurogenesis. Targeting a novel pathway regulating ependymal organization/assembly and showing its requirement for new neuron production, our results have important implications for environmental control of adult neurogenesis and harvesting NSCs for replacement therapy.