ASU Scholarship Showcase

Photosynthesis, a process catalysed by plants, algae and cyanobacteria converts sunlight to energy thus sustaining all higher life on Earth. Two large membrane protein complexes, photosystem I and II (PSI and PSII), act in series to catalyse the light-driven reactions in photosynthesis. PSII catalyses the light-driven water splitting process, which maintains the Earth’s oxygenic atmosphere. In this process, the oxygen-evolving complex (OEC) of PSII cycles through five states, S₀ to S₄, in which four electrons are sequentially extracted from the OEC in four light-driven charge-separation events. Here we describe time resolved experiments on PSII nano/microcrystals from Thermosynechococcus elongatus performed with the recently developed technique of serial femtosecond crystallography. Structures have been determined from PSII in the dark S₁ state and after double laser excitation (putative S₃ state) at 5 and 5.5 Å resolution, respectively. The results provide evidence that PSII undergoes significant conformational changes at the electron acceptor side and at the Mn₄CaO₅ core of the OEC. These include an elongation of the metal cluster, accompanied by changes in the protein environment, which could allow for binding of the second substrate water molecule between the more distant protruding Mn (referred to as the ‘dangler’ Mn) and the Mn₃CaO_x cubane in the S₂ to S₃ transition, as predicted by spectroscopic and computational studies. This work shows the great potential for time-resolved serial femtosecond crystallography for investigation of catalytic processes in biomolecules.

Neural progenitor cells (NPCs) derived from human pluripotent stem cells (hPSCs) are a multipotent cell population that is capable of nearly indefinite expansion and subsequent differentiation into the various neuronal and supporting cell types that comprise the CNS. However, current protocols for differentiating NPCs toward neuronal lineages result in a mixture of neurons from various regions of the CNS. In this study, we determined that endogenous WNT signaling is a primary contributor to the heterogeneity observed in NPC cultures and neuronal differentiation. Furthermore, exogenous manipulation of WNT signaling during neural differentiation, through either activation or inhibition, reduces this heterogeneity in NPC cultures, thereby promoting the formation of regionally homogeneous NPC and neuronal cultures. The ability to manipulate WNT signaling to generate regionally specific NPCs and neurons will be useful for studying human neural development and will greatly enhance the translational potential of hPSCs for neural-related therapies.

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

Although the majority of late-onset Alzheimer's disease (AD) patients are labeled sporadic, multiple genetic risk variants have been identified, the most powerful and prevalent of which is the e4 variant of the Apolipoprotein E (APOE) gene. Here, we generated human induced pluripotent stem cell (hiPSC) lines from the peripheral blood mononuclear cells (PBMCs) of a clinically diagnosed AD patient [ASUi003-A] and a non-demented control (NDC) patient [ASUi004-A] homozygous for the APOE4 risk allele. These hiPSCs maintained their original genotype, expressed pluripotency markers, exhibited a normal karyotype, and retained the ability to differentiate into cells representative of the three germ layers.

Nonsense-mediated RNA decay (NMD) is a highly conserved pathway that selectively degrades specific subsets of RNA transcripts. Here, we provide evidence that NMD regulates early human developmental cell fate. We found that NMD factors tend to be expressed at higher levels in human pluripotent cells than in differentiated cells, raising the possibility that NMD must be downregulated to permit differentiation. Loss- and gain-of-function experiments in human embryonic stem cells (hESCs) demonstrated that, indeed, NMD downregulation is essential for efficient generation of definitive endoderm. RNA-seq analysis identified NMD target transcripts induced when NMD is suppressed in hESCs, including many encoding signaling components. This led us to test the role of TGF-β and BMP signaling, which we found NMD acts through to influence definitive endoderm versus mesoderm fate. Our results suggest that selective RNA decay is critical for specifying the developmental fate of specific human embryonic cell lineages.

Previous proof-of-concept measurements on single-layer two-dimensional membrane-protein crystals performed at X-ray free-electron lasers (FELs) have demonstrated that the collection of meaningful diffraction patterns, which is not possible at synchrotrons because of radiation-damage issues, is feasible. Here, the results obtained from the analysis of a thousand single-shot, room-temperature X-ray FEL diffraction images from two-dimensional crystals of a bacteriorhodopsin mutant are reported in detail. The high redundancy in the measurements boosts the intensity signal-to-noise ratio, so that the values of the diffracted intensities can be reliably determined down to the detector-edge resolution of 4 Å. The results show that two-dimensional serial crystallography at X-ray FELs is a suitable method to study membrane proteins to near-atomic length scales at ambient temperature. The method presented here can be extended to pump–probe studies of optically triggered structural changes on submillisecond timescales in two-dimensional crystals, which allow functionally relevant large-scale motions that may be quenched in three-dimensional crystals.

It has been suggested that the extended intensity profiles surrounding Bragg reflections that arise when a series of finite crystals of varying size and shape are illuminated by the intense, coherent illumination of an x-ray free-electron laser may enable the crystal’s unit-cell electron density to be obtained ab initio via well-established iterative phasing algorithms. Such a technique could have a significant impact on the field of biological structure determination since it avoids the need for a priori information from similar known structures, multiple measurements near resonant atomic absorption energies, isomorphic derivative crystals, or atomic-resolution data. Here, we demonstrate this phasing technique on diffraction patterns recorded from artificial two-dimensional microcrystals using the seeded soft x-ray free-electron laser FERMI. We show that the technique is effective when the illuminating wavefront has nonuniform phase and amplitude, and when the diffraction intensities cannot be measured uniformly throughout reciprocal space because of a limited signal-to-noise ratio.

In the decade since Yamanaka and colleagues described methods to reprogram somatic cells into a pluripotent state, human induced pluripotent stem cells (hiPSCs) have demonstrated tremendous promise in numerous disease modeling, drug discovery, and regenerative medicine applications. More recently, the development and refinement of advanced gene transduction and editing technologies have further accelerated the potential of hiPSCs. In this review, we discuss the various gene editing technologies that are being implemented with hiPSCs. Specifically, we describe the emergence of technologies including zinc-finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 that can be used to edit the genome at precise locations, and discuss the strengths and weaknesses of each of these technologies. In addition, we present the current applications of these technologies in elucidating the mechanisms of human development and disease, developing novel and effective therapeutic molecules, and engineering cell-based therapies. Finally, we discuss the emerging technological advances in targeted gene editing methods.

The Dawn Framing Camera (FC) has imaged the northern hemisphere of the Asteroid (4) Vesta at high spatial resolution and coverage. This study represents the first investigation of the overall geology of the northern hemisphere (22–90°N, quadrangles Av-1, 2, 3, 4 and 5) using these unique Dawn mission observations. We have compiled a morphologic map and performed crater size–frequency distribution (CSFD) measurements to date the geologic units. The hemisphere is characterized by a heavily cratered surface with a few highly subdued basins up to ∼200 km in diameter. The most widespread unit is a plateau (cratered highland unit), similar to, although of lower elevation than the equatorial Vestalia Terra plateau. Large-scale troughs and ridges have regionally affected the surface. Between ∼180°E and ∼270°E, these tectonic features are well developed and related to the south pole Veneneia impact (Saturnalia Fossae trough unit), elsewhere on the hemisphere they are rare and subdued (Saturnalia Fossae cratered unit). In these pre-Rheasilvia units we observed an unexpectedly high frequency of impact craters up to ∼10 km in diameter, whose formation could in part be related to the Rheasilvia basin-forming event. The Rheasilvia impact has potentially affected the northern hemisphere also with S–N small-scale lineations, but without covering it with an ejecta blanket. Post-Rheasilvia impact craters are small (<60 km in diameter) and show a wide range of degradation states due to impact gardening and mass wasting processes. Where fresh, they display an ejecta blanket, bright rays and slope movements on walls. In places, crater rims have dark material ejecta and some crater floors are covered by ponded material interpreted as impact melt.

Oppia Quadrangle Av-10 (288–360°E, ±22°) is a junction of key geologic features that preserve a rough history of Asteroid (4) Vesta and serves as a case study of using geologic mapping to define a relative geologic timescale. Clear filter images, stereo-derived topography, slope maps, and multispectral color-ratio images from the Framing Camera on NASA’s Dawn spacecraft served as basemaps to create a geologic map and investigate the spatial and temporal relationships of the local stratigraphy. Geologic mapping reveals the oldest map unit within Av-10 is the cratered highlands terrain which possibly represents original crustal material on Vesta that was then excavated by one or more impacts to form the basin Feralia Planitia. Saturnalia Fossae and Divalia Fossae ridge and trough terrains intersect the wall of Feralia Planitia indicating that this impact basin is older than both the Veneneia and Rheasilvia impact structures, representing Pre-Veneneian crustal material. Two of the youngest geologic features in Av-10 are Lepida (∼45 km diameter) and Oppia (∼40 km diameter) impact craters that formed on the northern and southern wall of Feralia Planitia and each cross-cuts a trough terrain. The ejecta blanket of Oppia is mapped as ‘dark mantle’ material because it appears dark orange in the Framing Camera ‘Clementine-type’ color-ratio image and has a diffuse, gradational contact distributed to the south across the rim of Rheasilvia. Mapping of surface material that appears light orange in color in the Framing Camera ‘Clementine-type’ color-ratio image as ‘light mantle material’ supports previous interpretations of an impact ejecta origin. Some light mantle deposits are easily traced to nearby source craters, but other deposits may represent distal ejecta deposits (emplaced >5 crater radii away) in a microgravity environment.