A fixed-target platform for serial femtosecond crystallography in a hydrated environment.

For serial femtosecond crystallography at X-ray free-electron lasers, which entails collection of single-pulse diffraction patterns from a constantly refreshed supply of microcrystalline sample, delivery of the sample into the X-ray beam path while maintaining low background remains a technical challenge for some experiments, especially where this methodology is applied to relatively low-ordered samples or those difficult to purify and crystallize in large quantities. This work demonstrates a scheme to encapsulate biological samples using polymer thin films and graphene to maintain sample hydration in vacuum conditions. The encapsulated sample is delivered into the X-ray beam on fixed targets for rapid scanning using the Roadrunner fixed-target system towards a long-term goal of low-background measurements on weakly diffracting samples. As a proof of principle, we used microcrystals of the 24 kDa rapid encystment protein (REP24) to provide a benchmark for polymer/graphene sandwich performance. The REP24 microcrystal unit cell obtained from our sandwiched in-vacuum sample was consistent with previously established unit-cell parameters and with those measured by us without encapsulation in humidified helium, indicating that the platform is robust against evaporative losses. While significant scattering from water was observed because of the sample-deposition method, the polymer/graphene sandwich itself was shown to contribute minimally to background scattering.

Crystallization of ApoA1 and ApoE4 nanolipoprotein particles and initial XFEL-based structural studies.

Nanolipoprotein particles (NLPs), also called "nanodiscs", are discoidal particles with a patch of lipid bilayer corralled by apolipoproteins. NLPs have long been of interest due to both their utility as membrane-model systems into which membrane proteins can be inserted and solubilized and their physiological role in lipid and cholesterol transport via HDL and LDL maturation, which are important for human health. Serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs) is a powerful approach for structural biology of membrane proteins, which are traditionally difficult to crystallize as large single crystals capable of producing high-quality diffraction suitable for structure determination. To facilitate understanding of the specific role of two apolipoprotein/lipid complexes, ApoA1 and ApoE4, in lipid binding and HDL/LDL particle maturation dynamics and develop new SFX methods involving NLP membrane protein encapsulation, we have prepared and crystallized homogeneous populations of ApoA1 and ApoE4 NLPs. Crystallization of empty NLPs yields semi-ordered objects that appear crystalline and give highly anisotropic and diffuse X-ray diffraction, similar in characteristics to fiber diffraction. Several unit cell parameters were approximately determined for both NLPs from these measurements. Thus, low-background, sample conservative methods of delivery are critical. Here we implemented a fixed target sample delivery scheme utilizing the Roadrunner fast-scanning system and ultra-thin polymer/graphene support films, providing a low-volume, low-background approach to membrane protein SFX. This study represents initial steps in obtaining structural information for ApoA1 and ApoE4 NLPs and developing this system as a supporting scaffold for future structural studies of membrane proteins crystalized in a native lipid environment.

Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography.

Riboswitches are structural RNA elements that are generally located in the 5' untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time. Here we use femtosecond X-ray free electron laser (XFEL) pulses to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of 'mix-and-inject' time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

Epithelial cell dynamics in rabbit cecum and proximal colon P1.

The large intestine of mammals has long been viewed as an osmoregulatory organ, and evidence suggests that fluid and solute transport mechanisms within the intestine are heterogeneous, varying depending on the particular segment involved. Variations in function are often matched by morphological correlates, but despite the widespread use of rabbit large intestine as an experimental model, there is a lack of knowledge about the cellular makeup and dynamics in the colonic mucosal epithelium. The presence of mitotic figures and immunohistochemical localization of proliferating cell nuclear antigen (PCNA) were used to identify the proliferative zone(s). Cellular migration patterns were determined through the use of the thymidine analog 5-bromo-2-deoxyuridine (BrdU) over a 24-, 48-, and 72-hr period. Apoptotic nuclei were identified utilizing terminal deoxynucleotidyl transferase d-UTP nick-end labeling (TUNEL). Both cecum and the initial portion of the proximal colon (P1) exhibited a proliferative zone at or near the crypt base, and migration proceeded upwards toward the surface epithelium lining the intestinal lumen, where apoptosis occurred Turnover time of crypt columnar cells was determined to be about 3 days; that of mucous cells was estimated to be about 5 weeks. Rabbit cecum and proximal colon P1 are similar in their cellular morphology and epithelial cell kinetics. In both, the major proliferative zone is located at or near the crypt base, from which crypt columnar cells migrate toward the lumenal surface epithelium over a period of 3 days. Goblet cell turnover rate is much slower than that of columnar cells.

Col2-GFP reporter mouse--a new tool to study skeletal development.

Transgenic mice were generated that harbor a Col2-GFP reporter that marks chondrocytes and their immediate precursors during skeletal development. Cells engaged in chondrogenesis were identified by conventional fluorescence microscopy and confocal optical sectioning within their native environments in live embryos and in thick tissue slices. The use of these mice offers a novel approach for studying the role of chondrocytes in skeletal development.

Col2-GFP reporter marks chondrocyte lineage and chondrogenesis during mouse skeletal development.

Mice were generated in which a Col2-GFP transgene serves as a reporter for the chondrocyte lineage and for chondrogenesis in live embryos and newborn pups. Cells actively engaged in chondrogenesis were identified by confocal optical sectioning within their native environments in embryos and in thick tissue slices. Chondrocytes exhibiting GFP fluorescence were purified from rib cages by high-speed cell sorting of crude cell suspensions. Intensity of fluorescence correlated with biosynthesis of procollagen II in these cells. The use of these mice and their cells provides a novel approach for studying chondrocyte differentiation and chondrogenesis during skeletal development.

Proliferation of goblet cells and vacuolated cells in the rabbit distal colon.

Previous studies of colonic epithelial cell kinetics in mice and rats revealed a pattern similar to small intestine, where basally located stem cells proliferate, differentiating as they migrate towards the surface epithelium. Vacuolated and goblet cells are assumed to co-migrate at the same rate. The present study indicates that rabbit distal colon has more complicated epithelial cell kinetics. The zone of proliferation was detected immunohistochemically using proliferating cell nuclear antigen (PCNA) and confirmed with the use of colchicine to arrest dividing cells in metaphase. Migrating cells were tracked from the zero-hour position (PCNA labeling, mitosis) to positions 24, 48, 72 hrs by monitoring cell migration with the thymidine analog 5-Bromo-2-Deoxyuridine (BrdU). PCNA revealed a major proliferative zone in the upper third of the crypt column and the presence of mitotic figures after colchicine corroborated these results. Differentiated vacuolated cell proliferation was detected at three crypt sites: base, middle, and top of the crypt, while columnar cells arose from a population of dividing cells at the top of the crypt. Turnover of columnar and vacuolated cells occurred within 72 hrs. Goblet cells exhibited maximal proliferation at the crypt base and migrated at a much slower rate than the other cell types. In rabbit distal colon, populations of proliferating cells exist at multiple levels of the crypt column. Vacuolated and goblet cells differ in their labeling indices and migration rates, suggesting that the two cell types arise and migrate independently.

Epidermal growth factor modifies cell cycle control in A431 human squamous carcinoma cells damaged by ionizing radiation.

Epidermal growth factor (EGF) has been shown to radiosensitize A431 and other human squamous carcinoma cells with high numbers of surface EGF receptors. In this study of the mechanistic basis of EGF-induced radiosensitization, both EGF and ionizing radiation caused G1 phase arrests in cycling A431 cells, but only radiation caused a G2-M arrest. However, EGF enhanced the magnitude of this G2-M arrest, suggesting an interaction of signaling pathways involved in cellular responses to EGF and radiation damage. EGF and radiation also uniquely perturbed cyclin A and B1 mRNA levels during the time of maximum radiation-induced G2-M arrest. The effects of EGF on G2-M events probably originated in cells in G1. It is possible that aberrant EGF signal transduction in human squamous carcinoma cells may be exploited as a novel strategy for radiotherapy.

Selective down-regulation of integrin receptors in spheroids of squamous cell carcinoma.

Integrin expression in squamous cell carcinomas have been reported to be down-regulated in vivo. Because integrins are molecules whose functions include the reorganization of cytoskeleton in response to the surrounding extracellular matrix, we have examined the expression of integrins in the transformed cell line A431 when grown as multicellular spheroids or as monolayers. The spheroids were grown to sizes of approximately 100 microns and approximately 600 microns. Since larger A431 spheroids require epidermal growth factor for growth, we also investigated the effect of this growth factor on the expression of integrins in cells grown as monolayers or as small spheroids. Immunostaining studies using monoclonal antibodies specific for alpha 6, beta 1, and beta 4 subunits revealed a strong staining pattern in the periphery of the spheroids. The interior cells of the spheroids showed a moderate, positive reaction with the beta 1 antibody but significantly reduced from that at the periphery. Anti-alpha 2 antibody, on the other hand, revealed a uniform staining around the cells throughout the spheroids. Western blot analyses confirmed an overall diminution of alpha 6 and beta 1 protein levels in the spheroids compared with monolayers. Northern blot analyses showed that the low expression of integrin subunits alpha 6, beta 1, and beta 4 in spheroids was due to a reduction in mRNA transcripts. Northern blot analyses, however, showed no significant change in the expressions of alpha 2, alpha 5, or beta 5 mRNA. Conversely, the expression of alpha v was slightly reduced in spheroids. Epidermal growth factor increased the mRNA expression of alpha 2, alpha 6, beta 1, and beta 4 integrin subunits in cells grown either as monolayers or as spheroids whereas epidermal growth factor had no detectable effect on the expression of alpha v or beta 5. These results mimic the pattern of expression found in vivo and indicate that cell-cell contact and the microenvironments of cells within a spheroid regulate the expression and distribution of a subset of integrin molecules.

Enhancement of heme oxygenase expression and activity in A431 squamous carcinoma multicellular tumor spheroids.

We have investigated the effects of the growth of A431 human squamous carcinoma cells as three-dimensional aggregates (multicellular tumor spheroids) on the expression and enzyme activity of heme oxygenase (HO). We demonstrate that A431 squamous carcinoma cells grown as day 4 spheroids selectively increase the expression of heme oxygenase 1 (HO-1), caused, directly or indirectly, by three-dimensional cell-cell contact effects. Steady-state levels of both mRNA and protein are significantly enhanced in spheroids compared with day 4 monolayers (approximately 13-fold). Because of the similarity of apparent half-lives between monolayers (2.7 h) and spheroids (2.1 h), it appears that the increases are caused at least partly by altered transcriptional rates. Total HO enzyme activity, measured by carbon monoxide production, is also up-regulated (2.6-fold) in spheroids, compared to that in monolayers. This increase indicates that the up-regulation in HO-1 protein expression corresponds to an increase in functional enzyme levels. We propose that HO may play a more complex role in cellular metabolism than would be evident from studies using two-dimensional monolayer cultures.

Enhanced epidermal growth factor receptor synthesis in human squamous carcinoma cells exposed to low levels of oxygen.

Exposure of human A431 squamous carcinoma cells to levels of hypoxia found in some solid tumors causes 2-fold increases in epidermal growth-factor receptor (EGF-R) mRNA levels and rate of receptor protein synthesis compared with aerobic cells. Similar results are shown for receptor message from other squamous carcinoma cells, human keratinocytes, and human W138 fibroblasts. Less basal tyrosine phosphorylation of the receptor occurs in hypoxic compared with aerobic A431 cells. Scatchard analysis also shows that reoxygenated A431 cells display enhanced surface expression of the EGF-R compared with aerobic control cells. Possible mechanisms and implications for tumor therapy are discussed.

Enhancement of transforming growth factor-alpha synthesis in multicellular tumour spheroids of A431 squamous carcinoma cells.

Multicellular tumour spheroids are cellular aggregates that can be prepared from many types of tumour cells. These three-dimensional structures provide a model for analysing the effects of cell-cell contact and intercellular microenvironments on phenomena such as autocrine regulation of growth factor synthesis. Autoregulation of the synthesis of transforming growth factor-alpha (TGF-alpha) was investigated at the message and protein levels in spheroid and monolayer cultures prepared from the A431 human squamous carcinoma cell line. The epidermal growth factor receptor (EGF-R) of these monolayer A431 cells had an average surface density of 2.2 x 10(6)/cell. Constitutive expression of TGF-alpha mRNA was an average of 3-fold greater in A431 spheroids than in monolayers, even for densely packed, confluent monolayers. This effect did not depend on hypoxic stress within the spheroids. TGF-alpha protein synthesis was enhanced in comparison with that in monolayer culture, reaching a value of up to 2-fold greater on a per cell basis. These results are discussed in the context of a TGF-alpha/EGF-R autocrine loop operating within cells that produce high local concentrations of TGF-alpha in the three-dimensional architecture of a spheroid.