7A projection map of the S-layer protein sbpA obtained with trehalose-embedded monolayer crystals

Two-dimensional crystallization on lipid monolayers is a versatile tool to obtain structural information of proteins by electron microscopy. An inherent problem with this approach is to prepare samples in a way that preserves the crystalline order of the protein array and produces specimens that are sufficiently flat for high-resolution data collection at high tilt angles. As a test specimen to optimize the preparation of lipid monolayer crystals for electron microscopy imaging, we used the S-layer protein sbpA, a protein with potential for designing arrays of both biological and inorganic materials with engineered properties for a variety of nanotechnology applications. Sugar embedding is currently considered the best method to prepare two-dimensional crystals of membrane proteins reconstituted into lipid bilayers. We found that using a loop to transfer lipid monolayer crystals to an electron microscopy grid followed by embedding in trehalose and quick-freezing in liquid ethane also yielded the highest resolution images for sbpA lipid monolayer crystals. Using images of specimens prepared in this way we could calculate a projection map of sbpA at 7A resolution, one of the highest resolution projection structures obtained with lipid monolayer crystals to date.     

Shp2 regulates SRC family kinase activity and Ras/Erk activation by controlling Csk recruitment

The protein-tyrosine phosphatase Shp2 plays an essential role in growth factor and integrin signaling, and Shp2 mutations cause developmental defects and/or malignancy. Previous work has placed Shp2 upstream of Ras. However, the mechanism of Shp2 action and its substrate(s) are poorly defined. Additional Shp2 functions downstream of, or parallel to, Ras/Erk activation also are proposed. Here, we show that Shp2 promotes Src family kinase (SFK) activation by regulating the phosphorylation of the Csk regulator PAG/Cbp, thereby controlling Csk access to SFKs. In Shp2-deficient cells, SFK inhibitory C-terminal tyrosines are hyperphosphorylated, and the tyrosyl phosphorylation of multiple SFK substrates, including Plcgamma1, is decreased. Decreased Plcgamma1 phosphorylation leads to defective Ras activation on endomembranes, and may help account for impaired Erk activation in Shp2-deficient cells. Decreased phosphorylation/activation of other SFK substrates may explain additional consequences of Shp2 deficiency, including altered cell spreading, stress fibers, focal adhesions, and motility.     

Atomic-level characterization of disordered protein ensembles

The roles of unfolded states of proteins in normal folding and in diseases involving aggregation, as well as the prevalence and regulatory functions of intrinsically disordered proteins, have become increasingly recognized. The structural representation of these disordered states as ensembles of interconverting conformers can therefore provide critical insights. Experimental methods can be used to probe ensemble-averaged structural properties of disordered states and computational approaches generate representative ensembles of conformers using experimental restraints. In particular, NMR and small-angle X-ray scattering provide quantitative data that can readily be incorporated into calculations. These techniques have gleaned structural information about denatured, unfolded and intrinsically disordered proteins. The use of experimental data in different computational approaches, including ensemble molecular dynamics simulations and algorithms that assign populations to pregenerated conformers, has highlighted the presence of both local and long-range structure, and the occurrence of native-like and non-native interactions in unfolded and denatured states. Analysis of the resulting ensembles has suggested important implications of this fluctuating structure for folding, aggregation and binding.     

High-resolution imaging reveals indirect coordination of opposite motors and a role for LIS1 in high-load axonal transport

The specific physiological roles of dynein regulatory factors remain poorly understood as a result of their functional complexity and the interdependence of dynein and kinesin motor activities. We used a novel approach to overcome these challenges, combining acute in vivo inhibition with automated high temporal and spatial resolution particle tracking. Acute dynein inhibition in nonneuronal cells caused an immediate dispersal of diverse forms of cargo, resulting from a sharp decrease in microtubule minus-end run length followed by a gradual decrease in plus-end runs. Acute LIS1 inhibition or LIS1 RNA interference had little effect on lysosomes/late endosomes but severely inhibited axonal transport of large, but not small, vesicular structures. Our acute inhibition results argue against direct mechanical activation of opposite-directed motors and offer a novel approach of potential broad utility in the study of motor protein function in vivo. Our data also reveal a specific but cell type-restricted role for LIS1 in large vesicular transport and provide the first quantitative support for a general role for LIS1 in high-load dynein functions.     

An effector domain mutant of Arf6 implicates phospholipase D in endosomal membrane recycling

In this study, we investigated the role of phospholipase D (PLD) in mediating Arf6 function in cells. Expression of Arf6 mutants that are defective in activating PLD, Arf6N48R and Arf6N48I, inhibited membrane recycling to the plasma membrane (PM), resulting in an accumulation of tubular endosomal membranes. Additionally, unlike wild-type Arf6, neither Arf6 mutant could generate protrusions or recruit the Arf6 GTPase activating protein (GAP) ACAP1 onto the endosome in the presence of aluminum fluoride. Remarkably, all of these phenotypes, including accumulated tubular endosomes, blocked recycling, and failure to make protrusions and recruit ACAP effectively, could be recreated in either untransfected cells or cells expressing wild-type Arf6 by treatment with 1-butanol to inhibit the formation of phosphatidic acid (PA), the product of PLD. Moreover, most of the defects present in cells expressing Arf6N48R or N48I could be reversed by treatment with agents expected to elevate PA levels in cells. Together, these observations provide compelling evidence that Arf6 stimulation of PLD is required for endosomal membrane recycling and GAP recruitment.     

Differential signaling by adaptor molecules LRP1 and ShcA regulates adipogenesis by the insulin-like growth factor-1 receptor

The low density lipoprotein receptor-related protein (LRP1) is a transmembrane receptor that integrates multiple signaling pathways. Its cytoplasmic domain serves as docking sites for several adaptor proteins such as the Src homology 2/α-collagen (ShcA), which also binds to several tyrosine kinase receptors such as the insulin-like growth factor 1 (IGF-1) receptor. However, the physiological significance of the physical interaction between LRP1 and ShcA, and whether this interaction modifies tyrosine kinase receptor signaling, are still unknown. Here we report that LRP1 forms a complex with the IGF-1 receptor, and that LRP1 is required for ShcA to become sensitive to IGF-1 stimulation. Upon IGF-1 treatment, ShcA is tyrosine phosphorylated and translocates to the plasma membrane only in the presence of LRP1. This leads to the recruitment of the growth factor receptor-bound protein 2 (Grb2) to ShcA, and activation of the Ras/MAP kinase pathway. Conversely, in the absence of ShcA, IGF-1 signaling bifurcates toward the Akt/mammalian target of rapamycin pathway and accelerates adipocyte differentiation when cells are stimulated for adipogenesis. These results establish the LRP1-ShcA complex as an essential component in the IGF-1-regulated pathway for MAP kinase and Akt/mammalian target of rapamycin activation, and may help to understand the IGF-1 signaling shift from clonal expansion to growth-arrested cells and differentiation during adipogenesis.     

Isolation and characterisation of transposon-induced mutants of Erwinia carotovora subsp. atroseptica exhibiting reduced virulence

Summary The blackleg pathogen Erwinia carotovora subsp. atroseptica (Eca) causes an economically important disease of potatoes. We selected a genetically amenable Eca strain for the genetic analysis of virulence. Tn5 mutagenesis was used to generate nine mutants which exhibited reduced virulence (Rvi^-) of strain SCRI1043. Following physiological characterisation, mutants were divided into three classes: (1) auxotrophs; (2) extracellular enzyme mutants; and (3) a growth rate mutant. The isolation of these Rvi^- mutants has allowed us to consider some factors that affect Eca virulence.     

A Simple Dynamic Model of Respiratory Pump

To study the interaction of forces that produce chest wall motion, we propose a model based on the lever system of Hillman and Finucane (J Appl Physiol 63(3):951–961, 1987) and introduce some dynamic properties of the respiratory system. The passive elements (rib cage and abdomen) are considered as elastic compartments linked to the open air via a resistive tube, an image of airways. The respiratory muscles (active) force is applied to both compartments. Parameters of the model are identified in using experimental data of airflow signal measured by pneumotachography and rib cage and abdomen signals measured by respiratory inductive plethysmography on eleven healthy volunteers in five conditions: at rest and with four level of added loads. A breath by breath analysis showed, whatever the individual and the condition are, that there are several breaths on which the airflow simulated by our model is well fitted to the airflow measured by pneumotachography as estimated by a determination coefficient R ² ≥ 0.70. This very simple model may well represent the behaviour of the chest wall and thus may be useful to interpret the relative motion of rib cage and abdomen during quiet breathing.     

Coprolites as a source of information on the genome and diet of the cave hyena

We performed high-throughput sequencing of DNA from fossilized faeces to evaluate this material as a source of information on the genome and diet of Pleistocene carnivores. We analysed coprolites derived from the extinct cave hyena (Crocuta crocuta spelaea), and sequenced 90 million DNA fragments from two specimens. The DNA reads enabled a reconstruction of the cave hyena mitochondrial genome with up to a 158-fold coverage. This genome, and those sequenced from extant spotted (Crocuta crocuta) and striped (Hyaena hyaena) hyena specimens, allows for the establishment of a robust phylogeny that supports a close relationship between the cave and the spotted hyena. We also demonstrate that high-throughput sequencing yields data for cave hyena multi-copy and single-copy nuclear genes, and that about 50 per cent of the coprolite DNA can be ascribed to this species. Analysing the data for additional species to indicate the cave hyena diet, we retrieved abundant sequences for the red deer (Cervus elaphus), and characterized its mitochondrial genome with up to a 3.8-fold coverage. In conclusion, we have demonstrated the presence of abundant ancient DNA in the coprolites surveyed. Shotgun sequencing of this material yielded a wealth of DNA sequences for a Pleistocene carnivore and allowed unbiased identification of diet.     

Conformational Changes Relevant to Channel Activity and Folding within the first Nucleotide Binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator

Deletion of Phe-508 (F508del) in the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to defects in folding and channel gating. NMR data on human F508del NBD1 indicate that an H620Q mutant, shown to increase channel open probability, and the dual corrector/potentiator CFFT-001 similarly disrupt interactions between β-strands S3, S9, and S10 and the C-terminal helices H8 and H9, shifting a preexisting conformational equilibrium from helix to coil. CFFT-001 appears to interact with β-strands S3/S9/S10, consistent with docking simulations. Decreases in T(m) from differential scanning calorimetry with H620Q or CFFT-001 suggest direct compound binding to a less thermostable state of NBD1. We hypothesize that, in full-length CFTR, shifting the conformational equilibrium to reduce H8/H9 interactions with the uniquely conserved strands S9/S10 facilitates release of the regulatory region from the NBD dimerization interface to promote dimerization and thereby increase channel open probability. These studies enabled by our NMR assignments for F508del NBD1 provide a window into the conformational fluctuations within CFTR that may regulate function and contribute to folding energetics.