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The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP3)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP3 and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP3/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP3 and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.  相似文献   
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We combine total internal reflection fluorescence structured illumination microscopy with spatiotemporal image correlation spectroscopy to quantify the flow velocities and directionality of filamentous-actin at the T cell immunological synapse. These techniques demonstrate it is possible to image retrograde flow of filamentous-actin at superresolution and provide flow quantification in the form of velocity histograms and flow vector maps. The flow was found to be retrograde and radially directed throughout the periphery of T-cells during synapse formation.Many biological processes are now being visualized with the use of superresolution fluorescence microscopy techniques. However, localization-based techniques primarily rely on fixed or slow moving samples to permit the collection of structural information. The 10-fold gains in resolution afforded by these superresolution techniques are usually possible through sacrificing the factors that originally made microscopy such a powerful tool: the ability to image live cells. In the case of stimulated emission depletion imaging, the scanning approach associated with this technique may fail to detect faster molecular events when imaging whole cellular regions.Structured illumination microscopy (SIM) is an alternative to these methods (1). It increases the resolution of conventional fluorescence microscopy twofold; it has the advantage of using a wide-field system, providing fast acquisition speeds of whole cells with relatively low laser powers; and it is compatible with standard fluorophores. By using a physical grating to produce interference patterns from a laser, periodic illumination is created. This patterned illumination causes information from higher spatial frequencies to be downmodulated (i.e., shifted) into the optical transfer function (support region) of the lens, resulting in higher-resolution spatial information being captured than is ordinarily obtainable.To quantify the directional motion of intracellular molecules, spatiotemporal image correlation spectroscopy (STICS (2)) was applied. Using spatial image correlation in time, STICS measures the similarity of pixels with those surrounding in lagging frames via a correlation function. The correlation function provides information on both flow velocities and directionality, while discounting static structures through the immobile object filter, achieved by subtracting a moving average of pixel intensities.The formation of an immunological synapse between T cells and antigen-presenting cells is a process requiring many dynamic (3) and subdiffraction-limited clustering events (4–6) to take place. The polymerization of actin is important for the spreading of cells over their target antigen-presenting cells (7), as well as cell mobility and migration (8). Retrograde flow of densely meshed cortical actin is observed at the basal membrane of synapse-forming T cells, where it may have a role in the corralling and clustering of signaling molecules at the plasma membrane (9), as well as at the leading edge of migrating cells (10). Filamentous actin is an extremely dynamic (7), densely packed, and thin (7-nm) structure (11,12).Here, we perform STICS on SIM data acquired on a total internal reflection fluorescence (TIRF) microscope system, which generated an evanescent field of 75-nm depth for excitation. To our knowledge, this is the first demonstration of an image correlation approach to quantify molecular dynamics on subresolution length scales using wide-field microscopy. To demonstrate the technique, we analyze two-dimensional actin flows in CD4+ T cells during immunological synapse formation, performed after cross-linking of antigen T cell receptors on a coverslip coated with specific antibodies.Fig. 1 a shows a schematic of the TIRF SIM setup. Excitation light (488 nm) passes through a polarizing module and then a phase-grating block, producing diffracted beams. These are then passed through a diffraction filter module to isolate the −1 and +1 order laser beams. These first-order laser beams are angled through the objective to produce total internal reflection conditions at the glass-water interface. The two evanescent waves interfere at the sample, producing structured illumination. The setup then produces lateral and rotational shifts through three orientations, producing nine raw images containing higher spatial frequencies than can normally be acquired by an objective using standard light microscopy. Fig. 1 b demonstrates the increased resolution obtained from TIRF SIM. Shown are the collected Fourier frequencies compared to those of a conventional microscope (dotted red line). Resolution was also measured using sparse 100-nm diameter fluorescent beads. Fig. 1 c shows a magnified image of these beads from which a line profile was obtained (yellow arrow). The full width at half-maximum of this profile (Fig. 1 d) gives a lateral resolution for the system of 120 nm.Open in a separate windowFigure 1(a) Schematic of the TIRF SIM setup. (b) Demonstration of the doubling of spatial resolution of collected frequencies through a Fourier transform (superimposed red circle demonstrating regular spatial frequency limits). (c) SIM reconstructed image of 100-nm bead (scale bar 0.5 μm). (d) (Plotted line) Bead showing full width at half-maximum of 120 nm.We then applied STICS analysis to quantify actin flow in T cell synapses acquired using TIRF SIM (Fig. 2). Fig. 2 a shows a schematic of the STICS analysis. From the raw data, immobile objects are first filtered by subtracting a moving average of the pixel values. Vector maps were obtained from correlation analysis of the time-series as previously published in Hebert et al. (2) and Brown et al. (13). Fig. 2 b shows a reconstructed TIRF SIM image of a mature T cell immunological synapse, representative of a time-point derived from the time series acquired at 1.28 fps (see Movie S1 in the Supporting Material). From this reconstructed image, two representative regions have been selected. In these regions, pseudo-colored actin flow vectors are overlaid onto the fluorescence intensity image. These range in magnitude from 0.01 μm/min (blue) to 5.61 μm/min (red). It can be observed that all flow vectors are directed radially toward the synapse center. A histogram of this flow is shown in Fig. 2 c. The histogram shows a peak retrograde flow velocity of 1.91 ± 1.27 μm/min. These data are representative of n = 7 T-cell synapses imaged by TIRF SIM.Open in a separate windowFigure 2(a) STICS analysis, performed by isolating mobile from immobile structures through a moving average filter (i) and binning a subset of pixels into blocks of superpixels (ii); the STICS software correlates spatial fluorescence fluctuations through time (iii). The code then outputs vector maps showing directionality and flow velocities. (b) TIRF SIM image of actin flow in a T cell 5 min after contact with a stimulatory coverslip. (Zoomed regions) Retrograde actin flow at the synapse periphery. (c) Histograms showing flow speed statistics of vectors from T-cell synapses (n = 7).  相似文献   
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This work describes a quick semi-quantitative colony immunoassay (QSCI) method for immunoblot detection of intracellularly expressed proteins in both yeast and bacterial cells. After induction of protein expression, only 4.5 h is required for cell breakage, protein detection, and data analysis. This protocol was used to screen and unambiguously identify Saccharomyces cerevisiae cells efficiently overexpressing glutathione S-transferase (GST)-tagged Yih1 in addition to cells expressing the myc-tagged large 297-kDa Gcn1 protein. In addition, the method was used to identify Escherichia coli cells efficiently expressing His6-tagged Yih1 and a GST-tagged Gcn1 fragment, respectively. The protocol allows the use of both epitope-specific and protein-specific antibodies. The same colony immunoassay can also be used to determine the minimal concentration of inducing agent sufficient for induction of optimal protein expression (e.g., galactose for yeast, isopropyl β-d-1-thiogalactopyranoside [IPTG] for E. coli). To our knowledge, this is the first report on a rapid low-cost procedure that allows the calibration of inducing agent on solid medium.  相似文献   
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At the intersection of geological activity, climatic fluctuations, and human pressure, the Mediterranean Basin – a hotspot of biodiversity – provides an ideal setting for studying endemism, evolution, and biogeography. Here, we focus on the Roucela complex (Campanula subgenus Roucela), a group of 13 bellflower species found primarily in the eastern Mediterranean Basin. Plastid and low‐copy nuclear markers were employed to reconstruct evolutionary relationships and estimate divergence times within the Roucela complex using both concatenation and species tree analyses. Niche modeling, ancestral range estimation, and diversification analyses were conducted to provide further insights into patterns of endemism and diversification through time. Diversification of the Roucela clade appears to have been primarily the result of vicariance driven by the breakup of an ancient landmass. We found geologic events such as the formation of the mid‐Aegean trench and the Messinian Salinity Crisis to be historically important in the evolutionary history of this group. Contrary to numerous past studies, the onset of the Mediterranean climate has not promoted diversification in the Roucela complex and, in fact, may be negatively affecting these species. This study highlights the diversity and complexity of historical processes driving plant evolution in the Mediterranean Basin.  相似文献   
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Conventionally flood mapping typically includes only a static water level (e.g. peak of a storm tide) in coastal flood inundation events. Additional factors become increasingly important when increased water-level thresholds are met during the combination of a storm tide and increased mean sea level. This research incorporates factors such as wave overtopping and river flow in a range of flood inundation scenarios of future sea-level projections for a UK case study of Fleetwood, northwest England. With increasing mean sea level it is shown that wave overtopping and river forcing have an important bearing on the cost of coastal flood events. The method presented converts inundation maps into monetary cost. This research demonstrates that under scenarios of joint extreme surge-wave-river events the cost of flooding can be increased by up to a factor of 8 compared with an increase in extent of up to a factor of 3 relative to “surge alone” event. This is due to different areas being exposed to different flood hazards and areas with common hazard where flood waters combine non-linearly. This shows that relying simply on flood extent and volume can under-predict the actual economic impact felt by a coastal community. Additionally, the scenario inundation depths have been presented as “brick course” maps, which represent a new way of interpreting flood maps. This is primarily aimed at stakeholders to increase levels of engagement within the coastal community.  相似文献   
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