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Secret sabertooth
PaleoJoe, his eleven-year-old assistant Shelly, her classmate Dakota, and new friend Sarra fly to California to investigate a mystery involving Sarra's brother, saber-tooth fossils, and the La Brea Tar Pits.
Book
FerriTag is a new genetically-encoded inducible tag for correlative light-electron microscopy
A current challenge is to develop tags to precisely visualize proteins in cells by light and electron microscopy. Here, we introduce FerriTag, a genetically-encoded chemically-inducible tag for correlative light-electron microscopy. FerriTag is a fluorescent recombinant electron-dense ferritin particle that can be attached to a protein-of-interest using rapamycin-induced heterodimerization. We demonstrate the utility of FerriTag for correlative light-electron microscopy by labeling proteins associated with various intracellular structures including mitochondria, plasma membrane, and clathrin-coated pits and vesicles. FerriTagging has a good signal-to-noise ratio and a labeling resolution of approximately 10 nm. We demonstrate how FerriTagging allows nanoscale mapping of protein location relative to a subcellular structure, and use it to detail the distribution and conformation of huntingtin-interacting protein 1 related (HIP1R) in and around clathrin-coated pits.
Correlative light-electron microscopy (CLEM) pairs versatile fluorescence imaging with high resolution electron microscopy. Here, the authors develop a genetically-encoded, chemically-inducible tag that allows acute labeling of single proteins for CLEM.
Journal Article
Wall Displacement and Ground-Surface Settlement Caused by Pit-in-Pit Foundation Pit in Soft Clays
The number of pit-in-pit foundation pit is increasing quickly because of the continuous utilization of underground spaces in urban areas. Based on the co-construction project of Shanghai Museum of Natural History foundation pit and Shanghai Metro Line 13 foundation pit in Shanghai, China, the deformation characteristics of pit-in-pit foundation pit are researched by field observation and centrifugal model tests. The lateral wall displacement of inner foundation pit includes global deformation caused by the outer foundation pit excavation and deflection caused by the excavation of itself. The effect of inner foundation pit excavation on the lateral wall displacement of outer foundation pit and ground-surface settlement is smaller. The two factors affecting the deformation characteristics of pit-in-pit foundation pit, the distance between inner and outer foundation pits (
D
) and the excavation width of inner foundation pit (
W
in
), are analyzed by centrifugal model tests. The result shows that the maximum lateral wall displacements of inner and outer foundation pits decrease nearly linearly with the increase of
D
, but increase with the increase of
W
in
.
Journal Article
Field Study on Rectangular Inclined Deep Foundation Excavation in Soft Soils
Due to the twofold influence of irregular excavation and geometric effect, the deformation mechanism of foundation pits is complicated. To investigate the impact of uneven excavation and geometric differences on the deformation characteristics of deep foundation pits, a long tunnel Phase IV deep foundation pit project in Suzhou is used as a case. Through the field measured data, the special effects of inclined pit bottom and rectangular shape are analyzed, and the deformation characteristics of the pit are summarized, as well as compared against the statistical data. The results indicate that the overall deformation of the pit is small since the pit is in a semi-excavated state. The excavation depth has a significant impact on pit deformation, and irregular excavation will cause the distribution of pit deformation to be skewed towards the deeper side of the excavation depth. As a result of the uneven excavation, the actual working condition of each portion of the foundation trench is inconsistent, causing the unexcavated portion to disturb the excavated portion, resulting in an ongoing process of cumulative deformation. Under the influence of the geometric effect of the rectangular pit, the deformation distribution pattern along the longitudinal section of the pit is parabolic with peaks at the centre and minimal towards the ends. The results of this study can provide a reference for the excavation design of the same type of pit and the study of pit deformation characteristics.
Journal Article
Clathrin-adaptor ratio and membrane tension regulate the flat-to-curved transition of the clathrin coat during endocytosis
Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was long believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form flat structures that then bend while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical growth laws to study the ultrastructural rearrangements of the clathrin coat during endocytosis in BSC-1 mammalian cells. We confirm that clathrin coats initially grow flat and demonstrate that curvature begins when around 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the notion that BSC-1 mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.
The sequence of structural and molecular events during clathrin-mediated endocytosis is unclear. Here the authors combine correlative microscopy and simple mathematical growth laws to demonstrate that the flat patch starts to curve when around 70% of the final clathrin content is reached.
Journal Article
Actin dynamics counteract membrane tension during clathrin-mediated endocytosis
Kirchhausen and colleagues show that actin is required for clathrin-mediated endocytosis at membranes under tension—such as apical surfaces of polarized cells. Actin engages with Hip1R bound to clathrin light chain to complete the deformation of a clathrin-coated pit into an endocytic vesicle.
Clathrin-mediated endocytosis is independent of actin dynamics in many circumstances but requires actin polymerization in others. We show that membrane tension determines the actin dependence of clathrin-coat assembly. As found previously, clathrin assembly supports formation of mature coated pits in the absence of actin polymerization on both dorsal and ventral surfaces of non-polarized mammalian cells, and also on basolateral surfaces of polarized cells. Actin engagement is necessary, however, to complete membrane deformation into a coated pit on apical surfaces of polarized cells and, more generally, on the surface of any cell in which the plasma membrane is under tension from osmotic swelling or mechanical stretching. We use these observations to alter actin dependence experimentally and show that resistance of the membrane to propagation of the clathrin lattice determines the distinction between ‘actin dependent and ‘actin independent’. We also find that light-chain-bound Hip1R mediates actin engagement. These data thus provide a unifying explanation for the role of actin dynamics in coated-pit budding.
Journal Article
Pore constrictions in intervessel pit membranes provide a mechanistic explanation for xylem embolism resistance in angiosperms
• Embolism spreading in angiosperm xylem occurs via mesoporous pit membranes between vessels. Here, we investigate how the size of pore constrictions in pit membranes is related to pit membrane thickness and embolism resistance.
• Pit membranes were modelled as multiple layers to investigate how pit membrane thickness and the number of intervessel pits per vessel determine pore constriction sizes, the probability of encountering large pores, and embolism resistance. These estimations were complemented by measurements of pit membrane thickness, embolism resistance, and number of intervessel pits per vessel in stem xylem (n = 31, 31 and 20 species, respectively).
• The modelled constriction sizes in pit membranes decreased with increasing membrane thickness, explaining the measured relationship between pit membrane thickness and embolism resistance. The number of pits per vessel affected constriction size and embolism resistance much less than pit membrane thickness. Moreover, a strong relationship between modelled and measured embolism resistance was observed.
• Pore constrictions provide a mechanistic explanation for why pit membrane thickness determines embolism resistance, which suggests that hydraulic safety can be uncoupled from hydraulic efficiency. Although embolism spreading remains puzzling and encompasses more than pore constriction sizes, angiosperms are unlikely to have leaky pit membranes, which enables tensile transport of water.
Journal Article
Regulation of endocytic recycling by C. elegans Rab35 and its regulator RME-4, a coated-pit protein
Using
Caenorhabditis elegans
genetic screens, we identified receptor‐mediated endocytosis (RME)‐4 and RME‐5/RAB‐35 as important regulators of yolk endocytosis
in vivo
. In
rme‐4
and
rab‐35
mutants, yolk receptors do not accumulate on the plasma membrane as would be expected in an internalization mutant, rather the receptors are lost from cortical endosomes and accumulate in dispersed small vesicles, suggesting a defect in receptor recycling. Consistent with this, genetic tests indicate the RME‐4 and RAB‐35 function downstream of clathrin, upstream of RAB‐7, and act synergistically with recycling regulators RAB‐11 and RME‐1. We find that RME‐4 is a conserved DENN domain protein that binds to RAB‐35 in its GDP‐loaded conformation. GFP–RME‐4 also physically interacts with AP‐2, is enriched on clathrin‐coated pits, and requires clathrin but not RAB‐5 for cortical association. GFP–RAB‐35 localizes to the plasma membrane and early endocytic compartments but is lost from endosomes in
rme‐4
mutants. We propose that RME‐4 functions on coated pits and/or vesicles to recruit RAB‐35, which in turn functions in the endosome to promote receptor recycling.
Journal Article
Evolutionarily unique mechanistic framework of clathrin-mediated endocytosis in plants
In plants, clathrin mediated endocytosis (CME) represents the major route for cargo internalisation from the cell surface. It has been assumed to operate in an evolutionary conserved manner as in yeast and animals. Here we report characterisation of ultrastructure, dynamics and mechanisms of plant CME as allowed by our advancement in electron microscopy and quantitative live imaging techniques. Arabidopsis CME appears to follow the constant curvature model and the bona fide CME population generates vesicles of a predominantly hexagonal-basket type; larger and with faster kinetics than in other models. Contrary to the existing paradigm, actin is dispensable for CME events at the plasma membrane but plays a unique role in collecting endocytic vesicles, sorting of internalised cargos and directional endosome movement that itself actively promote CME events. Internalized vesicles display a strongly delayed and sequential uncoating. These unique features highlight the independent evolution of the plant CME mechanism during the autonomous rise of multicellularity in eukaryotes.
Journal Article
Monitoring the effects of open-pit mining on the eco-environment using a moving window-based remote sensing ecological index
Environmental problems caused by mines have been increasing. As one of the most serious types of mining damage caused to the eco-environment, open pits have been the focus of monitoring and management. Previous studies have obtained effective results when evaluating the ecological quality of a mining area by using the remote sensing ecological index (RSEI). However, the calculation of RSEI does not consider that the ecological environmental impact is limited under natural conditions. To overcome this shortcoming, this paper proposes an improved RSEI based on a moving window model, namely the moving window-based remote sensing ecological index (MW-RSEI). This improved index is more in agreement with the First Law of Geography than RSEI. This study uses Landsat ETM/OLI/TIRS images to extract MW-RSEI information of a case area in Zhengzhou City, Henan Province, central China, in 2009 and 2018. The results revealed that the average value of MW-RSEI declined from 0.668 to 0.611 from 2009 to 2018, and the main drivers of the deterioration of the eco-environment were land use/cover (LUCC) changes, most of which were derived from urban expansion and mining. The serious impact of open pits on the eco-environment in mining areas is mainly due to their low vegetation cover; therefore, some effectively managed open pits can have a positive impact on the mining environment. The use of MW-RSEI provides valuable information on the eco-environment surrounding the open pit, which can be used for the rapid and effective monitoring of the eco-environment in mining areas.
Journal Article