We designed an epi-illumination SPIM system which utilizes a single objective and has an identical sample interface as an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of sixteen cell lines and parallel recording of cellular responses to perturbations.
The shape of many eukaryotic cells depends on the actin cytoskeleton, and changes in actin assembly dynamics underlie many changes in cell shape. Ena/VASP-family actin polymerases, for example, modulate cell shape by locally accelerating actin filament assembly and slowing filament capping. When concentrated into discrete foci at the leading edge, VASP promotes filopodia assembly, and forms part of a poorly understood molecular complex that remains associated with growing filopodia tips. Here we identify precursors of this filopodia tip complex in migrating B16F1 cells: small leading-edge clusters of the adaptor protein lamellipodin (Lpd) which subsequently recruit VASP and initiate filopodia formation. Dimerization, membrane association, and VASP binding are all required for lamellipodin to incorporate into filopodia tip complexes, and over-expression of monomeric, membrane-targeted lamellipodin mutants disrupts tip complex assembly. Once formed, tip complexes containing VASP and lamellipodin grow by fusing with each other, but their growth is limited by a size-dependent, dynamic instability. Our results demonstrate that assembly and disassembly dynamics of filopodia tip complexes are determined, in part, by a network of multivalent interactions between Ena/VASP proteins, EVH1 ligands, and actin filaments. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]
Few chemical strategies for activating enzymes have been developed. Here we show that a biarsenical compound (FlAsH) can directly activate a rationally engineered protein tyrosine phosphatase (Shp2 PTP) via disruption of autoinhibitory interactions between Shp2’s N-terminal SH2 domain and its PTP domain. We find that introduction of a tri-cysteine motif at a loop of Shp2’s N-SH2 domain confers affinity for FlAsH; binding of FlAsH to the cysteine-enriched loop relieves Shp2’s inhibitory inter-domain interaction and substantially increases the enzyme’s PTP activity. Activation of engineered Shp2 is substrate-independent and is observed in the contexts of both purified enzyme and complex proteomes. A chemical means for activating Shp2 may be useful for investigating its roles in signaling and oncogenesis, and the loop-targeting strategy described herein may provide a blueprint for the development of target-specific activators of other autoinhibited enzymes.
In many areas of the world, overhead video collected from aircraft and other airborne vehicles is an important component of surveillance and security operations. A typical use scenario might involve realtime monitoring of video streams by human analysts with the goal of identifying patterns of vehicle movement that might be considered suspicious or dangerous. In performing such a task, analysts often have access to low-level image processing support, most notably automated extraction of vehicle tracks. The goal of this research is to investigate the hypothesis that analysts would benefit from higher-level automated support that provides an assessment of vehicle behavior on the basis of an analysis of track data. Experimental results were obtained in the context of a simulated environment that involves a relatively simple task for video analysis. The results show that automated support for the identification of suspicious vehicle behaviors significantly improved successful vehicle identification.
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