Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets—typically comprising thousands of particles—is a tedious and time-consuming process, numerous automatic particle pickers have been developed. However, non-ideal datasets pose a challenge to particle picking. Here we present the particle picking software crYOLO which is based on the deep-learning object detection system You Only Look Once (YOLO). After training the network with 200–2500 particles per dataset it automatically recognizes particles with high recall and precision while reaching a speed of up to five micrographs per second. Further, we present a general crYOLO network able to pick from previously unseen datasets, allowing for completely automated on-the-fly cryo-EM data preprocessing during data acquisition. crYOLO is available as a standalone program under http://sphire.mpg.de/ and is distributed as part of the image processing workflow in SPHIRE.

Selecting particles from digital micrographs is an essential step in single particle electron cryomicroscopy (cryo-EM). Since manual selection of complete datasets typically comprising many thousands of particles is a tedious and time-consuming process, many automatic particle pickers have been developed in the past few decades.However, non-ideal datasets pose a challenge to particle picking. Here, we present a novel automated particle picking software called crYOLO, which is based on the deep learning object detection system "You Only Look Once" (YOLO). After training the network with 500 -2,500 particles per dataset, it automatically recognizes particles with high recall and precision reaching a speed of up to five micrographs per second.Importantly, we demonstrate a powerful general network trained on more than 40 datasets to select previously unseen datasets, thus paving the way for completely automated "on-the-fly" cryo-EM data pre-processing during data acquisition. CrYOLO is available as a standalone program under http://sphire.mpg.de/ and will be part of the image processing workflow in SPHIRE.

House dust mites (HDMs) belong to the most potent indoor allergen sources worldwide and are associated with allergic manifestations in the respiratory tract and the skin. Here we studied the importance of the high-molecular-weight group 11 allergen from Dermatophagoides pteronyssinus (Der p 11) in HDM allergy. Sequence analysis showed that Der p 11 has high homology to paramyosins from mites, ticks, and other invertebrates. A synthetic gene coding for Der p 11 was expressed in Escherichia coli and rDer p 11 purified to homogeneity as folded, alpha-helical protein as determined by circular dichroism spectroscopy. Using antibodies raised against rDer p 11 and immunogold electron microscopy, the allergen was localized in the muscle beneath the skin of mite bodies but not in feces. IgE reactivity of rDer p 11 was tested with sera from HDM-allergic patients from Europe and Africa in radioallergosorbent test-based dot-blot assays. Interestingly, we found that Der p 11 is a major allergen for patients suffering from atopic dermatitis (AD), whereas it is only a minor allergen for patients suffering from respiratory forms of HDM allergy. Thus, rDer p 11 might be a useful serological marker allergen for the identification of a subgroup of HDM-allergic patients suffering from HDM-associated AD.

Edited by Velia M. FowlerMacrophage filopodia, finger-like membrane protrusions, were first implicated in phagocytosis more than 100 years ago, but little is still known about the involvement of these actin-dependent structures in particle clearance. Using spinning disk confocal microscopy to image filopodial dynamics in mouse resident Lifeact-EGFP macrophages, we show that filopodia, or filopodia-like structures, support pathogen clearance by multiple means. Filopodia supported the phagocytic uptake of bacterial (Escherichia coli) particles by (i) capturing along the filopodial shaft and surfing toward the cell body, the most common mode of capture; (ii) capturing via the tip followed by retraction; (iii) combinations of surfing and retraction; or (iv) sweeping actions. In addition, filopodia supported the uptake of zymosan (Saccharomyces cerevisiae) particles by (i) providing fixation, (ii) capturing at the tip and filopodia-guided actin anterograde flow with phagocytic cup formation, and (iii) the rapid growth of new protrusions. To explore the role of filopodia-inducing Cdc42, we generated myeloid-restricted Cdc42 knock-out mice. Cdc42-deficient macrophages exhibited rapid phagocytic cup kinetics, but reduced particle clearance, which could be explained by the marked rounded-up morphology of these cells. Macrophages lacking Myo10, thought to act downstream of Cdc42, had normal morphology, motility, and phagocytic cup formation, but displayed markedly reduced filopodia formation. In conclusion, live-cell imaging revealed multiple mechanisms involving macrophage filopodia in particle capture and engulfment. Cdc42 is not critical for filopodia or phagocytic cup formation, but plays a key role in driving macrophage lamellipodial spreading.Phagocytosis is a specialized form of endocytosis, which requires localized actin polymerization to engulf large particles (Ͼ ϳ0.5 m), and initially involves particle binding to phagocytic receptors, which triggers phagocytic cup formation through the activation of kinases, such as Syk, and Rho GTPases, such as the Cdc42 and Rac subfamilies (1). Although phagocytosis has been investigated in-depth, the involvement of filopodia (finger-like projections containing bundled actin filaments (2)) in actually capturing and clearing particles has not been conclusively demonstrated, except for the historical observations of Metschnikoff (3) and more recent work using brightfield microscopy (4 -7).Young et al. (4) observed using time-lapse differential interference contrast (DIC) 2 imaging that Escherichia coli expressing invasin, a transmembrane protein of Yersinia pseudotuberculosis, could enter Hep-2 (HeLa-derived) cells via filopodia. Using another approach, coating of magnetic microbeads with invasin, Vonna et al. (5) found that the adhesion of beads to filopodial tips induced pulling toward the cell body. Similarly, Kress et al. (6) reported that filopodia act as "phagocytic tentacles" and pulled IgG-coated beads in an optical trap with discrete steps, suggesting that a motor pro...

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