Microbe-independent entry of oomycete RxLR effectors and fungal RxLR-like effectors into plant and animal cells is specific and reproducible.

A wide diversity of pathogens and mutualists of plant and animal hosts, including oomycetes and fungi, produce effector proteins that enter the cytoplasm of host cells. A major question has been whether or not entry by these effectors can occur independently of the microbe or requires machinery provided by the microbe. Numerous publications have documented that oomycete RxLR effectors and fungal RxLR-like effectors can enter plant and animal cells independent of the microbe. A recent reexamination of whether the RxLR domain of oomycete RxLR effectors is sufficient for microbe-independent entry into host cells concluded that the RxLR domains of Phytophthora infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and nonhost plant and animal cells. Here, we present new, more detailed data that unambiguously demonstrate that the RxLR domain of Avr1b does show efficient and specific entry into soybean root cells and also into wheat leaf cells, at levels well above background nonspecific entry. We also summarize host cell entry experiments with a wide diversity of oomycete and fungal effectors with RxLR or RxLR-like motifs that have been independently carried out by the seven different labs that coauthored this letter. Finally we discuss possible technical reasons why specific cell entry may have been not detected by Wawra et al. (2013).

Epigenetically regulated digital signaling defines epithelial innate immunity at the tissue level.

To prevent damage to the host or its commensal microbiota, epithelial tissues must match the intensity of the immune response to the severity of a biological threat. Toll-like receptors allow epithelial cells to identify microbe associated molecular patterns. However, the mechanisms that mitigate biological noise in single cells to ensure quantitatively appropriate responses remain unclear. Here we address this question using single cell and single molecule approaches in mammary epithelial cells and primary organoids. We find that epithelial tissues respond to bacterial microbe associated molecular patterns by activating a subset of cells in an all-or-nothing (i.e. digital) manner. The maximum fraction of responsive cells is regulated by a bimodal epigenetic switch that licenses the TLR2 promoter for transcription across multiple generations. This mechanism confers a flexible memory of inflammatory events as well as unique spatio-temporal control of epithelial tissue-level immune responses. We propose that epigenetic licensing in individual cells allows for long-term, quantitative fine-tuning of population-level responses.

MAPK activity dynamics regulate non-cell autonomous effects of oncogene expression.

A large fraction of human cancers contain genetic alterations within the Mitogen Activated Protein Kinase (MAPK) signaling network that promote unpredictable phenotypes. Previous studies have shown that the temporal patterns of MAPK activity (i.e. signaling dynamics) differentially regulate cell behavior. However, the role of signaling dynamics in mediating the effects of cancer driving mutations has not been systematically explored. Here, we show that oncogene expression leads to either pulsatile or sustained ERK activity that correlate with opposing cellular behaviors (i.e. proliferation vs. cell cycle arrest, respectively). Moreover, sustained-but not pulsatile-ERK activity triggers ERK activity waves in unperturbed neighboring cells that depend on the membrane metalloprotease ADAM17 and EGFR activity. Interestingly, the ADAM17-EGFR signaling axis coordinates neighboring cell migration toward oncogenic cells and is required for oncogenic cell extrusion. Overall, our data suggests that the temporal patterns of MAPK activity differentially regulate cell autonomous and non-cell autonomous effects of oncogene expression.

In animals, the MAPK pathway is a network of genes that helps a cell to detect and then respond to an external signal by switching on or off a specific genetic program. In particular, cells use this pathway to communicate with each other. In an individual cell, the MAPK pathway shows fluctuations in activity over time. Mutations in the genes belonging to the MAPK pathway are often one of the first events that lead to the emergence of cancers. However, different mutations in the genes of the pathway can have diverse effects on a cell's behavior: some mutations cause the cell to divide while others make it migrate. Recent research has suggested that these effects may be caused by changes in the pattern of MAPK signaling activity over time. Here, Aikin et al. used fluorescent markers to document how different MAPK mutations influence the behavior of a human breast cell and its healthy neighbors. The experiments showed that cells with different MAPK mutations behaved in one of two ways: the signaling quickly pulsed between high and low levels of activity, or it remained at a sustained high level. In turn, these two signaling patterns altered cell behavior in different ways. Pulsed signaling led to more cell division, while sustained signaling stopped division and increased migration. Aikin et al. then examined the effect of the MAPK mutations on neighboring healthy cells. Sustained signaling from the cancerous cell caused a wave of signaling activity in the surrounding cells. This led the healthy cells to divide and migrate toward the cancerous cell, pushing it out of the tissue layer. It is not clear if these changes protect against or promote cancer progression in living tissue. However, these results explain why specific cancer mutations cause different behaviors in cells.

Endocytic Markers Associated with the Internalization and Processing of Aspergillus fumigatus Conidia by BEAS-2B Cells.

Aspergillus fumigatus is a ubiquitous mold that produces small airborne conidia capable of traversing deep into the respiratory system. Recognition, processing, and clearance of A. fumigatus conidia by bronchial airway epithelial cells are thought to be relevant to host defense and immune signaling. Using z-stack confocal microscopy, we observed that only 10 to 20% of adherent conidia from the AF293 clinical isolate are internalized by BEAS-2B cells 6 h postchallenge and not prior. Similar percentages of internalization were observed for the CEA10 clinical isolate. A large subset of both AF293 and CEA10 conidia are rendered metabolically inactive without internalization at 3 h postchallenge by BEAS-2B cells. A significantly larger percentage of CEA10 conidia are metabolically active at 9 and 12 h postchallenge in comparison to the AF293 isolate, demonstrating heterogeneity among clinical isolates. We identified 7 host markers (caveolin, flotillin-2, RAB5C, RAB8B, RAB7A, 2xFYVE, and FAPP1) that consistently localized around internalized conidia 9 h postchallenge. Transient gene silencing of RAB5C, PIK3C3, and flotillin-2 resulted in a larger population of metabolically active conidia. Our findings emphasize the abundance of both host phosphatidylinositol 3-phosphate (PI3P) and PI4P around internalized conidia, as well as the importance of class III PI3P kinase for conidial processing. Therapeutic development focused on RAB5C-, PIK3C3-, and flotillin-2-mediated pathways may provide novel opportunities to modulate conidial processing and internalization. Determination of how contacted, external conidia are processed by airway epithelial cells may also provide a novel avenue to generate host-targeted therapeutics.IMPORTANCE Conidia from the fungus Aspergillus fumigatus are notorious for their ability to stay airborne. This characteristic is believed to allow conidia to penetrate into the cleanest environments. Several hundred conidia are thought to be inhaled each day by a given individual and then expelled by mucociliary clearance. Given that airway epithelial cells make up a significant portion of the pulmonary-air interface, we set out to determine the percentage of conidia that are actually internalized after initial contact with airway epithelial cells. We determined this through an in vitro assay using an immortalized bronchial airway epithelial cell line known as BEAS-2B. Our results suggest a small fraction of conidia are internalized by BEAS-2B cells, while the majority stay adherent to the surface of cells or are washed away during sample processing. Internalization of conidia was observed at 6 h postchallenge and not prior. Our data also indicate conidia are rendered metabolically inactive within 3 h of challenge, suggesting BEAS-2B cells process a large number of conidia without internalization in this early time frame. We have also identified several host endocytosis markers that localize around internalized conidia as well as contribute to the processing of conidia. Understanding how these host endocytosis markers affect the processing of internal and/or external conidia may provide a novel avenue for therapeutic development.

NF-κB signaling dynamics is controlled by a dose-sensing autoregulatory loop.

Over the last decade, multiple studies have shown that signaling proteins activated in different temporal patterns, such as oscillatory, transient, and sustained, can result in distinct gene expression patterns or cell fates. However, the molecular events that ensure appropriate stimulus- and dose-dependent dynamics are not often understood and are difficult to investigate. Here, we used single-cell analysis to dissect the mechanisms underlying the stimulus- and dose-encoding patterns in the innate immune signaling network. We found that Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling dynamics relied on a dose-dependent, autoinhibitory loop that rendered cells refractory to further stimulation. Using inducible gene expression and optogenetics to perturb the network at different levels, we identified IL-1R-associated kinase 1 (IRAK1) as the dose-sensing node responsible for limiting signal flow during the innate immune response. Although the kinase activity of IRAK1 was not required for signal propagation, it played a critical role in inhibiting the nucleocytoplasmic oscillations of the transcription factor NF-κB. Thus, protein activities that may be "dispensable" from a topological perspective can nevertheless be essential in shaping the dynamic response to the external environment.

Correction: Central Presynaptic Terminals Are Enriched in ATP but the Majority Lack Mitochondria.

[This corrects the article DOI: 10.1371/journal.pone.0125185.].

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy.

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer's and Parkinson's diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of <200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

Central presynaptic terminals are enriched in ATP but the majority lack mitochondria.

Synaptic neurotransmission is known to be an energy demanding process. At the presynapse, ATP is required for loading neurotransmitters into synaptic vesicles, for priming synaptic vesicles before release, and as a substrate for various kinases and ATPases. Although it is assumed that presynaptic sites usually harbor local mitochondria, which may serve as energy powerhouse to generate ATP as well as a presynaptic calcium depot, a clear role of presynaptic mitochondria in biochemical functioning of the presynapse is not well-defined. Besides a few synaptic subtypes like the mossy fibers and the Calyx of Held, most central presynaptic sites are either en passant or tiny axonal terminals that have little space to accommodate a large mitochondrion. Here, we have used imaging studies to demonstrate that mitochondrial antigens poorly co-localize with the synaptic vesicle clusters and active zone marker in the cerebral cortex, hippocampus and the cerebellum. Confocal imaging analysis on neuronal cultures revealed that most neuronal mitochondria are either somatic or distributed in the proximal part of major dendrites. A large number of synapses in culture are devoid of any mitochondria. Electron micrographs from neuronal cultures further confirm our finding that the majority of presynapses may not harbor resident mitochondria. We corroborated our ultrastructural findings using serial block face scanning electron microscopy (SBFSEM) and found that more than 60% of the presynaptic terminals lacked discernible mitochondria in the wild-type mice hippocampus. Biochemical fractionation of crude synaptosomes into mitochondria and pure synaptosomes also revealed a sparse presence of mitochondrial antigen at the presynaptic boutons. Despite a low abundance of mitochondria, the synaptosomal membranes were found to be highly enriched in ATP suggesting that the presynapse may possess alternative mechanism/s for concentrating ATP for its function. The potential mechanisms including local glycolysis and the possible roles of ATP-binding synaptic proteins such as synapsins, are discussed.

Characterizing and measuring endocytosis of lipid-binding effectors in mammalian cells.

Pathogen-host interactions are mediated in part by secreted microbial proteins capable of exploiting host cells for their survival. Several of these manipulations involve, but are not limited to, suppression of defense responses, alterations in host vesicular trafficking, and manipulation of gene expression. The delivery of such molecules from microbe to host has been of intense interest in several microbe-host systems. Several well-studied bacterial effectors are delivered directly into host cells through a needle injection apparatus. Conversely, there have been several examples of secreted effectors and protein toxins from bacteria and eukaryotic microbes, such as fungi and oomycetes, being internalized into host cells by receptor-mediated endocytosis. In the following chapter, we discuss various techniques utilized to measure these endocytosed lipid-binding effectors that can be delivered in the absence of the pathogen.