Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking.

AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity.

A holistic approach to lead pipe scale analysis: Importance, methodology, and limitations.

With lead service lines (LSLs) remaining for decades to come, scale analyses are critical to helping limit lead exposure from drinking water. This laboratory has used an integrated suite of analytical techniques to characterize the elemental composition, mineral identification, and physical features of scales, helping the water industry to evaluate, predict, and reduce lead corrosion. The methods used in this laboratory to prepare and analyze the LSL scale, and guidance to achieving reliable and meaningful results, are described. Primary methods include the following: optical microscopy, powder X-ray diffraction, inductively coupled plasma spectroscopy, X-ray fluorescence, scanning electron microscopy with energy dispersive spectroscopy, combustion and coulometric analyses of C and S, and X-ray absorption spectroscopy. Examples of associated pitfalls and ways to avoid them are provided, including pipe excavation/transport, sample preparation, analysis, and data interpretation. Illustrative examples are presented of practical scale analysis questions that could be answered by combinations of pipe scale analyses.

Quantifying Molecular Dynamics within Complex Cellular Morphologies using LLSM-FRAP.

Quantifying molecular dynamics within the context of complex cellular morphologies is essential toward understanding the inner workings and function of cells. Fluorescence recovery after photobleaching (FRAP) is one of the most broadly applied techniques to measure the reaction diffusion dynamics of molecules in living cells. FRAP measurements typically restrict themselves to single-plane image acquisition within a subcellular-sized region of interest due to the limited temporal resolution and undesirable photobleaching induced by 3D fluorescence confocal or widefield microscopy. Here, an experimental and computational pipeline combining lattice light sheet microscopy, FRAP, and numerical simulations, offering rapid and minimally invasive quantification of molecular dynamics with respect to 3D cell morphology is presented. Having the opportunity to accurately measure and interpret the dynamics of molecules in 3D with respect to cell morphology has the potential to reveal unprecedented insights into the function of living cells.

When light meets biology - how the specimen affects quantitative microscopy.

Fluorescence microscopy images should not be treated as perfect representations of biology. Many factors within the biospecimen itself can drastically affect quantitative microscopy data. Whereas some sample-specific considerations, such as photobleaching and autofluorescence, are more commonly discussed, a holistic discussion of sample-related issues (which includes less-routine topics such as quenching, scattering and biological anisotropy) is required to appropriately guide life scientists through the subtleties inherent to bioimaging. Here, we consider how the interplay between light and a sample can cause common experimental pitfalls and unanticipated errors when drawing biological conclusions. Although some of these discrepancies can be minimized or controlled for, others require more pragmatic considerations when interpreting image data. Ultimately, the power lies in the hands of the experimenter. The goal of this Review is therefore to survey how biological samples can skew quantification and interpretation of microscopy data. Furthermore, we offer a perspective on how to manage many of these potential pitfalls.

Hexameric rings of the scaffolding protein CheW enhance response sensitivity and cooperativity in Escherichia coli chemoreceptor arrays.

The Escherichia coli chemoreceptor array is a supramolecular assembly that enables cells to respond to extracellular cues dynamically and with great precision and sensitivity. In the array, transmembrane receptors organized as trimers of dimers are connected at their cytoplasmic tips by hexameric rings of alternating subunits of the kinase CheA and the scaffolding protein CheW (CheA-CheW rings). Interactions of CheW molecules with the members of receptor trimers not directly bound to CheA-CheW rings may lead to the formation of hexameric CheW rings in the chemoreceptor array. Here, we detected such CheW rings with a cellular cysteine-directed cross-linking assay and explored the requirements for their formation and their participation in array assembly. We found that CheW ring formation varied with cellular CheW abundance, depended on the presence of receptors capable of a trimer-of-dimers arrangement, and did not require CheA. Cross-linking studies of a CheA~CheW fusion protein incapable of forming homomeric CheW oligomers demonstrated that CheW rings were not essential for the assembly of CheA-containing arrays. Förster resonance energy transfer (FRET)-based kinase assays of arrays containing variable amounts of CheW rings revealed that CheW rings enhanced the cooperativity and the sensitivity of the responses to attractants. We propose that six-membered CheW rings provide the additional interconnectivity required for optimal signaling and gradient tracking performance by chemosensory arrays.

The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson's disease.

Astrocytes are essential cells of the central nervous system, characterized by dynamic relationships with neurons that range from functional metabolic interactions and regulation of neuronal firing activities, to the release of neurotrophic and neuroprotective factors. In Parkinson's disease (PD), dopaminergic neurons are progressively lost during the course of the disease, but the effects of PD on astrocytes and astrocyte-to-neuron communication remain largely unknown. This study focuses on the effects of the PD-related mutation LRRK2 G2019S in astrocytes generated from patient-derived induced pluripotent stem cells. We report the alteration of extracellular vesicle (EV) biogenesis in astrocytes and identify the abnormal accumulation of key PD-related proteins within multivesicular bodies (MVBs). We found that dopaminergic neurons internalize astrocyte-secreted EVs and that LRRK2 G2019S EVs are abnormally enriched in neurites and fail to provide full neurotrophic support to dopaminergic neurons. Thus, dysfunctional astrocyte-to-neuron communication via altered EV biological properties may participate in the progression of PD.

Variability and sampling of lead (Pb) in drinking water: Assessing potential human exposure depends on the sampling protocol.

Lead (Pb) in drinking water has re-emerged as a modern public health threat which can vary widely in space and in time (i.e., between homes, within homes and even at the same tap over time). Spatial and temporal water Pb variability in buildings is the combined result of water chemistry, hydraulics, Pb plumbing materials and water use patterns. This makes it challenging to obtain meaningful water Pb data with which to estimate potential exposure to residents. The objectives of this review paper are to describe the root causes of intrinsic Pb variability in drinking water, which in turn impacts the numerous existing water sampling protocols for Pb. Such knowledge can assist the public health community, the drinking water industry, and other interested groups to interpret/compare existing drinking water Pb data, develop appropriate sampling protocols to answer specific questions relating to Pb in water, and understand potential exposure to Pb-contaminated water. Overall, review of the literature indicated that drinking water sampling for Pb assessment can serve many purposes. Regulatory compliance sampling protocols are useful in assessing community-wide compliance with a water Pb regulatory standard by typically employing practical single samples. More complex multi-sample protocols are useful for comprehensive Pb plumbing source determination (e.g., Pb service line, Pb brass faucet, Pb solder joint) or Pb form identification (i.e., particulate Pb release) in buildings. Exposure assessment sampling can employ cumulative water samples that directly capture an approximate average water Pb concentration over a prolonged period of normal household water use. Exposure assessment may conceivably also employ frequent random single samples, but this approach warrants further investigation. Each protocol has a specific use answering one or more questions relevant to Pb in water. In order to establish statistical correlations to blood Pb measurements or to predict blood Pb levels from existing datasets, the suitability of available drinking water Pb datasets in representing water Pb exposure needs to be understood and the uncertainties need to be characterized.

Orthophosphate Interactions with Destabilized PbO2 Scales.

This research presents two case studies in which a change in the disinfectant from free chlorine to chloramine caused an increase in lead corrosion. In both systems, the predominantly tetravalent lead (PbO2) scales destabilized as a result of disinfectant change. Orthophosphate corrosion control was used in both systems, and the effect of this treatment chemical on the destabilized PbO2 scales was examined. The absence of chemical reactivity between PbO2 and phosphorus is well known, and this research confirms that phosphorus does not interact with the legacy PbO2 scales. Instead, phosphorus and calcium were found to permeate through the destabilized PbO2 material and react with divalent lead [Pb(II)] at the surface of a basal litharge (PbO) layer. This reaction precipitated a crystalline lead phosphate in both systems, which could not be specifically identified by any known powder diffraction files. Further analysis suggested that the compound formed was not the typically modeled hydroxypyromorphite but rather a calcium-substituted hydroxypyromorphite. During scale formation, calcium is frequently bound to the Pb(II) phosphate crystal lattice structure, causing measurable crystal lattice distortion in powder X-ray diffraction patterns. The results of this study illustrate the longevity of legacy scales and how disequilibrium compounds persist long after treatment changes have been made.

Lead Particle Size Fractionation and Identification in Newark, New Jersey's Drinking Water.

Following a pH reduction in their drinking water over a span of more than 20 years, the City of Newark, New Jersey, has struggled with elevated lead (Pb) release from Pb service lines and domestic plumbing in the zone fed by the Pequannock Water Treatment Plant. In response, Newark initiated orthophosphate addition and provided faucet-mounted point-of-use (POU) filters and pitcher filters certified for Pb and particulate reduction under NSF/ANSI Standards 53 and 42 to residential homes in that zone. Water chemistry analysis and size fractionation sampling were performed at four of these houses. Analysis of the particulate material retained by the fractionation filters revealed that Pb was dominantly present in the water as fine Pb(II) orthophosphate particles. A considerable amount of the particulates occurred as a nanoscale fraction that sometimes passed through the POU faucet or pitcher filtration units. Scanning electron microscopy, transmission electron microscopy, and energy-dispersive spectroscopy analyses showed that the nanoparticles (<100 nm) and their aggregates were composed of Pb, phosphorus, and chlorine, which are consistent with pyromorphite, Pb5(PO4)3Cl. Electron diffraction and X-ray analyses supported the presence of hydroxypyromorphite and chloropyromorphite nanoparticles and the size range estimates from the imaging. This research confirmed that nonadherent Pb(II)-orthophosphate nanoparticles were an important form of Pb in drinking water in the Pequannock water quality zone of Newark.

Probability of Immobilization on Host Cell Surface Regulates Viral Infectivity.

The efficiency of a virus to establish its infection in host cells varies broadly among viruses. It remains unclear if there is a key step in this process that controls viral infectivity. To address this question, we use single-particle tracking and Brownian dynamics simulation to examine human immunodeficiency virus type 1 (HIV-1) infection in cell culture. We find that the frequency of viral-cell encounters is consistent with diffusion-limited interactions. However, even under the most favorable conditions, only 1% of the viruses can become immobilized on cell surface and subsequently enter the cell. This is a result of weak interaction between viral surface gp120 and CD4 receptor, which is insufficient to form a stable complex the majority of the time. We provide the first direct quantitation for efficiencies of these events relevant to measured HIV-1 infectivity and demonstrate that immobilization on host cell surface post-virion-diffusion is the key step in viral infection. Variation of its probability controls the efficiency of a virus to infect its host cells. These results explain the low infectivity of cell-free HIV-1 in vitro and offer a potential rationale for the pervasive high efficiency of cell-to-cell transmission of animal viruses.

Practical Considerations in Particle and Object Tracking and Analysis.

The rapid advancement of live-cell imaging technologies has enabled biologists to generate high-dimensional data to follow biological movement at the microscopic level. Yet, the "perceived" ease of use of modern microscopes has led to challenges whereby sub-optimal data are commonly generated that cannot support quantitative tracking and analysis as a result of various ill-advised decisions made during image acquisition. Even optimally acquired images often require further optimization through digital processing before they can be analyzed. In writing this article, we presume our target audience to be biologists with a foundational understanding of digital image acquisition and processing, who are seeking to understand the essential steps for particle/object tracking experiments. It is with this targeted readership in mind that we review the basic principles of image-processing techniques as well as analysis strategies commonly used for tracking experiments. We conclude this technical survey with a discussion of how movement behavior can be mathematically modeled and described. © 2019 by John Wiley & Sons, Inc.

Water quality-pipe deposit relationships in Midwestern lead pipes.

The conventional wisdom of lead-scale solubility has been built over the years by geochemical solubility models, experimental studies, and field sampling utilizing multiple protocols. Rarely, have the mineral phases from scales formed in real-world drinking water lead service lines (LSLs) been compared to theoretical predictions. In this study, model predictions are compared to LSL scales from 22 drinking water distribution systems. The results show that only nine of the 22 systems had LSL scales that followed model predictions. The remaining systems had unpredictable scales some with unknown lead release characteristics demonstrating that predicting scale formation and lead release solely by models cannot be relied on in all cases to protect human health. Therefore, for many systems with LSLs, pilot studies with existing LSL scales will be necessary to evaluate and optimize corrosion control, and correspondingly, appropriate residential water sampling will be needed to demonstrate consistent and optimal system corrosion control.

A bacterial immunomodulatory protein with lipocalin-like domains facilitates host-bacteria mutualism in larval zebrafish.

Stable mutualism between a host and its resident bacteria requires a moderated immune response to control bacterial population size without eliciting excessive inflammation that could harm both partners. Little is known about the specific molecular mechanisms utilized by bacterial mutualists to temper their hosts' responses and protect themselves from aggressive immune attack. Using a gnotobiotic larval zebrafish model, we identified an Aeromonas secreted immunomodulatory protein, AimA. AimA is required during colonization to prevent intestinal inflammation that simultaneously compromises both bacterial and host survival. Administration of exogenous AimA prevents excessive intestinal neutrophil accumulation and protects against septic shock in models of both bacterially and chemically induced intestinal inflammation. We determined the molecular structure of AimA, which revealed two related calycin-like domains with structural similarity to the mammalian immune modulatory protein, lipocalin-2. As a secreted bacterial protein required by both partners for optimal fitness, AimA is an exemplar bacterial mutualism factor.

Mineralogical Evidence of Galvanic Corrosion in Drinking Water Lead Pipe Joints.

Galvanic corrosion as a mechanism of toxic lead release into drinking water has been under scientific debate in the U.S. for over 30 years. Visual and mineralogical analysis of 28 lead pipe joints, excavated after 60+ years from eight U.S. water utilities, provided the first direct view of three distinct galvanic corrosion patterns in practice: (1) no evidence of galvanic corrosion; (2) galvanic corrosion with lead cathode; (3) galvanic corrosion with lead anode. Pattern 3 is consistent with empirical galvanic series (lead → brass → copper in order of increasing nobility) and poses the greatest risk of Pb exposure. Pattern 2 is consistent with galvanic battery reversion. The identification of copper-sulfate minerals (Pattern 2), and lead-sulfate and lead-chloride minerals (Pattern 3) in galvanic zones illustrated the migration of chloride and sulfate toward the anode. Geochemical modeling confirmed the required pH drop from the bulk water level to at least pH 3.0-4.0 (Pattern 2) and pH < 5.5 (Pattern 3) in order to form these minerals. Despite joints being over 60 years old, galvanic zones in Pattern 3 were active and possibly posed an important source of lead to drinking water. Importantly, Pattern 3 was not observed in samples from systems representing water qualities favoring PbO2 formation.

Noncritical Signaling Role of a Kinase-Receptor Interaction Surface in the Escherichia coli Chemosensory Core Complex.

In Escherichia coli chemosensory arrays, transmembrane receptors, a histidine autokinase CheA, and a scaffolding protein CheW interact to form an extended hexagonal lattice of signaling complexes. One interaction, previously assigned a crucial signaling role, occurs between chemoreceptors and the CheW-binding P5 domain of CheA. Structural studies showed a receptor helix fitting into a hydrophobic cleft at the boundary between P5 subdomains. Our work aimed to elucidate the in vivo roles of the receptor-P5 interface, employing as a model the interaction between E. coli CheA and Tsr, the serine chemoreceptor. Crosslinking assays confirmed P5 and Tsr contacts in vivo and their strict dependence on CheW. Moreover, the P5 domain only mediated CheA recruitment to polar receptor clusters if CheW was also present. Amino acid replacements at CheA.P5 cleft residues reduced CheA kinase activity, lowered serine response cooperativity, and partially impaired chemotaxis. Pseudoreversion studies identified suppressors of P5 cleft defects at other P5 groove residues or at surface-exposed residues in P5 subdomain 1, which interacts with CheW in signaling complexes. Our results indicate that a high-affinity P5-receptor binding interaction is not essential for core complex function. Rather, P5 groove residues are probably required for proper cleft structure and/or dynamic behavior, which likely impact conformational communication between P5 subdomains and the strong binding interaction with CheW that is necessary for kinase activation. We propose a model for signal transmission in chemotaxis signaling complexes in which the CheW-receptor interface plays the key role in conveying signaling-related conformational changes from receptors to the CheA kinase.

Dynasore inhibition on productive infection of HIV-1 in commonly used cell lines is independent of transferrin endocytosis.

The route of HIV-1 entry for productive infection in CD4+ host cells is a fundamental question for the molecular understanding of HIV-1 infection and transmission. Although direct fusion has long been thought to be the mode of entry, recent studies have suggested that productive entry of HIV-1 may actually occur through dynamin-dependent endocytosis. In several of these studies, dynasore, a noncompetitive inhibitor of the GTPase activity of dynamin, has been used to support this conclusion. Here we show that dynasore does produce inhibitory effects on the productive infection of HIV-1 in several commonly used cell lines. This effect is present regardless of the methods used to facilitate the infection of HIV-1. However, transferrin uptake remains fully functional in these cell lines upon dynasore treatment. Therefore, the inhibition on HIV-1 infection by dynasore in these cell lines is due to an effect that is independent of transferrin endocytosis. The use of dynasore in probing the role of endocytosis in HIV-1 infection should be corroborated by other methods.

Scale Formation Under Blended Phosphate Treatment for a Utility With Lead Pipes.

US corrosion control practice often assumes that the orthophosphate component of blended phosphate corrosion inhibitors causes the formation of low-solubility lead-orthophosphate solids that control lead release into drinking water. This study identified the solids that formed on the interior surface of a lead service line and a galvanized steel pipe excavated from a system using a proprietary blended phosphate chemical. The scale was analyzed by X-ray diffraction, X-ray fluorescence, and scanning electron microscopy/energy dispersive spectroscopy. Instead of crystalline lead-orthophosphate solids, a porous amorphous layer rich in aluminum, calcium, phosphorus, and lead was observed at the lead pipe scale-water interface. Thus, the mechanism inhibiting lead release into the water was not a thermodynamically predictable passivating lead-orthophosphate scale, but rather an amorphous barrier deposit that was possibly vulnerable to disturbances. Galvanized pipe scales showed relatively crystalline iron and zinc compounds, with additional surface deposition of aluminum, phosphorus, calcium, and lead.

Multibuilding Block Janus Synthesized by Seed-Mediated Self-Assembly for Enhanced Photothermal Effects and Colored Brownian Motion in an Optical Trap.

The asymmetrical features and unique properties of multibuilding block Janus nanostructures (JNSs) provide superior functions for biomedical applications. However, their production process is very challenging. This problem has hampered the progress of JNS research and the exploration of their applications. In this study, an asymmetrical multibuilding block gold/iron oxide JNS has been generated to enhance photothermal effects and display colored Brownian motion in an optical trap. JNS is formed by seed-mediated self-assembly of nanoparticle-loaded thermocleavable micelles, where the hydrophobic backbones of the polymer are disrupted at high temperatures, resulting in secondary self-assembly and structural rearrangement. The JNS significantly enhances photothermal effects compared to their homogeneous counterpart after near-infrared (NIR) light irradiation. The asymmetrical distribution of gold and iron oxide within JNS also generates uneven thermophoretic force to display active colored Brownian rotational motion in a single-beam gradient optical trap. These properties indicate that the asymmetrical JNS could be employed as a strong photothermal therapy mediator and a fuel-free nanoscale Janus motor under NIR light.

Optical manipulation of a single human virus for study of viral-cell interactions.

Although Ashkin and Dziedzic first demonstrated optical trapping of individual tobacco mosaic viruses in suspension as early as 1987, this pioneering work has not been followed up only until recently. Using human immunodeficiency virus type 1 (HIV-1) as a model virus, we have recently demonstrated that a single HIV-1 virion can be stabled trapped, manipulated and measured in physiological media with high precision. The capability to optically trap a single virion in suspension not only allows us to determine, for the first time, the refractive index of a single virus with high precision, but also quantitate the heterogeneity among individual virions with single-molecule resolution, the results of which shed light on the molecular mechanisms of virion infectivity. Here we report the further development of a set of microscopic techniques to physically deliver a single HIV-1 virion to a single host cell in solution. Combined with simultaneous epifluorescence imaging, the attachment and dissociation events of individual manipulated virions on host cell surface can be measured and the results help us understand the role of diffusion in mediating viral attachment to host cells. The establishment of these techniques opens up new ways for investigation of a wide range of virion-cell interactions, and should be applicable for study of B cell interactions with particulate antigens such as viruses.