Yeast biopanning against site-specific phosphorylations in tau.

The detection of site-specific phosphorylation in the microtubule-associated protein tau is emerging as a means to diagnose and monitor the progression of Alzheimer's Disease and other neurodegenerative diseases. However, there is a lack of phospho-specific monoclonal antibodies and limited validation of their binding specificity. Here, we report a novel approach using yeast biopanning against synthetic peptides containing site-specific phosphorylations. Using yeast cells displaying a previously validated phospho-tau (p-tau) single-chain variable region fragment (scFv), we show selective yeast cell binding based on single amino acid phosphorylation on the antigen. We identify conditions that allow phospho-specific biopanning using scFvs with a wide range of affinities (KD = 0.2 to 60 nM). Finally, we demonstrate the capability of screening large libraries by performing biopanning in 6-well plates. These results show that biopanning can effectively select yeast cells based on phospho-site specific antibody binding, opening doors for the facile identification of high-quality monoclonal antibodies.

Depth-multiplexed ptychographic microscopy for high-throughput imaging of stacked bio-specimens on a chip.

Imaging a large number of bio-specimens at high speed is essential for many biomedical applications. The common strategy is to place specimens at different lateral positions and image them sequentially. Here we report a new on-chip imaging strategy, termed depth-multiplexed ptychographic microscopy (DPM), for parallel imaging and sensing at high speed. Different from the common strategy, DPM stacks multiple specimens in the axial direction and images the entire z-stack all at once. In our prototype platform, we modify a low-cost car mirror for programmable steering of the incident laser beam. A blood-coated image sensor is then placed underneath the stacked sample for acquiring the resulting diffraction patterns. With the captured images, we perform blind recovery of the incident beam angle and model different layers of the stacked sample as different coded surfaces for object reconstruction. For in vitro experiment, we demonstrate time-lapse cell culture monitoring by imaging 3 stacked microfluidic channels on the coded sensor. For high-throughput cytometric analysis, we image 5 stacked brain sections with a 205-mm2 field of view in ∼50 s. Cytometric analysis is also performed to quantify the cellular proliferation biomarkers on the slides. The DPM approach adds a new degree of freedom for data multiplexing in microscopy, enabling parallel imaging of multiple specimens using a single detector. The demonstrated 6-mm depth of field is among the longest ones in microscopy imaging. The novel depth-multiplexed configuration also complements the miniaturization provided by microfluidics devices, offering a solution for on-chip sensing and imaging with efficient sample handling.

Degradation-driven protein level oscillation in the yeast Saccharomyces cerevisiae.

Generating robust, predictable perturbations in cellular protein levels will advance our understanding of protein function and enable the control of physiological outcomes in biotechnology applications. Timed periodic changes in protein levels play a critical role in the cell division cycle, cellular stress response, and development. Here we report the generation of robust protein level oscillations by controlling the protein degradation rate in the yeast Saccharomyces cerevisiae. Using a photo-sensitive degron and red fluorescent proteins as reporters, we show that under constitutive transcriptional induction, repeated triangular protein level oscillations as fast as 5-10 min-scale can be generated by modulating the protein degradation rate. Consistent with oscillations generated though transcriptional control, we observed a continuous decrease in the magnitude of oscillations as the input modulation frequency increased, indicating low-pass filtering of input perturbation. By using two red fluorescent proteins with distinct maturation times, we show that the oscillations in protein level is largely unaffected by delays originating from functional protein formation. Our study demonstrates the potential for repeated control of protein levels by controlling the protein degradation rate without altering the transcription rate.

High-specificity antibodies and detection methods for quantifying phosphorylated tau from clinical samples.

The ability to measure total and phosphorylated tau levels in clinical samples is transforming the detection of Alzheimer's disease (AD) and other neurodegenerative diseases. In particular, recent reports indicate that accurate detection of low levels of phosphorylated tau (p-tau) in plasma provides a reliable biomarker of AD long before sensing memory loss. Therefore, the diagnosis and monitoring of neurodegenerative diseases progression using blood samples is becoming a reality. These major advances were achieved by using antibodies specific to p-tau as well as sophisticated high-sensitivity immunoassay platforms. This review focuses on these enabling advances in high-specificity antibody development, engineering, and novel signal detection methods. We will draw insights from structural studies on p-tau antibodies, engineering efforts to improve their binding properties, and efforts to validate their specificity. A comprehensive survey of high-sensitivity p-tau immunoassay platforms along with sensitivity limits will be provided. We conclude that although robust approaches for detecting certain p-tau species have been established, systematic efforts to validate antibodies for assay development is still needed for the recognition of biomarkers for AD and other neurodegenerative diseases.

Optogenetics in Sinorhizobium meliloti Enables Spatial Control of Exopolysaccharide Production and Biofilm Structure.

Microorganisms play a vital role in shaping the soil environment and enhancing plant growth by interacting with plant root systems. Because of the vast diversity of cell types involved, combined with dynamic and spatial heterogeneity, identifying the causal contribution of a defined factor, such as a microbial exopolysaccharide (EPS), remains elusive. Synthetic approaches that enable orthogonal control of microbial pathways are a promising means to dissect such complexity. Here we report the implementation of a synthetic, light-activated, transcriptional control platform using the blue-light responsive DNA binding protein EL222 in the nitrogen fixing soil bacterium Sinorhizobium meliloti. By fine-tuning the system, we successfully achieved optical control of an EPS production pathway without significant basal expression under noninducing (dark) conditions. Optical control of EPS recapitulated important behaviors such as a mucoid plate phenotype and formation of structured biofilms, enabling spatial control of biofilm structures in S. meliloti. The successful implementation of optically controlled gene expression in S. meliloti enables systematic investigation of how genotype and microenvironmental factors together shape phenotype in situ.

High specificity of widely used phospho-tau antibodies validated using a quantitative whole-cell based assay.

Antibodies raised against defined phosphorylation sites of the microtubule-associated protein tau are widely used in scientific research and being applied in clinical assays. However, recent studies have revealed an alarming degree of non-specific binding found in these antibodies. In order to quantify and compare the specificity phospho-tau antibodies and other post-translational modification site-specific antibodies in general, a measure of specificity is urgently needed. Here, we report a robust flow cytometry assay using human embryonic kidney cells that enables the determination of a specificity parameter termed Φ, which measures the fraction of non-specific signal in antibody binding. We validate our assay using anti-tau antibodies with known specificity profiles, and apply it to measure the specificity of seven widely used phospho-tau antibodies (AT270, AT8, AT100, AT180, PHF-6, TG-3, and PHF-1) among others. We successfully determined the Φ values for all antibodies except AT100, which did not show detectable binding in our assay. Our results show that antibodies AT8, AT180, PHF-6, TG-3, and PHF-1 have Φ values near 1, which indicates no detectable non-specific binding. AT270 showed Φ value around 0.8, meaning that approximately 20% of the binding signal originates from non-specific binding. Further analyses using immunocytochemistry and western blotting confirmed the presence of non-specific binding of AT270 to non-tau proteins found in human embryonic kidney cells and the mouse hippocampus. We anticipate that the quantitative approach and parameter introduced here will be widely adopted as a standard for reporting the specificity for phospho-tau antibodies, and potentially for post-translational modification targeting antibodies in general. Cover Image for this issue: doi: 10.1111/jnc.14727.

Yeast surface display of full-length human microtubule-associated protein tau.

Microtubule-associated protein tau is an intrinsically disordered, highly soluble protein found primarily in neurons. Under normal conditions, tau regulates the stability of axonal microtubules and intracellular vesicle transport. However, in patients of neurodegeneration such as Alzheimer's disease (AD), tau forms neurofibrillary deposits, which correlates well with the disease progression. Identifying molecular signatures in tau, such as posttranslational modification, truncation, and conformational change has great potential to detect earliest signs of neurodegeneration and develop therapeutic strategies. Here, we show that full-length human tau, including the longest isoform found in the adult brain, can be robustly displayed on the surface of yeast Saccharomyces cerevisiae. Yeast-displayed tau binds to anti-tau antibodies that cover epitopes ranging from the N-terminus to the 4R repeat region. Unlike tau expressed in the yeast cytosol, surface-displayed tau was not phosphorylated at sites found in AD patients (probed by antibodies AT8, AT270, AT180, and PHF-1). However, yeast-displayed tau showed clear binding to paired helical filament (PHF) tau conformation-specific antibodies Alz-50, MC-1, and Tau-2. Although the tau possessed a conformation found in PHFs, oligomerization or aggregation into larger filaments was undetected. Taken together, yeast-displayed tau enables robust measurement of protein interactions and is of particular interest for characterizing conformational change.

A yeast display immunoprecipitation screen for targeted discovery of antibodies against membrane protein complexes.

Yeast display immunoprecipitation is a combinatorial library screening platform for the discovery and engineering of antibodies against membrane proteins using detergent-solubilized membrane fractions or cell lysates as antigen sources. Here, we present the extension of this method for the screening of antibodies that bind to membrane protein complexes, enabling discovery of antibodies that target antigens involved in a functional protein-protein interaction of interest. For this proof-of-concept study, we focused on the receptor-mediated endocytosis machinery at the blood-brain barrier, and adaptin 2 (AP-2) was chosen as the functional interaction hub. The goal of this study was to identify antibodies that bound to blood-brain barrier (BBB) membrane protein complexes containing AP-2. Screening of a nonimmune yeast display antibody library was carried out using detergent-solubilized BBB plasma membranes as an antigen pool, and antibodies that could interact with protein complexes containing AP-2 were identified. Downstream characterization of isolated antibodies confirmed targeting of proteins known to play important roles in membrane trafficking. This functional yeast display immunoprecipitation screen may be applied to other systems where antibodies against other functional classes of protein complexes are sought.

Multidimensional screening yields channelrhodopsin variants having improved photocurrent and order-of-magnitude reductions in calcium and proton currents.

Channelrhodopsins (ChRs) are light-gated ion channels in widespread use in neuroscience for mediating the genetically targetable optical control of neurons (optogenetics). ChRs pass multiple kinds of ions, and although nonspecific ChR-mediated conductance is not an issue in many neuroscience studies, conductance of calcium and protons, which can mediate diverse cellular signals, may be undesirable in some instances. Here, we turned our attention to the creation of ChRs that have high cation photocurrent but pass fewer calcium ions and protons. We developed an automated, time-resolved screening method capable of rapidly phenotyping channelrhodopsin-2 (ChR2) variants. We found substitution mutations throughout ChR2 that could boost current while altering ion selectivity and observed that the mutations that reduced calcium or proton conductance have additive effects. By combining four mutations, we obtained a ChR, ChromeQ, with improved photocurrent that possesses order-of-magnitude reductions in calcium and proton conductance and high fidelity in driving repetitive action potentials in neurons. The approach presented here offers a viable pathway toward customization of complex physiological properties of optogenetic tools. We propose that our screening method not only enables elucidation of new ChR variants that affect microbial opsin performance but may also reveal new principles of optogenetic protein engineering.

Directed evolution of a picomolar-affinity, high-specificity antibody targeting phosphorylated tau.

Antibodies are essential biochemical reagents for detecting protein post-translational modifications (PTMs) in complex samples. However, recent efforts in developing PTM-targeting antibodies have reported frequent nonspecific binding and limited affinity of such antibodies. To address these challenges, we investigated whether directed evolution could be applied to improve the affinity of a high-specificity antibody targeting phosphothreonine 231 (pThr-231) of the human microtubule-associated protein tau. On the basis of existing structural information, we hypothesized that improving antibody affinity may come at the cost of loss in specificity. To test this hypothesis, we developed a novel approach using yeast surface display to quantify the specificity of PTM-targeting antibodies. When we affinity-matured the single-chain variable antibody fragment through directed evolution, we found that its affinity can be improved >20-fold over that of the WT antibody, reaching a picomolar range. We also discovered that most of the high-affinity variants exhibit cross-reactivity toward the nonphosphorylated target site but not to the phosphorylation site with a scrambled sequence. However, systematic quantification of the specificity revealed that such a tradeoff between the affinity and specificity did not apply to all variants and led to the identification of a picomolar-affinity variant that has a matching high specificity of the original phosphotau antibody. In cell- and tissue-imaging experiments, the high-affinity variant gave significantly improved signal intensity while having no detectable nonspecific binding. These results demonstrate that directed evolution is a viable approach for obtaining high-affinity PTM-specific antibodies and highlight the importance of assessing the specificity in the antibody engineering process.

Rapid focus map surveying for whole slide imaging with continuous sample motion.

Whole slide imaging (WSI) has recently been cleared for primary diagnosis in the U.S. A critical challenge of WSI is to perform accurate focusing in high speed. Traditional systems create a focus map prior to scanning. For each focus point on the map, a sample needs to be static in the x-y plane, and axial scanning is needed to maximize the contrast. Here we report a novel focus map surveying method for WSI. In this method, we illuminate the sample with two LEDs and recover the focus points based on 1D autocorrelation analysis. The reported method requires no axial scanning, no additional camera and lens, works for stained and transparent samples, and allows continuous sample motion in the surveying process. By using a 20× objective lens, we demonstrate a mean focusing error of ∼0.08 µm in the static mode and ∼0.17 µm in the continuous motion mode. The reported method may provide a turnkey solution for most existing WSI systems due to its simplicity, robustness, accuracy, and high speed. It may also standardize the imaging performance of WSI systems for digital pathology and find other applications in high-content microscopy, such as time-lapse live-cell imaging.

Optogenetics: Basic Concepts and Their Development.

The discovery of light-gated ion channels and their application to controlling neural activities have had a transformative impact on the field of neuroscience. In recent years, the concept of using light-activated proteins to control biological processes has greatly diversified into other fields, driven by the natural diversity of photoreceptors and decades of knowledge obtained from their biophysical characterization. In this chapter, we will briefly discuss the origin and development of optogenetics and highlight the basic concepts that make it such a powerful technology. We will review how these enabling concepts have developed over the past decade, and discuss future perspectives.

An Optimized Calcium-Phosphate Transfection Method for Characterizing Genetically Encoded Tools in Primary Neurons.

In order to characterize genetically encoded tools under the most relevant conditions, the constructs need to be expressed in the cell type in which they will be used. This is a major hurdle in developing optogenetic tools for neuronal cells, due to the difficulty of gene transfer to these cells. Several protocols have been developed for transfecting neurons, focusing on improved transfection efficiency. However, obtaining healthy cells is as important. We monitored transfected cell health by measuring electrophysiological parameters, and used them as a guideline to optimize transfection. Here we describe an optimized transfection protocol that achieves reasonably high efficiency (10-20 %) with no discernable impact on cell health, as characterized by electrophysiology.

Roadmap on neurophotonics.

Mechanistic understanding of how the brain gives rise to complex behavioral and cognitive functions is one of science's grand challenges. The technical challenges that we face as we attempt to gain a systems-level understanding of the brain are manifold. The brain's structural complexity requires us to push the limit of imaging resolution and depth, while being able to cover large areas, resulting in enormous data acquisition and processing needs. Furthermore, it is necessary to detect functional activities and 'map' them onto the structural features. The functional activity occurs at multiple levels, using electrical and chemical signals. Certain electrical signals are only decipherable with sub-millisecond timescale resolution, while other modes of signals occur in minutes to hours. For these reasons, there is a wide consensus that new tools are necessary to undertake this daunting task. Optical techniques, due to their versatile and scalable nature, have great potentials to answer these challenges. Optical microscopy can now image beyond the diffraction limit, record multiple types of brain activity, and trace structural features across large areas of tissue. Genetically encoded molecular tools opened doors to controlling and detecting neural activity using light in specific cell types within the intact brain. Novel sample preparation methods that reduce light scattering have been developed, allowing whole brain imaging in rodent models. Adaptive optical methods have the potential to resolve images from deep brain regions. In this roadmap article, we showcase a few major advances in this area, survey the current challenges, and identify potential future needs that may be used as a guideline for the next steps to be taken.

Genetically encoded tools: bridging the gap between neuronal identity and function.

Genetically encoded tools are positioned to serve a unique and critical role in bridging the gap between the genetic identity of neurons and their functional properties. However, the use of these tools is limited by our current understanding of cell-type identity. As we make technological advances that focus on capturing functional aspects of neurons such as connectivity, activity, and metabolic states, our understanding of neuronal identity will deepen and may enable the use of genetically encoded tools for modulating disease-specific circuits for therapeutic purposes.

Natural photoreceptors and their application to synthetic biology.

The ability to perturb living systems is essential to understand how cells sense, integrate, and exchange information, to comprehend how pathologic changes in these processes relate to disease, and to provide insights into therapeutic points of intervention. Several molecular technologies based on natural photoreceptor systems have been pioneered that allow distinct cellular signaling pathways to be modulated with light in a temporally and spatially precise manner. In this review, we describe and discuss the underlying design principles of natural photoreceptors that have emerged as fundamental for the rational design and implementation of synthetic light-controlled signaling systems. Furthermore, we examine the unique challenges that synthetic protein technologies face when applied to the study of neural dynamics at the cellular and network level.

All-optical electrophysiology in mammalian neurons using engineered microbial rhodopsins.

All-optical electrophysiology-spatially resolved simultaneous optical perturbation and measurement of membrane voltage-would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell-derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

Independent optical excitation of distinct neural populations.

Optogenetic tools enable examination of how specific cell types contribute to brain circuit functions. A long-standing question is whether it is possible to independently activate two distinct neural populations in mammalian brain tissue. Such a capability would enable the study of how different synapses or pathways interact to encode information in the brain. Here we describe two channelrhodopsins, Chronos and Chrimson, discovered through sequencing and physiological characterization of opsins from over 100 species of alga. Chrimson's excitation spectrum is red shifted by 45 nm relative to previous channelrhodopsins and can enable experiments in which red light is preferred. We show minimal visual system-mediated behavioral interference when using Chrimson in neurobehavioral studies in Drosophila melanogaster. Chronos has faster kinetics than previous channelrhodopsins yet is effectively more light sensitive. Together these two reagents enable two-color activation of neural spiking and downstream synaptic transmission in independent neural populations without detectable cross-talk in mouse brain slice.

Cells and cell lysates: a direct approach for engineering antibodies against membrane proteins using yeast surface display.

Membrane proteins (MPs) are often desirable targets for antibody engineering. However, the majority of antibody engineering platforms depend implicitly on aqueous solubility of the target antigen which is often problematic for MPs. Recombinant, soluble forms of MPs have been successfully employed as antigen sources for antibody engineering, but heterologous expression and purification of soluble MP fragments remains a challenging and time-consuming process. Here we present a more direct approach to aid in the engineering of antibodies to MPs. By combining yeast surface display technology directly with whole cells or detergent-solubilized whole-cell lysates, antibody libraries can be screened against MP antigens in their near-native conformations. We also describe how the platform can be adapted for antibody characterization and antigen identification. This collection of compatible methods serves as a basis for antibody engineering against MPs and it is predicted that these methods will mature in parallel with developments in membrane protein biochemistry and solubilization technology.