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New Technologies for Glutamate Interaction Neurons and Glia 1st Edition Maria Kukley
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Series Preface
Experimental life sciences have two basic foundations: concepts and tools. The Neuromethods series focuses on the tools and techniques unique to the investigation of the nervous system and excitable cells. It will not, however, shortchange the concept side of things as care has been taken to integrate these tools within the context of the concepts and questions under investigation. In this way, the series is unique in that it not only collects protocols but also includes theoretical background information and critiques which led to the methods and their development. Thus it gives the reader a better understanding of the origin of the techniques and their potential future development. The Neuromethods publishing program strikes a balance between recent and exciting developments like those concerning new animal models of disease, imaging, in vivo methods, and more established techniques, including, for example, immunocytochemistry and electrophysiological technologies. New trainees in neurosciences still need a sound footing in these older methods in order to apply a critical approach to their results.
Under the guidance of its founders, Alan Boulton and Glen Baker, the Neuromethods series has been a success since its first volume published through Humana Press in 1985. The series continues to flourish through many changes over the years. It is now published under the umbrella of Springer Protocols. While methods involving brain research have changed a lot since the series started, the publishing environment and technology have changed even more radically. Neuromethods has the distinct layout and style of the Springer Protocols program, designed specifically for readability and ease of reference in a laboratory setting.
The careful application of methods is potentially the most important step in the process of scientific inquiry. In the past, new methodologies led the way in developing new disciplines in the biological and medical sciences. For example, Physiology emerged out of Anatomy in the nineteenth century by harnessing new methods based on the newly discovered phenomenon of electricity. Nowadays, the relationships between disciplines and methods are more complex. Methods are now widely shared between disciplines and research areas. New developments in electronic publishing make it possible for scientists that encounter new methods to quickly find sources of information electronically. The design of individual volumes and chapters in this series takes this new access technology into account. Springer Protocols makes it possible to download single protocols separately. In addition, Springer makes its print-on-demand technology available globally. A print copy can therefore be acquired quickly and for a competitive price anywhere in the world.
Wolfgang Walz
Foreword
Ionotropic glutamate receptors (iGluRs) are glutamate-gated ion channels that mediate fast excitatory neurotransmission in the central nervous system (CNS). They mediate the normal development and function of the CNS and play critical roles in numerous neurologic and psychiatric disorders. The already high and increasing burden of neurodegenerative diseases across the world has prompted national interest to better understand the structure and function of the nervous system. This ambitious desideratum will require more advanced knowledge of the structure, function, and biological activities of iGluRs.
The field of ligand-gated ion channels has its roots in the pharmacologic investigations of excitable tissues in the second part of the nineteenth century. The observations that drugs and poisons (atropine, nicotine, morphine, etc.) have dramatic effects on the electrical properties of tissues and organs (heartbeat, muscle twitch, etc.) were most parsimoniously explained by postulating the existence of chemically receptive proteins that form transmembrane pores. Since then, the evolution of theories and concepts in the field of ligand-activated channels has followed closely the development of technologies to apply and withdraw ligands (perfusion techniques), to record and interpret electrical signals (electrophysiology), and to access increasingly diverse biological preparations (tissue culture, molecular biology). Many of these concepts and theories were developed in the first part of the twentieth century, in advance of the first direct observation of a single-channel current and before the cells of the mammalian central nervous system were amenable to direct experimental investigation. In fact, these concepts evolved primarily from observations of the more easily accessible nerve-muscle synapse; because the ion channel responsible for the endplate current is the muscle acetylcholine receptor, to this day, in many textbooks and reviews, the term ligand-gated ion channel often refers to this founding receptor and its family of pentameric ligand-gated ion channels, despite the growing diversity of proteins in this class.
After the Second World War, two simultaneous technologic advances propelled the glutamate-gated ion channels to the experimentally accessible range. First, increased knowledge on how to prepare and maintain explants of central nervous tissue made it progressively feasible to investigate their molecular and cellular properties. In parallel, methods to record and measure electrical currents across biological membranes—first with sharp intracellular electrodes and then with patch-clamp microelectrodes—made it possible to observe and record the electrical properties of much smaller cells in the manner already used for the neuromuscular junction. With what was at the time the state of the art in electrophysiology, it was repeatedly demonstrated that glutamate and related amino acids excited central neurons. In fact, kainate, a glutamate analogue, had been a well-known neurotoxin and was used by neuroanatomists to trace neuronal circuits by staining degenerating axons. Still, up until the early 1980s, the pervasive thought was that glutamate is too important a metabolite, and its concentration in brain too high, for it to possibly be a neurotransmitter, and many scientists speculated that its excitatory effects had no physiologic meaning. More than two decades of careful pharmacologic
and neurophysiologic investigations were necessary to slowly bring into unanimous acceptance that in the CNS excitatory postsynaptic currents are largely the result of iGluR activations.
The molecular biology revolution of the late twentieth century allowed for the first time the preparation and thus the functional investigation of proteins of defined molecular identity. It also exposed an unanticipated multiplicity of ligand-gated channels, which are organized not only as the prototypical acetylcholine receptor as pentamers but also as tri-, tetra-, or hexameric proteins. Furthermore, it became clear that ligand-gated channels are not only expressed at synapses and on the surface of cells but also embedded in virtually every biological membrane. For iGluRs, as well, this has been a particularly exciting era. It was demonstrated during this time that functional iGluRs assemble as homo- or heterotetramers of homologous subunits, and the subsequent decoding of several genomes has established that mammalian iGluRs assemble from a family of 18 homologous subunits. These cluster further into three classes or types, corresponding to the already established pharmacologically based nomenclature as AMPA, kainate, and NMDA receptors.
The current volume compiles methods that have afforded important conceptual advances in the iGluR field within the past decade. Among these recent developments perhaps the most spectacular are several atomic structures for functional AMPA and NMDA receptors, and parts of kainate receptors. Along with these new structural benchmarks, modern means of identifying and sorting intramolecular motions seek to associate conformational changes with state lifetimes and ultimately with functional output. Thus, a common goal is to organize observed structural changes into a coherent chronologic sequence that narrates the molecular trajectories that produce function. Zooming out from the atomic to the molecular and cellular levels, methodologic advances described in this volume expose mechanisms that control receptor assembly, oligomerization, expression, and trafficking, and provide approaches to identify or count molecular assemblies expressed on cells or at specific synapses. Lastly, the volume would have been incomplete without giving a modern account of classic electrophysiologic approaches that evaluate receptor function following mutagenesis, pharmacologic treatment, and a variety of stimulation protocols, whether for recombinant or native receptors.
Taken together the chapters in this volume outline the contemporary landscape of iGluR technologies. They highlight exciting advances in the field in a manner designed to facilitate additional investigations along these newly forged tracks. Necessarily, they also illustrate that progress has been uneven across the three classes of iGluRs, most likely due to the specific experimental challenges associated with each receptor type. By assisting new and established investigators to adopt these technologies, the present volume may expedite the development of the next generation of approaches and techniques to produce a comprehensive understanding of how iGluRs work to fulfill their essential biologic functions in the CNS.
Buffalo, NY, USA
Gabriela K. Popescu
Preface
Glutamate is the principal excitatory neurotransmitter in the brain and spinal cord and its rapid action at more than 90 % of central synapses occurs through membrane receptors of the ionotropic glutamate receptor (iGluR) family. Since their molecular cloning in the early 1990s, the number of PubMed indexed articles focusing on these receptors’ structure, function, and role in health and disease has exploded in the 1990s and has held steady during the past 15 years at ~2,500 publications per year, with no sign of a decline.
This large number of publications reflects the constant and substantial advances in our collective understanding of these receptors but also the development of new technologies that allow scientists to address gaps in knowledge in this area. Increasingly, scientific journals that report primary research have moved to enforcing page limits for the articles they are willing to review and publish. This fact has resulted in deliberate abbreviation of the Methods section, usually accomplished by extensive referencing of previous literature or by relegating a major part of this section to Supplementary Material. This practice has made it cumbersome to follow the technical procedures and quite difficult to implement these in a lab with no previous experience with the particular technique. The chapters in this volume of Neuromethods describe techniques, methods, and approaches that are either specific to iGluRs or have advanced the field significantly in recent years. They are intended as detailed practical guides that will facilitate the implementation of these technologies in new or established laboratories.
Despite the critical roles of iGluRs in health and disease, much remains unknown about the operation, modulation, and the biological functions of iGluRs. The development, maintenance, and experience-dependent plasticity of excitatory CNS synapses depend critically on the activity of iGluRs; and iGluRs participate in fundamental aspects of development and behavior including learning and memory, information processing, and cognition. In addition, iGluRs mediate glutamate neurotoxicity, a key component of pathology in a number of neurodegenerative conditions. Newly delineated atomic models of functional iGluRs have galvanized the field with new information that had been previously difficult to obtain and have formulated new questions in iGluR research. In addition, recent national initiatives into the structure and function of the brain are sure to increase the demand for accessible techniques to evaluate the structure, function, and physiologic contributions of iGluRs. Helping investigators to implement successfully iGluR-specific methods will accelerate the pace of discovery in this important scientific area.
This volume compiles practical guides, organized as chapters, to technologies that are used currently to investigate iGluR structure, function, and physiology. Chapters focus on a particular approach that has been proven successful in revealing fundamental aspects of iGluRs’ involvement with health and disease. The first section includes methods that can help illuminate the assembly, trafficking, molecular composition, and subcellular location of iGluRs. The second section describes approaches used to understand the atomic organization of iGluRs and the intramolecular motions associated with function. The last section provides techniques to monitor receptor activity in real time, whether from single molecules or receptor populations, and approaches to assemble a storyboard of conformational
changes that underlie the observed electrical signal and ultimately the biological function. Each chapter includes the exposition of theoretical concepts as well as reagents, equipment, and step-by-step protocols to ensure successful replication in any research laboratory. The authors aim to facilitate the implementation of specific methods to iGluR investigations, and thus to accelerate the pace of discovery in this important scientific area.
Buffalo, NY, USA Gabriela
K. Popescu
Series Preface .
Foreword
Preface
1 Assaying AMPA Receptor Oligomerization .
Catherine L. Salussolia, Quan Gan, and Lonnie P. Wollmuth
2 A Step-by-Step Guide to Single-Subunit Counting of Membrane-Bound Proteins in Mammalian Cells.
Mark R.P. Aurousseau, Hugo McGuire, Rikard Blunck, and Derek Bowie
3 Counting NMDA Receptors at the Cell Surface
Martin Horak and Young Ho Suh
4 Electrophysiological Tagging of Ionotropic Glutamate Receptors
Andres Barria
5 Electron Microscopy Analysis of AMPA Receptors in Dendritic Spines
Audra A. Kramer, Amber N. Petersen, and Nashaat Z. Gerges
6 Functional Detection of Novel Triheteromeric NMDA Receptors
Sanjay S. Kumar PART II PROTEIN STRUCTURE
7 Expression, Purification, and Crystallization of Full Length Ionotropic Glutamate Receptors
Maria V. Yelshanskaya, Kei Saotome, Minfen Li, and Alexander I. Sobolevsky
8 NMR Approaches to Functional Dynamics of Genetically Separated iGluR Domains.
Christopher P. Ptak, Ahmed H. Ahmed, and Robert E. Oswald
9 Computing Conformational Free Energies of iGluR Ligand-Binding Domains
Alvin Yu, Tyler Wied, John Belcher, and Albert Y. Lau
10 LRET Methods for Investigating Conformational Changes in Functional Ionotropic Glutamate Receptors
Rita E. Sirrieh and Vasanthi Jayaraman
11 Assaying the Energetics of NMDA Receptor Pore Opening.
Rashek Kazi, Melissa Daniel, and Lonnie P. Wollmuth
PART III PROTEIN FUNCTION
12 Constructing a Rapid Solution Exchange System.
David M. MacLean
13 Assessing the Effects of Ligand-binding Mutations to AMPA and Kainate Receptor Kinetics. .
Mark W. Fleck
14 Analysis of Whole-cell NMDA Receptor Currents. .
Vojtech Vyklicky, Miloslav Korinek, Ales Balik, Tereza Smejkalova, Barbora Krausova, and Ladislav Vyklicky
15 Calcium Imaging to Study NMDA Receptor-mediated Cellular Responses .
Kelly A. Krogh and Stanley A. Thayer
16 Timing AMPA Receptor Activation with Laser-Pulse Photolysis.
Li Niu
17 Current Recording and Kinetic Analyses for Single AMPA Receptors.
Kinning Poon, Robert E. Oswald, and Linda M. Nowak
18 Extracting Rate Constants for NMDA Receptor Gating from One-Channel Current Recordings .
Kirstie A. Cummings, Gary J. Iacobucci, and Gabriela K. Popescu
Part I
Protein Assembly and Trafficking
Chapter 1
Assaying AMPA Receptor Oligomerization
Catherine L. Salussolia, Quan Gan, and Lonnie P. Wollmuth
Abstract
Functional AMPA receptors (AMPARs) are tetrameric complexes formed by four identical (homomeric) or similar (heteromeric) subunits. Variations in the number and composition of AMPARs on the plasma membrane impact synaptic strength, neurodevelopment, and brain disorders. While the mechanisms mediating oligomerization of AMPARs are not well understood, they form the template for defining the number and preferential assembly of AMPARs. In this chapter we describe the application of two methods, blue-native PAGE (BN-PAGE) and fluorescence-detection size-exclusion chromatography (FSEC), to delineate the oligomeric state of AMPAR complexes and factors that determine the oligomerization process.
Biogenesis of AMPARs involves the biosynthesis, folding, and oligomerization of subunits within the endoplasmic reticulum prior to the trafficking and insertion of receptors at the synaptic membrane. The oligomerization of individual subunits directly determines the number and nature of the reserve receptor pool available for activity-dependent trafficking, and is therefore of particular interest as a target for long-term modulation of glutamatergic signaling. Much work remains to be done before a complete mechanistic model of AMPAR assembly becomes available [1–3]. Until then, developing methods for efficiently assaying the oligomerization states of AMPARs will be essential to the advancement of this highly promising research field.
A diverse repertoire of methods has been used to assay the oligomerization state of iGluRs. The use of analytical ultracentrifugation (AUC) underlies the tremendous success in understanding the contribution of the ATD to oligomerization [4–6]. Singleparticle electron microscopy (EM) has also been used to infer the quaternary arrangement of subunits within an assembled receptor
complex [7]. While both AUC and EM are powerful tools, they suffer from the limitation that purified samples must be used. AUC has the additional limitation that the protein species being measured must be soluble, which precludes its application on fulllength iGluRs. In this chapter, we focus on two methods that circumvent the aforementioned limitations: blue native PAGE (BN-PAGE) [8, 9] (Fig. 1a) and fluorescent size-exclusion chromatography (FSEC) (also see Chap. 7) [10] (Fig. 1b), both being
Fig. 1 Assaying oligomerization states in AMPARs. (a) Blue native-PAGE (BN-PAGE) of untagged wild-type GluA2(R) or substitutions in the M4 interacting face. Note that G802W and E813W yield constructs where no tetramer band is detectable. For G802A, the tetramer band is greatly attenuated. The approximate locations of the tetramer (T), dimer (D), and monomer (M) (not shown in this gel) bands were identified using Apoferritin (Sigma) and NativeMark (Invitrogen) markers. (b) Fluorescence-detection size-exclusion chromatography (FSEC) of wild-type or tryptophan-substituted GluA2(Q) subunits. As in the BN-PAGE, G802W or E813W does not show a detectable tetramer peak. The magnitude of the peaks in the chromatograph oftentimes show considerable variability (cf., G802W versus E813W). However, the important quantitative differences are the ratio of the different oligomerization states (tetramer versus dimer versus monomer). For ease of comparison and quantification, we normalize all chromatographs to the tetramer peak in the wild type for that transfection cycle. (c) Upper panel, FSEC of wild-type GluA2(Q) or GluA2(Q) (E813W). Lower panel, BN-PAGE of fractions (time point indicated by dashed lines) from FSEC to verify that the peaks in the chromatograph correspond to tetramers and dimers. The outcome of these BN-PAGE gels and FSEC chromatographs are part of the evidence implicating the M4 segment as a key structural element in the dimer-to-tetramer transition [12]. All figures are adapted from Salussolia et al. (2013)
applicable to unpurified crude membrane fractions containing full-length receptors. BN-PAGE and FSEC have been used with great success to assay the oligomerization states of iGluRs [7, 11–13]. Here we describe the basis and rationale underlying BN-PAGE and FSEC, as well as how these techniques may be used to determine the oligomeric state of heteromeric AMPARs. When assaying the oligomeric state of a protein, one must do so under nonreducing and non-denaturing conditions. However, when working with higher order oligomeric proteins using gelbased techniques, some of the sample may accumulate in the loading wells without migrating into the gel, beckoning the question whether these proteins are grossly misfolded. To address this question, we have used FSEC. By analyzing the chromatograph of an individual sample, one can identify misfolded proteins, which elute as higher order form high-molecular-weight aggregates [10]. Further, FSEC allows for the collection of specific fractions, allowing one to discern the relative amounts of oligomeric species as well as their subunit compositions within one specific fractionation using BN-PAGE (Fig. 1c). Thus, interpretation of FSEC chromatographs in combination with antibody-probed BN-PAGE gels allows one to gain insight into subunit-specific oligomerization of AMPARs.
2 Materials
1. HEK 293 cells or other mammalian cell line transfected with desired construct(s).1
2. Solubilization buffer composed of 1–2 % N-dodecyl-α-dmaltopyranoside (DDM) [14] dissolved in phosphate buffer saline (PBS) containing protease inhibitors (0.8 μM aprotinin, 2 μg/mL leupeptin, 2 mM pepstatin A, and 1 mM phenylmethylsulfonyl fluoride).
3. Rotator or rotating platform.
4. Ultracentrifuge capable of achieving 70,000 RPM.
5. Beckman TLA-100 rotor with accompanying thick-wall centrifuge tubes.
6. 4× Native Buffer (Life Technologies) for sample preparation.
7. Dark and light cathode buffers (containing 0.25 % and 0.025 % Coomassie Brilliant Blue G-250, respectively) as well
1 Although HA or flagged tag can be attached, these tags may affect assembly given that the process is influenced by many structural elements throughout the whole receptor. Hence wherever possible, we recommend using untagged constructs.
2.1
Blue Native PAGE
Catherine L. Salussolia et al.
as the anode buffer prepared from commercially available NativePage buffer systems (Life Technologies)2 [8, 9].
20. Anti-mouse secondary antibodies conjugated to horseradish peroxidase (Santa Cruz).
2.2 FluorescenceDetection SizeExclusion
Chromatography
1. HEK 293 cells transfected with fluorescent-tagged construct.4
2. Solubilization buffer: Tris-buffered saline or TBS (20 mM Tris–HCl, pH 8.0, 200 mM NaCl) supplemented with 1 % DDM.5
3. Sonicator.
4. Rotator or rotating platform.
5. Ultracentrifuge capable of achieving 70,000 RPM.
6. Beckman TLA-100 rotor with accompanying thick-wall centrifuge tubes.
2 Buffers can be custom-made as well. All buffers should be pre-chilled at 4 °C.
3 Both antibodies are from mice and bind to epitopes in the ATD of the receptors.
4 Constructs tagged with GFP (or other appropriate fluorescent probe). It is notable that for AMPARs the fluorescent tag must be located at the C-terminal end. A non-dimerizing mutant of GFP is recommended to avoid complication of the data due to endogenous oligomerization of the fluorescent tag (see Sect. 4).
5 TBS must be less than a week old. Detergent can be added right before use. Same for the column chromatography buffer. Choice of DDM as the detergent is made according to the detergent screening originally performed by Kawate and Gouaux [10], where it produced minimal amounts of fluorescence in the void volume. However, it is advisable to perform a detergent screening for each protein of interest to determine the best option.
9. Chromatography system (Shimadzu HPLC with fluorometer).
3 Methods
1. Plate HEK 293 cells on 6 cm dishes to achieve 90 % confluence. Transfect cells with constructs 24 h after plating. Exogenous GFP can be added for the purpose of checking transfection efficiency but it is entirely optional.
2. 30–40 h after transfection, rinse cells twice with 2 mL PBS prechilled at 4 °C.
3. Prepare solubilization buffer (see Sect. 2.2). Add 250 μL solubilization buffer to each dish and scrape cells into an Eppendorf tube. Rotate at 4 °C for 1 h.
4. Centrifuge at 50,000 RPM on a Beckman TL-100 rotor for 40 min at 4 °C.6 Collect the supernatant, which contains the whole-cell membrane fraction.
5. Prepare samples to run on gels: 7 μL supernatant, 2.5 μL 4× Native Buffer (Life Technologies), and 0.5 μL 2.5 % Coomassie Brilliant Blue G-250. Use apoferritin and Native Mark (Life Technologies) as molecular weight ladders.
6. Add dark cathode buffer and anode buffer to the cathode and anode compartments of the blot apparatus, respectively.
7. Run gel at 105–115 V for 1 h at 4 °C.
8. Remove dark cathode buffer and replace with light cathode buffer. Run for approximately 1.5 h at 215 V, 4 °C, until samples are at the end of the gel.
9. Pre-rinse PVDF membranes in 100 % methanol to activate membrane.
10. Soak blotting pads, filter paper, and modules in transfer buffer. Do not soak for more than 10 min.
11. Remove gel from cassette and rinse in transfer buffer to remove extra Coomassie.
12. Place methanol-rinsed PVDF membrane in transfer buffer for 30–60 s.
13. Assemble transfer unit in the following order (all pre-soaked in transfer buffer): cathode of cassette, blotting pad, filter paper, gel, PVDF membrane, filter paper, blotting pad, and anode of
6 The exact speed of the ultracentrifuge depends on the rotor. It is preferable to spin samples at a speed greater than 100,000 × g. 3.1
BN-PAGE
Catherine L. Salussolia et al.
cassette. With the addition of each layer, roll out the bubbles. Close cassette and place in transfer apparatus chamber with the correct polarity.
14. Fill transfer chamber with transfer buffer.
15. Transfer gel at room temperature for 12–14 h at constant amperage (35 mA). Place a stir bar in the chamber and keep spinning throughout transfer for better heat dissipation.
16. After transfer, rinse membrane in 100 % methanol for 1–2 min twice to remove extra Coomassie.
17. Fix proteins onto the membrane by incubating in 8 % acetic acid for 15 min.
18. Rinse membrane twice with distilled water. Incubate membrane in 0.1 % Ponceau S stain to visualize molecular weight markers.
19. Rinse extra Ponceau S off with distilled water. Mark the positions of the ladder bands with pencil.
20. Rehydrate membrane with 100 % methanol.
21. Rinse membrane with TBS containing 0.05 % Tween 20 (TBST 0.05 %) for 15 min to remove all remaining Ponceau S.
22. Block in 5 % nonfat milk dissolved in TBS for 1 h at RT.
23. Incubate in 2 % milk containing primary antibody of appropriate concentration for 1 h at RT.
24. Rinse three times with TBST 0.05 %.
25. Incubate in HRP-conjugated secondary antibody dissolved in 2 % milk for 1 h at RT.
26. Wash three times with TBST 0.05 %.
27. Use chemiluminescence (Santa Cruz) to develop blot.
1. Plate HEK 293 cells on 10 cm dishes to achieve 90 % confluence. Transfect cells with GFP-tagged constructs 24 h after plating. Do NOT add exogenous GFP.
2. 30–40 h after transfection, rinse cells twice with 5 mL of cold PBS that has been pre-chilled at 4 °C.
3. Scrape cells into 1 mL of cold PBS and transfer to an Eppendorf tube.
4. Spin down cells at 5,000 RPM for 5 min at 4 °C.
5. Remove supernatant. At this point you can flash freeze samples in liquid nitrogen to save for later use or proceed to solubilization.
Solubilization and Chromatography
1. Thaw and resuspend cells in 250 μL of solubilization buffer containing DDM (see Sect. 2.1) by gently pipetting solution up and down to gently dislodge pellet from the side of the tube until it dissolves.
3.2 Cell Harvest for FSEC
3.3 FSEC
3.4 Data Analysis
2. Lyse cells. Sonicate samples at 4 °C 30 s on/30 s off for 2 min. Make sure that cells are well suspended and that the solution is not cloudy. If it is cloudy, add an additional 250 μL of solubilization buffer and sonicate again.
3. Rotate Eppendorf tubes at 4 °C for 1 h. Meanwhile, install the Superose six column in the chromatography system and equilibrate with TBS containing 0.05 % DDM. Run at a flow rate of 0.4 mL/min.
4. Centrifuge at 70,000 RPM on a Beckman TL-100 rotor for 10 min at 4 °C. Insoluble nuclear DNA, if still visibly present after the centrifugation, must be removed from the supernatant since it is highly viscous and could clog the column.7
5. Inject 300 μL of each sample into column and start eluting the column with TBS containing 0.05 % DDM at a flow rate of 0.4 mL/min. Use excitation and emission channels appropriate for your fluorophore (e.g., 475 nm excitation and 507 nm emission for EGFP). Time increment for signal collection: 0.5 s; integration time: 1 s; recording time: 0–4,500 s.
6. Monitor the elution profile in the fluorescent emission channel. If necessary, collect desired fractions in separate Eppendorfs for analysis by BN-PAGE (Fig. 1c).
Quantification of the tetramer-to-dimer (or monomer) ratio on BN-PAGE gels and FSEC chromatographs can provide information about the process of oligomerization. A critical question in the quantification of BN-PAGE gels is how stable the oligomeric states are under particular conditions. As illustrated in Fig. 2, the homomeric GluA1 is quite stable. For comparisons to be made, samples must be prepared under uniform detergent conditions (e.g., Fig. 1). Further, the effect of any manipulation must be referenced to a “wild type” collected under the same condition.
3.4.1 BN-PAGE Band Densitometry (Fig. 3)
1. Scan developed film into .tiff format at 300 dpi resolution with 16-bit grayscale. Open the file with ImageJ.
2. Invert the color and change the scale to “pixels.”
3. Measure the mean intensity (I) as well as the area (A) of each band of interest (define the area of interest using the “freehand selection” tool since the shapes of the bands may be irregular on BN-PAGE). Use an area on the image with no signal as background mean intensity (IB) (Fig. 3).
4. Calculate the cumulative intensity (C) of each band:
7 Addition of DNase in the solubilization buffer could help circumvent this problem [15] (though we have never encountered situations where this is necessary).
Catherine L. Salussolia et al.
Fig. 2 Stability of the GluA1 homomer. BN-PAGE of wild-type GluA1 using 20 mM DDM either alone (DDM) or with added SDS. SDS is a more denaturing detergent. The persistence of the tetramer band with the addition of up to 0.1 % SDS, where the dimer band becomes somewhat more prominent, highlights the stability of the homomer GluA1 under the harvesting conditions used (20 mM DDM)
Fig. 3 Quantification of a BN-PAGE gel. Left panel, scanned image of a BN-PAGE immunoblot film. Content of each lane is displayed below. Molecular weight markers in lanes M1 and M2 were visualized using Ponceau stain and positions of the bands were marked on the membrane with pencil prior to immunoblotting. Right panel, inverted image with the areas of interest selected using the “freehand selection” tool of ImageJ. The rectangular area at the bottom is used to measure the background mean intensity (IB). Cumulative intensity of each area of interest (CT, CD, etc.) is calculated as described
3.4.2 FSEC Area Under Curve Quantification (Fig. 4)
5. For each construct, calculate the tetramer-to-dimer ratio (RT−D) as well as percentage tetramer (%T) from the cumulative intensity of each band (CT, CD, and CM):
1. Load files (from Excel) into Igor Pro 6.2 or later (WaveMetrics).
2. Because the original chromatographs are long (typically 0–4,500 s in length) and are sampled at 2 Hz, we typically resample the chromatographs at 0.2 Hz to improve data handling.
3. Normalize all records from a transfection cycle to the tetramer peak (occurring between 1,700 and 2,000 s) for the wild-type control.
4. Load appropriate record into multi-peak fitting routine.
5. Using the Graph Cursors, set baseline at minimum points around peaks of interest, typically at 1,700 (prior to the tetramer peak) and between 2,200 and 2,400 (after the monomer peak) (green line in Fig. 4). In multi-peak routine set baseline to “linear.”
Fig. 4 Quantification of FSEC chromatographs. Upper panels, chromatographs of wild-type GluA2(Q) (left) or GluA2(G802A) (right). Black line, original data; green line, baseline; and red line is the sum of the individual fits. Lower panels, fraction of the total chromatograph corresponding to tetramer, dimer, or monomer
L. Salussolia et al.
6. All curves should be set to “Gaussian.”
7. Initially use “Auto-locate Peak Now” to identify most significant peaks (tetramer, dimer, monomer). Often “Do Fit” will fit the peaks well, yielding residuals of less than 0.02, which is the minimum quality fit.
8. For chromatographs, where the residuals are greater than 0.02, it reflects that the multi-fit routine failed to identify a minor peak, typically the monomer peak. In such instances, use a combination of “holding” the tetramer and dimer peaks and the editing function to add in the third peak.
4 Notes/Limitations
One of the disadvantages of FSEC is that for AMPARs the tag must be placed in the C-terminal end in order to acquire satisfactory signal levels [10] (unpublished data). Given that the C-terminal domain may modulate oligomerization (data not shown), the presence of the large and bulky GFP (or other fluorescent tag) in the CTD could alter oligomerization. Furthermore, due to the possible presence of PDZ-binding motifs at the extreme C-terminus of the receptor subunit [16], the necessity of C-terminal tagging makes it difficult to apply FSEC to the study of AMPAR oligomerization in neurons (neuronal cultures), where scaffolding proteins containing PDZ domains (e.g., SAP97) might influence that process. In addition, endogenous dimerization of the fluorescent tags could complicate the results. To avoid that, we used a GFP mutant (A206K) that is unable to dimerize [17]. In contrast to FSEC, BN-PAGE can be done without any tags as long as a high-affinity and high-specificity antibody against the AMPAR subunit of interest is available.
In some proteins certain mutations may incur aberrant electrophoretic mobility in BN-PAGE [18] although such cases are rare. However, soluble proteins and membrane proteins do have slightly different mass calibration curves in BN-PAGE [19], with membrane proteins generally migrating slower than soluble ones. Since the molecular weight markers used for these experiments usually contain soluble globular proteins, this creates difficulty for the accurate measurement of molecular weight using BN-PAGE. Instead, BN-PAGE is preferably used for relative size comparisons between different mutants of the same protein.
Another limitation associated with BN-PAGE and FSEC is that they are both ensemble assays, which makes them suboptimal for studying receptor subunit composition at high resolution. For example, if a protein band in BN-PAGE is detected by both anti-GluA1 and anti-GluA2 antibodies, it would still be uncertain whether that band contains GluA1/GluA2 heterotetramers or whether it contains
Catherine
a mixture of GluA1 and GluA2 homotetramers. To tackle that question, one would have to utilize single-molecule techniques such as subunit counting, which is discussed in another chapter of this book (Chap. 10).
The method for quantification of BN-PAGE we describe here suffers from a number of pitfalls that render it only semiquantitative [20]. This is further complicated by the fact that the shapes/ areas of bands can be highly variable in native gels. The method should therefore only be applied where the difference between the mutant and the control is highly significant and reproducible. As a possible alternative, the Odyssey™ Quantitative Infrared Westerns system from Li-COR could be used. The system uses infrared fluorescent secondary antibodies and produces signals that do not diminish over time and are directly proportional to the amount of target protein (Introduction to Quantitative Infrared Westerns, Li-COR), therefore avoiding many of the pitfalls involved in traditional Western blot and densitometry.
Despite the aforementioned limitations, the advance of BN-PAGE and FSEC used in conjunction has furthered our understanding of AMPAR biogenesis and offers promising avenues for future investigation into mechanisms mediating the assembly of AMPARs and how this process might be modulated by activity.
Acknowledgments
We thank Dr. Hiro Furukawa for helpful discussions and/or comments on the manuscript. Special thanks goes to Dr. Ingo Gregor for early guidance in establishing BN-PAGE. This work was supported by NIH RO1 grants from NIMH (MH066892, LPW), a SBU-CSHL Collaborative grant (LPW), an NIH NRSA from NINDS (NS073382) (CLS), and an American Heart Association pre-doctoral fellowship (QG).
References
1. Nakagawa T (2010) The biochemistry, ultrastructure, and subunit assembly mechanism of AMPA receptors. Mol Neurobiol 42(3): 161–184
2. Sukumaran M, Penn AC, Greger IH (2012) AMPA receptor assembly: atomic determinants and built-in modulators. Adv Exp Med Biol 970:241–264
3. Gan Q, Salussolia CL, Wollmuth LP (2015) Assembly of AMPA receptors: mechanisms and regulation. J Physiol 593(1):39–48. doi:10.1113/jphysiol.2014.273755
5. Rossmann M, Sukumaran M, Penn AC, Veprintsev DB et al (2011) Subunit-selective N-terminal domain associations organize the formation of AMPA receptor heteromers. EMBO J 30(5):959–971
6. Zhao H, Berger AJ, Brown PH, Kumar J et al (2012) Analysis of high-affinity assembly for AMPA receptor amino-terminal domains. J Gen Physiol 139(5):371–388
7. Shanks NF, Maruo T, Farina AN, Ellisman MH et al (2010) Contribution of the global subunit structure and stargazin on the
4. Jin R, Singh SK, Gu S, Furukawa H et al (2009) Crystal structure and association behaviour of the GluR2 amino-terminal domain. EMBO J 28(12):1812–1823
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8. Wittig I, Braun HP, Schagger H (2006) Blue native PAGE. Nat Protoc 1(1):418–428
9. Schagger H, Cramer WA, von Jagow G (1994) Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal Biochem 217(2):220–230
10. Kawate T, Gouaux E (2006) Fluorescencedetection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14(4):673–681
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12. Salussolia CL, Gan Q, Kazi R, Singh P et al (2013) A eukaryotic specific transmembrane segment is required for tetramerization in AMPA receptors. J Neurosci 33(23):9840–9845
13. Greger IH, Khatri L, Kong X, Ziff EB (2003) AMPA receptor tetramerization is mediated by Q/R editing. Neuron 40(4):763–774
14. Seddon AM, Curnow P, Booth PJ (2004) Membrane proteins, lipids and detergents: not
just a soap opera. Biochim Biophys Acta 1666(1–2):105–117
15. Structural Genomics C, China Structural Genomics C, Northeast Structural Genomics C, Graslund S et al (2008) Protein production and purification. Nat Methods 5(2):135–146
16. Sheng M, Hoogenraad CC (2007) The postsynaptic architecture of excitatory synapses: a more quantitative view. Annu Rev Biochem 76:823–847
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18. Brown HH, Borchelt DR (2014) Analysis of mutant SOD1 electrophoretic mobility by Blue Native gel electrophoresis; evidence for soluble multimeric assemblies. PLoS One 9(8), e104583
19. Wittig I, Beckhaus T, Wumaier Z, Karas M et al (2010) Mass estimation of native proteins by blue native electrophoresis: principles and practical hints. Mol Cell Proteomics 9(10):2149–2161
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A Step-by-Step Guide to Single-Subunit Counting of Membrane-Bound Proteins in Mammalian Cells
Mark R.P. Aurousseau, Hugo McGuire, Rikard Blunck, and Derek Bowie
Abstract
Determining the composition and stoichiometry of membrane-bound proteins has been a perennial problem that has plagued biology for a long time. The most recurring issue is that composition and subunit stoichiometry is commonly inferred from bulk biochemical assays that can only shed light on the “averaged” makeup of the protein complex. However, recent studies have been able to circumvent this issue by studying the stoichiometry of individual protein complexes. The most common approach has been to express GFP-tagged subunits in Xenopus laevis oocytes and then manually count the number of photobleaching steps to report mature protein stoichiometry. Although valuable, an important drawback of this technique is that the strict rules of mammalian protein assembly are not always adhered to in this surrogate expression system. Furthermore, manual counting of bleaching steps is subject to user bias and places practical limits on the amount of data that can be analyzed. In this chapter, we provide a step-by-step account of how we adapted the subunit counting method for mammalian cells to study the composition and stoichiometry of ionotropic glutamate receptors. Using custom-made software, we have automated the entire counting process so that it is much less time consuming and no longer subject to user bias. Given its universality, this methodological approach permits the elucidation of subunit number and stoichiometry for a wide variety of plasma-membrane-bound proteins in mammalian cells.
Key words Single-subunit counting, Single molecule, Automated step detection, Fluorescence spectroscopy, Ionotropic glutamate receptors, Superfolder GFP
1 Introduction
The vast majority of signaling proteins assemble as multimeric complexes including most, if not all, neurotransmitter receptor families found in the vertebrate CNS, such as the ionotropic glutamate receptor (iGluR) and cys-loop receptor families which form tetramers and pentamers, respectively [1, 2]. Insight into the stoichiometry of native receptors has been achieved using ensemble biochemical methods (such as blue native PAGE) or spectroscopic approaches (such as FRET). However, these techniques fall short in that they
are based on the underlying assumption that stoichiometry is fixed within the entire population. A simple way around this is to study proteins one by one. Consequently, several single-molecule approaches have been developed to determine subunit copy number and stoichiometry of individual protein complexes. Of these, the single-subunit counting method is particularly useful especially when applied to the study of integral membrane proteins.
To achieve this, researchers have used fluorescently labelled proteins and inferred the number of subunits per protein complex by counting the number of photobleaching steps. At the global or macroscopic level, where many fluorophores are present, photobleaching is described by an exponential decay in fluorescence intensity. In contrast, at the single-molecule level, photobleaching produces a rapid steplike decrease in fluorescence intensity as the fluorophore is extinguished. Originally, the concept of photobleaching fluorophores to count subunits was applied to Cy3labelled nucleotides incorporated into DNA [3] and was later extended to intact cells by Ulbrich and Isacoff to determine the stoichiometry of GFP-tagged ion channels that included NMDA type of iGluR [4].
Subunit counting is commonly performed in Xenopus laevis oocytes as it offers fine control of surface expression density as well as an excellent fluorescence signal-to-noise ratio (SNR). However, there are two problems when using this expression system for studying mammalian neurotransmitter receptors. First, this surrogate expression system may not properly assemble mammalian receptors. For example, nicotinic acetylcholine receptors have an altered stoichiometry in Xenopus laevis oocytes [5, 6]. Secondly, oocytes express subunits from many neuronal receptor families endogenously, including orthologs of all iGluR subunits [7]. While this potential lack of a fully homogenous population may be ignored in macroscopic measurements, it may significantly influence measurements at the low expression level required for single-molecule observation and become particularly problematic when attempting to interpret subunit counting data. To circumvent these problems, we adapted single-subunit counting to mammalian cells (HEK293). Unlike Xenopus laevis oocytes, HEK293 cells do not express iGluRs endogenously but share a number of characteristics with neurons, such as their mRNA expression profile [8].
An important drawback for single-molecule fluorescent imaging is the challenge of achieving a sufficiently high SNR. To realize this, subunit counting is performed using total internal reflection fluorescence (TIRF) microscopy, and fluorescence is detected using highly sensitive cameras. A second major difficulty is to reduce fluorophore-receptor expression density, which we achieved using the protocol described below [9]. From cell culture and transfection to optimizing imaging system components and analysis, we provide a step-by-step procedure describing how to perform
subunit counting experiments in HEK293 cells. Particular emphasis is placed on maximizing the SNR of the system and on reducing fluorophore-receptor expression. We also provide a guide to analyzing raw subunit counting data with Progressive Idealization and Filtering (PIF) software, an all-in-one analysis suite designed specifically for single-subunit counting [9].
2 Materials
1. Transfection-grade mammalian expression plasmid designed to express the fusion protein of interest. For iGluR subunits, fusions at the N-terminus should occur after the plasma localization signal. In this chapter, we describe the use of a monomeric version of the superfolder GFP (msfGFP) for subunit counting, but in theory, any fluorescent protein (FP) could be employed as long as it does not readily dimerize. Dimerization could influence the results. An ideal FP should be as bright as possible, be photostable for long periods of time, and have excitation/emission profiles that fall outside the spectra of autofluorescent components of the cell (see Note 1).
2. HEK293 or HEK293T cells (see Note 2).
3. Round 35 mm glass-bottom dishes. These can be purchased (MatTek Corp. or WPI) or made by hand in the lab (see Note 3). It is important to match cover slip thickness (usually #1 or #1.5) to the requirements of the TIRF objective being used.
4. Poly-d-lysine (molecular weight 70,000–150,000 Da) at 10 mg/mL in water. Filter-sterilize the solution with a 0.2 μm filter. Store at −20 °C for months.
5. DMEM (Life Technologies cat. #10564-011) supplemented with 2 % fetal bovine serum (see Note 4).
6. Phosphate-buffered saline (PBS) containing 100 μM each MgCl2 and CaCl2.
1. 1× and 2× concentrated PBS containing 100 μM each MgCl2 and CaCl2
2. 20 % EM-grade formaldehyde in H2O. This can be purchased in small volumes (5–10 mL; Ladd Research Industries) in sealed glass vials and should be stored in the dark at room temperature.
TIRF microscope systems are commercially available or can be built in the lab. The most common type is based on an inverted microscope using a prism-less (or through-the-objective) TIRF setup [10], similar to the setup depicted in Fig. 1. An objective with a numerical aperture larger than 1.42 is required for
2.1 Cell Culture and Transfection
2.2 Sample Fixation
2.3 Imaging
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no haze la merced tu voluntad. Si lo consientes iuzgandome desagradecido porque no me contento con el bien que me heziste en darme causa de tan ufano pensamiento, no me culpes, que avnque la voluntad se satisfaze, el sentimiento se querella. Si te plaze porque nunca te hize seruizio, no pude sobir los seruizios á la alteza de lo que mereces; que quando todas estas cosas y otras muchas pienso hallome que dexas de hazer lo que te suplico porque me puse en cosa que no pude merecer. Lo qual yo no niego; pero atreuime á ello pensando que me harias merced no segund quien la pedia mas segund tú que la auies de dar. Y tambien pense que para ello me ayudadaran virtud y compasion y piedad porque son acetas á tu condicion, que quando los que con los poderosos negocian para alcançar su gracia, primero ganan las voluntades de sus familiares; y pareceme que en nada hallé remedio. Busqué ayudadores para contigo y hallélos por cierto leales y firmes y todos te suplican que me ayas merced; el alma por lo que sufre, la vida por lo que padece, el coraçon por lo que pasa, el sentido por lo que siente. Pues no niegues galardon á tantos que
con ansia te lo piden y con razon te lo merecen. Yo soy el más sin ventura de los más desauenturados. Las aguas reuerdecen la tierra y mis lagrimas nunca tu esperança la qual cabe en los canpos y en las yeruas y arboles y no puede caber en tu coraçon.
Desesperado auria segund lo que siento si alguna vez me hallase solo, pero como siempre me acompañan el pensamiento que me das y el deseo que me ordenas y la contemplacion que me causas, viendo que lo vo á hazer consuelanme acordandome que me tienen conpañia de tu parte, de manera que quien causa las desesperaciones me tiene que no desespere. Si todavia te plaze que muera, hazmelo saber, que gran bien harás á la vida pues no será desdichada del todo. Lo primero della se pasó en inocencia y lo del conocimiento en dolor; a lo menos el fin será en descanso porque tú lo das, el qual, si ver no me quieres, será forçado que veas.
EL AUCTOR
Con mucha pena recibio Laureola la carta de Leriano y por despedirse dél onestamente
respondiole desta manera, con determinacion de iamas recebir enbaxada suya.
CARTA DE LAUREOLA Á LERIANO
El pesar que tengo de tus males te seria satisfacion dellos mismos si creyeses quanto es grande, y él solo tomarias por galardon sin que otro pidieses, avnque fuese poca paga segund lo que tienes merecido, la qual yo te daria como deuo si la quisieses de mi hazienda y no de mi onrra. No respondere á todas las cosas de tu carta porque en saber que te escriuo me huye la sangre del coraçon y la razon del iuycio. Ninguna causa de las que dizes me haze consentir tu mal sino sola mi bondad, porque cierto no estó dudosa del, porque el estrecho á que llegaste fue testigo de lo que sofriste. Dizes que nunca me hiziste seruicio. Lo que por mi has hecho me obliga á nunca oluidallo y sienpre desear satisfacerlo, no segund tu deseo mas segund mi onestad. La virtud y piedad y conpasion que pensaste que te ayudarian para comigo, aunque son aceptas á mi condicion, para en tu caso son enemigas de mi fama y por esto las hallaste contrarias. Quando
estaua presa saluaste mi vida y agora que estó libre quieres condenalla. Pues tanto me quieres, antes devrias querer tu pena con mi onrra que tu remedio con mi culpa; no creas que tan sanamente biuen las gentes, que sabido que te hablé, iuzgasen nuestras linpias intenciones, porque tenemos tienpo tan malo que antes se afea la bondad que se alaba la virtud; assi que es escusada tu demanda porque ninguna esperança hallarás en ella aunque la muerte que dizes te viese recebir, auiendo por mejor la crueldad onesta que la piedad culpada. Dirás oyendo tal desesperança que só mouible porque te comence á hazer merced en escreuirte y agora determino de no remediarte. Bien sabes tú quan sanamente lo hize y puesto que en ello uviera otra cosa, tan conuenible es la mudança en las cosas dañosas como la firmeza en las onestas. Mucho te ruego que te esfuerces como fuerte y te remedies como discreto. No pongas en peligro tu vida y en disputa mi onrra, pues tanto la deseas, que se dirá muriendo tú que galardono los seruicios quitando las vidas, lo que si al rey venço de dias se dirá al reues. Ternas en el reyno toda la parte que quisieres, crecere tu
onrra, doblaré tu renta, sobiré tu estado, ninguna cosa ordenarás que reuocada te sea, assi que biuiendo causarás que me iuzguen agradecida y muriendo que me tengan por mal acondicionada. Avnque por otra cosa no te esforçases, sino por el cuydado que tu pena me da lo devrias hazer No quiero mas dezirte porque no digas que me pides esperança y te do conseio. Plugiere á Dios que fuera tu demanda iusta, por que vieras que como te aconseió en lo vno te satisfiziera en lo otro; y assi acabo para sienpre de más responderte ni oyrte.
EL AUCTOR
Cuando Laureola vuo escrito dixome con proposito determinado que aquella fuese la postrimera vez que pareciese en su presencia porque ya de mis pláticas andaua mucha sospecha y porque en mis ydas auia mas peligro para ella que esperança para mi despacho. Pues vista su determinada voluntad, pareciendome que de mi trabaio sacaua pena para mí y no remedio para Leriano, despedime della con mas lágrimas que palabras y despues de besalle las manos salime de palacio con vn
nudo en la garganta que pense ahogarme, por encobrir la pasion que sacaua, y salido de la cibdad, como me vi solo, tan fuertemente comence á llorar que de dar bozes no me podía contener. Por cierto yo tuuiera por meior quedar muerto en Macedonia que venir biuo á Castilla; lo que deseaua con razon pues la mala ventura se acaba con la muerte y se acrecienta con la vida. Nunca por todo el camino sospiros y gemidos me fallecieron, y quando llegué á Leriano dile la carta, y como acabó de leella dixele que ni se esforçase, ni se alegrase, ni recibiese consuelo pues tanta razon auia para que deuiese morir. El qual me respondió que más que hasta alli me tenia por suyo porque le aconseiaua lo propio, y con boz y color mortal començo a condolerse. Ni culpaua su flaqueça, ni avergonçaua su desfallecimiento; todo lo que podie acabar su vida alabaua, mostrauase amigo de los dolores, recreaua con los tormentos, amaua las tristezas; aquellos llamaua sus bienes por ser mensaieros de Laureola y porque fuesen tratados segund de cuya parte venian, aposentólos en el coraçon, festeiólos con el sentimiento, convidólos con la memoria, rogauales que
acabasen presto lo que venian a hazer porque Laureola fuese seruida. Y desconfiando ya de ningun bien ni esperança, aquexado de mortales males, no podiendo sustenerse ni sofrirse vuo de venir á la cama, donde ni quiso comer ni beuer ni ayudarse de cosa de las que sustentan la vida, llamandose sienpre bienauenturado porque era venido á sazon de hazer seruicio á Laureola quitandola de enoios. Pues como por la corte y todo el reyno se publicase que Leriano se dexaua morir, ybanle a ueer todos sus amigos y parientes y para desuialle su proposito dezianle todas las cosas en que pensauan prouecho, y como aquella enfermedad se auia de curar con sabias razones, cada uno aguzaua el seso lo meior que podia; y como vn cauallero llamado Tefeo[276] fuese grande amigo de Leriano viendo que su mal era de enamorada pasion puesto que quien la causaua él ni nadie lo sabia dixole infinitos males de las mugeres y para fauorecer su habla truxo todas las razones que en disfamia dellas pudo pensar, creyendo por alli restituylle la vida. Lo qual oyendo Leriano, acordandose que era muger Laureola, afeó mucho á Tefeo porque tal cosa hablaua y
puesto que su disposicion no le consintiese mucho hablar, esforçando la lengua con la pasion de la saña començo a contradezille en esta manera.
LERIANO CONTRA TEFEO Y TODOS LOS QUE DIZEN MAL DE MUGERES
Tefeo, para que recibieras la pena que merece tu culpa, onbre que te tuuiera menos amor te auie de contradezir, que las razones mias mas te seran en exenplo para que calles que castigo para que penes. En lo qual sigo la condicion de verdadera amistad, porque pudiera ser, si yo no te mostrara por biuas causas tu cargo, que en qualquiera plaça te deslenguaras como aqui has hecho; asi que te será mas prouechoso emendarte por mi contradicion que auergonçarte por tu perseverança. El fin de tu habla fue segund amigo, que bien noté que la dexiste porque aborreciese la que me tiene qual vees, diziendo mal de todas mugeres, y como quiera que tu intencion no fue por remediarme, por la via que me causaste remedio tú por cierto me lo as dado, porque tanto me lastimaste con tus feas palabras, por ser muger quien me pena, que de pasion de auerte
oydo beuire menos de lo que creya, en lo qual señalado bien recebi, que pena tan lastimada meior es acaballa presto que sostenella más; assi que me truxiste alivio para el padecer y dulce descanso para ella acabar. Porque las postrimeras palabras mias sean en alabança de las mugeres, porque crea mi fe la que tuuo merecer para causalla y no voluntad para satisfazella.
Y dando comienço á la intencion tomada, quiero mostrar quinze causas porque yerran los que en esta nacion ponen lengua, y veynte razones porque les somos los onbres obligados, y diuersos enxenplos de su bondad. Y quanto a lo primero que es proceder por las causas que hazen yerro los que mal las tratan, fundo la primera por tal razon. Todas las cosas hechas por la mano de Dios son buenas necesariamente, que segun el obrador han de ser las obras; pues siendo las mugeres sus criaturas, no solamente á ellas ofende quien las afea, mas blasfema de las obras del mismo Dios. La segunda causa es porque delante dél y de los onbres no ay pecado más abominable ni más graue de perdonar quel desconocimiento; ¿pues quál lo puede ser mayor
que desconocer el bien que por Nuestra Señora nos vino y nos viene? Ella nos libró de pena y nos hizo merecer la gloria; ella nos salua, ella nos sostiene, ella nos defiende, ella nos guia, ella nos alumbra, por ella que fue muger merecen todas las otras corona de alabança. La tercera es porque a todo onbre es defendido segund virtud mostrarse fuerte contra lo flaco, que si por ventura los que con ellas se deslenguan pensasen recebir contradicion de manos, podria ser que tuuiesen menos libertad en la lengua. La quarta es porque no puede ninguno dezir mal dellas sin que a si mismo se desonrre, porque fue criado y traydo en entrañas de muger y es de su misma sustancia, y despues desto, por el acatamiento y reuerencia que a las madres deuen los hijos. La quinta es por la desobediencia de Dios, que dixo por su boca que el padre y la madre fuesen onrrados y acatados, de cuya causa los que en las otras tocan merecen pena. La sesta es porque todo noble es obligado a ocuparse en autos virtuosos assi en los hechos como en las hablas; pues si las palabras torpes ensusian la linpieza, muy a peligro de infamia tienen la onrra de los que en tales platicas gastan su vida. La setima
es porque quando se establecio la caualleria, entre las otras cosas que era tenudo a guardar el que se armaua cauallero era vna que a las mugeres guardase toda reuerencia y onestad, por donde se conosce que quiebra la ley de nobleza quien vsa el contrario della. La otaua es por quitar de peligro la onrra; los antiguos nobles tanto adelgazauan las cosas de bondad y en tanto la tenian que no auian mayor miedo de cosa que de memoria culpada; lo que no me parece que guardan los que anteponen la fealdad de la virtud poniendo macula con su lengua en su fama, que qualquiera se iuzga lo que es en lo que habla. La nouena y muy principal es por la condenacion del alma. Todas las cosas tomadas se pueden satisfazer y la fama robada tiene dudosa la satisfacion, lo que más conplidamente determina nuestra fé. La dezena es por escusar enemistad. Los que en ofensa de las mugeres despienden el tiempo hazense enemigos dellas y no menos de los virtuosos, que como la virtud y la desmesura diferencian la propiedad no pueden estar sin enemiga. La onzena es por los daños que de tal auto malicioso se recrecian, que como las palabras tienen
licencia de llegar á los oydos rudos tanbien como a los discretos, oyendo los que poco alcançan las fealdades dichas de las mugeres, arrepentidos de auerse casado danles mala vida o vanse dellas, o por ventura las matan. La dozena es por las murmuraciones, que mucho se deuen temer, siendo vn onbre infamado por disfamador en las plaças y en las casas y en los canpos y donde quiera es retratado su vicio. La trezena es por razon del peligro, que quando los maldizientes que son auidos por tales tan odiosos son a todos[277] que qualquier les es mas contrario, y algunas por satisffazer a sus amigos, puesto que ellas no lo pidan ni lo quieran[278] , ponen las manos en los que en todas ponen la lengua. La catorzena es por la hermosura que tienen, la qual es de tanta ecelencia que avnque copiesen en ellas todas las cosas que los deslenguados les ponen, más ay en vna que loar con verdad que no en todas que afear con malicia. La quinzena es por las grandes cosas de que han sido causa. Dellas nacieron onbres virtuosos que hizieron hazañas de dina alabança, dellas procedieron sabios que alcançaron a conocer qué cosa era Dios en cuya fé
somos saluos; dellas vinieron los inuentiuos que hizieron cibdades y fuerças y edeficios de perpetual ecelencia; por ellas vuo tan sotyles varones que buscaron todas las cosas necesarias para sustentacion del linage vmanal.
DA LERIANO VEYNTE RAZONES PORQUE
LOS ONBRES SON OBLIGADOS
Á LAS MUGERES
Tefeo, pues as oydo las causas porque soys culpados tú y todos lo que opinion tan errada seguis, dexada toda prolixidad, oye veynte razones por donde proferí a prouar que los onbres á las mugeres somos obligados. De las quales la primera es porque á los sinples y rudos disponen para alcançar la virtud de la prudencia y no solamente á los torpes hazen discretos mas á los mismos discretos mas sotyles, porque si de la enamorada pasion se catyuan, tanto estudian su libertad que abiuando con el dolor el saber dizen razones tan dulces y tan concertadas que alguna vez de compasion que les an se libran della: y los sinples de su natural inocentes quando en amar se ponen entran con rudeza y hallan el estudio del sentimiento tan agudo que diuersas vezes salen
sabios, de manera que suplen las mugeres lo que naturaleza en ellos faltó. La segunda razon es porque de la virtud de la iusticia tanbien nos hazen suficientes, que los penados de amor, aunque desygual tormento reciben, hanlo por descanso iustificandose porque iustamente padecen: y no por sola esta causa nos hazen goçar desta virtud mas por otra tan natural: los firmes enamorados para abonarse con las que siruen buscan todas las formas que pueden, de cuyo deseo biuen iustificadamente sin eceder en cosa de toda ygualdad por no infamarse de malas costunbres. La tercera porque de la tenplança nos hazen dinos, que por no selles aborrecibles para venir á ser desamados somos templados en el comer y en el beuer y en todas las otras cosas que andan con esta virtud. Somos tenplados en la habla, somos templados en la mesura, somos templados en las obras, sin que vn punto salgamos de la onestad. La quarta es porque al que fallece fortaleza gela dan, y al que la tiene gela acrecientan. Hacennos fuertes para sofrir, causan osadia para cometer, ponen coraçon para esperar; quando á los amantes se les ofrece peligro se les apareia la gloria, tienen las
afrentas por vicio, estiman mas ell alabança del amiga quel precio del largo beuir. Por ellas se comiençan y acaban hechos muy hazañosos, ponen la fortaleza en el estado que merece. Si les somos obligados aqui se puede iuzgar. La quinta razon es porque no menos nos dotan de las virtudes teologales que de las cardinales dichas. Y tratando de la primera ques la fé, avnque algunos en ella dudasen, siendo puestos en pensamiento enamorado creerian en Dios y alabarian su poder porque pudo hazer á aquella que de tanta ecelencia y hermosura les parece. Iunto con esto los amadores tanto acostumbran y sostienen la fe que de vsalla en el coraçon conocen y creen con más firmeza la de Dios, y porque no sea sabido de quien los pena que son malos cristianos, ques vna mala señal en el onbre, son tan deuotos católicos que ningun apostol les hizo ventaia. La sesta razon es porque nos crian en el alma la virtud del esperança, que puesto que los sugetos á esta ley de amores mucho penen, siempre esperan en su fé, esperan en su firmeza, esperan en la piedad de quien los pena, esperan en la condicion de quien los destruye, esperan en la ventura; ¿pues
quien tiene esperança donde recibe pasion, como no la terná en Dios que le promete descanso? Sin duda haziendonos mal nos apareian el camino del bien como por esperiencia de lo dicho parece. La setena razon es porque nos hazen merecer la caridad, la propiedad de la qual es amor Esta tenemos en la voluntad, esta ponemos en el pensamiento, esta traemos en la memoria, esta firmamos en el coraçon, y como quiera que los que amamos la vsemos por el prouecho de nuestro fin, dél nos redunda que con biua contricion la tengamos para con Dios, porque trayendonos amor á estrecho de muerte hazemos lymosnas, mandamos dezir misas, ocupamosnos en caritatiuas obras porque nos libre de nuestros crueles pensamientos: y como ellas de su natural son deuotas, participando con ellas es forçado que hagamos las obras que hazen. La otaua razon, porque nos hazen contenplatiuos: que tanto nos damos á la contemplacion de la hermosura y gracias de quien amamos y tanto pensamos en nuestras pasiones, que quando queremos contenplar la de Dios, tan tiernos y quebrantados tenemos los coraçones, que sus
llagas y tormentos parece que recebimos en nosotros mismos; por donde se conosce que tanbien por aquí nos ayudan para alcançar la perdurable holgança. La nouena razon es porque nos hazen contritos, que como siendo penados pedimos con lagrimas y sospiros nuestro remedio acostunbrado en aquello, yendo á confesar nuestras culpas assi gemimos y lloramos quel perdon dellas merecemos. La dezena es por el buen consejo que sienpre nos dan, que á las vezes acaece hallar en su presto acordar, lo que nosotros con[279] largo estudio y diligencias buscamos. Son sus conseios pacificos sin ningund escandalo, quitan muchas muertes, conseruan las pazes, refrenan la yra y aplacan la saña; sienpre es muy sano su parecer. La onzena es porque nos hazen onrrados: con ellas se alcançan grandes casamientos, muchas haziendas y rentas. Y porque alguno podria responderme que la onrra está en la virtud y no en la riqueza, digo que tanbien causan lo vno como lo otro. Ponen nos presunciones tan virtuosas que sacamos dellas las grandes onrras y alabanças que deseamos; por ellas estimamos más la verguença que la vida; por ellas estudiamos todas las obras
de nobleza, por ellas las ponemos en la cunbre que merecen. La dozena razon es porque apartandonos del auaricia nos iuntan con la libertad, de cuya obra ganamos las voluntades de todos; que como largamente nos hazen despender lo que tenemos, somos alabados y tenidos en mucho amor, y en qualquier necesidad que nos sobrevenga recebimos ayuda y seruizio; y no solo nos aprouechan en hazernos usar la franqueza como deuemos, mas ponen lo nuestro en mucho recaudo porque no ay lugar donde la hazienda esté mas segura que en la voluntad de las gentes. La trezena es porque acrecientan y guardan nuestros averes y rentas, las quales alcanzan los onbres por ventura y conseruanlas ellas con diligencia. La catorzena es por la limpieça que nos procuran asi en la persona, como en el vestir, como en el comer, como en todas las cosas que tratamos. La quinzena es por la buena criança que nos ponen, vna de las principales cosas de que los onbres tienen necesidad. Siendo bien criados vsamos la cortesya y esquiuamos la pesadumbre, sabemos onrrar los pequeños, sabemos tratar los mayores; y no solamente nos hazen bien criados mas bien
quistos, porque como tratamos á cada vno como merece, cada vno nos da lo que merecemos. La razon desiseys es porque nos hazen ser galanes. Por ellas nos desuelamos en el vestir, por ellas estudiamos en el traer, por ellas nos atauiamos de manera que ponemos por industria en nuestras personas la buena disposicion que naturaleza algunos negó. Por artificio se endereçan los cuerpos pidiendo[280] las ropas con agudeza y por el mismo se pone cabello donde fallece y se adelgazan ó engordan las piernas si conuiene hazello; por las mugeres se inuentan los galanes entretales, las discretas bordaduras, las nueuas inuenciones; de grandes bienes por cierto son causa. La dezisiete razon es porque nos conciertan la musica y nos hazen gozar de las dulcedumbres della: ¿por quién se asuenan las dulces canciones? ¿por quién se cantan los lindos romances? ¿por quién se acuerdan las bozes? ¿porquién se adelgazan y sotilizan todas las cosas que en el canto consisten? La dizeochena es porque crecen las fuerças á los braceros, y la maña á los luchadores, y la ligereza á los que boltean y corren y saltan y hazen otras
cosas semeiantes. La dezinueue razon es porque afinan las gracias. Los que como es dicho tañen y cantan por ellas, se desuelan tanto que suben á lo mas perfeto que en aquella gracia se alcança. Los trobadores ponen por ellas tanto estudio en lo que troban que lo bien dicho hazen parecer meior, y en tanta manera se adelgazan que propiamente lo que sienten en el coraçon ponen por nueuo y galan estilo en la cancion ó inuencion ó copla que quieren hazer. La veyntena y postrimera razon es porque somos hijos de mugeres, de cuyo respeto les somos mas obligados que por ninguna razon de las dichas ni de quantas se puedan dezir. Diuersas razones auía para mostrar lo mucho que á esta nacion somos los onbres en cargo, pero la disposicion mia no me da lugar á que todas las diga. Por ellas se ordenaron las reales iustas y los ponposos torneos y las alegres fiestas, por ellas aprouechan las gracias y se acaban y comiençan todas las cosas de gentileza; no sé causa porque de nosotros deuan ser afeadas. ¡O culpa merecedora de graue castigo, que porque algunas ayan piedad de los que por ellas penan les dan tal galardon! ¿A qué muger deste
