Analysis of Cell‐Cell Contact Mediated by Ig Superfamily Cell Adhesion Molecules

Esther T. Stoeckli1, Devrim Kilinc2, Beat Kunz1, Stefan Kunz3, Gil U. Lee2, Elena Martines2, Christoph Rader4, Daniel Suter5

1 University of Zurich, Zurich, 2 University College Dublin, Dublin, 3 University Hospital Center and University of Lausanne, Lausanne, 4 Scripps Florida, Jupiter, Florida, 5 Purdue University, West Lafayette, Indiana
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 9.5
DOI:  10.1002/0471143030.cb0905s61
Online Posting Date:  December, 2013
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Abstract

Cell‐cell adhesion is a fundamental requirement for all multicellular organisms. The calcium‐independent cell adhesion molecules of the immunoglobulin superfamily (IgSF‐CAMs) represent a major subgroup. They consist of immunoglobulin folds alone or in combination with other protein modules, often fibronectin type‐III folds. More than 100 IgSF‐CAMs have been identified in vertebrates and invertebrates. Most of the IgSF‐CAMs are cell surface molecules that are membrane‐anchored either by a single transmembrane segment or by a glycosylphosphatidylinositol (GPI) anchor. Some of the IgSF‐CAMs also occur in soluble form, e.g., in the cerebrospinal fluid or in the vitreous fluid of the eye, due to naturally occurring cleavage of the GPI anchor or the membrane‐proximal peptide segment. Some IgSF‐CAMs, such as NCAM, occur in various forms that are generated by alternative splicing. This unit contains a series of protocols that have been used to study the function of IgSF‐CAMs in vitro and in vivo. Curr. Protoc. Cell Biol. 61:9.5.1‐9.5.85. © 2013 by John Wiley & Sons, Inc.

Keywords: neural development; adhesive strength; cell‐cell adhesion; primary cell culture; axon; protein‐protein interaction

     
 
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Table of Contents

  • Introduction
  • Preparation of IgSF‐CAMs
  • Basic Protocol 1: Purification of IgSF‐CAMs by Immunoaffinity Chromatography
  • Support Protocol 1: Preparation of the Affinity Column
  • Support Protocol 2: Solubilization of Membrane Proteins
  • Support Protocol 3: Production of Recombinant CAMs by Transient Transfection of HEK293 Cells with Calcium Phosphate
  • Support Protocol 4: Detection of IgSF‐CAMs by the Dot Immunoblot Method
  • Functional Assays with Purified Proteins
  • Basic Protocol 2: Analysis of Protein Interactions with Fluorescent Microspheres
  • Support Protocol 5: Coupling Proteins to Fluorescent Microspheres
  • Support Protocol 6: Covalent Coupling of Proteins to Glutaraldehyde‐Activated Amino Beads
  • Basic Protocol 3: Analysis of Binding and Forced Unbinding of Homophilic IgSF‐CAM Interactions Using Magnetic Tweezers
  • Support Protocol 7: Conjugation of IgSF‐CAM to Particle and Microwell Surfaces
  • Support Protocol 8: Quantification of Protein on Particles and Microwell Surfaces
  • Basic Protocol 4: Measurement of apCAM‐apCAM Interactions by Single Molecule Force Spectroscopy with an Atomic Force Microscope
  • Support Protocol 9: Covalent Coupling of apCAM to Gold Substrates and Probes
  • Basic Protocol 5: Binding of Protein‐Conjugated Microspheres to Cultured Cells
  • Cell‐Based Assays for IgSF‐CAM Function
  • Basic Protocol 6: Trans‐Interaction Assay with Myeloma Cells
  • Support Protocol 10: Stable Transfection of Myeloma Cells by Protoplast Fusion
  • Basic Protocol 7: Detecting cis‐Interactions between IgSF‐CAMs by Chemical Cross‐Linking
  • Basic Protocol 8: Detecting cis‐Interactions Between IgSF‐CAMs by Antibody‐Induced Co‐Capping
  • Basic Protocol 9: Adhesion‐Induced Neuronal Growth in Restrained Bead Interaction Assay (RBI)
  • Support Protocol 11: Coupling of His‐tagged IgSF‐CAMs to Ni2+‐NTA Silica Beads
  • Basic Protocol 10: Neurite Outgrowth Assay
  • Basic Protocol 11: Inhibiting CAM‐CAM Interactions In Vitro
  • Coating of Culture Dishes
  • Support Protocol 12: Coating of Culture Dishes with IgSF‐CAMs
  • Support Protocol 13: Pre‐Coating Glass Surfaces with Nitrocellulose
  • Support Protocol 14: Pre‐Coating Culture Dishes with Polylysine
  • Support Protocol 15: Coating Culture Dishes with Collagen
  • Support Protocol 16: Coating Culture Dishes with Laminin
  • Support Protocol 17: Fixation of Cells for Immunohistochemical Staining Procedures Using Fluorescent Antibodies
  • Support Protocol 18: Fixation of Cells for Morphological Analysis, Neurite Length Measurement, and Immunohistochemical Staining Procedures Using Non‐Fluorescent Secondary Antibodies
  • In Vivo Assay for IgSF‐CAM Function
  • Basic Protocol 12: Analysis of IgSF‐CAM Function In Vivo by In Ovo RNAi
  • Support Protocol 19: Preparation of dsRNA Solution for In Ovo RNAi
  • Support Protocol 20: Whole‐Mount Staining of Chicken Embryos
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Purification of IgSF‐CAMs by Immunoaffinity Chromatography

  Materials
  • CNBr‐activated Sepharose 4B immunoaffinity column with monoclonal antibody against protein to be purified (see protocol 2)
  • Loading buffer: 0.5% CHAPS in PBS with Ca2+/Mg2+ (see recipe)
  • Elution buffer: 0.5% CHAPS in 50 mM diethylamine
  • Protein solution (see protocol 3)
  • 1 M Tris·Cl, pH 7.0 ( appendix 2A)
  • PBS with Ca2+/Mg2+ (see recipe)
  • 0.02% (v/v) merthiolate or equivalent bacteriostatic agent

Support Protocol 1: Preparation of the Affinity Column

  Materials
  • CNBr‐activated Sepharose 4B gel (GE Healthcare)
  • 1 mM HCl
  • Buffer I: 0.5 M NaCl in 0.1 M NaHCO 3, pH 8.3
  • Monoclonal antibody against the protein to be purified
  • 0.2 M glycine, pH 8.0
  • Buffer II: 0.5 M NaCl in 0.1 M sodium acetate, pH 4.0
  • Loading buffer (see protocol 1)
  • Sintered glass filter connected to a vacuum pump
  • U‐bottom polypropylene vial that can be closed tightly
  • End‐over‐end rotator
  • Column (e.g., Poly‐Prep column, Bio‐Rad)

Support Protocol 2: Solubilization of Membrane Proteins

  Materials
  • 14‐day‐old chicken embryo brains, freshly frozen in liquid nitrogen
  • Liquid nitrogen
  • Ca2+/Mg2+‐free buffer (CMF buffer; see recipe)
  • 0.8 M and 2.25 M sucrose in PBS (see recipe for PBS)
  • 1 M and 2 M NaCl in PBS (see recipe for PBS)
  • 50 mM triethylamine
  • 0.5% and 1% CHAPS in PBS (see recipe for PBS)
  • Mortar and pestle
  • Dounce homogenizer
  • Centrifuge tubes for Sorvall SS‐34 or equivalent rotor
  • 38‐ml polycarbonate tubes for ultra‐high‐speed centrifuge

Support Protocol 3: Production of Recombinant CAMs by Transient Transfection of HEK293 Cells with Calcium Phosphate

  Materials
  • HEK293 cells (HEK293 cells are used in many labs; however, they may not be of sufficient quality because they have been passaged for a long time—low‐passage‐number HEK293 cells can be obtained from ATCC).
  • Cell culture medium for HEK293 cells (see recipe)
  • Purified DNA of interest
  • CaCl 2 solution (see recipe)
  • HBS solution (see recipe)
  • 175‐cm2 tissue culture flasks
  • Additional reagents and equipment for trypsinizing cells (unit 1.1)

Support Protocol 4: Detection of IgSF‐CAMs by the Dot Immunoblot Method

  Materials
  • Protein solution of interest
  • TBS: 0.2 M NaCl in 50 mM Tris·Cl, pH 7.4 (see appendix 2A for Tris buffer)
  • Blocking solution: 2% (w/v) milk powder in TBS with or without 0.1% (w/v) Tween 20
  • Antibody against the protein of interest diluted in blocking solution
  • Secondary antibody coupled with horseradish peroxidase (HRP) diluted in blocking solution
  • 4‐chloro‐1‐naphthol solution (see recipe)
  • 0.2‐µm nitrocellulose membrane (e.g., GE Healthcare/Whatman)
  • 96‐well plates
  • Orbital shaker

Basic Protocol 2: Analysis of Protein Interactions with Fluorescent Microspheres

  Materials
  • Protein‐conjugated fluorescent microspheres; stock solutions contain 1011 beads/ml in 0.5% (w/v) BSA (see protocol 7; Polysciences; Bangs Laboratories; Thermo Fisher)
  • 0.5% (w/v) BSA solution (see recipe)
  • 0.5 mg/ml Fab fragments of antibodies against proteins of interest in PBS (optional)
  • 0.5% (w/v) trypsin (optional)
  • PBS (see recipe)
  • Water bath sonicator (Branson Ultrasonics)
  • Fluorescence microscope equipped with FITC and TRITC filters
  • 0.5‐ml microcentrifuge tube
  • Rotator
  • Glass microscope slides
  • Refrigerated microcentrifuge
  • Fluorescence‐activated flow cytometer

Support Protocol 5: Coupling Proteins to Fluorescent Microspheres

  Additional Materials (also see protocol 6)
  • 1011 unconjugated beads/ml fluorescent polystyrene microspheres, 0.5‐µm diameter (Polysciences; Bangs Laboratories; Thermo Fisher)
  • Biochemically purified IgSF‐CAMs (see protocol 1) in a phosphate‐based buffer system (either PBS or 20 mM sodium phosphate, pH 7.0)
  • 20 mM sodium phosphate, pH 7.0 ( appendix 2A)
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1)

Support Protocol 6: Covalent Coupling of Proteins to Glutaraldehyde‐Activated Amino Beads

  Additional Materials (also see protocol 6)
  • Amino‐functional microspheres (e.g., silica aminopropyl beads from Bangs Laboratories)
  • 0.1 M NaOH or HCl (optional)
  • 8% (v/v) EM‐grade glutaraldehyde, newly opened bottle
  • 400 µg/ml biochemically purified IgSF‐CAMs (see protocol 1) in a phosphate‐based buffer system: either PBS (see recipe) or 20 mM sodium phosphate, pH 7.0 (see appendix 2A)
  • Blocking solution (see recipe)

Basic Protocol 3: Analysis of Binding and Forced Unbinding of Homophilic IgSF‐CAM Interactions Using Magnetic Tweezers

  Materials
  • Epoxy glue
  • Protein‐conjugated magnetic microspheres (see protocol 10)
  • Fluorescent microspheres, 1 µm in diameter (Polysciences)
  • 55% (w/w) sucrose (Sigma) solution in H 2O
  • 25% (w/w) dextran (60 to 90 kDa; Sigma) solution in H 2O
  • Protein‐conjugated microwells (see protocol 10)
  • PBS (see recipe)
  • Magnets, and materials to prepare magnet holder, position control unit, and holder arm as described in steps 1 and 3 (also see Fig. )
  • Position control unit (see step 3)
  • Silicone isolators (1 mm depth, 9 mm diameter, adhesive on one side; Grace Bio‐Labs)
  • Rectangular microscope slides
  • Inverted epi‐fluorescence microscope equipped with phase contrast or differential interference contrast optics and FITC filter cube; a motorized stage is preferred
  • Cannon‐Fenske capillary viscometer (Sigma)
  • Water bath sonicator
  • Circular glass coverslips; 12 mm in diameter (Menzel Glass)
  • Image‐analysis software (e.g., NIH ImageJ with Particle Tracker plugin)
  • Protein‐conjugated microwells (see protocol 10))

Support Protocol 7: Conjugation of IgSF‐CAM to Particle and Microwell Surfaces

  Materials
  • Carboxylated superparamagnetic microspheres (see step 1)
  • 50 mM 4‐morpholinoethanesulfonic acid (MES) buffer in H 2O, pH 6.0
  • 1‐ethyl‐3‐[3‐dimethylaminopropyl] carbodiimide hydrochloride (EDC; Pierce)
  • N‐hydroxysulfosuccinimide (Sulfo‐NHS; Pierce)
  • 50 mM Na 2CO 3 buffer in H 2O, pH 8.2
  • Oxidation reagent: 4 mg/ml KMnO 4 in H 2SO 4
  • Polyethyleneimine (PEI; mol. wt., 1.3 kDa; Sigma)
  • Methoxy‐PEG‐NHS (mol. wt., 2kDa; Rapp Polymere, http://www.rapp‐polymere.com/)
  • BOC‐protected amine‐PEG‐NHS (mol. wt., 3 kDa; Rapp Polymere, http://www.rapp‐polymere.com/)
  • High‐salt Na 2CO 3 buffer: 0.6 M K 2SO 4 in 50 mM Na 2CO 3 buffer, pH 8.2
  • Sulfosuccinimidyl‐4‐(N‐maleimidomethyl)cyclohexane‐1‐carboxylate (Sulfo‐SMCC; Pierce)
  • Dimethylsulfoxide (DMSO; Sigma)
  • 5% (v/v) 2‐mercaptoethanol (Sigma) in PBS (see recipe for PBS)
  • PBST: 0.02% (v/v) Tween 20 in PBS (see recipe for PBS)
  • 50% (v/v) trifluoroacetic acid (TFA) in H 2O (Sigma)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 1 mg/ml methyl N‐succinimidyl adipate (MSA; Pierce) in PBS (see recipe for PBS)
  • Basic phosphate solution, pH 9.5 (see recipe)
  • 2 mg/ml N α,N α‐Bis(carboxymethyl)‐L‐lysine hydrate (NTA; Sigma) in PBS, pH 7.0
  • 250 µM NiSO 4 in PBS (see recipe for PBS)
  • 6xHis‐tagged recombinant protein encoding the extracellular portion of the IgSF‐CAM molecule to be tested (see protocol 4)
  • Magnetic separator (suitable for 1.5 ml microcentrifuge tubes)
  • End‐over‐end rotator
  • Bath sonicator
  • 96‐well plate covers (optical bottom plate lid with condensation rings and evaporation rim; Thermo Scientific, cat. no. 255983)
  • Plate shaker (e.g., IKA)

Support Protocol 8: Quantification of Protein on Particles and Microwell Surfaces

  Materials
  • 10 µg/ml horseradish peroxidase (HRP)‐conjugated secondary antibody (in BSA solution) specific for the primary antibody used
  • 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) diammonium salt (ABTS; Pierce)
  • 0.005% (w/v) NaN 3 solution in PBS (see recipe for PBS)
  • Protein‐conjugated magnetic particles and microwells (see protocol 10)
  • 0.5% (w/v) BSA solution (see recipe)
  • 10 µg/ml primary antibody (in BSA solution) specific for the IgSF‐CAMs of interest
  • Spectrophotometer

Basic Protocol 4: Measurement of apCAM‐apCAM Interactions by Single Molecule Force Spectroscopy with an Atomic Force Microscope

  Materials
  • 2% SDS in H 2O
  • apCAM‐functionalized gold substrates (substrates; see protocol 13)
  • Phosphate‐buffered saline (PBS), pH 7.4 (see recipe; filtered through a 0.2 µm filter ) or artificial sea water buffer (ASW buffer; see recipe)
  • apCAM‐functionalized gold AFM probes (probes; see protocol 13)
  • Nanowizard III (JPK Instruments, http://www.jpk.com/) or a MFP‐3D Bio (Asylum Research, http://www.asylumresearch.com/) atomic force microscope
  • Auxiliary AFM cell for holding samples in liquid (liquid cell; Fluid Cell Lite from Asylum Research, http://www.asylumresearch.com/)
  • 100‐ml glass or Teflon beakers
  • Ultrasonic cleaning bath
  • Tweezers #1, #3, or #6 (flat round tips are best for handling AFM chips)
  • Empty plastic box to cover liquid cell
  • Glass slides

Support Protocol 9: Covalent Coupling of apCAM to Gold Substrates and Probes

  Materials
  • Concentrated sulfuric acid (H 2SO 4)
  • 30% hydrogen peroxide (H 2O 2)
  • Nitrogen source
  • Chloroform, analytical grade
  • Gold‐coated TR400‐PB AFM probes (spring constant, 0.02 N/m; Olympus); referred to as “probes” in the protocol
  • Gold substrates, 1 cm × 1 cm, 200‐nm thick gold film evaporated onto 1‐mm glass slides with a 5 nm thick chromium underlayer (Lebow Company, http://www.lebowcompany.com/); referred to as “substrates” in the protocol
  • Carboxyl‐terminated heterobifunctional thiol poly‐ethylene glycol (COOH‐PEG‐SH or “COOH‐PEG”, mol. wt., 3.4 kDa) and methoxy‐terminated heterobifunctional thiol polyethylene glycol (CH 3O‐ PEG‐SH or “mPEG”, mol. wt., 2 kDa, Creative PEGWorks, http://www.creativepegworks.com/)
  • Absolute ethanol
  • 50 mM 2‐(N‐morpholino)ethanesulfonic acid (MES) buffer, pH 6.0
  • N‐hydroxysulfosuccinimide (sulfo‐NHS, referred to in the protocol as NHS)
  • 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.1% Tween 20 in PBS (see recipe for PBS)
  • BT‐PBS: 4% (w/v) BSA and 0.05% (v/v) Tween 20 in PBS
  • Low BT‐PBS: 0.2% (w/v) BSA and 0.1% (v/v) Tween 20 in PBS
  • Recombinant 6xHis‐tagged apCAM (see protocol 4, or Martines et al., )
  • Primary antibody: 4E8 monoclonal anti‐apCAM antibody or monoclonal anti‐His antibody
  • Secondary antibody: peroxidase‐conjugated human anti–mouse IgG
  • 1‐Step Ultra tetramethylbenzidine substrate (Pierce)
  • 2 M sulfuric acid
  • 1‐liter glass beakers
  • Wash bottle
  • Tweezers #1, #3, or #6 (flat round tips are best for handling AFM chips)
  • 35‐mm glass Petri dishes
  • UV/ozone cleaner (UVO; Jelight Co., http://www.jelight.com)
  • Clean glass cap vials with internal diameter 20‐ to 40‐mm and height 15‐ to 20‐mm
  • Glass wells with 10‐ to 15‐mm diameter (can be fabricated by cutting out the bottom of glass vials)
  • Clean Kimax weighing bottles, Type I, ∼60 ml volume
  • 100‐mm tissue culture dishes
  • Shaker
  • 96‐well microtiter plates
  • Microplate reader

Basic Protocol 5: Binding of Protein‐Conjugated Microspheres to Cultured Cells

  Materials
  • Primary cultures of neuronal and/or glial cells or other cells
  • Complete medium used for cell cultures
  • Serum‐free BSA‐containing cell culture medium
  • 1011 beads/ml protein‐conjugated fluorescent polystyrene microspheres; stock solutions in 0.5% BSA (see protocol 7)
  • 0.5 mg/ml Fab against protein of interest in serum‐free medium (optional)
  • Fixation solution (see recipe)
  • PBS (see recipe)
  • Mounting medium (see recipe)
  • Waterbath sonicator
  • 37°C, humidified incubator
  • Glass microscope slides
  • Fluorescence microscope
  • Additional reagents and equipment for immunofluorescence staining of cells (unit 4.3)

Basic Protocol 6: Trans‐Interaction Assay with Myeloma Cells

  Materials
  • Two populations of myeloma cell clones expressing the IgSF‐CAM(s) of interest
  • Selection medium, e.g., 5 mM L‐histidinol (see recipe for 50 mM) in DMEM supplemented with 10% (v/v) FBS (e.g., Life Technologies)
  • PBS with Ca2+/Mg2+ (see recipe)
  • Stock solution of green fluorogenic dye, e.g., 1 mM 2′,7′‐bis‐(2‐carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein acetoxymethyl ester (Life Technologies) in DMSO
  • Stock solution of red fluorogenic dye, e.g., 7.5 mM 5‐(and‐6)‐ carboxynaphthofluorescein diacetate (Life Technologies) in DMSO
  • 1% (v/v) FBS in PBS with Ca2+/Mg2+
  • 5 mg/ml p‐phenylenediamine (Sigma‐Aldrich) in 1% (v/v) FBS in PBS with Ca2+/Mg2+
  • 15‐ml conical polypropylene centrifuge tubes
  • Hemacytometer
  • V‐shaped 96‐well microtiter plate (e.g., Costar, Corning)
  • Centrifuge and rotor for microtiter plates
  • 22‐G needle attached to 1‐ml syringe
  • Glass microscope slide
  • Fluorescence microscope with appropriate filters for green and red fluorescence, e.g., FITC and Texas Red
  • Additional reagents and equipment for counting cells (unit 1.1)

Support Protocol 10: Stable Transfection of Myeloma Cells by Protoplast Fusion

  Materials
  • Glycerol stock of an E. coli strain 803 clone (ATCC #35581) transformed with a mammalian expression vector containing the cDNA of the IgSF‐CAM of interest (store at –80°C)
  • LB/ampicillin agar plates (see recipe)
  • DMEM supplemented with 10% (v/v) FBS (e.g., Life Technologies)
  • LB medium (see recipe), prewarmed to 37°C
  • 50 mg/ml ampicillin (Sigma‐Aldrich) (store at –20°C)
  • 60 mg/ml chloramphenicol (Sigma‐Aldrich) in ethanol (store at –20°C)
  • DMEM supplemented with 10% (w/v) sucrose and 10 mM MgCl 2, prewarm
  • 20% (w/v) sucrose in 50 mM Tris·Cl, pH 8.0, ice cold
  • 1 mg/ml lysozyme (Roche Applied Science), freshly dissolved 10 mg in 10 ml of 250 mM Tris·Cl, pH 8.0, and filtered through 0.22‐µm filter
  • 250 mM EDTA, pH 8.0, ice cold
  • 50 mM Tris·Cl, pH 8.0 ( appendix 2A), ice cold
  • 10 mg/ml DNase I (Roche Applied Science; store at –20°C)
  • DMEM
  • PEG 1500 in DMEM supplemented with DMSO (see recipe)
  • Mouse BALB/c myeloma cell line J558L (ECACC #88032902)
  • 50 mg/ml kanamycin (Sigma‐Aldrich)
  • 50 mM L‐histidinol (see recipe)
  • Polyclonal or monoclonal anti‐IgSF‐CAM antibody
  • Fluorescein‐conjugated secondary antibody
  • 25‐ml cell culture flasks
  • 12‐ml and 50‐ml polypropylene tubes
  • 15‐ and 50‐ml conical polypropylene centrifuge tubes
  • 37°C bacterial shaker
  • 500‐ml Erlenmeyer flask
  • Refrigerated tabletop centrifuge
  • 37°C water bath
  • Glass microscope slides
  • Microscope with 1000× magnification
  • Multichannel pipet trays
  • 24‐ and 96‐well tissue culture plates
  • Multichannel pipet
  • Plastic wrap (e.g., Saran)
  • 96‐well plates with V‐shaped wells
  • Additional reagents and equipment for indirect immunofluorescence (unit 4.3) and freezing cells (unit 1.1)

Basic Protocol 7: Detecting cis‐Interactions between IgSF‐CAMs by Chemical Cross‐Linking

  Materials
  • Cells of interest growing in tissue culture at low density
  • PBS with Ca2+/Mg2+ (see recipe)
  • 100 mM cross‐linking reagent (see recipe):
  • Bis(sulfosuccinimidyl)suberate (BS3) in H 2O
  • Disuccinimidyl tartrate (DST) in H 2O‐free DMSO
  • Disulfodisuccinimidyl tartrate (Sulfo‐DST) in H 2O
  • 3, 3′‐Dithiobis(sulfosuccinimidyl propionate) (DTSSP) in H 2O
  • 5 mM EDTA in Ca2+/Mg2+‐free PBS
  • 1 M glycine solution in H 2O, pH 8.0
  • Lysis buffer (see recipe)
  • Primary antibody: serum, purified immunoglobulin, or purified immunoglobulin immobilized on agarose or Sepharose matrix
  • Protein A or protein G coupled to agarose or Sepharose matrix (optional)
  • Wash buffer (see recipe)
  • Sample buffer for SDS‐PAGE ( appendix 2A)
  • 10‐cm tissue culture dishes precoated with polylysine combined with additional substrates, such as laminin (see Support Protocols protocol 2312 and protocol 2514)
  • Horizontal shaker
  • Cell scraper
  • 2‐ml microcentrifuge tubes
  • End‐over‐end rotator
  • 100‐ or 200‐µl and 500‐µl Hamilton syringe and 22‐G needle
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1), immunoblot analysis (unit 6.2), and immunoprecipitation (unit 7.2)

Basic Protocol 8: Detecting cis‐Interactions Between IgSF‐CAMs by Antibody‐Induced Co‐Capping

  Materials
  • Cells of interest growing in tissue culture (e.g., primary neurons or cell lines transfected with recombinant candidate molecules)
  • Cell culture medium (used for the cells of interest) without serum
  • PBS (see recipe)
  • Hank's balanced salt solution (HBSS; appendix 2A)
  • HBSS/1% FBS: HBSS containing 1% (v/v) fetal bovine serum (FBS)
  • Primary antibodies (serum or purified immunoglobulin) for molecules A and B
  • Secondary antibodies of the appropriate sources coupled to different fluorescent dyes
  • 4× fixative solution (see recipe)
  • Vectashield mounting medium for fluorescence (H‐1000, Vector Laboratories).
  • Vectashield mounting medium with 4′,6‐diamidino‐2‐phenylindole (DAPI; Vector Laboratories, cat. no. H‐1200) may be used
  • 10‐cm tissue culture dishes
  • 12‐mm no. 1 round glass coverslips, sterilized by autoclaving or soaking in 70% ethanol for 1 hr and precoated with polylysine (see protocol 25)
  • Watchmaker's forceps
  • 24‐well tissue culture plates
  • Microscope slides
  • Nail polish
  • Fluorescence microscope with 60× and/or 100× oil immersion objectives

Basic Protocol 9: Adhesion‐Induced Neuronal Growth in Restrained Bead Interaction Assay (RBI)

  Materials
  • Dispase II solution (see recipe)
  • Aplysia californica sea slugs, adult, 100 to 200 g (Marinus Scientific or the National Resource for Aplysia at the University of Miami)
  • 0.5 M MgCl 2, prepared with ultrapure H 2O (store at 4°C)
  • 6 N HCl
  • 200 µg/ml (10×) poly‐L‐lysine (70 to 150 kDa; Sigma, cat. no. P‐6282) stock solution prepared in sterile ultrapure H 2O (store at −20oC)
  • L15‐ASW medium (see recipe)
  • apCAM silica bead suspension ( protocol 20)
  • Phosphate‐buffered saline (PBS; see recipe), sterile
  • L15‐ASW medium (see recipe) containing 5 mg/ml BSA.
  • 60‐ml syringe with Luer‐lock tip (BD)
  • 18.5‐G sterile hypodermic needle, 2 in. long.
  • Styrofoam dissecting board
  • Micro dissecting tools:
    • Vannas Spring Scissor, 8.5 cm long, 7 mm blade (WPI, cat. no. 500086)
    • Regular dissecting scissors
    • Two pairs of forceps: Dumont tweezers #5―11 cm long (WPI, cat. no. 500341).
  • 22°C temperature‐controlled water bath
  • Coverslips: #1.5, 22 × 22 mm (acid‐cleaned as described below and store in 100% ethanol)
  • Dissecting microscope
  • 20‐µl (P‐20 or yellow) pipet tips
  • 20‐µl (P‐20) pipettor with reduced tension
  • 14oC incubator
  • 5‐µl calibrated pipets (Drummond Scientific, cat. no. 2‐000‐001)
  • Vertical micropipet puller (e.g., Narishige, cat. no. PP830)
  • Custom‐made open imaging chamber with aluminum base and brass inset (Suter, )
  • Narishige 3‐D hydraulic micromanipulator
  • Research‐grade inverted microscope, such as Nikon Eclipse TE2000 including motorized stage and filter wheel, CCD camera such CoolSnap HQ, computer, and imaging software such as MetaMorph 7.0

Support Protocol 11: Coupling of His‐tagged IgSF‐CAMs to Ni2+‐NTA Silica Beads

  Materials
  • 5% (w/v) NTA silica beads (diameter, 5 µm; Sicastar; micromod Partikeltechnologie GmbH, cat. no. 43‐11‐503); store at 4°C.
  • 0.1 M nickel sulfate (store at room temperature)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 6xHis‐tagged recombinant IgSF‐CAM used as adhesive substrate for growth cones, e.g., apCAM, 200‐400 µg/ml in PBS. apCAM was expressed in a baculovirus system.
  • Blocking solution: 5 mg/ml BSA in PBS (filter through 0.22‐µm sterile filter; store at 4°C)
  • Refrigerated tabletop centrifuge (e.g., Beckman‐Coulter Allegra X‐22R)
  • End‐over end rotator (e.g., Barnstead/Thermolyne Rotisserie Labquake)

Basic Protocol 10: Neurite Outgrowth Assay

  Materials
  • Chicken embryos (E8 to E10)
  • 0.5% (v/v) glucose in PBS
  • 0.25% (w/v) trypsin in PBS without Ca2+/Mg2+ (Life Technologies)
  • Chemically defined, serum‐free culture medium (see recipe)
  • Tissue culture dishes coated with substrate of choice (see protocol 23)
  • Sterile dissecting tools
  • 15‐ml centrifuge tubes
  • Fire‐polished Pasteur pipet, ∼0.3‐mm diameter bore
  • Neubauer chamber for cell counting (Fig. 1.1.1)
  • 35‐mm cell culture dishes
  • Image analysis software and required equipment
  • Additional reagents and equipment for counting cells (unit 1.1)

Basic Protocol 11: Inhibiting CAM‐CAM Interactions In Vitro

  Materials
  • 10‐day‐old (E10) chicken embryos
  • 0.5% (w/v) glucose in PBS
  • Chemically defined, serum‐free cell culture medium (see recipe)
  • Control Fab
  • Fab against CAM of interest
  • 15‐ml centrifuge tubes
  • 8‐well‐slide cell culture dishes (e.g., LabTek or Nunc/Thermo Fisher Scientific) coated with IgSF‐CAM (see protocol 23)
  • Pasteur pipet or automatic pipettor with 200‐µl tips

Support Protocol 12: Coating of Culture Dishes with IgSF‐CAMs

  Materials
  • Protein to be coated
  • PBS (see recipe)
  • 10 mg/ml bovine serum albumin (e.g., Albumax, Life Technologies) in PBS
  • Culture dish or other item to be coated

Support Protocol 13: Pre‐Coating Glass Surfaces with Nitrocellulose

  Materials
  • Methanol
  • Nitrocellulose (e.g., Protran BA‐83, Whatman), 0.2‐µm pore size
  • Coverslips, 22‐mm diameter (preclean glass with acetone before coating, dry, and autoclave)

Support Protocol 14: Pre‐Coating Culture Dishes with Polylysine

  Materials
  • 0.5 mg/ml polylysine stock (see recipe)
  • 150 mM sodium borate, pH 8.4
  • 35‐mm cell culture dishes

Support Protocol 15: Coating Culture Dishes with Collagen

  Materials
  • 2 mg/ml rat tail, type I collagen in 0.1% (v/v) acetic acid, sterile (Millipore, cat. no. 08‐115)
  • 0.1% (v/v) acetic acid, sterile
  • 35‐mm cell culture dishes
  • 60°C incubator

Support Protocol 16: Coating Culture Dishes with Laminin

  Materials
  • Fixation solution (see recipe)
  • Cell culture medium

Support Protocol 17: Fixation of Cells for Immunohistochemical Staining Procedures Using Fluorescent Antibodies

  Materials
  • Fertilized eggs from a local hatchery
  • 70% ethanol
  • Paraffin wax (Paraplast tissue embedding medium; e.g., Sigma‐Aldrich)
  • dsRNA injection solution ( protocol 31)
  • 4% (w/v) paraformaldehyde (see recipe for 10%)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Incubator(s) set at 38.5°C and 45% humidity (we use two incubators, one to incubate eggs before they are windowed, and a second one for incubation during the experiment; e.g., Heraeus/Kendro Model B12 from Thermo Scientific or Juppiter 576 Setter+Hatcher from FIEM, http://www.fiem.it/)
  • Drafter's knife or scalpel
  • Sterile syringe with 18‐G needle
  • Heating plate set at 80°C to melt paraffin
  • Scotch tape
  • Fine pointed scissors
  • Coverslips (24 × 24 mm; optionally, Scotch tape can be used)
  • Polyethylene tubing (Ø 1.24 mm)
  • Borosilicate glass capillaries (outer Ø/inner Ø: 1.2 mm/0.68 mm; World Precision Instruments, cat. no. 1B120F‐4)
  • Glass needle puller (Narishige, cat. no. PC‐10)
  • Paint brush
  • Soldering iron (when eggs are closed with coverslips)
  • Spring scissors (Fine Science Tools, cat. no. 15003‐08)
  • Dumont #5 forceps (Fine Science Tools, cat. no. 11252‐20)
  • Small scissors (e.g., Fine Science Tools, cat. no. 14040‐10)
  • Square wave electroporator (BTX ECM 830)
  • Platinum electrodes (4 mm length, 4 mm distance between cathode and anode; BTX Gentronics, http://www.btxonline.com/)

Support Protocol 18: Fixation of Cells for Morphological Analysis, Neurite Length Measurement, and Immunohistochemical Staining Procedures Using Non‐Fluorescent Secondary Antibodies

  Materials
  • Plasmid containing cDNA of IgSF‐CAM of interest flanked by T7 and T3 (or SP6) promoter
  • Restriction enzymes suitable to cut cDNA insertion site
  • dNTPs (Roche)
  • T7 and T3 (or SP6) polymerase
  • RNasin (Roche)
  • Appropriate transcription buffer
  • DNase I
  • 25:24:1 phenol:chloroform:isoamyl alcohol, pH 4.0
  • 24:1 chloroform:isoamyl alcohol
  • Ethanol
  • 0.4% (w/v) trypan Blue (0.4 % stock solution)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 95°C water bath or heat block
  • Additional reagents and equipment for basic molecular biology techniques (see appendix 3A and Ausubel et al., )

Basic Protocol 12: Analysis of IgSF‐CAM Function In Vivo by In Ovo RNAi

  Materials
  • Fixed embryos ( protocol 30)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 1% Triton‐X‐100 in PBS
  • 20 mM lysine in 0.1 M sodium phosphate buffer, pH 7.4 (see appendix 2A for phosphate buffer)
  • Blocking buffer (10% fetal bovine serum in PBS)
  • Primary antibody: mouse IgG anti‐neurofilament 160 kD (RMO270) diluted 1:1500 in blocking buffer
  • Secondary antibody: goat anti‐mouse IgG‐Cy3 (Jackson ImmunoResearch Labs), diluted 1:250 in blocking buffer
  • 25% methanol in H 2O
  • 50% methanol in H 2O
  • 75% methanol in H 2O
  • 100% methanol
  • BBBA (2:1 benzyl benzoate:benzyl alcohol)
  • Horizontal shaker
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Literature Cited

Literature Cited
  Andermatt, I. and Stoeckli, E.T. 2014. RNAi‐based gene silencing in chicken brain development. Methods Mol. Biol. 1082:253‐266.
  Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. 2013. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, N.J.
  Baeriswyl, T., Mauti, O., and Stoeckli, E.T. 2008. Temporal control of gene silencing by in ovo electroporation. Methods Mol. Biol. 442:231‐244.
  Bard, L., Boscher, C., Lambert, M., Mège, R.M., Choquet, D., and Thoumine, O. 2008. A molecular clutch between the actin flow and N‐cadherin adhesions drives growth cone migration. J. Neurosci. 28:5879‐5890.
  Bebbington, C.R., Renner, G., Thomson, S., King, D., Abrams, D., and Yarranton, G.T. 1992. High‐level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Biotechnology 10:169‐175.
  Bell, G. I. 1978. Models for the specific adhesion of cells to cells. Science. 200:618‐627.
  Billy, E., Brondani, V., Zhang, H., Mueller, U., and Filipowicz, W. 2001. Specific interference with gene expression induced by long, double‐stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc. Natl. Acad. Sci. U.S.A. 98:14428‐14433.
  Billy, E., Brondani, V., Zhang, H., Müller, U., Filipowicz, W. 2001. Specific interference with gene expression induced by long, double‐stranded RNA in mouse embryonal teratocarcinoma cell lines. Proc. Natl. Acad. Sci. U.S.A. 98:14428‐14433.
  Bourikas, D., Pekarik, V., Baeriswyl, T., Grunditz, A., Sadhu, R., Nardo, M., and Stoeckli, E.T. 2005. Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord. Nat. Neurosci. 8:297‐304.
  Buchstaller, A., Kunz, S., Berger, P., Kunz, B., Ziegler, U., Rader, C., and Sonderegger, P. 1996. Cell adhesion molecules NgCAM and axonin‐1 form heterodimers in the neuronal membrane and cooperate in neurite outgrowth promotion. J. Cell Biol. 135:1593‐1607.
  Bustamante, C., Chemla, Y.R., Forde, N.R., and Izhaky, D. 2004. Mechanical processes in biochemistry. Annu. Rev. Biochem. 73:705‐748.
  Carthew, R.W. 2001. Gene silencing by double‐stranded RNA. Curr. Opin. Cell Biol. 13:244‐248.
  Chang, S., Rathjen, F.G., and Raper, J.A. 1987. Extension of neurites on axons is impaired by antibodies against specific cell surface glycoproteins. J. Cell Biol. 104:355‐362.
  Domanitskaya, E., Wacker, A., Mauti, O., Baeriswyl, T., Esteve, P., Bovolenta, P., and Stoeckli, E.T. 2010. Sonic hedgehog guides post‐crossing commissural axons both directly and indirectly by regulating Wnt activity. J. Neurosci. 30:11167‐11176.
  Dubreuil, R.R., MacVicar, G., Dissanayake, S., Liu, C., Homer, D., and Hortsch, M. 1996. Neuroglian‐mediated cell adhesion induces assembly of the membrane cytoskeleton at cell contacts sites. J. Cell Biol. 133:647‐655.
  Evans, E. and Ritchie, K. 1997. Dynamic strength of molecular adhesion bonds. Biophys. J. 72:1541‐1555.
  Fire, A., Xu, S., Montgomery, M.K., Kostas, S.A., Driver, S.E., and Mello, C.C. 1998. Potent and specific genetic interference by double‐stranded RNA in Caenorhabditis elegans. Nature 391:806‐811.
  Fitzli, D., Stoeckli, E.T., Kunz, S., Siribour, K., Rader, C., Kunz, B., Kozlov, S.V., Buchstaller, A., Lane, R.P., Suter, D.M., Dreyer, W.J., and Sonderegger, P. 2000. A direct interaction of axonin‐1 with NgCAM‐related cell adhesion molecule (NrCAM) results in guidance, but not growth of commissural axons. J. Cell Biol. 149:951‐968.
  Frei, A.P., Jeon, O.Y., Kilcher, S., Moest, H., Henning, L.M., Jost, C., Pluckthun, A., Mercer, J., Aebersold, R., Carreira, E.M., and Wollscheid, B. 2012. Direct identification of ligand‐receptor interactions on living cells and tissues. Nat. Biotechnol. 30:997‐1001.
  Freigang, J., Proba, K., Leder, L., Diederich, K., Sonderegger, P., and Welte, W. 2000. The crystal structure of the ligand‐binding module of axonin‐1/TAG‐1 suggests a zipper mechanism for neural cell adhesion. Cell 101:425‐433.
  Gillies, S.D., Morrison, S.L., Oi, V.T., and Tonegawa, S. 1983. A tissue‐specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell 33:717‐728.
  Gillies, S.D., Dorai, H., Wesolowski, J., Majeau, G., Young, D., Boyd, J., Gardner, J., and James, K. 1989. Expression of human anti‐tetanus toxoid antibody in transfected murine myeloma cells. Biotechnology 7:799‐804.
  Graham, F.L. and van der Eb, A.J. 1973. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52:456‐467.
  Hamburger, V. and Hamilton, H.L. 1992. A series of normal stages in the development of the chick embryo. Dev. Dyn. 195:231‐272.
  Hermanson, G.T. 1996. Bioconjugate Techniques. Academic Press, New York.
  Hershfield, V., Boyer, H.W., Yanofsky, C., Lovett, M.A., and Helinski, D.R. 1974. Plasmid ColE1 as a molecular vehicle for cloning and amplification of DNA. Proc. Natl. Acad. Sci. U.S.A. 71:3455‐3459.
  Ji, T.H. 1983. Bifunctional reagents. Methods Enzymol. 91:580‐609.
  Jung, S.M. and Moroi, M. 1983. Crosslinking of platelet glycoprotein Ib by N‐succinimidyl(4‐azidophenyldithio) propionate and 3, 3′‐dithiobis(sulfosuccinimidyl propionate). Biochem. Biophys. Acta 761:152‐162.
  Kawano, M.M., Huang, N., Tanaka, H., Ishikawa, H., Sakai, A., Tanabe, O., Nobuyoshi, M., and Kuramoto, A. 1991. Homotypic cell aggregations of human myeloma cells with ICAM‐1 and LFA‐1 molecules. Br. J. Haematol. 79:583‐588.
  Kilinc, D., Blasiak, A., O'Mahony, J.J., Suter, D.M., and Lee, G.U. 2012. Magnetic tweezers‐based force clamp reveals mechanically distinct apCAM domain interactions. Biophys. J. 103:1120‐1129.
  Kuhn, T.B., Stoeckli, E.T., Condrau, M.A., Rathjen, F.G., and Sonderegger, P. 1991. Neurite outgrowth on immobilized axonin‐1 is mediated by a heterophilic interaction with L1(G4). J. Cell Biol. 115:1113‐1126.
  Kunz, B., Lierheimer, R., Rader, C., Spirig, M., Ziegler, U., and Sonderegger, P. 2002. Axonin‐1/TAG‐1 mediates cell‐cell adhesion by a cis‐assisted trans interaction. J. Biol. Chem. 277:4551‐4557.
  Kunz, S., Spirig, M., Ginsburg, C., Buchstaller, A., Berger, P., Lanz, R., Rader, C., Vogt, L., Kunz, B., and Sonderegger, P. 1998. Neurite fasciculation mediated by complexes of axonin‐1 and Ng cell adhesion molecule. J. Cell Biol. 143:1673‐1690.
  Lagenaur, C. and Lemmon, V. 1987. An L1‐like molecule, the 8D9 antigen, is a potent substrate for neurite extension. Proc. Natl. Acad. Sci. U.S.A. 84:7753‐7757.
  Lee, A.C. and Suter, D.M. 2008. Quantitative analysis of microtubule dynamics during adhesion‐mediated growth cone guidance, Dev. Neurobiol. 68:1363‐1377.
  Lee, A.C., Decourt, B., and Suter, D.M. 2008. Neuronal cell cultures from Aplysia californica for high‐resolution imaging of growth cones. J. Vis. Experim. (JoVE) 12. http://www.jove.com/video/662/neuronal‐cell‐cultures‐from‐aplysia‐for‐high‐resolution‐imaging.
  Lewis, R.V., Roberts, M.F., Dennis, E.A., and Allison, W.S. 1977. Photoactivated heterobifunctional cross‐linking reagents which demonstrate the aggregation state of phospholipase A2. Biochemistry 16:5650‐5654.
  Lomant, A.J. and Fairbanks, G. 1976. Chemical probes of extended biological structures: Synthesis and properties of the cleavable protein cross‐linking reagent [35S]dithiobis(succinimidyl propionate). J. Mol. Biol. 104:243‐261.
  Martines, E., Garcia, I., Marradi, M., Padro, D., and Penades, S. 2012a. Dissecting the carbohydrate specificity of the anti‐HIV‐1 2G12 antibody by single molecule force spectroscopy. Langmuir 28:17726‐17732.
  Martines, E., Zhong, J., Muzard, J., Lee, A.C., Akhremitchev, B.B., Suter, D.M., and Lee, G.U. 2012b. Single‐molecule force spectroscopy of the Aplysia cell adhesion molecule reveals two homophilic bonds. Biophys. J. 103:649‐657.
  Middaugh, C.R., Vanin, E.F., and Ji, T.H. 1983. Chemical crosslinking of cell membranes. Mol. Cell. Biochem. 50:115‐141.
  Müller, D.J. and Dufrêne, Y.F. 2008. Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology. Nat. Nanotechnol. 3:261‐269.
  Nakatani, T., Nomura, N., Horigome, K., Ohtsuka, H., and Noguchi, H. 1989. Functional expression of human monoclonal antibody genes directed against pseudomonal exotoxin A in mouse myeloma cells. Biotechnology 7:805‐810.
  Niederkofler, V., Baeriswyl, T., Ott, R., Stoeckli, E.T. 2010. Nectin‐like molecules/SynCAMs are required for post‐crossing commissural axon guidance. Development 137:427‐435.
  Pekarik, V., Bourikas, D., Miglino, N., Joset, P., Preiswerk, S., and Stoeckli, E.T. 2003. Gene silencing by RNAi as an in vivo screen for gene function. Nat. Biotechnol. 21: 93‐96.
  Perrin, F.E. and Stoeckli, E.T. 2000. The use of lipophilic dyes in studies of axonal pathfinding in vivo. Microsc. Res. Tech. 48:25‐31.
  Philipp, M., Niederkofler, V., Debrunner, M., Alther, T., Kunz, B., and Stoeckli, E.T. 2012. RabGDI controls axonal midline crossing by regulating Robo1 surface expression. Neural Develop. 7:36.
  Rader, C. and Sonderegger, P. 1998. Structural features of neural cell adhesion molecules belonging to the immunoglobulin superfamily. In Ig Superfamily Molecules in the Nervous System (P. Sonderegger, ed.) pp. 1‐22. Harwood Academic Publishers, Chur, Switzerland.
  Rader, C., Stoeckli, E.T., Ziegler, U., Osterwalder, T., Kunz, B., and Sonderegger, P. 1993. Cell‐cell adhesion by homophilic interaction of the neuronal recognition molecule axonin‐1. Eur. J. Biochem. 215:133‐141.
  Rader, C., Kunz, B., Lierheimer, R., Giger, R.J., Berger, P., Tittmann, P., Gross, H., and Sonderegger, P. 1996. Implications for the domain arrangement of axonin‐1 derived from the mapping of its NgCAM binding site. EMBO J. 15:2056‐2068.
  Rassoulzadegan, M., Binetruy, B., and Cuzin, F. 1982. High frequency of gene transfer after fusion between bacteria and eukaryotic cells. Nature 295:257‐259.
  Ray, C., Guo, S., Brown, J., Li, N., and Akhremitchev, B.B. 2010. Kinetic parameters from detection probability in single molecule force spectroscopy. Langmuir. 26:11951‐11957.
  Rojo, J.M., Saizawa, K., and Janeway, C.A. 1989. Physical association of CD4 and the T‐cell receptor can be induced by anti‐T‐cell receptor antibodies. Proc. Natl. Acad. Sci. U.S.A. 86:3311‐3315.
  Sandri‐Goldin, R.M., Goldin, A.L., Levine, M., and Glorioso, J.C. 1981. High‐frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion. Mol. Cell. Biol. 1:743‐752.
  Savoca, R., Ziegler, U., and Sonderegger, P. 1995. Effects of L‐serine on neurons in vitro. J. Neurosci. Methods 61:159‐167.
  Sbalzarini, I.F. and Koumoutsakos, P. 2005. Feature point tracking and trajectory analysis for video imaging in cell biology. J. Struct. Biol. 151:182‐195.
  Schaffner, W. 1980. Direct transfer of cloned genes from bacteria to mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 77:2163‐2167.
  Shang, H. and Lee, G. U. 2007. Magnetic tweezers measurement of the bond lifetime‐force behavior of the IgG‐protein A specific molecular interaction. J. Am. Chem. Soc. 129:6640‐6646.
  Shitara, K., Nakamura, K., Tokutake‐Tanaka, Y., Fukushima, M., and Hanai, N. 1994. A new vector for the high level expression of chimeric antibodies in myeloma cells. J. Immunol. Methods 167:271‐278.
  Smith, R.J., Capaldi, R.A., Muchmore, D., and Dahlquist, F. 1978. Cross‐linking of ubiquinone cytochrome c reductase (complex III) with periodate‐cleavable bifunctional reagents. Biochemistry 17:3719‐3723.
  Sonderegger, P., Lemkin, P.F., Lipkis, L.E., and Nelson, P.G. 1985. Differential modulation of the expression of axonal proteins by non‐neuronal cells of the peripheral and central nervous systems. EMBO J. 4:1395‐1401.
  Sonderegger, P., Kunz, S., Rader, C., Buchstaller, A., Berger, P., Vogt, L., Kozlov, S.V., Ziegler, U., Kunz, B., Fitzli, D., and Stoeckli, E.T. 1998. Discrete clusters of axonin‐1 and NgCAM at neuronal contact sites: Facts and speculations on the regulation of axonal fasciculation. Prog. Brain Res. 117:93‐104.
  Staros, J.V. 1988. Membrane‐impermeant cross‐linking reagents: Probes of the structure and dynamics of membrane proteins. Acc. Chem. Res. 21:435‐441.
  Staros, J.V. and Anjaneyulu, P.S.R. 1989. Membrane‐impermeant cross‐linking reagents. Methods Enzymol. 172:609‐628.
  Stoeckli, E.T., Kuhn, T.B., Duc, C.O., Ruegg, M.A., and Sonderegger, P. 1991. The axonally secreted protein axonin‐1 is a potent substratum for neurite growth. J. Cell Biol. 112:449‐455.
  Stoeckli, E.T. and Landmesser, L.T. 1995. Axonin‐1, Nr‐CAM, and Ng‐CAM play different roles in the in vivo guidance of chick commissural neurons. Neuron 14:1165‐1179.
  Stoeckli, E.T., Ziegler, U., Bleiker, A.J., Groscurth, P., and Sonderegger, P. 1996. Clustering and functional cooperation of NgCAM and axonin‐1 in the substratum‐contact area of growth cones. Dev. Biol. 177:15‐29.
  Stoeckli, E.T., Sonderegger, P., Pollerberg, G.E., Landmesser, L.T. 1997. Interference with axonin‐1 and NrCAM interactions unmasks a floor plate activity inhibitory for commissural axons. Neuron 18:209‐221.
  Suter, D.M. 2011. Live cell imaging of neuronal growth cone motility and guidance in vitro. In Cell Migration: Methods and Protocols, Methods in Molecular Biology, Second Edition (C. Wells and M. Parsons, eds.) pp. 65‐86. Springer, New York.
  Suter, D.M., Pollerberg, G.E., Buchstaller, A., Giger, R.J., Dreyer, W.J., and Sonderegger, P. 1995. Binding between the neural cell adhesion molecules axonin‐1 and Nr‐CAM/Bravo is involved in neuron‐glia interaction. J. Cell Biol. 131:1067‐1081.
  Suter, D.M., Errante, L.D., Belotserkovsky, V., and Forscher, P. 1998. The Ig superfamily cell adhesion molecule, apCAM, mediates growth cone steering by substrate‐cytoskeletal coupling, J. Cell Biol. 141:227‐240.
  Traunecker, A., Olivieri, F., and Karjalainen, K. 1991. Myeloma based expression system for production of large mammalian proteins. Trends Biotechnol. 9:109‐113.
  Turney, S.G. and Bridgman, P.C. 2005. Laminin stimulates and guides axonal outgrowth via growth cone myosin II activity. Nat. Neurosci. 8:717‐719.
  Vogel, V. 2006. Mechanotransduction involving multimodular proteins: Converting force into biochemical signals. Annu. Rev. Biophys. Biomol. Struct. 35:459‐488.
  von Philipsborn, A.C., Lang, S., Bernard, A., Loeschinger, J., David, C., Lehnert, D., Bastmeyer, M., and Bonhoeffer, F. 2006. Microcontact printing of axon guidance molecules for generation of graded patterns, Nat. Protoc. 1:1322‐1328.
  Wigler, M., Silverstein, S., Lee, L‐S., Pellicer, A., Cheng, Y., and Axel, R. 1977. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell 11:223‐232.
  Wilson, N. and Stoeckli, E.T. 2011. Cell type‐specific, traceable gene silencing for functional gene analysis during vertebrate neural development. Nucleic Acids Res. 39:e11.
  Wilson, N.H. and Stoeckli, E.T. 2012. In ovo electroporation of miRNA‐based plasmids in the developing neural tube and assessment of phenotypes by DiI injection in open‐book preparations. J. Vis. Exp. 68:e4384.
  Wilson, N.H. and Stoeckli, E.T. 2013. Sonic hedgehog regulates its own receptor on post‐crossing commissural axons in a Glypican1‐dependent manner. Neuron 79:478‐491.
  Wood, C.L. and O'Dorisio, M.S. 1985. Covalent cross‐linking of vasoactive intestinal polypeptide to its receptors on intact human lymphoblasts. J. Biol. Chem. 260:1243‐1247.
  Zhou, M.‐J., Todd, R.F., van de Winkel, J.G.J., and Petty, H.R. 1993. Cocapping of the leukoadhesin molecules complement receptor type 3 and lymphocyte function‐associated antigen‐1 with Fc gamma receptor III on human neutrophils. Possible role of lectin‐like interactions. J. Immunol. 150:3030‐3041.
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