We thank the Microscopy Imaging Facility and Visual Function and Morphology Core at West Virginia University for discussion and help with image analysis. This work is supported by the tenure-track startup funds from West Virginia University School of Medicine to R.L. This work is also supported by National Institute of General Medical Sciences (NIGMS) Visual Sciences Center of Biomedical Research Excellence (Vs-CoBRE) (P20GM144230), and the NIGMS West Virginia Network of Biomedical Research Excellence (WV-INBRE) (P20GM103434).

Recombinant Human Myosin-7a Production and Functional Characterization Protocol

The authors declare no conflict of interest.

Presented here is a detailed protocol for the production of recombinant human myosin-7a protein from insect cells. Although the Sf9/baculovirus system has been used to produce a variety of myosins40,41,42,43, only recently has the mammalian myosin-7a been successfully purified using the MultiBac baculovirus system14. Mammalian myosin-7a is found to associate with three t...

Myosins are molecular motor proteins that interact with actin to drive numerous cellular processes1,2,3,4. Humans possess 12 classes and 39 myosin genes5, which are involved in a wide range of physiological functions, such as muscle contraction6 and sensory processes7. Each myosin molecule is a multimeric complex composed of a heavy chain and light chains. The heavy chain is divided into head, neck, and tail regions. The head contains actin- and nucleotide-binding sites that are responsible for ATP hydrolysis and generating force on actin filaments2. The neck is formed by several &#945;-helical IQ motifs where a specific set of light chains are bound. They together function as a lever arm to amplify the motor&#39;s conformational changes into large movements8,9,10. The tail contains class-specific subdomains and plays a regulatory role in tuning myosin&#39;s motor activity and mediating interactions with cellular binding partners2,11.
Human myosin-7a, a member of class-7 myosins, is essential for auditory and visual processes12,13. The IQ motifs of human myosin-7a are associated with a unique combination of light chains, including the regulatory light chain (RLC), calmodulin, and calmodulin-like protein 4 (CALML4)14,15,16. Besides stabilizing the lever arm, these light chains regulate the mechanical properties of myosin-7a in response to calcium signaling, a feature that appears to be unique to the mammalian isoform14.
Defects in the gene encoding the myosin-7a heavy chain (MYO7A/USH1B) are responsible for Usher syndrome type 1, the most severe form of combined vision and hearing loss in humans17. Additionally, the light chain gene CALML4, is among the candidate genes mapped to contain the causative allele for USH1H, another variant of type 1 Usher syndrome15,18. In the retina, myosin-7a is expressed in the retinal pigment epithelium and photoreceptor cells13. It has been implicated in the localization of melanosomes in the retinal pigment epithelium (RPE)19 and phagocytosis of photoreceptor outer segment disks by the RPE cells20. In the inner ear, myosin-7a is primarily found in the stereocilia, where it plays a critical role in establishing hair bundles and in gating the mechano-electrical transduction process12,21,22.
While the importance of myosin-7a in sensory cells is well established, its functional mechanisms at the molecular level remain poorly understood. This gap in knowledge is partly due to the challenges in purifying the intact protein, especially the mammalian isoform. Recently, significant progress has been made using the MultiBac system to recombinantly express the complete human myosin-7a holoenzyme14. This advancement has enabled structural and biophysical characterizations of this motor protein, leading to the discovery of several unique properties of human myosin-7a that are specifically adapted for mammalian auditory functions14,23.
The MultiBac system is an advanced baculovirus/insect cell platform specifically designed for the expression of eukaryotic multimeric complexes24,25. A key feature of this system is its ability to host multiple gene expression cassettes, each encoding a subunit of the complex, within a single MultiBac baculovirus. The assembly of the multigene expression cassettes is facilitated through a so-called multiplication module: a homing endonuclease (HE) site and a matching designed BstXI site flanking the multiple cloning sites (MCS). This module enables the iterative assembly of a single expression cassette by restriction/ligation, leveraging the fact that the HE and BstXI restriction sites are eliminated upon their ligation. In this paper, human myosin-7a heavy chain, RLC, calmodulin, and CALML4 are each cloned into the multiplication module within the pACEBac1 vector (Figure 1A), which are then assembled into a multigene expression cassette through the iterative process (Figure 1B). The myosin-7a multigene cassette is integrated into the baculoviral genome (bacmid) through the transposition of the mini-Tn7 element from the pACEBac1 vector to the mini-attTn7 target site in the genome (Figure 1C). Following procedures for bacmid purification, baculovirus production, and amplification (Figure 1D,E), the recombinant myosin-7a MultiBac baculovirus is prepared and can be used for large-scale protein production (Figure 1F). Additionally, the myosin-7a light chains can be produced separately in E. coli and purified using a cleavable His6-SUMO tag26,27,28. The purified light chains are useful for studying their binding dynamics and regulation of myosin-7a.
The purified myosin-7a protein can be subjected to structural, biochemical, and biophysical studies to gain insights into the structural-functional regulation of this motor protein. Additionally, its interactions with the actin network and other binding proteins29 can be examined using a variety of in vitro reconstitution approaches. Findings from these analyses will inform the biophysical properties of this myosin, leading to a mechanistic understanding of how myosin-7a drives the cytoskeletal changes and ultimately shapes the unique morphology and function of sensory cells. In this paper, we detail a workflow for actin filament gliding assay that has been specifically adapted for mammalian myosin-7a. Actin filament gliding assay is a robust in vitro motility assay that quantitatively studies the movement of fluorescent actin filaments propelled by a large number of myosin motors immobilized on a coverslip surface30,31,32. The advantages of this assay include its simplicity of setup, minimal equipment requirements (a wide-field fluorescence microscope equipped with a digital camera), and high reproducibility. Additionally, because the motion of actin filaments is driven by a cluster of immobilized myosin motors, this assay is particularly useful for studying the motility of monomeric myosins such as myosin-7a14,33. The protocols include several modifications, from experimental procedures to imaging analysis, specifically tailored to the unique motile properties of mammalian myosin-7a. With the availability of intact myosin-7a protein and the functional characterization protocol outlined here, this paper lays the groundwork for further investigation into the molecular roles of myosin-7a in both physiological and pathological processes.

<table><tbody><tr><td>1.7 mL microcentrifuge tubes</td><td>VWR</td><td>87003-294</td><td></td></tr><tr><td>1X FLAG Peptide</td><td>GenScript</td><td>N/A</td><td>Custom peptide synthesis</td></tr><tr><td>22x22mm No. 1.5 coverslips</td><td>VWR</td><td>48366-227</td><td></td></tr><tr><td>250 mL Conical Centrifuge Tubes</td><td>Nunc</td><td>376814</td><td></td></tr><tr><td>250 mL Vented Erlenmyer Shaker Flask</td><td>IntelixBio</td><td>DBJ-SF250VP</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>VWR</td><td>M131</td><td></td></tr><tr><td>75x25x1 mm Vistavision microscope slides</td><td>VWR</td><td>16004-42</td><td></td></tr><tr><td>Actin Protein (&gt;99% Pure)</td><td>Cytoskeleton</td><td>AKL99</td><td></td></tr><tr><td>Amicon Ultra-0.5 Centrifugal Filter Unit</td><td>Millipore Sigma</td><td>UFC510024</td><td></td></tr><tr><td>Amicon Ultra-4 Centrifugal Filter Unit</td><td>Millipore Sigma</td><td>UFC801024</td><td></td></tr><tr><td>ANTI-FLAG M2 Affinity Gel</td><td>Millipore Sigma</td><td>A2220</td><td></td></tr><tr><td>ATP</td><td>Millipore Sigma</td><td>A7699</td><td></td></tr><tr><td>ATP</td><td>Millipore Sigma</td><td>A7699</td><td></td></tr><tr><td>Bio-Spin Disposable Chromatography Column</td><td>Bio-Rad</td><td>732-6008</td><td></td></tr><tr><td>BL21 Competent E. coli</td><td>New England Biolabs</td><td>C2530H</td><td></td></tr><tr><td>Bluo-Gal</td><td>Thermo Fisher</td><td>15519028</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Millipore Sigma</td><td>5470</td><td></td></tr><tr><td>BstXI Enzyme</td><td>New England Biolabs</td><td>R0113S</td><td></td></tr><tr><td>Calmodulin</td><td>Millipore Sigma</td><td>208694</td><td></td></tr><tr><td>Catalase</td><td>Millipore Sigma</td><td>C40</td><td></td></tr><tr><td>Champion pET-SUMO Expression System</td><td>Thermo Fisher</td><td>K30001</td><td></td></tr><tr><td>cOmplete, EDTA-free Protease Inhibitor Cocktail</td><td>Roche Diagnostics</td><td>5056489001</td><td></td></tr><tr><td>Cutsmart Buffer</td><td>New England Biolabs</td><td>B6004S</td><td></td></tr><tr><td>DL-Dithiothreitol</td><td>Millipore Sigma</td><td>DO632</td><td></td></tr><tr><td>DL-Dithiothreitol</td><td>Millipore Sigma</td><td>DO632</td><td></td></tr><tr><td>DNase I, Spectrum Chemical</td><td>Fisher Scientific</td><td>18-610-304</td><td></td></tr><tr><td>Double-Sided Tape</td><td>Office Depot</td><td>909955</td><td></td></tr><tr><td>EGTA, Molecular Biology Grade</td><td>Millipore Sigma</td><td>324626-25GM</td><td></td></tr><tr><td>EGTA, Molecular Biology Grade</td><td>Millipore Sigma</td><td>324626-25GM</td><td></td></tr><tr><td>Ethanol</td><td>Thermo Fisher</td><td>BP2818</td><td></td></tr><tr><td>ExpiFectamine Sf Transfection Reagent</td><td>Gibco</td><td>A38915</td><td></td></tr><tr><td>FAST program</td><td>http://spudlab.stanford.edu/fast-for-automatic-motility-measurements;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;Model 505 Sonic Dismembrator</td><td>Fisher Scientific</td><td>FB505110</td><td></td></tr><tr><td>Gentamicin Reagent Solution</td><td>Gibco</td><td>15710-064</td><td>10 mg/mL in distilled water</td></tr><tr><td>Glucose</td><td>Millipore Sigma</td><td>G5767</td><td></td></tr><tr><td>Glucose Oxidase</td><td>Millipore Sigma</td><td>G2133</td><td></td></tr><tr><td>Glycerol</td><td>Invitrogen</td><td>15514-011</td><td></td></tr><tr><td>HisPur Cobalt Resin</td><td>Thermo Fisher</td><td>89966</td><td></td></tr><tr><td>I-CeuI Enzyme</td><td>New England Biolabs</td><td>R0699S</td><td></td></tr><tr><td>Image Stabilizer Plugin</td><td>https://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html</td><td></td><td></td></tr><tr><td>ImageJ FIJI</td><td>https://imagej.net/Fiji/Downloads</td><td></td><td></td></tr><tr><td>Imidazole</td><td>Millipore Sigma</td><td>I2399</td><td></td></tr><tr><td>In-Fusion Snap Assembly Master Mix</td><td>TaKaRa</td><td>638948</td><td></td></tr><tr><td>IPTG</td><td>Thermo Fisher</td><td>15529019</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Scientific</td><td>A451SK</td><td></td></tr><tr><td>Kanamycin</td><td>Fisher Scientific</td><td>AAJ67354AD</td><td></td></tr><tr><td>Large Orifice Pipet Tips</td><td>Fisher Scientific</td><td>02-707-134</td><td>1-200uL</td></tr><tr><td>LB Agar, Ready-Made Powder</td><td>Thermo Fisher</td><td>J75851-A1</td><td></td></tr><tr><td>Leupeptin Protease Inhibitor</td><td>Thermo Fisher</td><td>78435</td><td></td></tr><tr><td>Magnesium chloride</td><td>Thermo Fisher</td><td>J61014.=E</td><td>1M</td></tr><tr><td>Magnesium chloride</td><td>Thermo Fisher</td><td>J61014.=E</td><td>1M</td></tr><tr><td>Max Efficiency DH10Bac Competent Cells</td><td>Gibco</td><td>10361012</td><td></td></tr><tr><td>Microcentrifuge Tubes, 1.7mL</td><td>VWR</td><td>87003-294</td><td></td></tr><tr><td>Microcentrifuge Tubes, 1.7mL</td><td>VWR</td><td>87003-294</td><td></td></tr><tr><td>Microcentrifuge Tubes, 1.7mL</td><td>VWR</td><td>87003-294</td><td></td></tr><tr><td>Microscope</td><td>Nikon</td><td>Model: Eclipse Ti with H-TIRF system with 100X TIRF objective</td><td></td></tr><tr><td>Microscope Camera</td><td>ORCA-Fusion BT</td><td></td><td></td></tr><tr><td>Microscope Laser Unit</td><td>Andor iXon Ultra</td><td></td><td></td></tr><tr><td>Miller&#039;s LB Broth</td><td>Corning</td><td>46-050-CM</td><td></td></tr><tr><td>MOPS</td><td>Millipore Sigma</td><td>M3183</td><td></td></tr><tr><td>MOPS</td><td>Millipore Sigma</td><td>M3183</td><td></td></tr><tr><td>NanoDrop One/OneC Microvolume UV-Vis Spectrophotometer</td><td>Thermo Fisher</td><td>ND-ONE-W</td><td></td></tr><tr><td>NanoDrop One/OneC Microvolume UV-Vis Spectrophotometer</td><td>Thermo Fisher</td><td>ND-ONE-W</td><td></td></tr><tr><td>NEB 5-alpha Competent E.coli (High Efficiency)</td><td>New England Biolabs</td><td>C2987H</td><td></td></tr><tr><td>NEBuffer r3.1</td><td>New England Biolabs</td><td>B6003S</td><td></td></tr><tr><td>NIS Elements</td><td>Nikon</td><td></td><td></td></tr><tr><td>NIS-Elements</td><td>Nikon</td><td></td><td></td></tr><tr><td>Nitrocellulose</td><td>LADD Research Industries</td><td>53152</td><td></td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>31985070</td><td></td></tr><tr><td>pACEBac1 Vector</td><td>Geneva Biotech</td><td></td><td></td></tr><tr><td>Parafilm</td><td>Millipore Sigma</td><td>P7793</td><td></td></tr><tr><td>PMSF</td><td>Millipore Sigma</td><td>78830</td><td></td></tr><tr><td>PureLink RNase A (20 mg/mL)</td><td>Invitrogen</td><td>12091021</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit (250)</td><td>QIAGEN</td><td>27106</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit (50)</td><td>QIAGEN</td><td>28704</td><td></td></tr><tr><td>QIAquick PCR Purification Kit (50)</td><td>QIAGEN</td><td>28104</td><td></td></tr><tr><td>Quick CIP</td><td>New England Biolabs</td><td>M0525S</td><td></td></tr><tr><td>Rhodamine phalloidin</td><td>Invitrogen</td><td>R415</td><td></td></tr><tr><td>S.O.C. Medium</td><td>Invitrogen</td><td>15544034</td><td></td></tr><tr><td>SENP2 protease</td><td></td><td>PMID:17591783</td><td>Purified in the lab</td></tr><tr><td>Sf9 cells</td><td>Thermo Fisher</td><td>11496015</td><td></td></tr><tr><td>Sf-900 III SFM (1X) - Serum Free Media Complete</td><td>Gibco</td><td>12658-027</td><td></td></tr><tr><td>Slide-A-Lyzer G3 Dialysis Cassettes, 10K MWCO, 3 mL</td><td>Thermo Fisher</td><td>A52971</td><td></td></tr><tr><td>Sodium chloride</td><td>Millipore Sigma</td><td>S7653</td><td></td></tr><tr><td>Sodium chloride</td><td>Millipore Sigma</td><td>S7653</td><td></td></tr><tr><td>Stericup Quick Release Vacuum Driven Disposable Filtration System</td><td>Millipore Sigma</td><td>S2GPU01RE</td><td></td></tr><tr><td>Superdex 75 Increase 10/300 GL</td><td>Cytiva</td><td>29148721</td><td></td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>T4 DNA Ligase Buffer - 10X with 10mM ATP</td><td>New England Biolabs</td><td>B0202A</td><td></td></tr><tr><td>Tetracycline Hydrochloride</td><td>Millipore Sigma</td><td>T7660-5G</td><td></td></tr><tr><td>Tris</td><td>Millipore Sigma</td><td>10708976001</td><td></td></tr><tr><td>Triton X</td><td>American Bioanalytical</td><td>9002-93-1</td><td></td></tr></tbody></table>

multibac system-based purification and biophysical characterization of human myosin-7a

Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common and severe form of deaf-blindness in humans. It is hypothesized that myosin-7a forms a transmembrane adhesion complex with other Usher proteins, essential for the structural-functional integrity of photoreceptor and cochlear hair cells. However, due to the challenges in obtaining pure, intact protein, the exact functional mechanisms of human myosin-7a remain elusive, with limited structural and biomechanical studies available. Recent studies have shown that mammalian myosin-7a is a multimeric motor complex consisting of a heavy chain and three types of light chains: regulatory light chain (RLC), calmodulin, and calmodulin-like protein 4 (CALML4). Unlike calmodulin, CALML4 does not bind to calcium ions. Both the calcium-sensitive, and insensitive calmodulins are critical for mammalian myosin-7a for proper fine-tuning of its mechanical properties. Here, we describe a detailed method to produce recombinant human myosin-7a holoenzyme using the MultiBac Baculovirus protein expression system. This yields milligram quantities of high-purity full-length protein, allowing for its biochemical and biophysical characterization. We further present a protocol for assessing its mechanical and motile properties using tailored in vitro motility assays and fluorescence microscopy. The availability of the intact human myosin-7a protein, along with the detailed functional characterization protocol described here, paves the way for further investigations into the molecular aspects of myosin-7a in vision and hearing.

The purified myosin-7a complex and light chain proteins can be evaluated by SDS-PAGE gel electrophoresis, as shown in Figure 3. The band above the 200 kDa marker corresponds to the myosin-7a heavy chain (255 kDa). The three bands migrating between the 22 and 14 kDa markers from top to bottom are RLC (20 kDa), calmodulin, and CALML4, respectively. While calmodulin and CALML4 have a similar molecular weight of approximately 17 kDa, the two proteins can be separated using a 16% Tris-Glycine gel...

Author Spotlight: Unraveling the Role of Myosin-7a and Usher Proteins in Hearing and Human Disease

This protocol details the procedures for recombinantly producing the human myosin-7a holoenzyme using the MultiBac Baculovirus system and for studying its motility using a tailored in vitro filament gliding assay.

MultiBac System-Based Purification and Biophysical Characterization of Human Myosin-7a

Myosin-7a is an actin-based motor protein vital for auditory and visual processes. Mutations in myosin-7a lead to Usher syndrome type 1, the most common ...

multibac-system-based-purification-biophysical-characterization-human

Research

JoVE Journal

Biochemistry

368 Views.  West Virginia University. This protocol details the procedures for recombinantly producing the human myosin-7a holoenzyme using the MultiBac Baculovirus system and for studying its motility using a tailored in vitro filament gliding assay. 

Video: Author Spotlight: Unraveling the Role of Myosin-7a and Usher Proteins in Hearing and Human Disease

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis is an obligate pathogen that causes fatal inhalational anthrax. Bacillus cereus is considered a useful model for studying B. anthracis due to its close evolutionary relationship. The gene cluster ba1554 - ba1558 of B. anthracis is highly conserved with the bc1531- bc1535 cluster in B. cereus, as well as with the bt1364-bt1368 cluster in Bacillus thuringiensis, indicating the critical role of the associated genes in the Bacillus genus. This manuscript describes methods to prepare and characterize a protein product of the first gene (ba1554) from the gene cluster in B. anthracis using a recombinant protein of its ortholog in B. cereus, bc1531.

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Bacillus anthracis is the obligate pathogen of fatal inhalational anthrax, so studies of its genes and proteins are strictly regulated. An alternative approach is to study orthologous genes. We describe biophysicochemical studies of a B. anthracis ortholog in B. cereus, bc1531, requiring minimal experimental equipment and lacking serious safety concerns.

To overcome safety restrictions and regulations when studying genes and proteins from true pathogens, their homologues can be studied. Bacillus anthracis ...

Expression and Purification of Mammalian Bestrophin Ion Channels

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- channel (CaCC) predominantly expressed in retinal pigment epithelium (RPE). The physiological and pathological significance of BEST1 is highlighted by the fact that over 200 distinct mutations in the BEST1 gene have been genetically linked to a spectrum of at least five retinal degenerative disorders, such as Best vitelliform macular dystrophy (Best disease). Therefore, understanding the biophysics of bestrophin channels at the single-molecule level holds tremendous significance. However, obtaining purified mammalian ion channels is often a challenging task. Here, we report a protocol for the expression of mammalian bestrophin proteins with the BacMam baculovirus gene transfer system and their purification by affinity and size-exclusion chromatography. The purified proteins have the potential to be utilized in subsequent functional and structural analyses, such as electrophysiological recording in lipid bilayers and crystallography. Importantly, this pipeline can be adapted to study the functions and structures of other ion channels.

The purification of ion channels is often challenging, but once achieved, it can potentially allow in vitro investigations of the functions and structures of the channels. Here, we describe the stepwise procedures for the expression and purification of mammalian bestrophin proteins, a family of Ca2+-activated Cl- channels.

The human genome encodes four bestrophin paralogs, namely BEST1, BEST2, BEST3, and BEST4. BEST1, encoded by the BEST1 gene, is a Ca2+-activated Cl- ...

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content. In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca2+ release units (CRUs). In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools. In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions.

In cardiac myocytes, tubular membrane structures form intracellular networks. We describe optimized protocols for i) isolation of myocytes from mouse heart including quality control, ii) live cell staining for state-of-the-art fluorescence microscopy, and iii) direct image analysis to quantify the component complexity and the plasticity of intracellular membrane networks.

In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. ...

Bioengineering

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include&#160;cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric complexes. They interact with other cytoskeletal components and the cell membrane to participate in important cellular functions such as migration and cell division. Due to the complexity of septins' many interactions, the large number of septin genes (13 in humans), and the ability of septins to form hetero-oligomeric complexes with different subunit compositions, cell-free reconstitution is a vital strategy to understand the basics of septin biology. The present paper first describes a method to purify recombinant septins in their hetero-oligomeric form using a two-step affinity chromatography approach. Then, the process of quality control used to check for the purity and integrity of the septin complexes is detailed. This process combines native and denaturing gel electrophoresis, negative stain electron microscopy, and interferometric scattering microscopy. Finally, a description of the process to check for the polymerization ability of septin complexes using negative stain electron microscopy and fluorescent microscopy is given. This demonstrates that it is possible to produce high-quality human septin hexamers and octamers containing different isoforms of septin_9, as well as Drosophila septin hexamers.

In vitro reconstitution of cytoskeletal proteins is a vital tool to understand the basic functional properties of these proteins. The present paper describes a protocol to purify and assess the quality of recombinant septin complexes, which play a central role in cell division and migration.

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Biology

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force.
By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...

MultiBac System-Based Purification and Biophysical Characterization of Human Myosin-7a

Summary

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Chapters in this video

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG

Expression and Purification of Mammalian Bestrophin Ion Channels

Analysis of Tubular Membrane Networks in Cardiac Myocytes from Atria and Ventricles

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution

The MultiBac Protein Complex Production Platform at the EMBL

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy