Name: multibac system-based purification and biophysical characterization of human myosin-7a
Uploaded: 2024-08-23T14:30:00.000+00:00
Duration: 9 min 17 s
Description: 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 ...

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 types of light chains, all of which are essential for the protein&#39;s structural and functional integrity14. This is in contrast to its invertebrate homolog and most other myosins, which typically bind to only one or two light chain types44,45. This means that to synthesize the human myosin-7a holoenzyme, at least four different genes must be expressed simultaneously in each Sf9 cell. In this case, the MultiBac system offers significant benefits over co-infection with multiple baculoviruses because it ensures reproducible ratios of the myosin-7a complex subunits in each infected cell. In fact, Belyaev et al. have statistically demonstrated this: as the number of virus types increases, the likelihood of cells receiving an equal virus ratio decreases dramatically46. For example, with two virus types, only 12.78% of cells achieve an equal ratio, while this percentage drops to 0.29% when four virus types are used, assuming each virus type is distributed between cells independently. This variability can be problematic when the subunit proteins need to assemble at a consistent ratio to produce the maximum yield of the complex. While this paper focuses on mammalian myosin-7a, it is increasingly evident that the MultiBac system offers a more optimized approach for producing multimeric complexes than the co-infection method, particularly for proteins with more subunits and produced in low yields.
Several factors are critical for achieving high-yield production of myosin-7a protein using this method. First, it is crucial to determine the optimal timing for harvesting Sf9 cells. This allows maximum protein production while minimizing the detrimental effects of cell lysis and death caused by the virus. MultiBac-based protein complex production often exhibits a delayed peak in expression compared to monocistronic baculovirus systems or when expressing smaller proteins. We found that the best time to harvest cells infected with the myosin-7a MultiBac virus is between 60 - 65 h post-infection. Continuous monitoring of protein expression levels throughout the process is strongly recommended. This can be accomplished using fluorescent microscopy if a fluorescent protein tag is fused or through SDS-PAGE analysis otherwise. Additionally, it is important to monitor cell viability concurrently to identify the optimal timing for achieving the highest protein yield with minimal cell death.
Myosin-7a is a large monomeric myosin susceptible to protein degradation14. To prevent degradation during the purification process, it is important to ensure that all buffers are pre-chilled and all procedures are conducted at 4 &#730;C. In addition, minimizing the exposure of myosin-7a to proteases is critical. Besides utilizing protease inhibitor cocktails, we recommend keeping the incubation of the cell lysate with FLAG-resin to just 1 h. Extending this duration has not been shown to significantly improve resin binding or increase total protein yield and may pose a higher risk of protein degradation.
A distinctive feature of mammalian myosin-7a, compared to isoforms from lower species44, is its combination of unique light chain components. These light chains play important regulatory roles in the function of myosin-7a. For instance, calmodulin dynamically interacts with the myosin-heavy chain in a Ca2+-dependent manner47, modulating its motility and mechanical output, a mechanism that seems specifically adapted to the mammalian auditory hair cells14. While the exact binding affinities of calmodulin to individual IQ motifs have yet to be determined in the context of the whole molecule, we have observed that some calmodulin gradually dissociates as the excess light chains in the cell lysate are removed during purification. This may alter the mechanical properties of myosin-7a. To mitigate this, we employ spin columns combined with gentle centrifugation for the elution step. This allows proteins to be eluted in a small volume and at a high concentration, which can obviate the need for concentration steps. This practice shortens the total protein purification process and minimizes the risk of light chain dissociation. In actin gliding assays, we include excess light chain proteins in the final buffer to ensure that the native light chains remain bound with the myosin-7a heavy chain.
The requirement for excess light chains represents one of several differences between the actin gliding assay with myosin-7a and that of other well-studied myosins such as myosin-2. Myosin-2 assays are typically performed at low ionic strength (e.g., 50 mM). As ionic strength is raised towards physiological (150 mM), actin filaments tend to detach, and motility slows. In the case of myosin-7a, motility is stalled at low ionic strength, and gliding is only observed at greater than or equal to 150 mM. Due to the autoinhibition of full-length myosin-7a, it is applied to the coverslip in a high ionic strength solution in a manner akin to how myosin filaments are dissolved before application to allow proteins to bind in an orientation and conformation that allows subsequent gliding.
The low velocity observed in actin gliding assays with myosin-7a introduces some challenges in acquiring and analyzing motility data. Indeed, the translocation is several orders of magnitude slower compared to skeletal muscle myosin, which was used when this assay was originally developed30. This low velocity can lead to difficulty in accurate measurement and an overestimation of velocity that scales with frame rate48. The localization precision of any tracking method is finite, and even a static object has an apparent shift in position between successive images. As the sampling rate increases, this leads to an artificially high value for velocity. This effect is true for any type of motility assay, but the relative effect is very small for assays where a large movement of several pixels occurs between frames. By taking data points at very long intervals (every 60 s, 90 s, etc.), it can be verified that the measured velocity is accurate since the value should be unaffected by the sampling rate. Since the recording interval for myosin-7a is so long, the delay can be taken advantage of to record from several positions during the same acquisition. This effectively allows for the recording of several movies at one time. A disadvantage of this method is that mechanical imperfections with the stage will lead to additional drift due to the switching of positions. This can be accounted for using image stabilization as described above.
In the original Python2 version of the FAST software (github.com/turalaksel/FASTrack), each output data point represents a velocity for a filament within an N-frame window (5 frames by default). A modified Python3 version of the software (github.com/NeilBillington/FASTrack3) includes an additional output in which data points are on a filament-by-filament basis. The default plots produced by the software are based on the N window-type dataset. Both output types are equally valid, and although the original output will produce many more data points per movie, we typically use the filament-based data since this is more directly comparable to existing methods for quantifying filament gliding and yields more intuitive information about the numbers of filaments with specific velocities in a particular movie. Note that even in this filament-type analysis, the number of data points will not correspond exactly to the true number of filaments since tracking stops when filaments cross paths, and new tracks are then detected as they emerge after crossing.
Limitations of the methods described in this paper are that mammalian protein is being expressed in insect cells. Although many such proteins, including this one, have been successfully expressed in this way, there are other expression systems that may more closely mimic the native environment of the protein. Mammalian expression systems could introduce important post-translational modifications to the protein which are absent in the insect system. The same is true to an even greater extent for expression in a bacterial system, as used here for producing myosin light chains. Nevertheless, we believe the relative simplicity and high yield in these cases outweigh the potential limitations. The in vitro motility assay that is used to characterize the protein is limited in how much information it can yield about the protein. For example, many aspects of myosin regulation are masked or circumvented by the assay and thus cannot be investigated using this technique. Many other assay types, such as single-molecule motility, optical trapping, and biochemical and biophysical techniques, exist to investigate the properties of myosin in vitro, but the filament gliding assay is chosen here as a method to measure myosin activity because it requires less protein than many biochemical assays, is simple to perform and where successful motility can be demonstrated, tells us that the protein is both biochemically and mechanically active.
The workflow described here represents a series of methods for producing high-quality myosin-7a protein and characterizing its mechanical properties. Although these methods are specifically tailored to this myosin class, the expression and purification procedures are more generally useful as a guideline for how to produce this type of labile protein, consisting of several distinct polypeptides and with a tendency for dissociation and degradation. In addition, the characterization methods are useful for all types of motor proteins, and in particular, the considerations of data acquisition and analysis parameters are intended to be useful for anyone measuring the translocation rate of low-velocity molecular motors.

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.

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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.
Video 1 and Figure 5 demonstrate a characteristic actin filament gliding assay with mammalian myosin-7a. An immediate feature revealed by this assay is the slow motility of myosin-7a. The video is played back at 500 times normal speed to better visualize the movement of actin filaments driven by this myosin. This assay can be modified to further study how external factors such as temperature, ionic strength, and solution viscosity influence the activity of myosin-7a. Additionally, myosin-7a binding proteins can be introduced into the flow cell to investigate their effects on actomyosin interactions and motility.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67135/67135fig01.jpg" /> 
Figure 1: Workflow for MultiBac-system-based myosin-7a holoenzyme production. (A) Insert the cDNAs encoding the myosin-7a heavy chain, RLC, CALML4, and calmodulin into the multiple cloning sites (MCS) of the pACEBac1 vector. (B) Construct the multigene expression cassette for encoding the myosin-7a complex by iteratively ligating the pACEBac1 vectors that contain the cDNAs of the myosin-7a heavy chain, RLC, CALML4, and calmodulin. (C) Integrate the myosin-7a multigene cassette into the baculovirus genome through the transposition of the mini-Tn7 element from the pACEBac1 vector to the mini-attTn7 target site in the genome. (D) Produce baculoviruses expressing the myosin-7a complex by transfecting Sf9 cells with the recombinant bacmid containing the myosin-7a multigene cassette. (E) The baculovirus can be amplified by inoculating the P0 virus into an Sf9 cell suspension culture and collecting the supernatant. The resulting product, the P1 virus, can be used for large-scale protein expression. (F) Workflow for the FLAG-affinity column-based purification of the myosin-7a complex. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67135/67135fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67135/67135fig02.jpg" /> 
Figure 2: Representative fluorescent images of Sf9 cells transfected with bacmid expressing myosin-7a with a C-terminal GFP tag. The images show the normal progression of baculovirus infection: cells begin to express myosin-7a around day 4 post-transfection, with nearly all cells becoming infected within one week. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67135/67135fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67135/67135fig03.jpg" /> 
Figure 3: SDS-gels of purified myosin-7a complex and light chain proteins. (A)&#160;Purified full-length human myosin-7a holoenzyme using the MultiBac system. The heavy chain (HC) and light chains are indicated. (B) Purified RLC and CALML4 using the His6-SUMO system. Calmodulin (CaM) is purchased (see Table of Materials) and purified from the Bovine brain. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67135/67135fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67135/67135fig04.jpg" /> 
Figure 4: Workflow for flow chamber assembly and actin gliding assays. A nitrocellulose-treated coverslip is attached to a microscope slide using two pieces of double-sided tape. This creates a flow chamber with a volume of approximately 10 &#181;L. Proteins are added sequentially to the chamber, while excess solutions are wicked through the channel using tissue paper. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67135/67135fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67135/67135fig05.jpg" /> 
Figure 5: Representative results of gliding actin filament assays and the tracking analysis. (A)&#160;Example frame from a timelapse recording showing actin filament movement driven by surface-decorated myosin-7a motors. (B) Filament tracking image output from the FAST program for the same field-of-view as shown in (A). (C) Representative histogram of the actin gliding velocity generated by mammalian myosin-7a: 4.2 &#177; 1.4 nm/s (mean &#177; standard deviation; the number of tracks = 550). (D) Example of autogenerated output from the FAST program showing the calculated filament length and velocities. %STUCK shows the percentage of filaments that are deemed to be stuck based on the user supplied minimum velocity cutoff. MVEL represents the mean velocity of all non-stuck filaments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67135/67135fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Actin filament gliding assay performed with myosin-7a. Purified full-length human myosin-7a is attached to the surface. This movie was acquired at 1 frame every 30 s with 200 ms exposure. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67135/JOVE_Video1_New.mp4" target="_blank">Please click here to download this Video.</a>

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

427 Views.  West Virginia University. We proberen te beantwoorden waarom mutaties in myosinemotoren ziekten bij de mens veroorzaken en welke rol deze eiwitten spelen in het gehoorproces. Ons onderzoek richt zich op het begrijpen van de moleculaire mechanismen van Myosin-7a en hoe het werkt met Usher-eiwitten om de cellulaire machinerie die nodig is voor het gehoor te assembleren en te onderhouden. Het begrijpen van de structurele en functionele mechanismen van Myosin-7a vereist intact eiwit van h...

Video: Auteur in de schijnwerpers: het ontrafelen van de rol van myosine-7a en usher-eiwitten bij gehoor en menselijke ziekten

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

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