The authors acknowledge Howard Berg for the gift of the bead-tracking microscope/photomultipliers and the Texas A&amp;M Engineering Experiment Station for funds.

1. Cell Preparation
<ol>
	<li>Grow overnight cultures of the desired strain carrying the sticky fliC allele 15,22 in Tryptone Broth (TB, 1% Peptone, 0.5% NaCl) followed by inoculation at 1:100 dilution in 10 mL fresh TB. Grow the culture at 33 &#176;C in a shaker incubator until OD600 = 0.5.</li>
	<li>Pellet the cells at 1,500 x g for 5 - 7 min and re-disperse the pellet vigorously in 10 mL of filter sterilized motility buffer (MB; 10 mM phosphate buffer: 0.05-0.06 M NaCl, 10-4 M EDTA, 1 &#181;M methionine, pH 7.0).</li>
	<li>Repeat step 1.2 two more times and re-disperse the final pellet in 1 mL MB.</li>
	<li>Shear the suspension by passing back and forth ~ 75 times between two syringes with 21 to 23 gauge adapters connected by polyethylene tubing (7 - 12 cm long, 0.58 mm inner diameter). Limit the total time for shearing to 30 - 45 s.</li>
	<li>Centrifuge the sheared cells at 1,500 x g for 5-7 min and re-disperse the pellet in 100 - 500 &#181;L of MB.</li>
</ol>
2. Slide Preparation
<ol>
	<li>Prepare an imaging chamber by sandwiching two double-sided adhesive tapes between a cover-slip and a microscope slide. For chemotaxis assays, employ any microfluidic chamber that enables the exchange of MB and chemical stimulants.</li>
	<li>Add 0.01% poly-L-lysine solution in the chamber and after 5 min gently rinse the surfaces with MB (80 - 100 &#181;L).</li>
	<li>Add 40 &#181;L of the cell suspension into the chamber and allow sufficient time for attachment to the glass surface (7 - 8 min). Flow out unstuck cells by adding 100 &#181;L MB on one side of the chamber, while wicking the solution with a filter paper from the other side.</li>
	<li>Add 10 - 15 &#181;L of latex beads into the chamber and allow the beads adequate time to settle and attach to the cells (7 - 8 min). Gently rinse with 100 &#181;L of MB, as described in step 2.3, to remove unstuck beads. Use a range of bead-sizes for the experiments so long as a good contrast is available.</li>
</ol>
3. Bead Tracking
<ol>
	<li>Place the sample on a microscope stage and scan the surface for beads attached to motors. Use a 40X phase objective to make observations although phase microscopy is not necessary. Alternatively, employ bright-field imaging so long as sufficient contrast is maintained to clearly distinguish a bright bead on a dark background.</li>
	<li>Once a bead has been selected, move the stage laterally to position the bead in a pre-determined corner as shown in Figure 1B. Position beads at the same corner to ensure that the direction of rotation of the bead is correctly known. The ideal bead trajectory is approximately circular but elliptical trajectories are admissible.</li>
	<li>Maintain the sampling frequency higher than twice the rotational frequency of the motor to avoid errors associated with aliasing. In this work, use a motor that was rotating at 50 Hz and sample at frequencies that were 10 times higher (500 Hz), to obtain a smooth signal.</li>
</ol>
4. Data Analysis
<ol>
	<li>Center and scale the PMT output voltages and correct ellipticity in the trajectories with affine transformations if needed 23. Use a power-spectrum analysis to determine rotation rates 17.</li>
	<li>Determine polar angles, &#952;(t) = atan (y(t)/x(t)). Determine the variations in motor speeds and switching over time by calculating &#969; <img alt="Equation 1" src="/files/ftp_upload/55240/55240eq1.jpg" /> 14.</li>
	<li>Employ a median filter to smooth the motor speed data. A filter window over two full rotations is recommended 23,24.</li>
</ol>

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P., Roland, T., Shrivastava, A., Chen, Y. H., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+flagellar+motor+of+Caulobacter+crescentus+generates+more+torque+when+a+cell+swims+backwards.">The flagellar motor of Caulobacter crescentus generates more torque when a cell swims backwards.</a> Nat Phys. 12 (2), 175-178 (2016).</li><li>Lele, P. P., Shrivastava, A., Roland, T., Berg, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+thresholds+in+bacterial+chemotaxis.">Response thresholds in bacterial chemotaxis.</a> Sci Adv. 1 (9), e1500299 (2015).</li><li>Berg, H. 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The authors have nothing to disclose.

In order to facilitate tethered bead-tracking and correct estimation of motor-torques, the following information should be reviewed. When performing these measurements with flagellated cells, shearing is a critical step. Shearing reduces the flagellar filament to a mere stub, thereby ensuring that the viscous load on the motor is predominantly due to the bead and can be estimated within 10% error 16. Shearing also improves the chances of finding circular trajectories with tightly distributed eccentricities (&#60; bead diameter 14). Improper shearing results in wayward trajectories, which compounds errors in tracking and in the calculation of viscous drags, as well as resulting in poor signal-noise ratios. The use of an oscilloscope allows rapid elimination of such data. Since the biomechanical properties of flagellar filaments are expected to vary with species, shearing methods will likely need to be adapted to ensure adequate shearing in the bacteria of interest. An effective way of reducing errors associated with shearing is to work with cells that lack the genes that encode the filament proteins. Probe beads can then be attached directly to the hook via anti-hook antibodies.
Finding a bead that is appropriately tethered can be challenging. This is because most beads in the field of view will either be stuck to the cell bodies or the glass surface. Such beads can be readily brought into sharp focus. Other beads will appear to vibrate or rotate visibly with large amplitudes or large eccentricities (&#62; 1.5 - 2x bead diameter). These are typically tethered to flagellar filaments that have not been fully sheared or rotate in a plane that is inclined to the focal plane. Sampling of such beads will typically result in higher noise, and time-variations in viscous loads may result in an underestimation of motors torques for a given bead-size. A small fraction of tethered beads will undergo random motion; these are merely undergoing Brownian rotation. A fraction of the beads will appear blurred and cannot be brought in focus easily. These are most likely to be motors which have been tethered appropriately and are the motors of interest.
Among the limitations of single-motor tracking such as the one described here is the inability to conduct high-throughput experiments. A high-speed camera that images a larger region of interest can be advantageous in this regard. Other limitations include errors associated with multiple signals arising out of closely spaced rotating beads in the field of view of the PMTs. Finally, errors in determination of the correct position of the recorded bead with respect to the two photomultiplier slits will result in imprecise estimation of the switching dynamics.
Advantages of the setup described here include the ability to track the rotation of the beads over long durations and in real-time. This enables rapid elimination of error-prone trajectories, something that maybe difficult to achieve with ultrafast cameras. Additionally, with a few modifications this setup can be integrated with assays designed to subject cells to a variety of stimuli. Combined with thermoelectric cooling 26, the technique can be employed to measure the responses of individual motors to thermal stimuli. Integration with optical tweezers can enable the measurements of remodeling of individual motors in response to mechanical stimuli, as has been done recently 6. Finally, the adaptation of the motor to chemical stimulants can be measured with the use of an appropriate perfusion chamber and pumps 11.
A majority of known bacterial species are motile and flagellar-mediated motility is predominant in nature. The methods demonstrated here are expected to continue to aid in the development of insights into structural-remodeling and the adaptability of the flagellar motor.

Flagellar motors enable cells to swim by rotating helical extracellular filaments. The amount of torque the motor can generate for a given length of the flagellum (i.e., the viscous load) determines the swimming speeds. On the other hand, its ability to switch the direction of rotation controls cell migration in response to chemicals, a process known as chemotaxis. Chemotaxis and motility being virulence factors 1-3, flagellar motors have been well-characterized over the years 4. Mounting evidence now suggests that the motor acts as a mechanosensor &#8212; it mechanically detects the presence of solid substrates 5,6. This ability likely helps in triggering surface colonization and infections 5,7. As a result, the mechanisms whereby the motor senses surfaces and initiates signaling are of significance 8,9.
The flagellar motor can be readily studied by tethering the flagellum to a substrate and observing cell rotation. Such tethering was first achieved by Silverman and Simon, who worked with a polyhook mutant in E. coli and successfully attached hooks to glass substrates with anti-hook antibodies 10. The tethered-cell assay enabled researchers to study the responses of the motor-switch to a variety of chemical stimuli. For example, Segall and co-workers chemically stimulated tethered cells with the aid of iontophoretic pipettes. The corresponding changes in CWbias (the fraction of the time motors spin clockwise, CW) enabled them to measure the kinetics of adaptation in the chemotaxis network 11,12. While the tethered cell assay was effective in studying switch responses, it was only able to offer insights into motor mechanics over a limited range of viscous loads 13. To overcome this problem, Ryu and co-workers tethered spherical, latex beads to filament stubs on cells stuck to surfaces. The beads were then tracked using back-focal interferometry with weak optical traps 14. By working with beads of different sizes, researchers could study the motor over a much wider range of loads. This assay was later improved by Yuan and Berg, who developed a photomultiplier-based bead-tracking technique combined with laser dark-field illumination. Their method enabled tracking of tethered gold nanobeads that were so tiny (~ 60 nm) that the external viscous resistances were lower compared to the internal viscous resistances to rotation 15,16. This led to the measurements of the maximum achievable speeds in E. coli (~ 300 Hz). In V. alginolyticus, similar bead assays enabled measurements of the spinning rates at intermediate viscous loads (~ 700 Hz) 17. By enabling measurements of motor responses over the entire possible range of viscous loads (from zero-load to near-stall), the bead-assays provided an important biophysical tool to understand the torque-generation process 18,19.
Recently, we modified the Yuan-Berg assay to include optical tweezers that enabled us to apply precise mechanical stimuli to individual motors 6. Using this technique, we showed that the force-generators that rotate the motor are dynamic mechanosensors &#8212; they remodel in response to changes in viscous loads. It is possible that such load-sensing triggers cell differentiation into swarming bacteria, although the mechanisms remain unclear. It is also likely that the flagellar motors in other species are also mechanosensitive 20, although direct evidence is lacking. Here, we discuss the photomultiplier-based (PMT) approach for tracking the rotation of latex beads tethered to flagellar filaments 15. In comparison to tracking with ultrafast cameras, the photomultiplier-setup is advantageous because it is relatively straightforward to track single beads in real-time and over long durations. It is particularly useful when studying long-time remodeling in flagellar motor complexes due to environmental stimuli 21. Though we detail protocols specifically for E. coli, they can be readily adapted for studying flagellar motors in other species.

<table><tbody><tr><td>Poly-L-lysine Solution (0.1%)</td><td>Sigma-Aldrich</td><td>P8920</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en&amp;amp;region=US</td></tr><tr><td>Polybead Microspheres</td><td>Polysciences, Inc.</td><td>7307</td><td>http://www.sigmaaldrich.com/catalog/product/sigma/p8920?lang=en&amp;amp;region=US</td></tr><tr><td>1 mL Luer Slip Tip Syringe</td><td>Exel Int.</td><td>26048</td><td>http://www.exelint.com/tuberculin_syringes.php</td></tr><tr><td>Clay Adams Intramedic Luer-Stub Adapter 23-gauge</td><td>Becton, Dickinson and Company</td><td>427565</td><td>http://www.bd.com/ds/productCenter/ES-LuerStubAdaptors.asp</td></tr><tr><td>Polyethylene tubing</td><td>Harvard Apparatus</td><td>59-8325</td><td>http://www.harvardapparatus.com/laboratory-polye-polyethylene-non-sterile-tubing.html</td></tr><tr><td>Photomultiplier Tubes</td><td>Hamamatsu</td><td>R7400U-20</td><td>Spectral response range of 300 to 920 nm, Peak wavelength 630 nm,&amp;nbsp; 0.78 ns response time&amp;nbsp;&lt;br/&gt; http://pdf1.alldatasheet.com/datasheet-pdf/view/212308/HAMAMATSU/R7400U-20.html</td></tr><tr><td>3 x 1 mm precision slits</td><td>Edmund Optics</td><td>NT39-908</td><td>2 slits mounted at right angles to one another on photomultiplier tubes</td></tr><tr><td>Oscilloscope</td><td>Tektronix</td><td>TBS 1032B</td><td>Alternative brands are acceptable. Digital Oscilloscope, TBS 1000B Series, 2 Analogue, 30 MHz, 500 MSPS, 2.5 kpts&amp;nbsp;&lt;br/&gt; http://www.tek.com/oscilloscope/tbs1000b-digital-storage-oscilloscope</td></tr><tr><td>8 Pole LP/HP Filter</td><td>Krohn-Hite</td><td>3384</td><td>Alternative brands are acceptable. A frequency range from 0.1 Hz to 200 kHz is recommended.&amp;nbsp;&amp;nbsp;&amp;nbsp;&lt;br/&gt; http://www.krohn-hite.com/htm/filters/PDF/3384Data.pdf</td></tr><tr><td>Optiphot microscope</td><td>Nikon</td><td>NA</td><td>Any upright or inverted phase microscope can be used.&lt;br/&gt; https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=754</td></tr><tr><td>50:50 (R:T) Cube Beamsplitter</td><td>ThorLabs</td><td>BS013</td><td></td></tr></tbody></table>

biophysical characterization of flagellar motor functions

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel in response to a variety of environmental stimuli and are among the triggers for surface colonization and infections. The precise mechanisms by which motors remodel and promote cellular adaptation likely depend on key motor attributes. The photomultiplier-based bead-tracking technique presented here enables accurate biophysical characterization of motor functions, including adaptations in motor speeds and switch-dynamics. This approach offers the advantage of real-time tracking and the ability to probe motor behavior over extended durations. The protocols discussed can be readily extended to study flagellar motors in a variety of bacterial species.

The photomultiplier setup is shown in Figure 1A. It is important that the PMTs have high sensitivities over the range of wavelengths scattered by the beads of interest. The PMTs employed here operate in the visible and near-infrared ranges, and were able to detect light scattered by beads illuminated by a halogen light source. The optimum lighting conditions and supply voltages will vary from one setup to another. For the setup used in this work, a PMT gain ~ 104 - 105 proved adequate. Each photomultiplier was covered except for a 3 x 1 mm slit positioned in front of the photomultiplier. The slits limit the region in the cell-sample from which light can enter the photomultipliers, and the two slits are orthogonal to one another. When a rotating bead is positioned at the correct location (Figure 1B), the amount of light entering the photomultiplier increases as the bead comes in the view and decreases as its circular path takes it away from the view. The frequencies of the sinusoidal PMT voltage outputs indicate the speed of rotation and the phase differences between the two signals indicate the direction of rotation. The use of an oscilloscope to display the PMT outputs enables visualization of bead trajectories in real-time.
The time-varying PMT signals, y(t) and x(t), from a representative motor are shown in Figure 2A. The orthogonality of the two slits introduces a phase lag between the two signals. The signal amplitudes depend on the signal-to-noise ratio as well as the eccentricity of rotation. The corresponding trajectories of the bead are indicated in Figure 2B.
A histogram of the speeds measured from a representative motor in a cheY- deleted strain is shown in Figure 3A. The deletion ensures that flagellar motors cannot switch and rotate exclusively counterclockwise (CCW). The average speed from this particular motor was 60 Hz and the measured speeds are consistent with previous reports for motors tethered with 1 &#181;m beads 25. The bead was first positioned at the lower right corner, as seen in the schematic in Figure 1B. The corresponding angular speed is shown in Figure 3B (top panel). Positioning the bead to the adjacent lower left corner resulted in inversion of the sign on the motor speeds (bottom panel). Thus, moving the bead to an adjacent corner will change the observed direction of motor rotation. In this regard, diagonally opposite corners are identical. It is therefore crucial to know the location of the bead during measurements to correctly determine the switching dynamics. Figure 3C shows repeated transitions of a wild-type motor between the two directions of rotation.
Custom codes for data-acquisition software were adapted from prior work to record the data on a computer 15. The PMT output was AC-coupled and low-pass filtered with a cutoff frequency of 100 Hz. Real-time tracking was enabled by connecting the filtered outputs to an oscilloscope.
<img alt="Figure 1" src="/files/ftp_upload/55240/55240fig1.jpg" /> 
Figure 1: Bead-tracker Setup. A) Schematic of the PMT-based tracking setup. B) The ideal position of the bead (black sphere) relative to the two orthogonal slits. The trajectory is indicated by the dotted lines. The eccentricity e is the radius of the dotted circle. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55240/55240fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55240/55240fig2.jpg" /> 
Figure 2: PMT Outputs. A) Low-pass filtered outputs from the two photomultipliers, after centering/scaling. B) The bead trajectories obtained from the PMT data, sampled over 3 s. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55240/55240fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55240/55240fig3.jpg" /> 
Figure 3: Bead Trajectories. A) Histogram of CCW-only speeds of a representative motor. B) Rotational speeds of a CCW-only motor imaged at lower right corner (top panel). Rotational speeds of the same motor when positioned at the lower left corner (bottom panel). C) Switching in a representative wild-type motor. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55240/55240fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Biophysical Characterization of Flagellar Motor Functions at JoVE.com

Biophysical Characterization of Flagellar Motor Functions

Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

The role of flagellar motors in bacterial motility and chemotaxis is well-understood. Recent discoveries suggest that flagellar motors are able to remodel ...

biophysical-characterization-of-flagellar-motor-functions

Nanyang Technological University

Research

JoVE Journal

Biology

8.2K Views.  Texas A&M University. Recent findings suggest that bacterial flagellar motors sense a variety of environmental signals and remodel in response. The bead-assays discussed here are expected to help explain the role of remodeling in cellular adaptation to environmental stressors.

Video: Biophysical Characterization of Flagellar Motor Functions

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

Biochemistry

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 ...

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of catalytic motor domains, located at opposite ends of the active complex. This unique architecture enables kinesin-5 motors to crosslink and slide apart antiparallel spindle microtubules (MTs), thus providing the outwardly-directed force that separates the spindle poles apart. Previously, kinesin-5 motors were believed to be exclusively plus-end directed. However, recent studies revealed that several fungal kinesin-5 motors are minus-end directed at the single-molecule level and can switch directionality under various experimental conditions. The Saccharomyces cerevisiae kinesin-5 Cin8 is an example of such bi-directional motor protein: in high ionic strength conditions single molecules of Cin8 move in the minus-end direction of the MTs. It was also shown that Cin8 forms motile clusters, predominantly at the minus-end of the MTs, and such clustering allows Cin8 to switch directionality and undergo slow, plus-end directed motility. This article provides a detailed protocol for all steps of working with GFP-tagged kinesin-5 Cin8, from protein overexpression in S. cerevisiae cells and its purification to in vitro single-molecule motility assay. A newly developed method described here helps to differentiate between single molecules and clusters of Cin8, based on their fluorescence intensity. This method enables separate analysis of motility of single molecules and clusters of Cin8, thus providing the characterization of the dependence of Cin8 motility on its cluster size.

Motility of Single Molecules and Clusters of Bi-Directional Kinesin-5 Cin8 Purified from S. cerevisiae Cells

The bi-directional mitotic kinesin-5 Cin8 accumulates in clusters that split and merge during their motility. Accumulation in clusters also changes the velocity and directionality of Cin8. Here, a protocol for motility assays with purified Cin8-GFP and analysis of motile properties of single molecules and clusters of Cin8 is described.

The mitotic bipolar kinesin-5 motors perform essential functions in spindle dynamics. These motors exhibit a homo-tetrameric structure with two pairs of ...

Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative analysis of motor/cargo associations at the single vesicle level. The goal of this protocol is to use quantitative fluorescence microscopy to correlate (&#8220;map&#8221;) the position and directionality of movement of live cargo to the composition and relative amounts of motors associated with the same cargo. &#8220;Cargo mapping&#8221; consists of live imaging of fluorescently labeled cargoes moving in axons cultured on microfluidic devices, followed by chemical fixation during recording of live movement, and subsequent immunofluorescence (IF) staining of the exact same axonal regions with antibodies against motors. Colocalization between cargoes and their associated motors is assessed by assigning sub-pixel position coordinates to motor and cargo channels, by fitting Gaussian functions to the diffraction-limited point spread functions representing individual fluorescent point sources. Fixed cargo and motor images are subsequently superimposed to plots of cargo movement, to &#8220;map&#8221; them to their tracked trajectories. The strength of this protocol is the combination of live and IF data to record both the transport of vesicular cargoes in live cells and to determine the motors associated to these exact same vesicles. This technique overcomes previous challenges that use biochemical methods to determine the average motor composition of purified heterogeneous bulk vesicle populations, as these methods do not reveal compositions on single moving cargoes. Furthermore, this protocol can be adapted for the analysis of other transport and/or trafficking pathways in other cell types to correlate the movement of individual intracellular structures with their protein composition. Limitations of this protocol are the relatively low throughput due to low transfection efficiencies of cultured primary neurons and a limited field of view available for high-resolution imaging. Future applications could include methods to increase the number of neurons expressing fluorescently labeled cargoes.

Intracellular transport of cargoes, such as vesicles or organelles, is carried out by molecular motor proteins that track on polarized microtubules. This protocol describes the correlation of the directionality of transport of individual cargo particles moving inside neurons, to the relative amount and type of associated motor proteins.

Understanding the mechanisms by which molecular motors coordinate their activities to transport vesicular cargoes within neurons requires the quantitative ...

Neuroscience

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 ...

Probing Myosin Ensemble Mechanics in Actin Filament Bundles Using Optical Tweezers

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle contraction. To understand their force-generating mechanisms, myosin II has been investigated both at the single-molecule (SM) level and as teams of motors in vitro using biophysical methods such as optical trapping.
These studies showed that myosin force-generating behavior can differ greatly when moving from the single-molecule level in a three-bead arrangement to groups of motors working together on a rigid bead or coverslip surface in a gliding arrangement. However, these assay constructions do not permit evaluating the group dynamics of myosin within viscoelastic structural hierarchy as they would within a cell. We have developed a method using optical tweezers to investigate the mechanics of force generation by myosin ensembles interacting with multiple actin filaments.
These actomyosin bundles facilitate investigation in a hierarchical and compliant environment that captures motor communication and ensemble force output. The customizable nature of the assay allows for altering experimental conditions to understand how modifications to the myosin ensemble, actin filament bundle, or the surrounding environment result in differing force outputs.

Formation of actomyosin bundles in vitro and measuring myosin ensemble force generation using optical tweezers is presented and discussed.

Myosins are motor proteins that hydrolyze ATP to step along actin filament (AF) tracks and are essential in cellular processes such as motility and muscle ...

Bioengineering

Generation of Null Mutants to Elucidate the Role of Bacterial Glycosyltransferases in Bacterial Motility

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans constitute a strain-specific barcode. The associated glycans show higher diversity in sugar composition and structure than those of eukaryotes and are important in bacterial-host recognition processes and interaction with the environment. In pathogenic bacteria, glycoproteins have been involved in different stages of the infectious process, and glycan modifications can interfere with specific functions of glycoproteins. However, despite the advances made in the understanding of glycan composition, structure, and biosynthesis pathways, understanding of the role of glycoproteins in pathogenicity or interaction with the environment remains very limited. Furthermore, in some bacteria, the enzymes required for protein glycosylation are shared with other polysaccharide biosynthetic pathways, such as lipopolysaccharide and capsule biosynthetic pathways. The functional importance of glycosylation has been elucidated in several bacteria through mutation of specific genes thought to be involved in the glycosylation process and the study of its impact on the expression of the target glycoprotein and the modifying glycan. Mesophilic Aeromonas have a single and O-glycosylated polar flagellum. Flagellar glycans show diversity in carbohydrate composition and chain length between Aeromonas strains. However, all strains analyzed to date show a pseudaminic acid derivative as the linking sugar that modifies serine or threonine residues. The pseudaminic acid derivative is required for polar flagella assembly, and its loss has an impact on adhesion, biofilm formation, and colonization. The protocol detailed in this article describes how the construction of null mutants can be used to understand the involvement of genes or genome regions containing putative glycosyltransferases in the biosynthesis of a flagellar glycan. This includes the potential to understand the function of the glycosyltransferases involved and the role of the glycan. This will be achieved by comparing the glycan deficient mutant to the wild-type strain.

Here, we describe the construction of null mutants of Aeromonas in specific glycosyltransferases or regions containing glycosyltransferases, the motility assays, and flagella purification performed to establish the involvement and function of their encoded enzymes in the biosynthesis of a glycan, as well as the role of this glycan in bacterial pathogenesis.

The study of glycosylation in prokaryotes is a rapidly growing area. Bacteria harbor different glycosylated structures on their surface whose glycans ...

Immunology and Infection

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

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 ...

Visualizing Bacterial Motility Based on a Color Reaction

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or dissemination of many pathogenic species. Therefore, it is important to detect bacterial motility. Bacterial growth conditions, such as oxygen, pH, and temperature, can affect bacterial growth and the expression of bacterial flagella. This can lead to reduced motility or even loss of motility, resulting in the inaccurate evaluation of bacterial motility. Based on the color reaction of 2,3,5-triphenyl tetrazolium chloride (TTC) by intracellular dehydrogenases of living bacteria, TTC was added to traditional semisolid agar for bacterial motility detection. The results showed that this TTC semisolid agar method for the detection of bacterial motility is simple, easy to operate, and does not involve large and expensive instruments. The results also showed that the highest motility was observed in semisolid medium prepared with 0.3% agar. Compared with the traditional semisolid medium, the results are easier to evaluate and more accurate.

Here, we present a protocol to detect bacterial motility based on a color reaction. Key advantages of this method are that it is easy to evaluate and more accurate, and does not require specialized equipment.

Bacterial motility is crucial for bacterial pathogenicity, biofilm formation, and drug resistance. Bacterial motility is crucial for the invasion and/or ...

Biophysical Characterization of Flagellar Motor Functions

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