We thank Ge Yang, Gaudenz Danuser, Khuloud Jaqaman, and Daniel Whisler for assistance with the adaptation and development of the software to quantitate cargo mapping analyses, and Emily Niederst for help in making the microfluidic devices. This work was supported in part by NIH-NIA grant AG032180 to L.S.B.G., and the Howard Hughes Medical Institute. L.S. was supported in part by a NIH Bioinformatics Training Grant T32 GM008806, S.E.E. was supported by a Damon Runyon Cancer Research Foundation Fellowship, NIH Neuroplasticity Training Grant AG000216, and by a grant from The Ellison Medical Foundation New Scholar in Aging Award, G.E.C was supported by an NIH/NCATS&#160;1 TL1 award TR001114 and by the Achievement Rewards for College Scientists foundation.

All experiments were conducted following approved protocols and according to institutional guidelines for the humane care of research animals. Neonate mice were euthanized by decapitation.
1. Preparation of Microfluidic Devices for Cell Culture
<ol>
	<li>Prepare polydimethyl siloxane (PDMS) microfluidic devices for growth of hippocampal neurons as described by Harris and colleagues17-19. Below are some modifications that were adapted to the cargo mapping protocol. 
	NOTE: Microfluidic devices are also commercially available (Materials List), thus access to a fabrication facility is not necessary.</li>
	<li>Prepare No. 1&#189; cover glasses (24 x 40 mm) by washing them three times with acetone, followed by three washes with 100% ethanol and three washes of water. Store coverslips in water at 4 &#176;C until use. These treatments help ensure that coverslips are clean from debris that might interfere with plating of cells, contamination, and survival. 
	NOTE: Acetone and ethanol are flammable and hazardous in case of skin contact (irritant), of eye contact (irritant), of ingestion, of inhalation. Dispose according to official regulations.</li>
	<li>When ready to use, place one cover glass in a 60 mm cell culture dish per microfluidic device, and let it air dry uncovered for 45 min inside a biosafety cabinet to avoid contamination.</li>
	<li>After devices are cut out from the masters and punches have been cut to create the reservoirs (Figure 1A, B), place them with the microfluidic channels side-up inside a biosafety cabinet equipped with an UV lamp for 45 min for sterilization. 
	NOTE: Leaving devices under UV treatment for longer than 2 hr might affect the integrity of the PDMS.</li>
	<li>Assemble the devices by placing one device onto each cover glass inside the 60 mm plastic cell culture dish so that the microfluidic channels face down. Apply gentle pressure to the top of the device (avoid touching the microchannels) to provide a non-permanent seal of the device to the cover glass. Cover each 60 mm cell culture dish with a lid.</li>
	<li>Using a micropipette, hydrate the devices by adding cell culture grade water to the top two microfluidic reservoirs (1 and 2 in Figure 1A), allowing water to flow through. Remove water with a micropipette and add 1 mg/ml poly-L-lysine into microfluidic reservoir 1 (200 &#181;l) and 2 (100 &#181;l). The volume differential will allow for the poly-L-lysine to flow through the microchannels (Figure 1A, B).</li>
	<li>To ensure that the devices inside the 60 mm cell culture dishes do not topple over inside a 37 &#176;C incubator, place three 60 mm cell culture dishes containing the devices into a 150 mm plastic cell culture dish and cover the dish with a lid. Incubate O/N in a 37 &#176;C incubator with 5% CO2. Typically more than 90% of the channels are usable per device and do not contain any air bubbles or physical blockage.</li>
	<li>On the next day, replace poly-L-lysine with water and leave device in incubator for at least 1 hr. Repeat this washing three times. Remove water and add Neurobasal-A growth medium (containing 2% B-27 supplement and 500 &#181;M GlutaMAX) to each device. Leave O/N in a 37 &#176;C incubator with 5% CO2.</li>
	<li>On the next day, plate hippocampal cells as described below.</li>
</ol>
2. Plating of Mouse Hippocampal Primary Neurons in Microfluidic Devices
NOTE: The preparation of cultured hippocampal neurons from neonate rats has been previously published in JoVE25. Below are some modifications that were adapted to plate mouse hippocampal neurons in microfluidic devices, and that were optimal for transfections.
<ol>
	<li>Before mouse brain dissection, pre-warm Dulbecco&#8217;s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS; without antibiotics), Mixture A (125 mg DL-cysteine, 125 mg bovine serum albumin (BSA) and 3.125 g of D-glucose in 500 ml of phosphate buffered saline (PBS); filter sterilize through a 0.22 &#181;m filter), and deoxyribonuclease I solution (DNase I: 50 mg of DNase I and 0.5 ml of a 1.2 M MgSO4 solution in 100 ml Hank&#8217;s Balanced Salt Solution (HBSS) filter sterilize through a 0.22 &#181;m filter) in a 37 &#176;C water bath.
	<ol>
		<li>Pre-warm Neurobasal-A growth medium inside a 37 &#176;C incubator with 5% CO2 to equilibrate the pH.</li>
	</ol>
	</li>
	<li>Dissect hippocampi from 1-3 day old neonate mice according to established protocols26. Keep hippocampi in a 15 ml conical tube containing 10 ml dissection buffer (HBSS containing 10 mM HEPES pH 7.4, 50 mM glucose, 100 U/ml penicillin and 100 &#181;g/ml streptomycin), on ice. Put up to 4 hippocampi per 15 ml conical tube.</li>
	<li>Perform the remaining protocol is under sterile conditions inside a biosafety cabinet.</li>
	<li>Dilute 45 units of papain in 5 ml of Mixture A, vortex, and incubate for 5 min at 37 &#176;C until papain powder is dissolved. Add 1 ml of DNase I solution. Filter the mix through a 0.22 &#181;m syringe filter unit.</li>
	<li>Carefully aspirate HBSS&#8217; from 15 ml conical tube containing hippocampi. Add 10 ml cold (4 &#176;C) HBSS to hippocampi and let them settle to the bottom of the tube. Carefully aspirate HBSS from 15 ml conical tube.</li>
	<li>Add 1 ml papain/DNase I mix to up to 4 hippocampi and incubate for 20-30 min at 37 &#176;C. Tilt conical tube every 5 min to bathe hippocampi with mix. Alternatively place hippocampi in a 37 &#176;C water bath shaker with gentle shaking (~100 rpm).</li>
	<li>Aspirate papain/DNase I mix and wash hippocampi twice with pre-warmed DMEM (37 &#176;C) containing 10% FBS.</li>
	<li>After second wash, add 2 ml of pre-warmed DMEM containing 10% FBS to hippocampi and manually triturate hippocampi by pipetting up and down ~10-12 times using a 1 ml pipette tip to dissociate neurons.</li>
	<li>Let triturate settle for ~1 min as this will allow non-dissociated neurons to settle to the bottom of the conical tube. Transfer supernatant containing dissociated neurons to a new 15 ml conical tube avoiding transfer of larger tissue that has settled at the bottom of the tube.</li>
	<li>Spin cells at 1,000 x g for 2 min and carefully remove supernatant without disturbing the cell pellet. Resuspend cells in 80 &#956;l Neurobasal-A growth medium by pipetting gently.</li>
	<li>Remove medium from microfluidic reservoir 1 and 1&#8217;. Apply 20 &#181;l of cells (~275,000) to microfluidic reservoir 1, and allow them to flow through. Place device in 37 &#176;C incubator with 5% CO2 for 20 min.</li>
	<li>Look under microscope to ensure that cells have adhered to the cover glass. Add Neurobasal-A growth medium to top off all reservoirs. Return device with cells to a 37 &#176;C incubator with 5% CO2.</li>
	<li>Check devices every ~2-3 days and top off reservoirs with Neurobasal-A containing 2% B-27 supplement (without GlutaMAX) to avoid evaporation. Neurons start to extend their axons through microfluidic channels ~2-3 days after plating. Subject neurons to transfection typically 7-10 days after plating.</li>
</ol>
3. Transfection of Hippocampal Neurons Grown in Microfluidic Device
<ol>
	<li>Per device, prepare a mix of 1.2 &#181;l Lipofectamine 2000 in 30 &#181;l Neurobasal-A, and a mix of 0.5 &#181;g fluorescent cargo fusion plasmid in 30 &#181;l Neurobasal-A and incubate 5 min at RT. Add Lipofectamine mix to DNA mix and incubate for 20 min at RT. Add 60 &#181;l of Neurobasal-A containing 4% B-27 supplement to the DNA/Lipofectamine mix and mix by flicking the tube.</li>
	<li>Remove all media from microfluidic reservoir 2 and 2&#8217; and carefully fill up wells with Neurobasal-A containing 2% B-27 supplement without spilling over the medium into the other compartments.</li>
	<li>Remove media from microfluidic reservoir 1 and 1&#8217;. Add 120 &#181;l of DNA/Lipofectamine mix to microfluidic reservoir 1 and let it flow. Return device to 37 &#176;C incubator with 5% CO2 for 3-4 hr.</li>
	<li>Remove all media from microfluidic reservoir 1 and 1&#8217; and replace with Neurobasal-A containing 2% B-27 supplement for 24 hr (or until ready to image). Typically, 5-7 axons that grow through microchannels are transfected under those conditions, which represent approximately 1-3% transfection efficiency consistent with efficiencies published for hippocampal cultured cells 26.</li>
</ol>
4. &#8220;Cargo Mapping&#8221; Analyses
<ol>
	<li>Live Imaging of cargo movement
	<ol>
		<li>Perform the imaging on an inverted epifluorescence light microscope equipped with a 37 &#176;C incubator and a CO2 control chamber.</li>
		<li>Mount the coverslip with hippocampal neurons grown in a microfluidic device onto the microscope stage. To avoid neuronal death, work expeditiously to maintain the samples at the microscope for no longer than 1 hr.</li>
		<li>Using a high-resolution objective (100X), find axons of transfected cells that extend through channels of microfluidic chambers and record the number of the channels in which the transfected axon was found. Count the number of channels using a hand tally counter.
		<ol>
			<li>For optimal recording of live movement and subsequent colocalization analysis, choose axons that grow relatively flat through the channels so that imaging of most vesicles can be performed in focus.</li>
		</ol>
		</li>
		<li>Remove most of the medium from reservoirs 2 and 2&#8217; with a 1 ml plastic transfer pipette. To facilitate subsequent alignment of fixed images with movement trajectories on kymographs, align the right edge of the microchannels with the field of view during imaging (Figure 1B, C).</li>
		<li>To facilitate fixing of the samples during imaging, perform live imaging with two people. Alternatively, if live imaging is performed by a single person, use a microscope system equipped with drift correction.</li>
		<li>Start imaging live cargo movement with time-lapse specifications specific to the transport dynamics of the cargo being analyzed (microscope specifications and settings for imaging YFP-PrPC are specified in the legend of Figure 2 and Materials List).</li>
		<li>After sufficient live movement data have been collected, have one person fill up reservoirs 2 and 2&#8217; with pre-warmed 4% paraformaldehyde in PBS containing 0.04 g/ml sucrose to fix the cells. Avoid touching the microfluidic device, as this will disrupt the focus of the live imaging. Have the other person adjust the focus while the fixative is added. Fixation is successful when immediate stop of movement is observed. Add fixative to reservoir 1 and 1&#39; afterwards. 
		NOTE: Temporary photobleaching of the cargo fluorescence might also be observed, but the fluorescence persists after IF staining for motor proteins is completed (next step). 
		NOTE: Paraformaldehyde is harmful by inhalation, in contact with skin, and if swallowed. Irritating to eyes, respiratory system and skin. Dispose according to official regulations.</li>
	</ol>
	</li>
	<li>IF staining of motor proteins for colocalization analysis 
	NOTE: For quantitation of relative levels of motor proteins (as determined by antibody staining), associated to individual moving cargoes, validate and titrate antibodies to ensure that the antibody-antigen binding is saturated. This is done by determining the specificity of the antibody using neurons in which the protein of interest has been either knocked-out or knocked-down, and by performing IF analysis with increasing antibody concentrations. For negative controls, neurons are stained with secondary antibodies in the absence of primary antibodies. More detailed description of antibody validation can be found in Encalada et al.16, and Szpankowski et al.20.
	<ol>
		<li>Remove the coverslip with hippocampal neurons grown in microfluidic device from the microscope stage. Do not detach the microfluidic device from the coverslip, as the microchannels need to remain intact in order to superimpose the live and fixed images. Perform the following steps in a humidity chamber.</li>
		<li>To ensure the flow of solutions into the microchannels, maintain a buffer volume differential between reservoirs 1, 1&#8217;, and 2, 2&#8217; of at least 30 &#181;l. in all subsequent steps. For example, add 100 &#181;l media volume to microfluidic reservoir 2, 2&#8217;, and 70 &#181;l media volume to microfluidic reservoir 1, 1&#8217;.</li>
		<li>Remove fixative from chambers of the microfluidic device and wash cells three times with PBS waiting 10 min between washes.</li>
		<li>Permeabilize cells by adding 0.1% Triton X-100 diluted in PBS for 5 min to all reservoirs.</li>
		<li>Remove solution from all reservoirs and wash with PBS. Repeat washing three times waiting 10 min between washes.</li>
		<li>Remove solution from all reservoirs and apply blocking buffer containing 10% Normal Donkey Serum and 3% protease free and immunoglobulin G (IgG)-free BSA in PBS and incubate for at least 30 min at RT.</li>
		<li>Spin primary antibody stock solution at 20,000 x g for 5 min to remove precipitates. Dilute antibody to an appropriate concentration in blocking buffer. Replace the blocking buffer from microfluidic device with antibody solution. Incubate samples for 2 hr at RT or O/N at 4 &#176;C.</li>
		<li>Remove solution from all reservoirs and wash three times with PBS, waiting 10 min between washes.</li>
		<li>Spin secondary antibody stock solution at 20,000 x g for 5 min to remove precipitates. Dilute antibody to an appropriate concentration in blocking buffer. Replace PBS with secondary antibody solution. Incubate samples for 1 hr at RT.</li>
		<li>Remove solution from all reservoirs and wash three times with PBS, waiting 10 min between washes.</li>
		<li>Replace PBS with ~10-20 &#181;l mounting medium by directly pipetting mounting medium into the opening of the channels connecting the reservoirs. Let mounting media dry for ~20 min &#8211; 1 hr at RT.</li>
		<li>Store devices at 4 &#176;C protected from light to avoid photobleaching, and image axons within two days (maximum one week) of staining.</li>
		<li>After staining is completed, mount coverslip with hippocampal neurons grown in microfluidic device onto the microscope stage. Locate the region of the transfected axon imaged in step 4.1.1-4.1.7.</li>
		<li>Acquire Z-stack images (300 nm steps) for each of the three channels: cargo, motor 1, motor 2, using a high-resolution objective (e.g., a high numerical aperture oil or water objective). 
		NOTE: Acquisition of Z-stacks is important as axons often do not grow perfectly flat in microchannels and therefore not all fluorescent puncta are in the same focal plane.</li>
	</ol>
	</li>
	<li>Cargo Mapping
	<ol>
		<li>Generate a kymograph of cargo movement using image analysis software. The ImageJ plugin developed by Rietdorf and Seitz (EMBL) is recommended for generating kymographs (for download see Materials List). 
		NOTE: ImageJ is a public domain, Java-based image processing program developed at the National Institutes of Health27,28 (for download see Materials List).</li>
		<li>Align the fixed images of the fluorescent cargo (generated in step 4.2.14) manually by superimposing them onto the position of the trajectories in the kymograph at the point of fixation (Figure 2A, B), using a commercially available graphing software program (Materials List). Manually align by first superimposing the fixed cargo image onto the kymograph and manually choosing the cargo puncta that corresponds to a specific trajectory on the kymograph. For best alignment results, use the Z slice that is in best focus for the cargo mapped. Several Z slices may be used.</li>
		<li>For each image in the Z-stack (cargo, motor 1 and motor 2; generated in step 4.2.14) determine the X-Y coordinates and intensity amplitudes for each fluorescent puncta using an established algorithm to fit 2D Gaussians to the point spread function that represents each point source in each of the three channels16,20,21 (Figure 2D). 
		NOTE: To obtain the X-Y coordinates, a custom built MATLAB software package called &#8220;Motor Colocalization&#8221; was developed16,20, which incorporates a Gaussian fitting algorithm developed by Jaqaman, Danuser and colleagues21-23. The software package and detailed instructions for its use can be obtained upon request.</li>
		<li>For each of the individually cargo puncta that were mapped onto the mobile or stationary trajectories on the kymograph, select the X-Y coordinates and intensity amplitudes from the results of the &#8220;Motor Colocalization&#8221; program (these cargoes are individually labeled in Figure 2B). Use several Z-stack images acquired in step 4.2.14) to map more cargoes that are found on different focal planes.</li>
		<li>Compare the individual X-Y coordinates for the mapped cargo to those obtained for each of the motor channels in the corresponding Z slice (Figure 2D). Determine and select the X-Y motor coordinates that are within a 300 nm radius of the cargo. For cargoes and motors that have signal within the same focal plane, use the &#8220;Motor Colocalization&#8221; software package to determine the puncta that are located within a 300 nm radius.</li>
	</ol>
	</li>
</ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ImageJ+for+microscopy.">ImageJ for microscopy.</a> Biotechniques. 43, 25-30 (2007).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nat. Methods. 9, 671-675 (2012).</li><li>Moore, D. S., McCabe, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introduction to the Practice of Statistics. , (2005).</li></ol>

The authors have nothing to disclose.

The protocol presented here enables the correlation of directionality of movement of individual fluorescent microtubule-based moving cargo particles with the relative type and amount of associated motor proteins in live neurons. Previously, the total motor composition of axonal vesicular cargoes was assayed on heterogeneous populations of biochemically purified vesicles and organelles9,15. However, characterizing motor composition for a single type of cargo inside cells has been chal...

Intracellular transport is critical in all cell types for the delivery of proteins, membranes, organelles, and signaling molecules to various cellular domains1. Neurons are highly specialized cells with long, polarized projections that critically depend on intracellular transport of essential cargoes for their long-distance delivery to various axonal microdomains. This transport is mediated by kinesins and dyneins &#8212; two large families of molecular motor proteins &#8212; that bind to cargoes and track along polarized microtubules in anterograde and retrograde directions, respectively. While retrograde movement is mainly mediated by dynein, movement in the anterograde direction is facilitated by a large, functionally diverse family of kinesin motors. Consequently, anterograde transport of axonal cargoes could be mediated by various family members of the kinesin superfamily1-5. Though some cargoes move persistently in either direction, most cargoes move bidirectionally and reverse frequently on their way to their final destinations1,5-13. Furthermore, it has been shown that motors of opposing directionality associate simultaneously to cargoes, raising the question as to how regulated movement of cargoes is coordinated by opposite-polarity motors5-7. Together, transport of axonal cargoes is a concerted process that is regulated by the composition of motors and their specific biochemical activities, which in turn are dependent on various adaptors and regulatory binding partners14.
To faithfully describe the mechanism of axonal transport for a specific cargo and to uncover the underlying regulation of that transport, it is paramount to determine the composition of motor proteins and their regulatory binding partners associated with individual cargoes during their live transport. Other methods, for example biochemical approaches, provide estimates of average motor compositions on purified heterogeneous vesicle populations, but these estimates do not reveal the type or amounts of motors associated to single moving vesicles. Also, reconstitution of vesicle transport along preassembled microtubules in vitro enabled measuring the amount of one type of motor on a single vesicle level15. However, these experiments did not directly correlate the amount of motors with the transport characteristics of those vesicles, and measured transport in the absence of cellular regulatory factors.
A protocol is presented here, which determines the motor composition (type and relative amount of motors) of individual moving vesicles from immunofluorescence (IF) data measuring endogenously expressed motor proteins, and correlates these parameters to the live transport of the exact same vesicles in neurons16. This method entails precise mapping of IF-to-live cargo movement data. This is accomplished by growing hippocampal mouse neurons in microfluidic devices following established protocols17-19. These devices allow for the identification and correlation (&#8220;mapping&#8221;) of axons and single moving cargoes in fixed and live light microscopy modalities (Figure 1). Cultured neurons are transfected with fluorescently labeled cargo proteins whose transport is imaged at high spatial and temporal resolution to obtain detailed movement information that is plotted in kymographs. During the course of imaging, neurons are fixed with paraformaldehyde, and subsequently stained with antibodies against endogenous motor proteins. Fixed cargo and motor images are superimposed onto live movement kymographs to &#8220;map&#8221; (colocalize) them to the live cargo movement trajectories16. To correlate the live movement of cargoes with the association of motor proteins, colocalization is analyzed using a custom made MATLAB software package called &#8220;Motor Colocalization&#8221;16,20. Fluorescently labeled cargoes and motors generate diffraction-limited punctate features that can partially overlap. To resolve the position of overlapping puncta, the software first automatically fits Gaussian functions to each point spread function, representing individual fluorescent puncta, to determine their precise X-Y sub-pixel position coordinates and intensity amplitudes21-23. The positions of motors and cargoes are subsequently compared to each other to determine colocalization16,20. Therefore, this method more precisely assigns colocalization between fluorescent puncta as compared to other methods24.
The strength of this method is the ability to assess the colocalization of motors with individual cargo in fixed cells, for which live movement trajectories (e.g., the direction in which they were moving at the time of fixation) have been recorded. With this method, kinesins and dyneins were found to associate simultaneously to vesicles that carry the normal prion protein (PrPC-cellular), a neuronally enriched cargo that moves bidirectionally or remains stationary in axons16. This analysis allowed the formulation of a working model for the regulation of PrPC vesicle movement in which anterograde (kinesin) and retrograde (dynein) motors coordinate their activities in order to move the vesicles in either direction or to remain stationary while associated to the cargo. Another strength of this method is its potential broad applicability for characterizing colocalization/association of many fluorescently labeled cargoes that move in virtually any cell type, with any other protein(s) of interest. Thus, live/fixed correlation could potentially allow for the detection of transient protein-cargo interactions, as many individual fluorescently labeled moving particles can be analyzed over a desired period of time. Given the broad applicability and the type of questions that this method can address, this protocol will be of interest to a wide audience of cell biologists including those studying trafficking and transport in neurons or in other cell types.

<table><tbody><tr><td>poly-L-lysine</td><td>Sigma</td><td>P5899-20mg</td><td></td></tr><tr><td>D-MEM (Dulbecco&amp;rsquo;s Modified Eagle Medium) High Glucose, w/ L-Glutamine, w/o Sodium Pyruvate (1x)</td><td>Life Technologies</td><td>11965092</td><td></td></tr><tr><td>FBS (Fetal Bovine Serum)</td><td>Life Technologies</td><td>10082147</td><td></td></tr><tr><td>Neurobasal-A Medium (1x)</td><td>Life Technologies</td><td>10888022</td><td></td></tr><tr><td>B-27 Serum Free Supplement</td><td>Life Technologies</td><td>10888022</td><td></td></tr><tr><td>GlutaMAX I Supplement (100x)</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>HBSS (1x) (Hank&amp;rsquo;s Balanced Salt Solution)</td><td>Life Technologies</td><td>24020117</td><td></td></tr><tr><td>DPBS (Dulbecco&amp;rsquo;s Phosphate Buffered Saline, no Magnesium, no Calcium (1x)</td><td>Life Technologies</td><td>14190250</td><td></td></tr><tr><td>Penicillin/Streptomycin (100x)</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>Corning cellgro Water for Cell Culture&amp;nbsp;</td><td>Fisher Scientific</td><td>MT46000CM</td><td></td></tr><tr><td>Papain</td><td>USB Corporation</td><td>19925</td><td></td></tr><tr><td>DL-cysteine HCl</td><td>Sigma-Aldrich</td><td>C9768</td><td></td></tr><tr><td>BSA (bovine serum albumin)</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G6152</td><td></td></tr><tr><td>DNAse I grade II</td><td>Roche Applied Sciences</td><td>10104159001</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Life Technologies</td><td>&amp;nbsp;11668027</td><td></td></tr><tr><td>Formaldehyde Solution 16% EM Grade</td><td>Fisher Scientific</td><td>50980487</td><td>Caution: Harmful by inhalation, in contact with skin and if swallowed. Irritating to eyes, respiratory system and skin. Dispose according to official regulations.</td></tr><tr><td>Sucrose</td><td>Fisher Scientific</td><td>S5-500</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H-3375</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Jackson Immuno Research</td><td>017-000-0121</td><td></td></tr><tr><td>BSA fatty acid and IgG free</td><td>Jackson Immuno Research</td><td>001-000-162</td><td></td></tr><tr><td>Acetone</td><td>Fisher Scientific</td><td>BP2403-4</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818-100</td><td></td></tr><tr><td>ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36934</td><td></td></tr><tr><td>Cover Glass 1 1/2. 24 x 40 mm</td><td>Corning</td><td>2980-244</td><td></td></tr><tr><td>Axis microfluidic device, 450 &amp;micro;m</td><td>Millipore</td><td>AX450</td><td></td></tr><tr><td>Adobe Photoshop</td><td>Adobe</td><td></td><td></td></tr><tr><td>Nikon Eclipse TE2000-U&amp;nbsp;</td><td>Nikon</td><td></td><td></td></tr><tr><td>Coolsnap HQ camera&amp;nbsp;</td><td>Roper Scientific</td><td></td><td></td></tr><tr><td>60 mm cell culture dish</td><td>Fisher Scientific</td><td>12-565-95</td><td></td></tr><tr><td>150 mm cell culture dish</td><td>Fisher Scientific</td><td>12-565-100</td><td></td></tr><tr><td>Antibodies Used:</td><td></td><td></td><td></td></tr><tr><td>Anti-Kinesin light chain, V-17</td><td>Santa Cruz&amp;nbsp;</td><td>sc-13362</td><td>specificity verified using KLC1-/- neurons (Ref. 16), recommended dilution 1:100.</td></tr><tr><td>Anti Dynein Heavy Chain 1, R-325</td><td>Santa Cruz&amp;nbsp;</td><td>sc-9115</td><td>specificity verified using shRNA against DYN1HC1 (Ref. 16), recommended dilution 1:100</td></tr><tr><td>Alexa Fluor 568 Donkey Anti-Rabbit IgG Antibody</td><td>Life Technologies</td><td>A10042</td><td>&amp;nbsp;recommended dilution 1:200.</td></tr><tr><td>Alexa Fluor 647 Donkey Anti-Rabbit IgG (H+L) Antibody</td><td>Life Technologies</td><td>A31573</td><td>&amp;nbsp;recommended dilution 1:200.</td></tr><tr><td>Alexa Fluor 568 Donkey Anti-Goat IgG (H+L) Antibody IgG Antibody</td><td>Life Technologies</td><td>A11057</td><td>&amp;nbsp;recommended dilution 1:200.</td></tr><tr><td>Alexa Fluor 647 Donkey Anti-Goat IgG (H+L) Antibody</td><td>Life Technologies</td><td>A21447</td><td>&amp;nbsp;recommended dilution 1:200.</td></tr><tr><td colspan="4">Plugins and Macros</td></tr><tr><td>ImageJ</td><td></td><td></td><td>&lt;a href=&quot;http://imagej.nih.gov/ij/index.html&quot;&gt;&amp;nbsp;http://imagej.nih.gov/ij/index.html.&amp;nbsp;&lt;/a&gt;</td></tr><tr><td>ImageJ Kymograph Plugin</td><td></td><td></td><td>&lt;a href=&quot;http://www.embl.de/eamnet/html/body_kymograph.html&quot;&gt;http://www.embl.de/eamnet/html/body_kymograph.html.&lt;/a&gt;</td></tr></tbody></table>

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.

Figure 1 shows an overview of the microfluidic device used to grow hippocampal neurons (Figure 1A, B). Neurons are plated in reservoir 1. The size of the microchannels prevents the diffusion of cell bodies (soma) into the axonal compartment while the length of the channels prevents dendritic projections from crossing all the way to the axonal compartment. After ~2-3 days in culture, neurons start extending their axons across microchannels into the axonal compartment (Figure 1B, C...

Watch this Scientific Journal Video about Characterizing the Composition of Molecular Motors on Moving Axonal Cargo Using "Cargo Mapping" Analysis at JoVE.com

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

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 ...

characterizing-composition-molecular-motors-on-moving-axonal-cargo

Research

JoVE Journal

Neuroscience

9.3K Views.  The Scripps Research Institute. 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.

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

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

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 ...

Cargo Loading onto Kinesin Powered Molecular Shuttles

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.
These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.
Building on the classic inverted motility assay 2, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.
The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4, quantum dots 5 or a wide variety of antigens via biotinylated antibodies 4-6.

Molecular shuttles consisting of functionalized microtubules gliding on surface-adhered kinesin motor proteins can serve as a nanoscale transport system. Here, the assembly of a typical shuttle system is described.

Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport ...

Biology

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 ...

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human neurons, which may contribute to neurodegeneration in various disease states. Although it is challenging to examine mitochondrial dynamics in live human nerves, such paradigms are critical for studying the role of mitochondria in neurodegeneration. Described here is a protocol for analyzing mitochondrial transport and mitochondrial morphology in forebrain neuron axons derived from human induced pluripotent stem cells (iPSCs). The iPSCs are differentiated into telencephalic glutamatergic neurons using well-established methods. Mitochondria of the neurons are stained with MitoTracker CMXRos, and mitochondrial movement within the axons are captured using a live-cell imaging microscope equipped with an incubator for cell culture. Time-lapse images are analyzed using software with &#34;MultiKymograph&#34;, &#34;Bioformat importer&#34;, and &#34;Macros&#34; plugins. Kymographs of mitochondrial transport are generated, and average mitochondrial velocity in the anterograde and retrograde directions is read from the kymograph. Regarding mitochondrial morphology analysis, mitochondrial length, area, and aspect ratio are obtained using the ImageJ. In summary, this protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology to facilitate studies of neurodegenerative diseases.

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.

Axonal transport is a crucial mechanism for motor neuron health. In this protocol we provide a detailed method for tracking the axonal transport of acidic compartments and mitochondria in motor neuron axons using microfluidic chambers.

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal ...

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study of this movement in situ was only possible using radioisotopic pulse-labeling, which permits analysis of axonal transport in whole nerves with a temporal resolution of days and a spatial resolution of millimeters. To study neurofilament transport in situ with higher temporal and spatial resolution, we developed a hThy1-paGFP-NFM transgenic mouse that expresses neurofilament protein M tagged with photoactivatable GFP in neurons. Here we describe fluorescence photoactivation pulse-escape and pulse-spread methods to analyze neurofilament transport in single myelinated axons of tibial nerves from these mice ex vivo. Isolated nerve segments are maintained on the microscope stage by perfusion with oxygenated saline and imaged by spinning disk confocal fluorescence microscopy. Violet light is used to activate the fluorescence in a short axonal window. The fluorescence in the activated and flanking regions is analyzed over time, permitting the study of neurofilament transport with temporal and spatial resolution on the order of minutes&#160;and microns, respectively. Mathematical modeling can be used to extract kinetic parameters of neurofilament transport including the velocity, directional bias and pausing behavior from the resulting data. The pulse-escape and pulse-spread methods can also be adapted to visualize neurofilament transport in other nerves. With the development of additional transgenic mice, these methods could also be used to image and analyze the axonal transport of other cytoskeletal and cytosolic proteins in axons.

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Neurofilament protein polymers move along axons in the slow component of axonal transport at average speeds of ~0.35-3.5 mm/day. Until recently the study ...

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal transport have devastating consequences for individual neurons and their networks, and contribute to a plethora of neurological disorders. As many of these conditions involve both cell autonomous and non-autonomous mechanisms, and often display a spectrum of pathology across neuronal subtypes, methods to accurately identify and analyze neuronal subsets are imperative.
This paper details protocols to assess in vivo axonal transport of signaling endosomes and mitochondria in sciatic nerves of anesthetized mice. Stepwise instructions are provided to 1) distinguish motor from sensory neurons in vivo, in situ, and ex vivo by using mice that selectively express fluorescent proteins within cholinergic motor neurons; and 2) separately or concurrently assess in vivo axonal transport of signaling endosomes and mitochondria. These complementary intravital approaches facilitate the simultaneous imaging of different cargoes in distinct peripheral nerve axons to quantitatively monitor axonal transport in health and disease.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Axonal transport maintains neuronal homeostasis by enabling the bidirectional trafficking of diverse organelles and cargoes. Disruptions in axonal ...

Measuring Axonal Cargo Transport in Mouse Primary Cortical Cultured Neurons

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient bidirectional transport by motor proteins. Various reports have suggested that defects in axonal transport are associated with neurodegenerative diseases. Also, the mechanism of the coordination of multiple motor proteins has been an attractive topic. Since the axon has uni-directional microtubules, it is easier to determine which motor proteins are involved in the movement. Therefore, understanding the mechanisms underlying the transport of axonal cargo is crucial for uncovering the molecular mechanism of neurodegenerative diseases and the regulation of motor proteins. Here, we introduce the entire process of axonal transport analysis, including the culturing of mouse primary cortical neurons, transfection of plasmids encoding cargo proteins, and directional and velocity analyses without the effect of pauses. Furthermore, the open-access software "KYMOMAKER" is introduced, which enables the generation of a kymograph to highlight transport traces according to their direction and allow easier visualization of axonal transport.

The present protocol describes the whole procedure of analysis for axonal transport. In particular, the calculation of transport velocity, not including the pausing, and the visualization method using open-access software "KYMOMAKER" are shown here.

Neuronal cells are highly polarized cells that stereotypically harbor several dendrites and an axon. The length of an axon necessitates efficient ...

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear transcription factor c-MYC for proteosomal degradation in the cytoplasm. The method described here involves the study of a motorless KIF5B mutant for fluorescence microscopy. The wild-type and motorless KIF5B proteins are tagged with the fluorescent protein tdTomato. The wild-type tdTomato-KIF5B appears homogenously in the cytoplasm, while the motorless tdTomato-KIF5B mutant forms aggregates in the cytoplasm. Aggregation of the motorless KIF5B mutant induces aggregation of its cargo c-MYC in the cytoplasm. Hence, this method provides a visual means to identify the cargos of Kinesin-1. A similar strategy can be utilized to identify cargos of other motor proteins.

Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

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

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