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

The overall goal of cargo mapping is to determine the relative amount and type of molecular motor proteins that associate with and move vesicular cargoes and to correlate this composition of motors with a directionality of live cargo movement. This is accomplished by first growing primary mouse hippocampal neurons in microfluidic devices so that axons grow relatively straight and by recording the live intracellular movement of fluorescently labeled cargo proteins in the axons of transiently transfected neurons. The second step is to fix neurons during the course of live cargo imaging and subsequently stain the axons with antibodies against molecular motor proteins such as kinesin and dines, followed by immunofluorescence microscopy imaging.

Next, the fixed cargo and motor images are aligned to the plots of cargo movement over time, also called krafts in order to correlate the live cargo movement with the stained associated motors as determined by the immunofluorescent staining. The final step is to determine the subpixels positions of cargo and motor proteins using a custom developed MATLAB software package called motor colocalization. This software provides the precise XY coordinates of each cargo and motor position, as well as the relative amount of fluorescence intensities associated with cargoes and motors.

Ultimately, correlation of fluorescence microscopy of live and fixed cargoes and motors at the sub pixel resolution level provides information on the types of motors that are associated with specific cargoes for which the directionality of movement is known. So this method provides insight into the mechanism of exon transport. It can be applied to other studies correlating life microscopy with immunofluorescent staining, such as the Association of Proteins to microtubials during microt tubal dynamics or the recruitment of proteins during endocytosis.

Visual demonstration of this method will help to perform the imaging fixation and analysis steps, which might otherwise be difficult to learn as they require a specialized setup. The mouse hippocampal primary neurons for this procedure are prepared as ascribed in the protocol text and resuspended in neuro basal egg growth. Medium to plate the neurons in the microfluidic device.

Remove medium from microfluidic reservoir one and one prime. Apply 20 microliters of cells to the microfluidic reservoir one and allow them to flow through. Place the device in a 37 degrees Celsius incubator with 5%CO2 for 20 minutes.

Look under the microscope to ensure that the cells have adhere to the covered glass. Add neuro basal, a growth medium to top off all the reservoirs. Return the device with cells to the incubator by day nine.

In culture, the neurons extend axons through the micro channels and are ready for transfection for the expression of fluorescent proteins. The live imaging of the transfected mouse hippocampal primary neurons is performed on an inverted epi fluorescence light microscope, equipped with a 37 degrees Celsius incubator and a CO2 control chamber. To begin this procedure, select the 100 x oil objective.

Apply oil to the objective and mount the cover slip with the 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 one hour. Find axons of transfected cells that extend through the channels of the microfluidic chamber.

Record the number of the channels in which the transfected axon was found. Count the number of channels using a hand tally counter 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.

To facilitate subsequent alignment of fixed images with movement trajectories on chm graft. Align the right edge of the micro channels with the field of view during imaging. In order to successfully fix the cells during live imaging, the procedure is best performed with two people, one person to add the fixative and another person to adjust the focus.

It's also important to have all the reagents ready and within reach. Using a one milliliter plastic transfer pipette. Remove most of the medium from reservoirs two and two Prime Start imaging live cargo movement with time lapse specifications specific to the transport dynamics of the cargo being analyzed.

After sufficient live movement data have been collected, have one person fill up reservoirs two and two prime with prewarm fixative. 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.

In the same way, fill up the reservoirs one and one prime with the fixative fixation is successful when immediate cessation of movement is observed. Subsequently, the fixed neurons are stained with antibodies against endogenous motor proteins and imaged as described in the protocol text. To begin cargo mapping generate a chromograph of the cargo movement using image analysis software.

The Image J plugin developed by Rich Dorf Insights is recommended for generating Kraft using commercially available graphics software manually align the immunofluorescence image of cargo to the CHM graph of cargo movement, superimposed the cargo puncta onto the position of the trajectories in the chm graph at the point of fixation such that each cargo puncta corresponds to a specific trajectory on the CHM graph. For best alignment results, use the Z slice that is in best focus for the cargo mapped. Several Z slices may be used for each image in the Z stack.

Determine the XY coordinates and intensity amplitudes for each fluorescent puncta using an established algorithm to fit 2D gaussians to the point spread function that represents the point sources in each of the three channels. To obtain the XY coordinates, a custom built MATLAB software package called motor colocalization was developed, which incorporates a Gaussian fitting algorithm developed by Yakima DONA and colleagues for each of the individual cargo puncta that were mapped onto the mobile or stationary trajectories. On the Kog graph, select the XY coordinates and intensity amplitudes from the results of the motor colocalization program.

Use several Zack images to map more cargoes that are found in different focal planes. Compare the individual XY coordinates for the mapped cargo to those obtained for each of the motor channels In the corresponding Z slice. Determine and select the XY motor coordinates that are within a 300 nanometer radius of the cargo for cargoes and motors that have signaled within the same focal plane.

Use the motor colocalization software package to determine the puncta that are located within the 300 nanometer radius. Cargo mapping was used to correlate the transport of vesicles carrying normal prion protein tagged with yellow fluorescent protein or Y-F-P-P-R-P-C with relative amounts of kinesin and dine motors in axons. This graph shows F-P-P-R-P-C vesicle movement and the dotted line indicates the time of fixation during live imaging.

Fluorescence images of fixed Y-F-P-P-R-P-C vesicles as well as the corresponding immunofluorescence images of Kinesin light chain one and dine are aligned to the chromograph. This enlargement shows immunofluorescent signals of each of the three channels with 2D gian function assignments. This graph represents the quantification of COLOCALIZATION between Y-F-P-P-R-P-C vesicles, kinesin light chain one and dine in wild-type neurons and in neurons lacking KINESIN heavy chain.

The relative amount of Kinesin light chain one and dine associated with Y-F-P-P-R-P-C vesicles is obtained from the Gaussian amplitudes and plotted to scrutinize possible correlations between relative motor amounts and directionality of movement. This example from wildtype neurons indicates that Y-F-P-P-R-P-C vesicles, which are moving or stationary associate with either KINESIN light chain one or dine or both Once mastered this technique can be performed within three days. In addition to approximately 10 days of pre-EM imaging preparation of the micro fillic devices and the primary neuron, within three days, you will be able to perform live imaging imaging of immunofluorescent staining and data analysis.

After watching this video, you should have a good understanding of how to record live movement of fluorescently labeled cargoes and neurons that were grown in microfluidic devices. How to fix C cells during imaging, how to analyze colocalization as sub pixel resolution, and how to correlate the colocalization of a cargoes and molecular motor proteins with cargo movement.