The objective of this procedure is to assess the microtubule organization and dynamics in vivo. Neurons are polarized cells with distinct axonal, dendritic, and synaptic compartments, maintained by their underlying cytoskeleton. Microtubules form majority of the neuronal cytoskeleton, and the dynamics and orientation determine key events during neuronal development and maturation.
Microtubules are comprised of, alpha and beta tubulin heterodimers, and are inherently polarized with highly dynamic plus-ends and stable minus-ends. During polymerization at the plus-ends, a multi-molecular complex of end-binding proteins, transiently associate with the protofilaments and promote the assembly of tubulin dimers. This technique will help us to visualize neural microtubules in vivo.
Using this technique, one can determine the orientation and other parameters of dynamic microtubules during development and regeneration of C.elegans neuron. We typically use this reporter to visualize microtubule dynamics in sensory neuron. However, one can express this reporter in other cell types such as muscles and skin to visualize dynamic microtubules in these cells.
Fluorescently labeled end-binding proteins like EBP to GFP here, appear as comets. Dynamics of these comets has been correlated with concomitant growth of the microtubules, and a key indicator to gauge microtubule dynamics and orientation. In order to observe these comets in a specific cell type, transgene with DNA of EBP-2 and GFP are expressed under a specific promoter.
For expression in the PLM neurons, this transgene is expressed under the make for promoter. These transgenic worms can be viewed under a stereo microscope, with fluorescence set up before sorting or mounting. For mounting the worms for imaging EBP comets, we make a 10%Argos in M9 buffer.
and place a drop on a glass slide. Another slide is used to press the drop into a film that solidifies into a pad. We use 0.1 micron, polystyrene beads as a mounting medium, a few worms are picked and re-suspended in the bead solution.
A cover slip is poached to immobilize the worms and taken for imaging. The mounted ones can be imaged on any fluorescence microscope with a camera for high resolution imaging. The setup is equipped with a spinning disk unit for a faster acquisition and reduced photo bleaching and photo toxicity.
The slide is kept on the stage and the field of worms is focused and centered using the bright-field illumination. A low magnification objective like 5X or 10X can be used for this purpose. The worms can be re-focused at a high magnification objective like 63X using the same illumination.
This objective provides an optimum spatial resolution for the imaging of the comets. A fixed centering of the PLM neuron is done under the fluorescence illumination to prevent undue light exposure and photo bleaching of the prum. For the image acquisition we use send software off sites.
Using live imaging configuration in the brightfield channel, We focus the tail region of the worm and center it in the view field. This is followed by focusing of the PNM neuron using light imaging configuration with 488 nanometer excitation light. For a time-lapse acquisition, an experimental setting is created where exposure time, duration of the time lapse and the interval between the frames define the temporal skills of imaging.
For quantitative measurements some movies of EBP comets are to be analyzed on Image J, which is an open source software. The acquired time-lapse opens as a multi-image strand, that can be previewed as a movie. In this particular time-lapse, comets can be seen in the cell body, anterior and posterior process of the PLM neuron.
The comets moving away from the cell body are classified as plus-end-out, and those moving towards the cell body are classified as minus-end-out. To begin the analysis a segmented line is drawn over the region of interest, which in this case is the interior process of the PLM neuron. This line segment is converted into a kymograph using the reslice function of images.
A kymograph is a distance time image with diagonal creases representing the moving comets. Straight line segments can be drawn on these traces and measurement parameters can be set using the analyze menu. These parameters can be converted to standard measurable quantities and units in the data analysis program like Excel.
The width of the trace corresponds to the growth length. The hight represents of growth duration, and the angle of the crease gives the direction of the comets. These comets can further be classified into plus-end-out and minus-end-out, to find the relative orientation of microtubules.
Transgenic expression of EBTA GFP can be adapted to observe microtubule dynamics in various cellular context, such as new regeneration. To observe the microtubule dynamics in the region rating axons, the axon is injured using a femtosecond laser, which can sever the axon precisely without causing massive collateral damage. Following the injury, the worms can be recovered onto seeded engine plates for later observations.
PLM neurons show robust exoneree regeneration, which can be observed as early as six hours after injury. To prevent phototoxicity to regenerating new rights, live imaging is carried out in a spinning disk microscope. Time-lapse acquisitions of the regenerating axons can later be converted into kymographs for the analysis of comets.
The distance duration and direction of the comets can then be interpreted in terms of microtubule orientation and dynamics. My critical orientation and dynamics are key determiners of civil cellular processes like cell division, migration and maintenance of cellular architecture. Although there are many tools available for the measurement of microtubule dynamics in vivo measurement can be challenging.
Autoflorescence, photo toxicity, over expression artifact, and variable reported intensities are some of the difficulties encountered during live observation of end-binding proteins, to assess the microtubule dynamics. By using an integrated transgenic with low concentration of deported DNA and low illumination imaging, this study has addressed methods to mitigate some of these challenges. The imaging and analysis modules described here can be extended towards other microtubule based reporters and protein transport.
This is not only applicable to C.elegance neurons, but can be applied to other cell types and model systems.
