The overall goal of the following experiment is to study the motions of protein molecules and protein coated beads with the ends of depolymerizing microtubules. This is achieved by assembling a reusable flow chamber with a clean sized cover slip. Next microtubules seeds are prepared attached to the cover slip and used to nucleate microtubules, which are then stabilized temporarily with photo destructible caps.
Then GFP labeled proteins or protein coated beads are introduced to the flow chamber and bind to segmented microtubules. Micro tub depolymerization is then triggered by removing the caps by photo ablation. Images are analyzed with CH to determine whether the labeled proteins exhibit tip tracking behavior, which is movement with the ends of depolymerizing microtubules.
The main advantage of this technique is that it allows triggering microtubule ization with high temporal and special resolution. The visual demonstration of this method is critical. The protocol begins with flow chamber preparation and assembly Using ultrasonic milling.
Modify regular microscope slides to have two grooves. Make the grooves 14 millimeters apart. Give or take one millimeter, which is optimal for chambers assembled using square 22 millimeter cover slips.
Next place a 100 millimeter long polyethylene tube in each groove in the slide, leaving about five millimeter overhangs at the inner ends of the grooves. Fix the tubes inside the grooves with cyanoacrylate glue, embedding the tubes completely inside the grooves. Carefully fill the grooves with epoxy glue without spilling.
Allow it to dry for a day. The next day. Use a sharp razor blade to cut the solidified glue, three to four millimeters from the distal end of each attachment site.
Removing the parts proximal to the center of the slide. The tubes should remain in their grooves and the cuts should make flat surfaces with tube openings. Next, fill a syringe with water and test whether the tubes are working properly.
If the liquid flows freely, put a drop of epoxy glue about five millimeters in diameter at the outer ends of the grooves and allow it to dry for about a day. The completed flow chambers are thus durable and can be used repeatedly for many months. This three hour protocol prepares 12 cover slips.
It requires a fume hood, one ceramic cover slip holder, three 15 milliliters, staining jars, and 1 250 milliliter glass jar with a lid. The staining jars must fit four, regular number 1 22 millimeter cover slips in a stack. Start with a stack of 12 pre-cleaned and dried cover slips in the ceramic holder.
Fill the jar with plus one repel silene solution and slowly lower the slides in. Close the jar and incubate it for five minutes at room temperature. The most critical step to ensure uniform cover slip civilization is to avoid any water traces during the incubation of the cover slips in Selene solution.
This step is essential for tight attachment of micro tubal seeds. Later in a protocol, When the cover slips are ready, remove the ceramic holder from the jar. Then transfer the cover slips individually using Teflon coated tweezers from the ceramic holder to methanol loaded staining jars.
Now load the three jars into the water reservoir of a sonic bath with a metal or glass pedestal in it. The water level should be at two thirds the height of the jars sonicate the jars at 70 watts for 20 minutes. During the sonication, change the methanol solution every five minutes.
When the sonication is complete, rinse, the cover slips clean 10 times with deionized water. If the siloization worked properly, the cover slips will appear dry When removed from the water, regardless of appearance, thoroughly remove any residual water. Using nitrogen gas transfer, the dried cover slips to a nitrogen dried sealable container.
Interlay Kim wipes between the cover slips. They can be stored this way for several weeks at room temperature. This protocol requires one to two hours, one flow chamber and prepared G-M-P-C-P-P stabilized micro tubial seeds.
First, assemble the flow chamber. Attach two pieces of double-sided tape along the central five millimeter wide area of the prepared slide, and firmly press the cover slip onto the tape. Using a one milliliter syringe, fill the chamber with 100 microliters of B rrb 80 solution via a tube.
Next, squeeze a small drop of two colored quick cast sealant on top of a small plastic Petri dish. Mix it quickly and thoroughly with a toothpick. It will turn green.
Apply the sealant immediately along all the edges of the cover slip. Check if the sealant penetrates too deeply under the cover slip. If a tube is blocked, unplug the syringe and lift the tube to apply gentle pressure to prevent sealant from leaking in After 10 minutes, make sure the solution flow is not restricted.
Then transfer the chamber to a warmed microscope stage at 32 degrees Celsius. Attach one of the tubes to a pump for solution removal. Immerse the free end of the inlet tube in a 0.5 millimeter vial with warm BRB 80 buffer.
Set the pump to 100 microliters per minute and as needed, use it to gently push out air bubbles. This can also be done manually. Now use the pump to load 30 microliters of warm antigen antibodies.
Turn off the pump and wait five minutes. Turn off the pump during all incubations. Next, wash the chambers with about 100 microliters of warm b rrb 80.
Then perfuse 50 microliters of 1%onic F1 27 in BRB 80. Wait 10 minutes for it to block the hydrophobic surface of the siloized cover slip. Now wash the chambers with 100 microliters of motility buffer.
Next, reduce the pump speed to 15 microliters per minute and perfuse the chamber with 30 to 40 microliters of microtubules seeds diluted one to 200 to one to 600 in motility buffer. Incubate the seeds for 15 minutes, then wash the chamber with 400 microliters of motility buffer at 100 microliters per minute to remove any unbound material. Next, reduce the pump speed to 30 microliters per minute and perfuse the chamber with prewarm unlabeled tubulin solution.
The most critical aspects of this protocol are to avoid air bubbles. To use freshly prepared tubulin solutions and to follow exact timing of the protocol, especially during the micro tubular caping. Monitor the micro tubial growth using DIC optics during the five to seven minute incubation.
The microtubules usually grow about 10 microns quickly exchange the solution with pre-war Rumine Tubulin Mix and allow eight to 10 minutes more incubation time. Wash the chamber well with 100 microliters of motility buffer at 20 microliters per minute to remove turbulence and nucleotides, as well as soluble micro tubule fragments. Next, perfuse the chamber with a warmed solution of GFP labeled protein or protein coated beads and proceed with imaging yeast kinetico component DAM one labeled with GFP was purified from bacterial cells and used as a positive control for tip tracking assays.
Such proteins track with the depolymerizing end of a micro tubule and steadily move towards the cover slip attached seed. These proteins appear as an oblique line on a corresponding graph, which can then be analyzed to determine the number of protein molecules in the tip tracking complex. This analysis is detailed in the text protocol.
In contrast, proteins that fail to tip track do not show an enrichment in fluorescence signal at the disassembling micro end. This is seen with the human NDC 80 GFP protein. Binding between protein coated beads and microtubules is usually quite strong, so when beads were added to the microscopy chamber, they bound readily to the segmented cover slip attached microtubules.
When the bead tracking is processive, many observations can be made. Beads that attached to one microtubule showed an arc like motion as microtubules pivot slightly around the site of their growth. When the stabilizing cap is illuminated, if only one red segment can be seen distal from the seed and bead, then the bead is attached to one micro tubule.
The cap can often be seen following the beads alike motion until the cap is bleached The bead motion shortly after bleaching appears consistent with microtubule orientation. As the micro tubial shortens and the bead moves towards the seed, the amplitude of the arc like oscillations decreases. If the beads fail to form lasting attachments to the stable microtubules.
Laser trapping can be used beads with different protein constructs of plus end directed kinesin SE E required this technique to launch the beads on the segmented micro tubial walls in the presence of a TP.These beads moved rapidly towards the capped micro tubial plus ends on a graph. This motion resulted in an oblique line directed towards the cap after the cap was destroyed. The beads coated with truncated spy protein detached from the micro tubule as pointed out by the green arrow.
In contrast, beads with full length B protein could sustain motion in the reverse direction. This is seen as a zigzag pattern on the chm graph due to motion in opposing directions to either the plus and or minus end of the microtubules. After watching this video, you should have a good understanding of how to prepare flow chambers and grow segmented microtubules to study motions driven by micro tubal disassembly.
