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The cell's nuclear envelope contains approximately 2000 nuclear PO complexes that allow the transport of water-soluble molecules such as RNA, ribosomes certain proteins, carbohydrates, and signal molecules by adding labeled cargo molecules to permeable cells, the movement of single molecules through the nuclear pore can be visualized by fluorescence microscopy. Time-lapse images are required to determine localization as well as spatial temporal characteristics such as transport time, import, export efficiency, and entrance frequency. Hi, I'm Alex ov, PhD student from laboratory of single molecule microscopy in the Department of Biology at the Bowling Green State University.

I'm Asha P Shema master student also from the single molecule microscope lab. I'm Juma. I'm a postdoc fellow here as single molecule microscopic lab.

Today we'll show you a procedure of single molecule imaging of nuclear transport through the nuclear PO complexes in the helo cells. We use this procedure in our lab to study the translocation of various molecules through nuclear PO complex. So let's get started.

To prepare the proteins for the nuclear transport experiment conventionally transform JM 1 0 9 E coli expressing the genes responsible for production of the nuclear cargo proteins are grown in LB media and induce with IPTG. Once the cells are in log phase with an optical density of 0.7 to 0.8, spin the culture at 6, 000 times gravity for 15 minutes at four degrees Celsius, discard the supinate, weigh the pellet. Then Reese suspend the cells in 10 milliliters of Celtic B per gram to extract protein suitable for affinity purification and analysis.

Next, add the appropriate amount of protease cocktail in midsole DNAs one and beta me capto ethanol. Stir the mixture in the dark on ice for 30 minutes. Then spin at 25, 000 times gravity for 20 minutes at four degrees Celsius.

Following the spin, transfer the supinate to a new bottle in preparation for chromatography. Place 400 microliters of nickel NTA and acetone into four micro centrifuge tubes. Spin at 16, 000 times gravity for three minutes.

Discard the super nascent. Then add 400 microliters of lysis wash buffer. Repeat the previous wash steps three times following the final wash.

Re suspend the nickel NTA in lysis buffer and add all four tubes to the bottle containing the cell supinate. Stir the resultant mixture slowly in a dark container on ice for 60 minutes while the mixture is stirring. Prepare an ecno pack chromatography column by wrapping it in foil to prevent photobleaching of fluorescent proteins.

Also place 12 collection tubes into a rack underneath the column. Once the mixture has stirred for 60 minutes, load 10 milliliters of the SNA nickel NTA mixture and let it completely pass through. Begin to collect fractions.

Next, add 10 milliliters of lysis wash buffer. Let it pass completely. Then add 10 milliliters of IZO buffer and let it pass.

Continue collecting fractions. Next, flow through 0.5 milliliters of elution buffer and allow it to pass through repeat flowing elution buffer until all 12 fractions have been collected. To determine which fraction yield the highest concentration of the protein, run four microliters of each fraction on a page gel and kamasi stain.

According to standard procedures, combine the most concentrated fractions and add 500 microliters to a nano step Filter with a pore size of 10 kilo daltons that has been pre rinsed with elucian buffer. Concentrate the protein by spinning at 16, 000 times gravity for five minutes. Discard the buffer, stir the remaining protein fraction and add another 500 microliters to the filter centrifuge.

Again, repeat for all of the selected fractions. Next, bring the final volume of the concentrated protein to about 50 microliters with elucian buffer. Then while gently stirring with a micro pipet collected from the surface of the filter, continue to run and iron exchange and size exclusion columns to obtain super pure protein, determine the final protein concentration.

Next, label the cargo molecules with an excessive assisting reactive organic dye such as Alexa 5 55 or Alexa 6 47 maide. This is done because the intrinsic green fluorescence of GFP cannot be used for single molecule imaging of nuclear transport due to its poor photo stability and overlapping spectrum with cell autofluorescence, the labeled protein should be split into several fractions of 25 microliters and stored at minus 80 degrees Celsius one day before the experiments. Place an autoclave cover slip in a sterile 35 millimeter Petri dish and pipette nine milliliters of DMEM onto it.

Next, pipette one milliliter of cell culture and drop it uniformly onto the cover slip the cells used. Here were previously genetically engineered to express A GFP labeled poor membrane protein. POM 1 21, place the dishes in the carbon dioxide incubator for 12 hours.

On the day of the experiment, start preparing the flow chambers. Next retriever heel, our cell coated slide from the incubator drip, dry the slide, then mount it using the vacuum greases glue. In a home machined aluminum frame, it is essential to make sure the cells don't dry out during the preparation.

Using a 10 milliliter plastic syringe, add two lines of silicon on both sides of a cover slip using tweezers. Pick up the small prepared cover slip with the two lines of silicon applied inverted over the cell coated slide and press down lightly to form the flow chamber. If two separate chambers are prepared on one cell coated slide, it is essential to ensure a good ceiling to prevent cross contamination.

So apply several lines of the silicon grease between the chambers from the top to the bottom of the slide to seal them apart. Also, place several lines of grease on the outer sides of the chambers. Add DMEM to the flow chamber to keep the cells from drying out.

Next, using a cotton tipped swab, wash the bottom of the slide twice with water and then twice with 100%ethanol. Mount the frame on the microscope. Wash twice with 25 microliters of transport buffer by adding it to the flow chamber and wicking it.

Using a small piece of filter paper applied to the exit side, perme the cells once for about two minutes with 25 microliters of digit toin, permeation should be monitored in real time using brightfield. Finally, wash the cells with 25 microliters of transport buffer, supplemented with 1.5%polyvinyl pyro or PVP to prevent osmotic swelling of the nuclei. Find the permease cells onto the microscope and identify a bright green fluorescent ring.

Then focus on the equator of the cell and capture an image before adding fluorescent cargo molecules. Eliminate any autofluorescence by selecting the region of the nuclear envelope and illuminating with the 6 33 nanometer helium neon laser until it is completely photo bleached. Typically, over two minutes is required.

Add the labeled dextran sample diluted in transport buffer with 1.5%PVP to the flow chamber in a volume of 25 microliters and float through the chamber by applying a wick of filter paper. Cur should be taken upon addition of the import cargo proteins, the permeable cells, since the focus can easily be disrupted by any vigorous movement. Engage the optical chopper at approximately five hertz to avoid photobleaching and to allow a new portion of non-photo bleach single molecules to approach the nuclear envelope.

A series of videos of single molecule translocation should be acquired as soon as the sample molecules are added translocation through the nuclear pore complexes can be captured. Use the 6 33 nanometer helium neon laser to illuminate the transport trajectories of transing molecules performed correctly. This protocol allows for the collection of a series of images demonstrating the translocation of single cargo molecules interacting with the nuclear pore complex.

In perme, he r pom 1 21 cells. By tracking the spatial trajectories of individual transing dextran molecules, the fine steps of diffusion through the nuclear envelope can be distinguished. A cytoplasm to nucleus event is shown here in which a single dextron molecule shown as a red spot started from the cytoplasm, interacted with the nuclear envelope shown as the green pixel line and ended in the nucleus here.

The dextran molecule started from the cytoplasm, interacted with the nuclear envelope and returned back to the cytoplasm. In this example, the dextran molecule started from the nucleus, interacted with the nu nuclear envelope and returned back to the nucleus. And here the dextran molecule started from the nucleus, interacted with the nuclear envelope and ended in the cytoplasm.

By tracking the spatial trajectories of individual transing dextran molecules, the fine steps of diffusion through the nuclear envelope can be distinguished. The trajectories of the event shown as blue lines and open dots were determined and superimposed with the nuclear envelope shown as a black line. The green and red lines represent 100 nanometer distance from the nuclear envelope on the cytoplasm and the nuclear side.

We have just shown you how to visualize the translocation of single molecules through the nuclear pore complex with the resolution of 10 nanometer at 400 microseconds. When during this procedure, it is important to remember the necessity to achieve a maximum possible signal noise ratio and also be really careful while performing the single molecule experiment because the focus can be easily disrupted. So that's it.

Thanks for watching and good luck with your experiments.