The overall goal of this procedure is to measure microtubule dynamics in yeast cells. This method can help answer key questions in the microtubule field, such as how mutations in tubules and microtubule regulators regulate microtubule dynamics in a living cell. The main advantage of this technique is that living yeast cells are stabilized in the image.
Rather, high time-resolution for ten minutes, or low time-resolution for several hours. Demonstrating the procedure will be Colby Fees, and Cassi Estrem, two graduate students from my laboratory. The yeast cells used in this experiment have been constructed to constitutively express GFP labeled tubulin.
Inoculate about one microliter of the yeast cells from a single colony on a plate in three milliliters of synthetic complete medium. Allow the cells to grow overnight at 30 degrees celsius with shaking at 250 RPM. On the following day, dilute the saturated culture to an OD600 of less than 0.1 in the medium.
Return the diluted culture to the 30 degree Celsius shaker for three to four hours, or until the OD600 is between 0.4, and 0.8. Begin the preparation of flow chamber slides by cutting a sheet of paraffin film into a piece measuring four inches wide, and seven inches long. Place a standard microscope slide lengthwise on the four inch width of paraffin film and wrap the film around the slide three to four times such that the slide is covered in film that is three to four layers thick.
Press the layers together to ensure that there is a reasonable seal, but avoid pressing too hard or causing the film to wrinkle. Using a clean box cutter razor, and another slide to provide a straight edge for cutting, cut the paraffin film along the outside edges of the slide. Peel the excess film off the working slide and discard.
Using the second slide as a straight edge, cut the stacked film layers along the long axis of the slide into approximately 10 strips, each two to four millimeters in width. Next, cut the stacked paraffin film layers perpendicularly to the previous cuts along the short axis of the slide to create strips that are two to four millimeters in width, 23 to 24 millimeters in length, and three to four layers thick. Use a razor at a 45 degree angle to peel the cut film strips.
Then use forceps to evenly distribute the cut strips on a clean microscope slide to create the desired number of chambers. In this example, four film strips will be used to create three chambers. Place a single cover slip on top of the strips of film, and move it into position with forceps.
Place the prepared slide, cover slip up, on a hot plate set at about 95 degrees celsius, and heat the slide for approximately three minutes until the film strips are translucent. Seal the slide and cover slip together by using tweezers to gently press on the regions of the cover slip that are directly above the paraffin film strips. Use forceps to remove the slide from the hot plate, and set it aside to cool with the cover slip side facing up.
As the slide cools, the film will return to a white opaque coloration. To begin this procedure, thaw a frozen 200 microliter aliquot of Concanavalin A at room temperature. Add approximately 100 microliters of thawed Concanavalin A to each chamber of the prepared slide by pipetting into one of the open ends.
Then hang the slide, cover slip down, between two microfuge tube racks for five minutes to allow the Concanavalin A to adhere to the cover slip. Return the slide to a cover slip-up position, and remove the Concanavalin A by washing the chamber with synthetic complete medium. Flow two times the chamber volume of medium through the chamber by using the corner of a paper towel to draw fluid out of one end of the chamber, while simultaneously pipetting fresh fluid into the other end of the chamber.
Load cells by flowing two times the chamber volume of cell suspension using the directed flow method. Hang the slide, cover slip down, for 10 minutes to allow the cells to adhere to the cover slip. Flush out any non-adhering cells by flowing through four times the chamber volume of synthetic complete medium.
Carefully dry each end of the chamber with a clean corner of a paper towel, bringing the meniscus to the edge of the cover slip. Seal each end of the chamber with warm sealant. Image acquisition will be performed with an inverted microscope equipped with a 1.45 NA 100X CFI Plan Apo objective, a Piezoelectric stage, a spinning disc confocal scanning unit, an illumination laser, and an EMCCD camera.
Maintain the temperature of the stage at room temperature during image acquisition. Bring the cells into focus and search the slide for cells grouped together to ensure that the acquisition field has at least five to six cells clustered together on the same focal plane. Program a multidimensional acquisition to collect multiple zSeries over time.
Adjust the settings within the active acquisition settings to the following:Total Z depth of six micrometers to encompass the entire yeast cell, Z step size of 300 nanometers to maximize light collection without oversampling, total time duration of 10 minutes, time intervals of four seconds between zSeries, and exposure time of 90 milliseconds for each Z plane. Begin the acquisition by clicking Run, and allow it to run for the full 10 minute duration. The data is saved as an image series, and an example result of time lapse imaging is shown here.
Start the image analysis by opening ImageJ and dragging the acquired image stack directly onto the control panel. The bio formats imported will start automatically. Select Okay"to load the image stack as a hyperstack.
Using the ZProject function, collapse each zSeries into a maximum intensity of two dimensional projection. Apply a medium filter to the image stack to reduce speckling noise by selecting the process menu, the filter sub menu, and the median feature. Enter 2.0 pixels into the radius box and click Okay"Identify pre-NFA cells in the field.
These are characterized by medium sized buds and a short mitotic spindle about one to one point five micrometers in length. Starting at the first time point of the image series, use the segmented line tool to draw a line along a single astral microtubule from the located at the junction between the astral microtubule, and the more intensely labeled spindle microtubules, to the plus end, which is in the cytoplasm. Double click on the end of the microtubule to complete the segmented line.
Record the measurement in the results table using the measure feature by pressing the M button on the keyboard. After measurements have been made for all time points, copy the data from the results table, paste them into a spreadsheet, and select Save As"to select the data. This graphic displays a time series of astral microtubule dynamics in a wild type cell, and a mutant strain previously shown to have more stable microtubules.
Microtubules are labeled the GFP tagged alpha tubulin to visualize microtubule length. The red arrows trace the total length of an astral microtubule, with the arrowheads denoting the plus ends. Microtubule lengths were measured at each time point for eight minutes, and these compiled lengths are depicted in life plots.
The green points denote polymerization, and the pink points denote depolymerization. Catastrophe events are indicated by arrowheads. These plots show that the microtubule in the mutant is longer, and exhibits fewer catastrophes than the wild type microtubule.
In addition, the polymerization and depolymerization rates determined from the slopes of the ascending and descending microtubule lengths are decreased in the mutant compared to the wild type. Measurements of microtubule dynamics also reveal that the microtubule in the mutant cell has decreased dynamicity compared to the wild type. Once mastered, the slide preparation, cell loading, and image acquisition can be performed in 30 minutes, which means that many different samples can be imaged in a single day.
The slide chambers can be made and stored in advance prior to adding Concanavalin A.After watching this video, you should have a good understanding of how to stabilize yeast cells in simple slide chambers, image in four dimensions, and measure microtubule dynamics in living cells. In addition to microtubules, this procedure can be modified to measure the dynamics of other fluorescently labeled proteins in living yeast cells across time scales ranging from seconds to hours.
