Lab module 6b Receptor-mediated endocytosis

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1 Goal for the module Lab module 6b Receptor-mediated endocytosis To follow the movement of a degraded ligand, LDL, and a recycled ligand, transferrin, as they undergo endocytic processing. Pre-lab homework Read about receptor-mediated endocytosis and other forms of internalization in Alberts Molecular Biology of the Cell, available free online from the National Institutes of Health ( Introduction You will continue with examination of receptor-mediated endocytosis in this lab module. Last time you looked at the time and temperature dependence of the internalization of low density lipoprotein (LDL), which is brought into cells after binding to its cell surface receptor. The LDL binds with high affinity to the LDL receptor under conditions of the extracellular space but is released from the LDL-R in the acidic conditions of the endosome. The LDL is sent to lysosomes for degradation while the LDL-R is recycled to the cell surface to bring more LDL into the cell. Another ligand that binds its cognate cell surface receptor is transferrin (Tf). Transferrin is an iron-transport protein that binds two Fe 3+ ions. This iron-bound Tf has high affinity for its cognate cell surface receptor, the transferrin receptor (Tf-R). This receptor and its ligand are internalized via coated pits just as the LDL-R is. In the acidic conditions of the endosome, the receptor-bound iron-tf has a reduced affinity for the Fe 3+ ion, which dissociates from the Tf (that is still bound to the Tf-R). The Tf-R recycles back to the cell surface, but in contrast to the LDL-R, the iron-free Tf (called apotf) is carried along with the receptor. When the apotf/tf-r complex reaches the cell surface, the apotf dissociates because it has low affinity for its receptor under extracellular condtions. This freed apotf then can bind two more Fe 3+ ions, bind to another Tf-R and repeat delivery of iron into the cell. See Fig

2 Fig The receptor-mediated endocytosis of transferrin (Tf, blue) follows the recycling pathway. Tf binds to its cell surface receptor (green) when Tf is bound to one or two Fe 3+ ions (yellow). The ligand-receptor complex is internalized via coated pits to coated vesicles which uncoat to form early endosomes. As the endosomes acidify, the receptor-bound Tf releases its Fe 3+ ions which are transported to ferritin for storage. The Tf/Tf receptor complex is brought to the sorting endosome where it is placed as cargo in recyling endosomes destined to fuse with the plasma membrane, thus recycling both the Tf ligand and the Tf receptor. The endocytosis process involves movement of internalized receptors and ligands through acidified endosomes (see Fig. 6.3 in Module 6A). This acidification serves several purposes, including regulation of the binding of ligands to receptors. Inhibition of the acidification process in the endosomes will block processing of early endosomes to sorting and transport endosomes. The acidification process occurs by activation of a proton pump in the endosome membrane that pumps H + ions into the endosomes. There are inhibitors of this pump, which will block endosome acidification. It is also possible to block endosome acidification by applying ammonia (NH 3 ) to cells. The ammonia crosses the cell membrane and the endosome membrane. Inside the endosome, the ammonia binds a hydrogen ion to make ammonium ion: + + NH 3 + H NH 4. This reaction is reversible, such that an excess of ammonium ion produces ammonia in solution. You will use ammonia (applied in the form of ammonium ion) to de-acidify endosomes. The ammonium will convert to NH 3 which, being uncharged, will cross cell 6.13

3 membranes. When the NH 3 enters the endosome, the high H + ion concentration will drive formation of NH 4 +, thus reducing endosomal ph. This treatment will block the release of LDL from its receptor and Fe 3+ from Tf, thus disrupting the processing of both of these ligands. Early endosomes are transported along microtubules. A microtubule-specific motor protein is bound to the endosome, and this motor drives the endosome inward. Thus, drugs that disrupt microtubules will disrupt endosomal traffic. You will use nocodazole to disassemble microtubules, and thus to block the movement of endosomes. This treatment will block transport of both LDL and Tf to sorting endsosomes and beyond. You will use the same fluorescent analog of LDL to follow LDL processing that you used in Module 6A. To follow transferrin processing, you will use a fluorescently labeled human Tf. The fluorescent label on the Tf is Alexa Fluor 488, which is a bright fluorophore, Nevertheless, the labeing of the Tf is much less than the labeling of the LDL, so the Tf signal will be much dimmer than that for LDL. You can find the spectral properties of the AF488-Tf from the Molecular Probes website. In this module you will explore the differential processing of LDL and Tf by using continuous incubation and pulse-chase incubation protocols. You will also inhibit endocytosis by using ammonium ion to de-acidify endosomes or nocodazole to depolymerize microtubules. Materials 3T3 mouse fibroblasts on cover slips LLC-PK pig epithelial cells on cover slips dii-ldl at 10 μg/ml and Alexa Fluor 488-transferrin at 1 μm in Fe-PBS plus 1 mg/ml BSA (ice temperature) Fe-PBS plus 10 mm NH 4 Cl (to de-acidify endosomes) Fe-PBS plus 1 μm nocodazole (to depolymerize microtubules) Fine forceps Small beakers Humid chamber(s) Glass slides Fixative solution (PBS plus 3.7% paraformaldehyde) Mounting medium Ice bucket Procedures The dii-ldl and transferrin (Tf) reagents that you will be working with are produced from human blood serum. Both have been tested and found negative for viruses. Nevertheless, you should wear gloves while working with the labeling solution. Rinse solutions should be disposed of in a bottle that we will autoclave. Anything that may have contacted cells or solutions exposed to dii-ldl or Alexa Fluor 488-Tf (Petri dishes, 6.14

4 gloves, Parafilm, tips, filter paper, etc. should be placed in a biohazard bag for autoclaving). Used glass slides can be disposed of in the glass trash. They have been sterilized by the formaldehyde fixation. We do all of this just to be on the safe side. Each lab group will do a slightly different experiment on three separate cover slips, and the data will be shared among groups. We will look at the differences in distribution of dii-ldl and Alexa Fluor 488-Tf (AF488-Tf) endocytic vesicles with continuous incubation for 30 minutes and with a 15 minute pulse and 15 minute chase. This will be the basic experiment. Some groups will do this at 37 C and others will do this at room temperature. Some groups will repeat these experiments in the presence of inhibitors of endocytosis, ammonium ion and nocodazole. Groups 1, 2 and 3 will use LLC-PK endothelial cells. Groups 4, 5 and 6 will use 3T3 fibroblasts. The following table gives the protocols that are common among all groups. Be sure to use the correct temperature, cell type and inhibitor for your group. Exp. Inhibitor preincubation incubation Second 15 Ligand binding 15 incubation # 1 no 20 on ice yes, RT or 37 C absence of ligands 2 no 20 on ice yes, RT or 37 C presence of ligands 3 yes 20 on ice yes, RT or 37 C presence of ligands Groups 1 and 4 will do incubations at 37 C. You will use ammonium ion as the inhibitor. Groups 2 and 5 will do incubations at 37 C. You will use nocodazole as the inhibitor. Groups 3 and 6 will do incubations at room temperature. You will use either nocodazole or ammonium as the inhibitor (your choice). Preliminary setup Make three humid chambers for labeling and subsequent incubation as you did in Module 6a. Use ice-cold buffer and place the chambers on ice in an ice bucket. This will prechill the chamber surfaces. Label each chamber with your initials or other identifying mark. The dishes will be incubated in the presence or absence of inhibitor and also with ligands continuously or using a pulse-chase protocol. Label each chamber so that you can keep track of what s what. Place one 50 μl drop of the dii- LDL/AF488-Tf labeling solution on each of the two humid chamber Parafilm sheets that will be for cells not exposed to inhibitor. Place one 50 μl drop of the dii-ldl/af488-tf labeling solution plus your inhibitor, ammonium ion or nocodazole (see above) on the other humid chamber Parafilm sheet. BE SURE THAT YOU KNOW WHICH 6.15

5 INHIBITOR YOU ARE USING SEE THE NOTES BELOW THE TABLE ON THE PREVIOUS PAGE. Only after you have this set up should you proceed to the next step. You will use the Petri dishes that the cells are in for subsequent steps. Label each dish with your initials or other identifying mark plus the same labels that you used for the humid chambers. One cover slip at a time, remove the growth medium from a cell Petri dish and rinse 3 times with ice-cold buffer. Leave the last buffer rinse in the Petri dish and put it into the ice bucket to keep the cells cold. This will prechill the cells to 4 C. Inhibitor treatment Remove the medium buffer from the cover slip that will be treated with ammonium ion or nocodazole (see above for your inhibitor). Add ~1 ml cold inhibitor solution to this cover slip and incubate it on ice for 30 min. When you have finished this incubation, proceed on to the labeling and incubation steps below. BE SURE THAT YOU KNOW WHICH PROTOCOL YOU ARE USING. ALSO BE SURE THAT THE LABELING AND INCUBATION STEPS INCLUDE THE INHIBITOR FOR THIS COVER SLIP. Ligand binding When you have all three cover slips rinsed and chilled (and one treated with inhibitor), quickly transfer them to the humid chambers to bind dii-ldl to the LDL receptors and AF488-Tf to transferrin receptors on the cell surface. Blot the excess buffer from the cover slips before placing them on the dii-ldl/af488-tf drop. Be very sure that you put the correct cover slip that has been pretreated with inhibitor on the labeling solution that contains the same inhibitor. It is important to keep the cells cold. Do not delay in the transfer of the rinsed cells to the humid chamber. Incubate the cells in the ice bucket for 20 minutes. This will allow time for the dii-ldl and AF488-Tf to bind to their respective receptors. Incubations After the initial 20 minute ligand binding phase is complete, your cover slips will be incubated for 15 minutes to permit ligand-receptor internalization. Take note of your incubation temperature. Move your humid chambers to RT or 37 C, according to the protocol you are following. After the initial 15 minute incubation, one of your uninhibited cover slips will be rinsed with RT or 37 C Fe-PBS 3 times (using the same temperature rinse as you used to incubate). Do the rinses in the appropriate original Petri dish (cell side up). Leave the third rinse with the cover slip in the Petri dish and continue incubating it for another 15 minutes to chase the ligands through the endocytic pathway without adding more ligands to the endocytic pathway. 6.16

6 The other two cover slips will incubate at your experimental temperature for a total of 30 minutes. When they have completed incubation, rinse each 3 times in ice-cold Fe-PBS to remove ligands and inhibitors and to chill the cells. Do the rinses in the appropriate original Petri dish (cell side up). Leave the last rinse on the cells and put the Petri dishes in the ice bucket. Rinse the pulse-chase cover slip once with ice-cold Fe-PBS to chill the cells. Leave the rinse on the cells and put the Petri dish in the ice bucket. Keep the cells on ice until you can fix them in formaldehyde. Do this in the fume hood wearing gloves. The hood is downstairs in room 107. (One group at a time in the hood we will bring you there and show you what to do.) Remove the buffer and replace with 1 ml of the fixative. Fix for 15 minutes. As the cells are now dead, you may mount them on clean labeled slides: place a small drop of mounting media on the slide, and then invert the cover slip onto the drop. Blot excess PBS from the cover slip prior to mounting it in Vectashield mounting medium. Seal with nail polish. Now proceed on to the imaging section below. All groups: imaging The goal of this Module is to examine differences in the processing of a ligand (LDL) that is sent for degradation via the lysosomal pathway and a ligand (Tf) that is recycled to the cell surface. Additionally, we are looking at the effects of two inhibitors of internalization, ammonium ion and nocodazole. The dii-ldl ligand should proceed toward lysosomes in uninhibited cells. The AF488- Tf ligand should internalize into endosomes and sort back to the cell surface where it will be released to the extracellular medium. Thus, the continuous incubation protocol should have dii-ldl at all stages of the lysosomal pathway and should have AF488-Tf throughout the stages of the recycling pathway. The pulse-chase protocol should not have dii-ldl or AF488-Tf in the early stages of their respective pathways. This means that the cell content of dii-ldl should be concentrated in late endosomes and lysosomes. The cell content of AF488-Tf should be depleted to the extent that recycling endosomes have dumped the iron-free Tf back into the extracellular medium. Inhibited cells should show initial formation of early endosomes, but no further processing of ligands. To get at these points you will observe the distribution of dii-ldl and AF488-Tf on the surfaces and in the cytoplasm of your cells. You will take through-focal image series to observe the distribution of these ligands throughout the cell. You will compare the distribution of these ligands under the various different treatment protocols. Observe your cells. For each cover slip of cells, observe by eye and using the camera where dii-ldl and AF488-Tf is detected. DiI has an excitation peak around 540 nm and 6.17

7 an emission peak around 580 nm. The green excitation/red emission fluorescence filter cube will work well for dii-ldl imaging. Alexa Fluor 488-Tf has an excitation peak around 488 nm and an emission peak around 535 nm. The blue excitation/green emission fluorescence filter cube will work well for AF488-Tf imaging. You should get a feel for the distribution of dii-ldl and AF488-Tf in the cells and the variation among cells before storing images. Endosomes will be in the cytoplasm of the cells. Lysosomes are much larger (i.e. brighter) than endosomes and are often found near the cell nucleus. Try to minimize fluorophore photobleaching by shutting off fluorescence excitation between your observations and also use the shutter when taking single pictures with the camera. This is particularly important for the AF488-Tf labeling because the number of fluorophores per transferring is much smaller than the number of fluorophores per LDL, making the AF488-Tf quite a bit dimmer than the dii-ldl. Cell surface and cytoplasmic distribution of dii-ldl and AF488-Tf. Find areas in the cytoplasm that contain endosomes and lysosomes. Take images of the distribution of dii-ldl and AF488-Tf at the same focal plane. Aim to get the brightness of the dii-ldland AF488-Tf-containing vesicles about the same. The goal of this exercise is to see if the two ligands are in the same internalization sites or if they are in different locations. Try to minimize photobleaching of the two fluorophores. Repeat this for all three sets of cover slips. It will be best if you can leave the illumination and exposure settings constant for all three cover slips, but adjust them if you run into a camera overexposure situation. Take phase contrast images of the fields of view that you choose for fluorescence images. Are there differences in the distribution of the two ligands? What is the effect of removal of ligand on the distributions? How does your inhibitor affect what you see? Through-focal series. You should take a series of images from the bottom part of the cell to the top of the cell of the distribution of both fluorophores. The 100X, 1.3 NA lens is best for this purpose. The through-focal imaging can be done in a semi-quantitative way by using the fine focus knob markings. One full rotation of the fine focus control corresponds to 100 μm of movement in the z-direction. Thus, each mark on the fine focus knob equals 1 μm. Find a group of cells that you think is representative of the cell population. Locate the bottom of the cells by focusing on it. While one partner observes the cells, the other partner should change the focus using the fine focus control. Count the number of fine focus marks are needed to scan all the way to the top of the cells. This is the thickness of the cells in μm. (Not all cells will be the same thickness. You should scan to the top of the thickest one in your field of view. The dii- LDL label will be easier to visualize than the AF488-Tf label.) You need to keep the exposure of each of the images in the through-focal series the same as all the rest. You don t want to overexpose very bright elements of any image, so find the brightest area in your image stack and set the fluorescence illumination and shutter exposure time to keep the brightest pixels below the maximum camera signal value (4095). Probably keeping below 3500 is a safe bet in case you missed some 6.18

8 other very bright feature in one of your slices. These exposure settings will be different for the two fluorophores. You can take the through-focal series manually or using the time-lapse collection feature of MicroManager. Because you will process these images as image stacks later on, the images you take should be numbered sequentially so that ImageJ can read them in the correct stack order. Take the through-focal series of images at 1 μm z-steps from the bottom to the top of the cells, taking first a dii-ldl image and then an AF488-Tf image at each z-position. You can do this by taking each pair of images manually between increments in the fine focus knob or you can do this by setting up a time lapse image series (with enough images to cover twice the number of focal steps that you will need). Start the first image at the bottom of the cells with a dii-ldl image. Change to AF488-Tf illumination and take the second image. Move the fine focus up by 1 μm and take the next pair of images. Repeat this until you reach the top of the cells. Having 10 sec between images should give you enough time to change filter cubes and focus after one image has been taken and before the next one is taken. Be sure to use the shutter for pulsed fluorescence illumination to minimize photobleaching of the fluorophores. Note that you will be restricted to only one exposure time if you use the time lapse method, so you will probably need to adjust the fluorescence illumination neutral density filters to balance the exposure of the dii-ldl and AF488-Tf image brightness. Take a phase-contrast image at one of the focal planes to have a reference image for this fluorescence image series. Repeat this for all three sets of cover slips. You might need to reset the illumination level and exposure time. Analysis Cell surface and cytoplasmic distribution of dii-ldl and AF488-Tf. A standard way to compare the relative distributions of two different fluorescent labels is to make a merge image. You have done this already in other modules. With red and green fluorescent probes as you have in this experiment, it is standard to put the red fluorophore (dii-ldl) in the red channel and the green fluorophore (Af488-Tf) in the green channel, leaving the blue channel turned off. To do this, open the two images in ImageJ, then select Image>Color>RGB merge. Through-focal series: To examine the 3-D distributions of dii-ldl and AF488-Tf, read in the through-focal images into two image stacks (File>Import>Image sequence ). If you have numbered the images sequentially as described above, you can get the dii- LDL stack by starting with image 1 and importing every other image. You can get the AF488-Tf stack by starting with image 2 and importing every other image. Set the pixelto-distance conversion in the images with the Set scale menu item. 6.19

9 Now make 3-D projections of the two stacks by using the Image>Stacks>Reslice [/] menu item. The input spacing is 1 μm. Set the output spacing to be μm (otherwise the 3-D projection will use too much memory). Remember that your first slice was at the bottom of the cells, and set the start point accordingly. You should end up with a z-x image stack that lets you look at the interior of the cells and their surfaces in cross section. Make merge images of both the two original x-y image stacks and also the z-x image stack. This can be done by using the ImageJ Image>Color>RGB Merge menu item. It will merge single images or image stacks. To merge the x-y stack, have the dii-ldl image stack and the AF488-Tf stack open at the same time and use them as inputs for the RGB Merge command. Same thing for the z-x stacks. Correlation images: There are image processing methods to quantitate the degree to which two images have features that overlap. Several can be done using ImageJ. One compares the brightness of two images at each pixel of both images and plots this comparison as brightness of one image versus brightness of the other image. This correlation can be done by downloading a plugin for MicroManager from The plugin is called Image_Correlator.class. You should put this plugin in the Micro- Manager1.1/plugins/Micro-Manager folder. Once you do that, you need to quit ImageJ if it is open. When you restart, this plugin will be found in the ImageJ menu item Plugins>Micro-Manager>Image Correlator. Open the two images you want to compare. Both must be converted to so-called 8-bit images, where the maximum value in each pixel is 2 8, or 256. This can be done by selecting the image and the selecting 8-bit using the Image>Type pulldown menu. Do this for both images, enter them into the Image Correlator plugin dialog box as Image 1 and Image 2, and then click OK. The resultant image will give you the correlation between the two images. Points along the diagonal are from pixels where the images are similar. Points off the diagonal are where image pixels are different in value (a bright pixel in image 1 would have a large value in the x-direction but a small value in the y-direction, i.e. it would be at the lower right hand side of such a plot). There are other ways to do quantitative image correlations. Feel free to do some searching to see what other ways you can come up with. 6.20