Modelling intrinsic apoptosis In this part of the course we will model the steps that take place inside a cell when intrinsic apoptosis is triggered. The templates and instructions for preparing the models before you begin this course can be found below. NOTE: you can use Blu-Tack in the place of Velcro dots: MAKE A MITOCHONDRION TO PLACE INSIDE YOUR CELL (see image at bottom of page) 1. 7 x cytochrome C molecules: Cut out and add Velcro or Blu-Tack as shown in the image here. Place cytochrome C molecules inside the mitochondria before you attach paper over the top. 2. One mitochondrion: a container that can easily fit inside your cell with paper covering the top. Use a rubber band or tape to secure the paper over the top of the container. 3. 3 x Bcl-2 protein: Use a jumbo clip or some device that can hold onto Bax protein on the outer mitochondria membrane. Colour it red to indicate that it is an anti-apoptosis protein. 4. 3 x Bax protein: use a pen lid or similar and colour it green to indicate it is a pro-apoptosis protein. Add a piece of Blu-tack to each as you will need to join 3 together when you model intrinsic apoptosis. Place each of these proteins into the jumbo clip handles as they are being held onto by the Bcl-2 anti-apoptosis protein. 1
MAKE SOME PRO-APOPTOSIS PROTEINS 3 x Pro-apoptosis protein: Needs to be similar shape to our Bax protein. Place these in their own zip lock bag and do not put into the cell yet. MAKE AN INACTIVE p53 MOLECULE 1. 1 x p53 protein with a phosphate molecule attached to it by Velcro or Blu-Tack MAKING THE PARTS OF AN APOPTOSOME 1. Make an apoptosome bracelet to place around your wrist. You will add pieces to this during the modelling process so this is why it has Velcro dots (or Blu=Tack) on it. 2. 7 x inactive APAF proteins (pro-apoptosis proteins): Cut out 7 shown on next page. Add a Velcro dot (or Blu-Tack) onto it so cytochrome C can attach if it escapes from the mitochondria. Also add a Velcro dot (or Blu-Tack) onto its foot so it can attach to the apoptosome bracelet if activated. Place these individually inside the cell. 2
Cut out and prepare these APAF pro-apoptosis proteins 3
This is what an activated apoptosome looks like. You will make this as part of modelling intrinsic apoptosis. PREPARE INACTIVE CASPASES 1. 3 x inactive caspase 9 (active site is blocked) 2. 3 x inactive caspase 3 (active site is blocked) PREPARE INACTIVE DNase Two x inactive DNase (active site is blocked) 4
5
6
PREPARE A NUCLEUS TO GO INSIDE THE CELL 1. Nucleus plastic container with lid 2. DNA strands to place inside nucleus PREPARE A CELL TO USE IN MODELLING INSTRINSIC APOPTOSIS Cell membrane large zip-lock bag or clear plastic bag Before you start modelling intrinsic apoptosis place the following inside your cell (large zip-lock bag): 1. One mitochondria (see instructions on page 1) 2. One nucleus containing DNA strands (see instructions on page 7) 3. 7 x inactive APAF proteins (see instructions on page 2 & 3) 4. One deactivated p53 molecule (see instructions on page 2) 5. 3 x inactive caspase 9 (see instructions on page 4 & 5) 6. 3 x inactive caspase 3 (see instructions on page 4 & 5) 7. 2 x inactive DNAse (see instructions on page 4 & 6) 8. Cell cytoskeleton (make out of paper or cardboard so you can rip up with caspases when they become activated) see image on page 6 7
MODELLING INSTRUCTIONS As you watch the animation on intrinsic apoptosis you can pause the video and model the steps in the process of intrinsic apoptosis. A) Make sure you have the following prepared i. Model cell ii. iii. 3 pro-apoptosis proteins in a bag Apoptosome bracelet on your wrist B) Empty the contents of your cell onto a large space. You are now ready to watch the video and model intrinsic apoptosis. Step 1: When UV radiation hits the cell p53 becomes activated. Activate p53 by removing the phosphate group Step 2: p53 moves into the cell nucleus and binds to a promotor region near a pro-apoptosis gene. Model this by opening the nucleus, and attach activated p53 to the promotor region found near the pro-apoptosis gene. Step 3: Gene expression of the pro-apoptosis gene begins. As a result, pro-apoptosis proteins are made. To model this, add 3 pro-apoptosis proteins to the cell (these are located in a bag that was not inside the cell) Step 4: These pro-apoptosis proteins replace the Bax proteins that are being held onto by the Bcl-2 proteins on the mitochondria membrane. To model this, release the Bax proteins from the jumbo clips and replace with new pro-apoptosis proteins. Step 5: the released Bax proteins clump together. To show this, stick 3 Bax proteins together using the Blu-tack you placed onto them. Step 6: the clump of Bax proteins start to perforate the mitochondria membrane. To model this you can use your clump of Bax proteins to punch holes into the paper part of your mitochondria. Step 7: Cytochrome C molecules leak out of the mitochondria through holes in the membrane. Model this by tipping cytochrome C molecules out of the mitochondria Step 8: Cytochrome C molecules attach to inactive APAF proteins. This activates the pro-apoptosis APAF proteins. Model this by sticking your cytochrome C molecules to each of the 7 APAF proteins using Velcro (or Blu-Tack) Step 9: Activated APAF proteins group together to form an apoptosome. Model this by sticking the activated APAF proteins to your apoptosome bracelet. Do this by sticking the feet of the APAF protein to the Velcro or Blu-Tack on the apoptosome bracelet on your wrist. Step 10: The apoptosome activates caspase 9. Model this by ripping off the piece on each caspase 9 to reveal its active site. The active site is where the teeth can be seen. We ve shown them as teeth as this enzyme rips parts of the cell apart. 8
Step 11: Caspase 9 activates caspase 3. Model this by exposing the active site of each caspase 3. Step 12: Caspase 3 activates DNAse. Model this by exposing the active site of each DNAse. Step 13: Activated DNAse rips DNA into fragments. Model this by ripping DNA apart. Step 14: Activated caspases dismantle the cell cytoskeleton. Model this by ripping the cell cytoskeleton into pieces. Step 15: The cell blebs. Model this by arranging parts of the dismantled cell into groups. Step 16: Phagocytes remove and recycle the cell blebs. Model this by using your plastic bag to pick up the pieces of the cell. 9