Biol110L-Cell Biology Lab Spring Quarter 2012 Module 1-4 Friday April 13, 2012 (Start promptly; work fast; the protocols take ~4 h)

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1 Biol110L-Cell Biology Lab Spring Quarter 2012 Module 1-4 Friday April 13, 2012 (Start promptly; work fast; the protocols take ~4 h) A. Microscopic Examination of the Plasma Membrane and Its Properties B. Cellular Fractionation C. Protein Topology Studies Summary: Cells are surrounded by a phospholipid bilayer or plasma membrane. They also contain additional membrane-enclosed organelles distributed throughout their cytoplasm. Biological membranes are semi-permeable, allowing small non-polar molecules across (such as gases, ethanol), but preventing any charged molecules (such as ions) from moving across. Water molecules however move freely and quickly across biological membranes through aquaporin channels and equilibrate cellular osmolarity with environmental osmolarity (i.e. the concentration of solutes in both sides of the plasma membrane). Osmolarity is tightly regulated in cells and cell integrity depends on it. Today you will use sudden osmolarity changes in the environment to burst open (or lyse) yeast spheroplasts in a controlled fashion. When yeast spheroplasts burst, their cytoplasmic proteins, ribosomes and membrane vesicles escape cells through openings in the plasma membrane leaving the spheroplasts looking like ghosts. You will also have a chance to see under the microscope what happens to cells when the environmental osmolarity is suddenly increased using hypertonic buffers. Cellular fractionation can be used to determine the location and topology of a protein within the cell. For example, it can help determine if a protein of interest is soluble, or associated with a cellular component such as a membranous organelle. It can also help determine if a protein is peripherally associated with a membrane (a peripheral membrane protein) or whether it resides in the membrane (i.e. an integral membrane protein). The use of salts and detergents coupled to differential centrifugation can differentiate between these possibilities. Detergents such as Triton X-100 are synthetic, lipid-like molecules used in cell biology research to lyse cells by dissolving their membranes. Triton X-100 is routinely used to extract integral membrane proteins from membranes as a first step in their purification for biochemical analyses. You will explore the above principles using the yeast spheroplasts you prepared, which carry a GFP fusion protein. You will follow its fate as the cells are lysed and fractionated, using Western Blot analysis (next week). Preparative work done by TA: Align and check all microscopes (leave in Ph3 settings if using phase). Have microscopes on benches with oil and lens paper out. Instruct students on its proper use, cleaning, and storage. Provide each group with two live yeast suspensions (~2 OD 600 units of cells in 100 ul media for each group). One suspension prepared from a log-phase culture in fresh 1x YPD media (inoculate >4 h prior to use), and the other from a saturated growth culture in 1x YP media (no glucose). Stock solutions needed for solutions below: 1M Hepes ph 6.8; 2.8 M Sorbitol; 5 M KOAc; 1M Mg(OAc) 2 ; 0.5 M EGTA. Present solutions #1-3 on ice (4 C). Solution #1: Iso osmotic buffer: Make 100 ml (10 ml per group) of 20 mm Hepes ph 6.8, 400 mm sorbitol, 150 mm KOAc, 2 mm Mg(OAc) 2, 0.5 mm EGTA. Solution #2: Low osmotic support buffer (low salt): Make 50 ml total (5 ml per group) of 20 mm Hepes ph 6.8, 50 mm sorbitol, 50 mm KOAc, 2 mm Mg(OAc) 2, 0.5 mm EGTA.

2 Solution #3: High osmotic support buffer (high salt): Make 10 mls (1 ml per group) of 20 mm Hepes ph 6.8, 1.2 M sorbitol, 600 mm KOAc, 2 mm Mg(OAc) 2, 0.5 mm EGTA. Prepare solutions of 5 M NaCl; 10% Triton X-100; 75% 1,6 hexanediol, and 10% SDS in water; keep at room temperature. Aliquot 0.5 ml of each into labeled micro-centrifuge tubes. One set = 4 tubes per group. Bring frozen spheroplasts to lab in dry ice (-70 C). Materials and equipment: 4 micro-centrifuges (2 in the cold room); Tabletop ultracentrifuge with TLA rotor and thick wall polycarbonate tubes ; Light microscopes (16), slides, cover slips, and oil; ice buckets (8). Little buckets with dry ice (-70 C) (8). Top loading balances A. Cell lysis and fractionation 0) Label every tube containing cell fractions with your YS# in addition to any other label. Keep all buffers on ice (4 C). determine when it has melted. Place the tube on ice (4 C) when it does. Save a 10-ul aliquot in a microcentrifuge tube labeled #1-PCL and freeze in dry ice: this is the Pre Cell-Lysis sample. 2) Add 500 ul of chilled iso-osmotic buffer to the (~190 ul) cell slurry, mix gently by tube inversion, and sediment the spheroplasts at 5,000 rpm in the micro-centrifuge for 30 sec. Immediately place back on ice (4 C) and aspirate off the supernatant using the vacuum trap lines. 3) Resuspend the spheroplasts (by gentle pipetting up and down) in 500 ul of chilled iso-osmotic buffer. Sediment the cells again as before, and decant the supernatant by aspiration. 4) Resuspend the cell pellet with 400 ul of Low-osmotic support buffer (Solution #2), and exactly 90 sec after initiating this resuspension add 50 ul of High osmotic support buffer (Solution #3) and mix very well by pipetting. Save a 25 ul aliquot of this cell suspension in a microcentrifuge tube labeled #2-WCE, freeze in dry ice. This represents your Whole Cell Extract, and contains all cell contents after lysis, but prior to fractionation. 5) Sediment the remaining perforated spheroplasts at 2,000 rpm for 2 min, place on ice (4 C), and carefully remove the Low Speed Supernatant; avoid disturbing the pellet (keep the tip close to the meniscus). Place this supernatant into a new microcentrifuge tube labeled LSS. This LSS fraction is largely devoid of perforated or intact cells. 6) Resuspend the Low Speed Pellet with 450 ul of iso-osmotic buffer. Aliquot 25 ul of this fraction into a separate microcentrifuge tube labeled #3-LSP and freeze in dry ice. 7) Centrifuge the remaining LSS fraction at 13,000 rpm (full speed) for 10 min in the microcentrifuge in the adjacent cold room. Then place the tube on your ice bucket and promptly collect the Medium Speed Supernatant (avoid disturbing the pellet); transfer it into a new microcentrifuge tube labeled MSS. 8) Resuspend the Medium Speed Pellet with 200 ul of iso-osmotic buffer. Save 10 ul of this fraction in a microcentrifuge tube labeled #4-MSP and freeze it in dry ice. 9) Finally, transfer 200 ul of MSS to a labeled (mini) ultracentrifuge tube and place the tube in the TLA100.2 rotor. TA: After carefully balancing the tube against another from another group (to within ± 0.01 g) (they should all have ~200 ul), spin the samples at 250,000x g for 10 min at 4 C. Use the TLA100.2 rotor and the Tabletop Ultracentrifuge in the Rexach lab at a speed of 85,000 rpm. When done, transfer the rotor to ice (4 C). Students collect the High Speed Supernatant, avoiding the pellet, and transfer it to a new microcentrifuge tube labeled HSS.

3 10) If the HSP is visible and even if it is not, resuspend it with 20 ul of iso-osmotic buffer and transfer to a tube labeled #5-HSP; then freeze in dry ice. Then, go back to the HSS and save a 20 ul aliquot in a separate microcentrifuge tube labeled #6-HSS and freeze it in dry ice. B. Protein Topology Studies 0) Label every tube containing cell fractions with your YS# in addition to any other label. Keep the 5 M NaCl; 10% Triton X-100; and 10% SDS solutions at room temperature. determine when it has melted. Place the tube on ice (4 C) as soon as it does. 2) Add 500 ul of chilled iso-osmotic buffer, mix gently by tube inversion, and sediment the spheroplasts at 5,000 rpm in the micro-centrifuge for 30 sec. Immediately place back on ice and aspirate off the supernatant using the vacuum trap lines. 3) Resuspend the spheroplasts (by gentle pipetting up and down) in 500 ul of chilled iso-osmotic buffer. Sediment the cells again as before, and decant the supernatant by aspiration. 4) Resuspend the cell pellet with 400 ul of Low-osmotic support buffer (Solution #2), and exactly 90 sec after initiating this resuspension add 50 ul of High osmotic support buffer (Solution #3) and mix very well by pipetting. 5) Aliquot 100 ul portions of this whole cell extract into each of four microcentrifuge tubes containing: a) 25 ul of iso-osmotic buffer b) 25 ul of 5M NaCl c) 25 ul of 10% Triton X-100 d) 25 ul of 75% 1,6 hexanediol (or 50% if needed) 6) After 5 min on ice (4 C) subject the extract to centrifugation at full speed for 10 min in a microcentrifuge in the cold room. 7) Promptly collect 100 ul of the medium speed supernatant fraction from each of the tubes (avoiding contact with the medium speed pellet) and place them into a separate microcentrifuge tubes pre-labeled #7-MSSa, #8-MSSb, #9-MSSc and #10-MSSd. Also mark your YS# on the tubes. Freeze your samples in dry ice. You can toss the MSP's. C. Microscopic examination of biological membranes and their properties: Use your benchtop light microscope; one per person. Follow manual for their proper use and care. Part I- Visualization of intact yeast 1) Prepare a microscope-slide containing two different types of live yeast, which will be provided as a culture by the TA. One represents yeast during log phase of growth and the other yeast at saturated phase. For each, place 5 ul of yeast suspension unto a slide as indicated and gently place the cover slip on top. Make sure not to press the cover slip against the slide; that would crush the cells. 2) Place one drop of oil on top of each cover slip, carefully mount the slide on the microscope and examine the sample under the microscope using the 100x objective (Ph3 setting/annular stop 3; match the number with the white line). Familiarize yourself with the microscope; ask for help if needed. Do not crush the cover slip with the objective; that would crush the cells, break the glass and could ruin the objective. Ideally, the yeast density should be high enough to see many cells, but separate from each other. If there are too many cells, or too few, let the TA know as they can be adjusted.

4 3) Write down notes on the shape of the yeast, and whether they have buds or not and their size relative to the mother cell. Also note the opaqueness of the cytosol and the light halo around each cell, if any. Draw their shapes in your notebook. Part II- Visualization of yeast spheroplasts (one lab partner start at step 1, the other at step 4) determine when it has melted. Place the tube on ice (4 C) when it does. 2) Add 500 ul of chilled iso-osmotic buffer, mix gently by tube inversion, and sediment the spheroplasts at 5,000 rpm in the micro-centrifuge for 30 sec. Aspirate off the supernatant and place tube on ice (4 C). 3) Resuspend the spheroplasts gently in 500 ul of chilled iso-osmotic buffer. Sediment the cells as before, decant the supernatant by aspiration, and re-suspend the cells with 100 ul of chilled iso-osmotic buffer. This will serve as your stock of INTACT yeast spheroplasts for most of the experiments below; keep it on ice (4 C)-do not toss until the very end. 4) Thaw another aliquot of your spheroplasts by holding the tube in your hand. Gently tap on the side to determine when it has melted. Place the tube on ice (4 C) as soon as it does. 5) Add 500 ul of chilled iso-osmotic buffer, mix gently by tube inversion, and sediment the spheroplasts at 5,000 rpm in the micro-centrifuge for 30 sec. Place back on ice (4 C). 6) Aspirate the supernatant and resuspend the spheroplasts gently in 500 ul of chilled iso-osmotic buffer. 7) Sediment the cells again as before, decant the supernatant by aspiration, and quickly re-suspend the cell pellet with 400 ul of low-osmotic support buffer (Solution #2), and exactly 90 sec after initiating this resuspension, add 50 ul of High osmotic support buffer (Solution #3) and mix well by pipetting up and down. Sediment the cells again as before and resuspend with 100 ul of iso-osmotic buffer. This will serve as your stock of PERFORATED yeast spheroplasts. 8) Prepare one microscope slide containing both intact and perforated yeast spheroplasts, side by side. (Yeast settle easily by gravity; mix well before taking aliquots). Take 5 ul of the resuspended spheroplasts (this contains ~3 OD 600 units of cells), dilute with 10 ul of iso-osmotic buffer in a microcentrifuge tube, transfer 5 ul of this to the microscope slide, and GENTLY place the cover slip on top. Do not press on the coverslip or you ll crush the spheroplasts (very fragile). Compare the morphological differences between intact spheroplasts and perforated spheroplasts. Note any notable differences between them based on appearance and shape and draw them in your notebook. 9) Also compare the morphology of live yeast (from before) against the yeast spheroplasts. Write down your observations, note major differences between them based on what you can see (shape, buds, opaqueness of cytosol, and light halo around the cells, if any). Draw the shape of the yeast observed in your notebook. Part III- Effect of hyper-osmotic solutions (high salt) on yeast spheroplasts 1) Label two micro-centrifuge tubes (one NaCl and the other buffer ) and add 10 ul of 5 M NaCl and 10 ul of iso-osmotic buffer (in the corresponding tube). 2) To each tube, add 5 ul of the yeast spheroplast suspension (from part II step #3, mix it well) for a ~15 ul total volume, and mount 5 ul of each mix on one microscope slide. 3) Place the cover slips, the oil, and visualize under the microscope as before. Note what happens to the spheroplasts in the hyper-osmotic environment. Explain why. Draw your observations in your notebook.

5 Part IV- The effect of hypo-osmotic solutions (low salt) on cells 1) Mix 3 ul of the yeast spheroplast suspension (from part II, step 3) with 12 ul of water in a tube, and mix well. 2) Spot 5 ul of this suspension on a microscope slide, put a cover slip on it, a drop of oil, and focus on the cells. Note what happens to the spheroplasts in this hypo-osmotic environment. Explain why. Draw your observations in your notebook. Part V- The effect of ionic detergents (SDS), non-ionic detergents (Triton X-100) and aliphatic alcohols (1,6 hexanediol) on cell membranes. 1) Mix 3 ul of the yeast spheroplast suspension (from part II, step 3) with12 ul of 10% Triton X100; or with 12 ul of 10% SDS; or with 12 ul of 25% 1,6 hexanediol in separate tubes. 2) After mixing, spot 5 ul of each mixture on a microscope slide, put a cover slip on each, add a drop of oil, and focus on the cells. Note what happens to the spheroplasts in the presence of each chemical. Explain why. Draw your observations in your notebook.