Answer Key: Cellular Organization

BIOL230W, Week 1 – Cellular Organization Day 1 Learning Objectives 1. Explore and outline the symptoms of cystic fibrosis. 2. Hypothesize the mechanism that results in cystic fibrosis symptoms as related to structures in the cell. Small Group Activity: Symptoms of Cystic Fibrosis Below is a list of some of the symptoms associated with cystic fibrosis. Please use this list and Wikipedia/Google to answer the next questions.         A persistent cough that produces thick mucus Difficulty breathing Repeated lung and sinus infections Chronic constipation Foul-smelling and/or greasy feces due to clogged pancreatic ducts Female infertility associated with thickened cervical mucus Malnutrition Hypoxia (lack of oxygen delivery to tissues) 1. Do you notice any similarities and/or relationships between these symptoms? Malnutrition is likely related to female infertility since fertility is linked to nutrition. Additionally, clogged pancreatic ducts may be related to the constipation. 2. What type of tissue lines the lungs, digestive tract and makes the pancreas: epithelial, muscle, dense connective, or nervous? Epithelial tissue is what lines the lungs. The digestive tract is lined by several different types of tissue that comprise the mucosa (innermost layer of tract): epithelial, connective, and muscular. Finally, the pancreas is lined by epithelium. 3. Can you hypothesize a common mechanism for these symptoms related to cells of epithelial tissue and water transport? Cystic fibrosis may be caused by difficulties of the cells to transport water, which leads to problems with the excretory and respiratory systems since waste is not as easy to remove. Upon research and looking at class notes, cystic fibrosis is actually caused by a gene defect leading to epithelial tissue not exporting water to thin out mucus. Instead, it becomes too thick and causes blockages. Day 2 Learning Objectives 1. Identify the different parts of a phospholipid. 2. Relate how the parts of the phospholipid contribute to its amphipathic characteristic and how the characteristics affect the formation of a phospholipid bilayer. 3. Determine what types of molecules can diffuse through a membrane and why. 1 BIOL230W, Week 1 – Cellular Organization 4. Discuss the properties of a membrane that allow it to be selectively permeable. 5. Define the process of osmosis. Small Group Activity: Cell Membrane Structure and Transport Last class we learned that one symptom of cystic fibrosis is a thick mucus covering the epithelial cells associated with our respiratory tract. The epithelial cells are surrounded by a cell membrane composed of a phospholipid bilayer. 1. Draw a representation of a phospholipid (phosphate group, glycerol, fatty acids) and phospholipid bilayer (phosphate group, fatty acids, hydrophilic regions, hydrophobic region) in the space below. Label the listed parts of each structure. PHOSPHOLIPID PHOSPHOLIPID BILAYER 2. What characteristics of phospholipids cause them to assemble into a bilayer in an aqueous environment? The head is charged, which makes it hydrophilic. The tails are not charged, which makes them hydrophobic. Therefore, they arrange so that the hydrophilic heads are on the outside, and the hydrophobic tails are on the inside. 3. What feature(s) of the membrane allow it to be “selectively permeable” (i.e., allow some molecules to diffuse through and other not)? Hydrophilic heads face outwards, and hydrophobic tails face inwards. This creates a hydrophobic core of the membrane. Therefore, certain things can pass through the hydrophobic core (nonpolar molecules, or molecules that are small and small charge) 4. Using the structure of the bilayer drawn above, discuss if the following molecules can diffuse across a membrane.   2 A small non-polar molecule: Yes A small polar molecule: Yes BIOL230W, Week 1 – Cellular Organization   A large polar molecule: No An ion: No 5. Can water diffuse across a cell membrane? Please explain your answer. Yes, but it can diffuse slowly. It is small so it can fit, but it will have difficulty passing the hydrophobic core. Osmosis is the diffusion of water through a semipermeable membrane from an area of low solute concentration to an area of high solute concentration. The solute is typically polar or charged molecules. Cells use the transport of solutes to move water; there is no cellular mechanism to actively transport water. A cell surrounded by extracellular fluid that has a higher concentration of solutes compared to the cytoplasm is said to be hypertonic, and water molecules tend to diffuse into the hypertonic environment. In contrast, a solution that has a lower concentration of solutes than another solution is said to behypotonic, and water molecules tend to diffuse out of a hypotonic solution. Cells in a hypotonic solution will take on too much water and swell with the risk of eventually bursting. 6. In the space below, draw a representation of a cell in a hypertonic extracellular fluid and a cell in a hypotonic extracellular fluid. Draw an arrow representing the diffusion of water for each cell. HYPERTONIC HYPOTONIC 7. Hypothesize how the process of osmosis in epithelial cells plays a role in the thick mucus found in cystic fibrosis patients. (Hint: Why would mucus produced and deposited on the outside of cells be thicker than normal?) 3 BIOL230W, Week 1 – Cellular Organization Water is hindered from osmosis, since the mucus is too thick. This means there is not enough water able to diffuse into the mucus, so it is too hypertonic (too little water, too much solute). 8. If ions cannot pass through a membrane, how are they transported into or out of the cell to drive osmosis? Ions are transported via channels embedded in the cell membrane that open or close based on if the solution is hypertonic or hypotonic outside the cell. Summary Exercise: In the space below, please describe how a cell can regulate the water concentration of the extracellular environment using the transport of other molecules. To make a solution outside of the cell less hypotonic, a cell may open ion channels in the cell membrane to allow ions to enter the extracellular fluid. In contrast, if the outside fluid is too hypertonic, the cell may close those channels once the solution becomes isotonic to maintain fluid-ion balance. Day 3 Learning Objectives: 1. Define the three major categories of organelles and list example organelles. 2. Describe the general evolutionary process of the nucleus and endomembrane system as related to FECA and LECA. 3. Describe the major functions of the organelles of the endomembrane system. 4. Relate the different cellular locations of the endomembrane system with function in gene expression (i.e., where does transcription/translation occur). 5. Use a micrograph to analyze information related to the endomembrane system. We have already examined how phospholipids form bilayers to make cell membranes. Phospholipid bilayers are also the main structural component of several other organelles. Today we will focus on organelles of the endomembrane system and their relationship to protein synthesis in the cell. Mini-Lecture Questions 1. List three categories of organelles and underline the two that require membrane manipulation via proteins for formation? Membrane-bound, proteinaceous, and endosymbiotic 2. Compare and contrast the FECA and the LECA with respect to the mitochondria, the nucleus, and the chloroplast. FECA is the first eukaryotic common ancestor and demonstrates the first eukaryotic cell to exist. The FECA existed right at the entrance of nucleus into the prokaryote, and mitochondria formed right after it. The LECA is the last eukaryotic common ancestor and describes the last eukaryotic cell that existed 4 BIOL230W, Week 1 – Cellular Organization prior to divergence of species. The LECA had nuclei and mitochondria prior to divergence, but chloroplast was formed at divergence. Small Group Activity: Protein Localization in Cells Scientists have identified a protein that when mutated leads to the symptoms of cystic fibrosis. Below are several micrographs of cells thatdo not express the protein (image A – referred to as CFPAC cells), cells that express the normal protein (image B – referred to as Wildtype or WT) or cells that express amutated form of the protein (image C and D – referred to as ∆F508 CFTR). It may be helpful to label the images to make it easy to interpret them. *Labels difficult see areand highlighted in awith whitean outline. Each that cellare type wasto fixed treated antibody that is specific to the identified protein. The antibody binds to and labels the WT or mutated form of the protein with a dark stain. Please use the caption, knowledge from class or the Google/Wikipedia to answer the following questions. 3. Transmission electron microscopy was used in this experiment. What information can you visualize using this technique vs. a simple light microscope commonly used in lab? Answer based on your past experiences. The image is much higher resolution with the transmission microscope as compared to a light microscope, so you can see smaller things and you can see them more clearly. The transmission microscopes use electrons to visualize, and light uses visual light. 5 BIOL230W, Week 1 – Cellular Organization 4. Several organelles are labeled in the image with abbreviations. Please list the organelles shown and include major categories (i.e., proteinaceous, membrane-bound, or endosymbiotic) and function based on what we discussed in class. Mitochondria – endosymbiotic; Endoplasmic reticulum – membrane-bound; Nucleus – membranebound; Golgi complex – membrane-bound; plasma membrane – membrane-bound; multivesicular body – membrane-bound 5. CFPAC cells are derived from a cystic fibrosis patient that produces little or none of the identified protein. What is the significance of showing the staining pattern of these cells (image A) in this experiment? (Hint – Why stain cells that are known to NOT express the protein?) Dyes are used on control groups as well to get “background” which is non-specific binding. In other words, it is used to show what may stain that is not the protein of interest, since the stain may bind to things that are not the protein of interest. Therefore, it allows us to identify what is and what is not the protein. 6. What organelles does the protein localize to in Image B? Remember you are looking for a dark staining pattern to indicate the location of the protein. You may need to you Image A as a comparison. In image B (wild type), the protein accumulates by the plasma membrane. 7. What organelle does the protein localize to in Image C and D? The protein is accumulating in the endoplasmic reticulum. 8. How do proteins move between organelles/plasma membrane? Vesicles 9. Summarize the major difference between localization of the wildtype and the mutated protein? In the wild type (normal), the protein accumulates by the plasma membrane (increases solute concentration outside cell, ion transport) whereas in the mutated cells it accumulates by the endoplasmic reticulum instead (can’t transport ions across membrane. 6 BIOL230W, Week 1 – Cellular Organization 10. Thinking about the general synthesis and transport of proteins, what might cause the differential localization pattern? (Hint – please consider where the normal protein in translated, cytoplasm vs. ER and how proteins move between organelles/plasma membrane after translation). After translation, proteins are transported using vesicles that are not bound in the cytoplasm and can move freely. Proteins are normally translated in ribosomes which are also freely moving in the cytoplasm. However, in cells with the mutation, the proteins are transported in the ER which is membrane-bound and therefore they are stuck there instead of being free to move to the membrane. 11. We have been discussing protein synthesis, processing, and transport. Considering what you know about gene expression (i.e., the Center Dogma of Biology), what has occurred prior to the events we discussed today. Information transcribed into RNA and transported into endoplasmic reticulum Image from Yang, Yiping, et al. "The common variant of cystic fibrosis transmembrane conductance regulator is recognized by hsp70 and degraded in a preGolgi nonlysosomal compartment." Proceedings of the National Academy of Sciences90.20 (1993): 9480-9484. Summary Exercise 1. Drawing from what you learned about the Central Dogma of biology and protein transport in Biol110, draw a flow diagram indicating the order of events in these processes. Information in DNA is transcribed into RNA  RNA travels from nucleus to cytoplasm via vesicles  RNA is translated in ribosomes not bound in cytoplasm  proteins are formed and transported to plasma membrane via vesicles 2. Draw an arrow to the step that is disrupted in the mutants discussed above. 7