FREEMAN MEDIA INTEGRATION GUIDE Chapter 7: Inside the Cell

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1 FREEMAN MEDIA INTEGRATION GUIDE Chapter 7: Inside the Cell All media is on the Instructors Resource CD/DVD JPEG Resources Figures, Photos, and Tables PowerPoint Resources Chapter Outline with Figures Lecture Notes with Key Figures Instructor Animations Video Clips CRS/In-Class Questions 90 JPEGs 134 slides 164 slides 5 animations 5 video clips 5 questions INSTRUCTOR ANIMATIONS FOR CHAPTER The Nuclear Envelope: Transport Into and Out of the Nucleus, p The Endomembrane System: Manufacturing and Shipping Proteins, p. 145 From Web Tutorial 7.1 Transport into the Nucleus From Web Tutorial 7.2 A Pulse-Chase Experiment Transport into the Nucleus: Instructor version of student Web Tutorial 7.1 that shows how the pores of the nuclear envelope regulate both passive and active nuclear transport. Descriptions of experiments reveal how a nuclear localization signal on the protein nucleoplasmin was found to target the nucleus delivery of the protein. (A full description including script of the narration is on the Web Tutorial Worksheet found below.) Run time: 3:00 Membrane Transport of Polypeptides: Nuclear Localization Signal Experiment: Experimental demonstration of a nuclear localization signal on polypeptides transported into the nucleus. Nuclear, cytoplasmic, and hybrid proteins are followed after injection into the cell cytoplasm. Run time: 1:15 A Pulse-Chase Experiment: Instructor version of student Web Tutorial 7.2 that shows how pulse-chase experiments using newly synthesized radioactive proteins revealed the pathway of secretory proteins from their synthesis on the RER to their export at the cell membrane. (A full description including script of the narration is on the Web Tutorial Worksheet found below.) Run time: 4:10 Transport of Secreted Proteins from the ER to the Golgi Apparatus: This animation shows how protein transport through the endomembrane system was deduced by following radioactively labeled proteins in a pulse-chase experiment. The proteins chosen were destined for secretion from the cell. Run time: 0:45

2 A Cellular View of the Pulse-Chase Experiment: Using an illustration of a living cell, this animation shows the pulse phase and the chase phase of this experiment and discusses the data obtained from it. Run time: 1:15 VIDEO CLIPS FOR CHAPTER The Endomembrane System: Manufacturing and Shipping Proteins, p The Dynamic Cytoskeleton, p The Dynamic Cytoskeleton, p The Dynamic Cytoskeleton, p. 149 Tracking Vesicle Transport Between the ER and the Golgi: Transport of a fluorescently labeled protein from peripherally located endoplasmic reticulum to a centrally located Golgi Body is seen in this time lapse movie. Proteins, presumably carried in vesicles (not obvious), seem almost to race with darting movements to the target organelle. The movie clearly shows the movement of labeled protein in the secretory pathway similar to what would be seen in a pulse-chase type of experiment. See text Fig 7.26 and Fig Run time: 0:11 Actin-Based Motility in Cell Crawling: Dynamic meshwork of actin filaments forms and disintegrates along the leading edge of crawling keratocytes in this time-lapse video. Phase-contrast microscopy was used to visualize the diagonal cytoskeleton meshwork in the pseudopodia during cell crawling. This movie clearly implicates the cytoskeleton as essential to cell crawling. See text Fig Run time: 0:05 The Actin-Myosin Contraction Cycle: The cycle of attachment and powerstroke are shown in this threedimensional molecular animation based on X-ray crystallographic imagery. Muscle myosin is shown in blue (head piece) and the myosin lever arm changes from yellow to red during the power stroke. The binding site on the actin filament is colored green. The animation begins with the attachment of the myosin head to actin and proceeds with the release of pre-hydrolyzed Pi, the power stroke, the release of ADP, the binding of new ATP, and the cocking of the head with concomitant hydrolysis of the ATP. The actin filament clearly is moved laterally as a result of the cycle. See text Fig Run time: 0:30 Motor Protein: An Animated Model of Kinesin "Walking" Along a Microtubule: This clip offers a threedimensional animated model of kinesin walking, based on X-ray crystallographic imagery. The process begins as one of the two kinesin heads (blue) binds to a microtubule (green and white), releasing its bound ADP, and binding a new molecule of ATP to the "neck linker" region (causing a red to yellow color change in the linker). Subsequently the second head binds and exchanges ADP for ATP as the first head hydrolyzes its ATP and releases Pi. ATP hydrolysis causes the first head to detach from the microtubule and swing forward in what has been described as a "cartoon duckwalk" and rebind to the microtubule. The kinesin heads wobble and flap when unbound in realistic Brownian motion. See text Fig Run time: 0:45

3 7.4 The Dynamic Cytoskeleton, p. 149 Mitochondria Move Along Microtubule Tracks: Tubular mitochondria labeled with Enhanced Yellow Fluorescent Protein (EYFP) move along the invisible cytoskeleton in this time-lapse recording of a mouse embryonic fibroblast. Many of the mitochondria appear localized to the nuclear membrane, the large circular black body in the upper central part of the movie. This clip is useful in demonstrating that mitochondria and other organelles are not fixed in position but can move along microtubule "tracks" with the aid of microtubulebased motor proteins such as kinesin. See text Fig 7.36 and Fig Run time: 0:35 STUDENT WEB TUTORIALS FOR CHAPTER The Nuclear Envelope: Transport Into and Out of the Nucleus, p The Endomembrane System: Manufacturing and Shipping Proteins, p. 145 Web Tutorial 7.1 Transport into the Nucleus: How does a protein molecule too large to pass through the nuclear pore complex enter a cell's nucleus? This tutorial presents several types of experiments that clarified why some proteins can enter the nucleus and others cannot. (A full description including script of the narration is on the Web Tutorial Worksheet found below.) Run time: 3:00 Web Tutorial 7.2 A Pulse-Chase Experiment: How does a pulse-chase experiment allow a scientist to track protein movement in a cell? This tutorial explores an experiment that was used to determine the path of secreted proteins through a pancreas cell. (A full description including script of the narration is on the Web Tutorial Worksheet found below.) Run time: 4:10

4 Web Tutorial 7.1 Transport into the Nucleus Textbook sections 7.2 The Nuclear Envelope: Transport Into and Out of the Cell Nucleus (p. 142) How Are Molecules Imported into the Nucleus? After reading the text material, you should be able to Briefly describe the nuclear membrane and the nuclear pore complex. Explain how the nuclear localization signal (NLS) allows certain large proteins to enter the nucleus of a cell. After completing this tutorial, you should be able to Describe the experiments demonstrating that a protein s size determines whether or not it can enter the cell nucleus. Describe the experiments that clarified why some large proteins can pass into the nucleus and others cannot. NARRATION Transport of Small vs. Large Proteins The nuclear envelope is studded with pores that allow some proteins to enter the nucleus of a cell while restricting the passage of other proteins. The following experiments test whether the ability of a protein to enter the nucleus depends on its size. In this experiment, an investigator injects a solution of small proteins into the cytoplasm of a cell. These small proteins have a molecular weight of less than 60,000 daltons (or grams per mole). Although proteins this small normally would be invisible, they can be detected because they have been labeled with fluorescent molecules, which emit light represented here as stars. The proteins diffuse through the cytoplasm and are small enough to pass through the nuclear pores. After a while, the proteins are equally distributed throughout the cell. The diffusion of small proteins across the nuclear envelope does not require energy and is an example of passive transport. In another experiment, an investigator injects a solution of large, fluorescently labeled proteins into a cell s cytoplasm. These proteins have a molecular weight much greater than 60,000 daltons. Proteins this large cannot diffuse through channels in the nuclear pores. To pass through, they need an active transport mechanism one that requires energy. Transport of Large Proteins: Nuclear vs. Cytoplasmic Although some large proteins cannot enter the nucleus, others can. What is the difference between those that can enter and those that cannot?

5 The following experiments help us understand the difference. These cells will be injected with various types of large, fluorescently labeled proteins. In the first experiment, an investigator injects a solution containing a protein called nucleoplasmin. Even though nucleoplasmin is a large protein, it can enter the nucleus, where it accumulates. How is nucleoplasmin, which is a nuclear protein, different from a cytoplasmic protein? In the second experiment, an investigator injects a large protein that is normally located in the cytoplasm. This protein, called pyruvate kinase, moves randomly throughout the cell but cannot enter the nucleus. In the third experiment, an investigator injects an artificial fusion protein consisting of pyruvate kinase linked to a 17-amino-acid-long segment from nucleoplasmin. Pyruvate kinase can now cross into the nucleus. The 17-amino-acid-long segment contains a nuclear localization signal required for nuclear entry. The transport of these large proteins into the nucleus costs the cell energy in the form of guanosine triphosphate, or GTP a molecule similar to ATP. This transfer is a type of active transport. KEY TERMS & CONCEPTS active transport The movement of molecules or ions across a cell membrane against a concentration gradient (from a region of lower concentration to a region of higher concentration). Such a transfer requires energy. cytoplasm All of the contents of a cell, excluding the nucleus of eukaryotic cells. nuclear envelope A complex double membrane that encloses the nucleus of eukaryotic cells. nuclear localization signal (NLS) A specific amino acid sequence that allows the transport of large molecules through the nuclear pores. nuclear pore An opening in the nuclear envelope that connects the inside of the nucleus with the cytoplasm and allows molecules in the cytoplasm to enter the nucleus. nucleus A large, highly structured organelle in eukaryotic cells that is enclosed by a complex double membrane called the nuclear envelope. passive transport The movement of substances across a cell membrane without the expenditure of energy.

6 Web Tutorial 7.2 A Pulse-Chase Experiment Textbook section 7.3 The Endomembrane System: Manufacturing and Shipping Proteins (p. 145) After reading the text material, you should be able to Describe how a pulse-chase experiment works. After completing this tutorial, you should be able to Describe the steps of protein synthesis, from the protein s origin in the rough endoplasmic reticulum to its secretion into the extracellular fluid. Explain how researchers used a pulse-chase experiment to determine the secretory pathway of protein synthesis. NARRATION Molecular View of a Pulse-Chase Experiment In a pulse-chase experiment, an investigator tracks the progression of a radiolabeled molecule, such as an amino acid, through a cell. Before the experiment begins, protein molecules are being synthesized at a steady state through the translation of mrna by ribosomes. The pulse phase of the experiment begins when investigators add a large dose of a radioactive amino acid in this case, leucine to a cell s culture medium. Thereafter, the radioactive amino acids are incorporated into the proteins manufactured during protein synthesis. The chase phase of the experiment begins when a very large amount of nonradioactive leucine is added to the sample. After the beginning of the chase, no more radioactive proteins are made. This is the basic design of a pulse-chase experiment. The experiment results in a short period of production of radiolabeled molecules, which can then be tracked within the cell. Tracking the Radioactivity In 1955, scientist George Palade and his colleagues used a pulse-chase experiment to determine the roles of the rough endoplasmic reticulum, or rough ER, and Golgi apparatus in the production and secretion of proteins. They studied pancreatic cells that specialize in producing and secreting digestive enzymes.

7 Let s look at the experiment as it would appear to a researcher. To begin the experiment, the researcher adds a large dose of radioactive leucine for the pulse phase. The investigator tracks the positions of radioactive proteins by fixing a sample of cells at different times during the experiment. The fixing process effectively freezes the protein molecules in their locations at the moment in time when the cell is fixed. The cells are prepared for microscopy and overlaid with a photographic emulsion, after which the samples are developed. The radioactive proteins produce black spots on the emulsion s gray background, revealing the locations of the proteins in the cell. If the researcher fixes the sample a few minutes after the pulse begins, radioactivity is observed only in the rough ER. The researcher then adds a large dose of nonradioactive leucine for the chase phase of the experiment. Samples are fixed over the next two hours to track the progression of the black spots, which represent the radioactive proteins. Using this type of experiment, researchers learned that secreted proteins move from their site of manufacture in the rough ER to the Golgi apparatus, then to the secretory vesicles, and finally to the cell s exterior. A Cellular View of the Pulse-Chase Experiment Now let s interpret the data from this pulse-chase experiment using an illustration of a living cell. The green triangles represent digestive enzymes destined for secretion. The pulse phase of the experiment begins with the addition of a large dose of radioactive leucine to the cell s culture medium. The radioactive amino acids enter the cell and are incorporated into new proteins, indicated by the red triangles. The chase phase of the experiment begins with the addition of a very large dose of nonradioactive leucine to the culture medium. Newly synthesized proteins now lack the radiolabeled amino acid. The radiolabeled proteins, which began in the rough ER, have traveled to the Golgi apparatus within 10 minutes. The proteins then move through the cisternae of the Golgi apparatus. After a few hours, they are shuttled in secretory vesicles to the plasma membrane, where they are released outside the cell. This pulse-chase experiment established the path that secreted proteins take from their synthesis in the rough ER to their release outside the cell. KEY TERMS & CONCEPTS cisterna (plural: cisternae) One of the flattened, membrane-bound compartments of the Golgi apparatus. fixing A process in which a cell is killed quickly with a fixative chemical, thereby producing a snapshot of the contents of the cell at the moment it was fixed.

8 Golgi apparatus A stack of flattened membranous sacs in eukaryotic cells that processes the proteins and lipids that will be secreted or directed to other organelles. mrna (messenger RNA) An RNA molecule that carries the encoded information, transcribed from DNA, for building a protein. plasma membrane A membrane that surrounds the cell, separating it from the external environment and selectively regulating passage of molecules and ions into and out of the cell. radiolabeled Marked with a radioactive atom or substance. ribosome A complex of ribosomal RNA (rrna) and proteins that mediates protein synthesis from mrna strands. rough endoplasmic reticulum (rough ER) A type of endoplasmic reticulum that is dotted with ribosomes. secretory vesicle A small membrane-enclosed granule formed from the Golgi apparatus and containing a highly concentrated protein destined for secretion. translation The process by which proteins and peptides are synthesized from messenger RNA molecules.