FREEMAN MEDIA INTEGRATION GUIDEChapter 7: Inside the Cell

JPEG ResourcesFigures, Photos, and Tables 90 JPEGs

PowerPoint® ResourcesChapter Outline with Figures 134 slidesLecture Notes with Key Figures 164 slidesInstructor Animations 5 animationsVideo Clips 5 video clipsCRS/In-Class Questions 5 questions

All media is on the Instructors Resource CD/DVD

INSTRUCTOR ANIMATIONS FOR CHAPTER 7•Transport into the Nucleus: Instructor version of stu-dent Web Tutorial 7.1 that shows how the pores of thenuclear envelope regulate both passive and activenuclear transport. Descriptions of experiments revealhow a nuclear localization signal on the protein nucleo-plasmin was found to target the nucleus delivery of theprotein. (A full description including script of the narra-tion is on the Web Tutorial Worksheet found below.)Run time: 3:00

•Membrane Transport of Polypeptides: NuclearLocalization Signal Experiment: Experimental demon-stration of a nuclear localization signal on polypeptidestransported into the nucleus. Nuclear, cytoplasmic, andhybrid proteins are followed after injection into the cellcytoplasm. Run time: 1:15

From Web Tutorial 7.1Transport into theNucleus

Text Section7.2The NuclearEnvelope:Transport Intoand Out of theNucleus,p. 142

•A Pulse-Chase Experiment: Instructor version of stu-dent Web Tutorial 7.2 that shows how pulse-chaseexperiments using newly synthesized radioactive pro-teins revealed the pathway of secretory proteins fromtheir synthesis on the RER to their export at the cellmembrane. (A full description including script of thenarration is on the Web Tutorial Worksheet foundbelow.) Run time: 4:10

•Transport of Secreted Proteins from the ER to theGolgi Apparatus: This animation shows how proteintransport through the endomembrane system wasdeduced by following radioactively labeled proteins ina pulse-chase experiment. The proteins chosen weredestined for secretion from the cell. Run time: 0:45

From Web Tutorial 7.2A Pulse-ChaseExperiment

Text Section7.3TheEndomembraneSystem:Manufacturingand ShippingProteins,p. 145

•A Cellular View of the Pulse-Chase Experiment:Using an illustration of a living cell, this animationshows the pulse phase and the chase phase of thisexperiment and discusses the data obtained from it.Run time: 1:15

VIDEO CLIPS FOR CHAPTER 7•Tracking Vesicle Transport Between the ER and theGolgi: Transport of a fluorescently labeled protein fromperipherally located endoplasmic reticulum to a central-ly 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 tar-get organelle. The movie clearly shows the movementof labeled protein in the secretory pathway similar towhat would be seen in a pulse-chase type of experi-ment. See text Fig 7.26 and Fig 7.27. Run time: 0:11

Text Section7.3TheEndomembraneSystem:Manufacturingand ShippingProteins,p. 145

•Actin-Based Motility in Cell Crawling: Dynamicmeshwork of actin filaments forms and disintegratesalong the leading edge of crawling keratocytes in thistime-lapse video. Phase-contrast microscopy was usedto visualize the diagonal cytoskeleton meshwork in thepseudopodia during cell crawling. This movie clearlyimplicates the cytoskeleton as essential to cell crawling.See text Fig 7.34. Run time: 0:05

Text Section7.4The DynamicCytoskeleton,p. 149

•The Actin-Myosin Contraction Cycle: The cycle ofattachment and powerstroke are shown in this three-dimensional molecular animation based on X-ray crys-tallographic imagery. Muscle myosin is shown in blue(head piece) and the myosin lever arm changes fromyellow to red during the power stroke. The binding siteon the actin filament is colored green. The animationbegins with the attachment of the myosin head to actinand proceeds with the release of pre-hydrolyzed Pi, thepower stroke, the release of ADP, the binding of newATP, and the cocking of the head with concomitanthydrolysis of the ATP. The actin filament clearly ismoved laterally as a result of the cycle. See text Fig 7.34.Run time: 0:30

Text Section7.4The DynamicCytoskeleton, p. 149

•Motor Protein: An Animated Model of Kinesin"Walking" Along a Microtubule: This clip offers a three-dimensional animated model of kinesin walking, basedon X-ray crystallographic imagery. The process beginsas one of the two kinesin heads (blue) binds to a micro-tubule (green and white), releasing its bound ADP, andbinding a new molecule of ATP to the "neck linker"region (causing a red to yellow color change in the link-er). Subsequently the second head binds and exchangesADP for ATP as the first head hydrolyzes its ATP andreleases Pi. ATP hydrolysis causes the first head todetach from the microtubule and swing forward inwhat has been described as a "cartoon duckwalk" andrebind to the microtubule. The kinesin heads wobbleand flap when unbound in realistic Brownian motion.See text Fig 7.37. Run time: 0:45

Text Section7.4The DynamicCytoskeleton, p. 149

STUDENT WEB TUTORIALS FOR CHAPTER 7

•Mitochondria Move Along Microtubule Tracks:Tubular mitochondria labeled with Enhanced YellowFluorescent Protein (EYFP) move along the invisiblecytoskeleton in this time-lapse recording of a mouseembryonic fibroblast. Many of the mitochondria appearlocalized to the nuclear membrane, the large circularblack body in the upper central part of the movie. Thisclip is useful in demonstrating that mitochondria andother organelles are not fixed in position but can movealong microtubule "tracks" with the aid of microtubule-based motor proteins such as kinesin. See text Fig 7.36and Fig 7.37. Run time: 0:35

Text Section7.4The DynamicCytoskeleton, p. 149

•Web Tutorial 7.1 Transport into the Nucleus: Howdoes a protein molecule too large to pass through thenuclear pore complex enter a cell's nucleus? This tutori-al presents several types of experiments that clarifiedwhy some proteins can enter the nucleus and otherscannot. (A full description including script of the narra-tion is on the Web Tutorial Worksheet found below.)Run time: 3:00

Text Section7.2The NuclearEnvelope:Transport Intoand Out of theNucleus, p. 142

•Web Tutorial 7.2 A Pulse-Chase Experiment: Howdoes a pulse-chase experiment allow a scientist to trackprotein movement in a cell? This tutorial explores anexperiment that was used to determine the path ofsecreted proteins through a pancreas cell. (A fulldescription including script of the narration is on theWeb Tutorial Worksheet found below.) Run time: 4:10

Text Section 7.3TheEndomembraneSystem:Manufacturingand ShippingProteins, p. 145

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 whetheror not it can enter the cell nucleus.

• Describe the experiments that clarified why some large proteins can pass into thenucleus and others cannot.

NARRATIONTransport of Small vs. Large ProteinsThe nuclear envelope is studded with pores that allow some proteins to enter the nucleusof a cell while restricting the passage of other proteins. The following experiments testwhether 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 cytoplasmof a cell. These small proteins have a molecular weight of less than 60,000 daltons (orgrams per mole). Although proteins this small normally would be invisible, they can bedetected because they have been labeled with fluorescent molecules, which emit light—represented here as stars. The proteins diffuse through the cytoplasm and are smallenough to pass through the nuclear pores. After a while, the proteins are equally distrib-uted throughout the cell. The diffusion of small proteins across the nuclear envelope doesnot require energy and is an example of passive transport.

In another experiment, an investigator injects a solution of large, fluorescently labeled pro-teins into a cell’s cytoplasm. These proteins have a molecular weight much greater than60,000 daltons. Proteins this large cannot diffuse through channels in the nuclear pores. Topass 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 differencebetween those that can enter and those that cannot?

The following experiments help us understand the difference. These cells will be injectedwith various types of large, fluorescently labeled proteins.

In the first experiment, an investigator injects a solution containing a protein called nucle-oplasmin. Even though nucleoplasmin is a large protein, it can enter the nucleus, where itaccumulates. How is nucleoplasmin, which is a nuclear protein, different from a cytoplas-mic protein?

In the second experiment, an investigator injects a large protein that is normally located inthe cytoplasm. This protein, called pyruvate kinase, moves randomly throughout the cellbut cannot enter the nucleus.

In the third experiment, an investigator injects an artificial fusion protein consisting ofpyruvate kinase linked to a 17-amino-acid-long segment from nucleoplasmin. Pyruvatekinase can now cross into the nucleus. The 17-amino-acid-long segment contains a nuclearlocalization signal required for nuclear entry.

The transport of these large proteins into the nucleus costs the cell energy in the form ofguanosine triphosphate, or GTP—a molecule similar to ATP. This transfer is a type ofactive transport.

KEY TERMS & CONCEPTSactive transport The movement of molecules or ions across a cell membrane against a con-centration gradient (from a region of lower concentration to a region of higher concentra-tion). 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 eukaryoticcells.

nuclear localization signal (NLS) A specific amino acid sequence that allows the transportof large molecules through the nuclear pores.

nuclear pore An opening in the nuclear envelope that connects the inside of the nucleuswith 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 com-plex double membrane called the nuclear envelope.

passive transport The movement of substances across a cell membrane without the expen-diture of energy.

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 roughendoplasmic reticulum to its secretion into the extracellular fluid.

• Explain how researchers used a pulse-chase experiment to determine the secreto-ry pathway of protein synthesis.

NARRATIONMolecular View of a Pulse-Chase Experiment

In a pulse-chase experiment, an investigator tracks the progression of a radiolabeled mol-ecule, such as an amino acid, through a cell.

Before the experiment begins, protein molecules are being synthesized at a steady statethrough the translation of mRNA by ribosomes.

The pulse phase of the experiment begins when investigators add a large dose of aradioactive amino acid—in this case, leucine—to a cell’s culture medium. Thereafter, theradioactive amino acids are incorporated into the proteins manufactured during proteinsynthesis.

The chase phase of the experiment begins when a very large amount of nonradioactiveleucine is added to the sample. After the beginning of the chase, no more radioactive pro-teins are made.

This is the basic design of a pulse-chase experiment. The experiment results in a short peri-od 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 deter-mine the roles of the rough endoplasmic reticulum, or rough ER, and Golgi apparatus inthe production and secretion of proteins. They studied pancreatic cells that specialize inproducing and secreting digestive enzymes.

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 atdifferent times during the experiment. The fixing process effectively “freezes” the proteinmolecules 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, afterwhich the samples are developed. The radioactive proteins produce black spots on theemulsion’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 isobserved only in the rough ER.

The researcher then adds a large dose of nonradioactive leucine for the chase phase of theexperiment.

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 theirsite 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 liv-ing 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 radioactiveleucine to the cell’s culture medium. The radioactive amino acids enter the cell and areincorporated into new proteins, indicated by the red triangles.

The chase phase of the experiment begins with the addition of a very large dose of nonra-dioactive leucine to the culture medium. Newly synthesized proteins now lack the radio-labeled amino acid.

The radiolabeled proteins, which began in the rough ER, have traveled to the Golgi appa-ratus within 10 minutes. The proteins then move through the cisternae of the Golgi appa-ratus. 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 theirsynthesis in the rough ER to their release outside the cell.

KEY TERMS & CONCEPTScisterna (plural: cisternae) One of the flattened, membrane-bound compartments of theGolgi apparatus.

fixing A process in which a cell is killed quickly with a fixative chemical, thereby produc-ing a snapshot of the contents of the cell at the moment it was fixed.

Golgi apparatus A stack of flattened membranous sacs in eukaryotic cells that processesthe proteins and lipids that will be secreted or directed to other organelles.

mRNA (messenger RNA) An RNA molecule that carries the encoded information, tran-scribed from DNA, for building a protein.

plasma membrane A membrane that surrounds the cell, separating it from the externalenvironment and selectively regulating passage of molecules and ions into and out of thecell.

radiolabeled Marked with a radioactive atom or substance.

ribosome A complex of ribosomal RNA (rRNA) and proteins that mediates protein syn-thesis from mRNA strands.

rough endoplasmic reticulum (rough ER) A type of endoplasmic reticulum that is dottedwith ribosomes.

secretory vesicle A small membrane-enclosed granule formed from the Golgi apparatusand containing a highly concentrated protein destined for secretion.

translation The process by which proteins and peptides are synthesized from messengerRNA molecules.