1. MG5607, Cell Biology Instructor: Paul K. Herman 842 Riffe Building Office hours: Wed 4-5 PM E-mail: herman.81@osu.edu Mid-Term Exams (In class): Mon. Sept. 29 th (25%) Mon. Nov. 12 th (25%) Final Exam: Mon. Dec. 11 th (50%) Same room; from 4:00 - 5:45 PM

2. Molecular Genetics 5607, Cell Biology (14) DateTopics CoveredReading Assignment Aug. 27 (W)HermanOverview/ Membrane StructureChapter 10 Aug. 29 (F)HermanMembrane TransportChapter 11 Sept. 1 (M)*******No Class - Labor Day Sept. 3 (W)HermanMembrane Transport Sept. 5 (F)HermanMembrane Transport Sept. 8 (M)HermanProtein ImportChapter 12 Sept. 10 (W)Herman Import into Mitochondria Sept. 12 (F) Herman Import into ER Sept. 15 (M)HermanVesicular traffic: Coated vesiclesChapter 13 Sept. 17 (W)HermanVesicular traffic Sept. 19 (F)HermanProtein folding & degradationChapter 6 Sept. 22 (M)HermanCell SignalingChapter 15 Sept. 24 (W)HermanCell Signaling Sept. 26 (F)HermanReview Sept. 29 (M)HermanMIDTERM EXAM-1 Oct. 1 (W)HermanCell Signaling Oct. 3 (F)HermanCell Signaling Oct. 6 (M)FiskHow Cells are StudiedChapter 9 Oct. 8 (W)FiskHow Cells are Studied Oct. 10 (F)HermanCell Signaling Oct. 13 (M)Herman ApoptosisChapter 18 Oct. 15 (W)Herman CancerChapter 20 Oct. 17 (F)HermanCancer

3. Required Text: Alberts et al., 5th Edition Molecular Biology of the Cell Reading Assignment I: Ch. 12 Compartmentalization(pp. 695 - 699) Ch. 10 Membrane Structure(pp. 617 - 640) Ch. 11 Membrane Transport(pp. 651 - 683) Ch. 12 Protein Sorting(pp. 699-704, 713-745) Ch. 13 Vesicular Traffic(pp. 749 - 766) Ch. 6 Protein Folding(pp. 387-398) Ch. 15 Cell Signaling(pp. 879-937) Ch. 18 Apoptosis(TBA) Ch. 20 Cancer(TBA)

4. Figure 12-1 Molecular Biology of the Cell (© Garland Science 2008)

5. Figure 12-2 Molecular Biology of the Cell (© Garland Science 2008)

6. Cell Compartmentalization * Eukaryotic cells are subdivided into a set of functionally- distinct, membrane-enclosed compartments * Internal compartments comprise ~50% of cell volume * Plasma membrane contains ~5% of total cell membrane * Organelles are nonrandomly distributed within the cell

7. Questions that we will be addressing: * What are the major organelles and their functions? * How are the individual compartments formed? How are the relevant proteins delivered to the correct target organelle? * Why and how did the eukaryotic cell become compartmentalized? * How do small and large molecules pass through the membrane barrier? * How do cells communicate with each other across their respective plasma membranes? * How are extracellular signals recognized & processed by the cell?

8. Cell Compartments Nucleus: - contains DNA genome - site of DNA and RNA synthesis Cytoplasm: - comprises ~50% of cell volume - site of protein synthesis and intermediary metabolism Endoplasmic Reticulum (ER): - made up of smooth and rough ER - site of entry into the secretion pathway - major site of lipid biosynthesis - comprises > 50% of total cell membrane Golgi Complex: - made up of stacks of disc-like compartments - an intermediate compartment of the secretion pathway

9. Cell Compartments (cont’d) Mitochondria: - responsible for most of the ATP production in eukaryotic cells - enclosed within a double membrane Lysosomes: - contains a variety of digestive or degradative enzymes - responsible for degradation of cellular macromolecules, endocytosed material and defunct organelles (autophagy) Endosomes: - a series of compartments that serve as an intermediate destination for endocytosed material Peroxisomes: - vesicular compartments that carry out a variety of oxidative reactions

10. Evolution of eukaryotic cells * The precursor to first eukaryotic cells likely resembled bacterial cells. * Membrane-dependent functions were carried out by plasma membrane proteins. * The proliferation of internal membranes may have come about, in part, as a response to an increase in cell size. * The evolution of internal membranes likely occurred concomitantly with the specialization of membrane function. The cell size problem: As cells became larger, the surface-to-volume ratio decreases. As a consequence, the plasma membrane would no longer be sufficient to carry out those membrane reactions essential for cell viability. The Solution? Increase membrane area by accumulating specific membranes within the interior of the cell.

11. Figure 12-4a Molecular Biology of the Cell (© Garland Science 2008) Possible origin of the ER and nucleus

12. Figure 12-4b Molecular Biology of the Cell (© Garland Science 2008) Possible origins of the mitochondria

13. Membrane Structure * Lipid molecules in biological membranes are arranged as continuous double layer, known as a "bilayer". * All lipids in cell membranes are amphipathic (amphiphilic). * Phospholipids are the most abundant cellular lipids. * Phospholipids possess a polar head group and two hydrophobic hydrocarbon tails. * Lipid bilayers are two-dimensional fluids; proteins and lipids both exhibit rapid lateral diffusion. * Fluidity of a lipid bilayer depends on its composition.

14. Figure 10-1 Molecular Biology of the Cell (© Garland Science 2008) Part A shows an electron micrograph of a plasma membrane (of human RBC) seen in cross section. Parts B & C show two- and three-dimensional views of a cell membrane. The Lipid Bilayer

15. Figure 10-2 Molecular Biology of the Cell (© Garland Science 2008) The parts of a phospholipid molecule:

16. Figure 10-7 Molecular Biology of the Cell (© Garland Science 2008) Packing arrangements of lipid molecules in an aqueous environment

17. Figure 10-8 Molecular Biology of the Cell (© Garland Science 2008) The spontaneous closure of a phospholipid bilayer to form a sealed compartment.

18. Liposomes 100 nm

19. Phospholipid mobility: Figure 10-11b Molecular Biology of the Cell (© Garland Science 2008)

20. Lateral phase separation in artificial lipid bilayers (Fig. 10-13) Lipid rafts are transient domains within a bilayer that form as a result of the physical properties of some lipid molecules Particular proteins can be concentrated within lipid rafts (Fig. 10-14)

21. The structure of cholesterol Figure 10-4 Molecular Biology of the Cell (© Garland Science 2008)

22. Schematic drawing of a cholesterol molecule interacting with two phospholipid molecules in one monolayer of a lipid bilayer. The presence of cholesterol tends to decrease the permeability of the bilayer. Figure 10-5 Molecular Biology of the Cell (© Garland Science 2008)

23. The four major phospholipids (A-D) in mammalian plasma membranes Figure 10-3 Molecular Biology of the Cell (© Garland Science 2008)

24. Membrane Proteins * Most of the functions of the membrane bilayer are performed by associated proteins. * Proteins associate with membrane bilayers in several different ways. * Transmembrane proteins have amino acid stretches embedded within the lipid bilayer. * The membrane-spanning regions tend to be  -helical, especially in single-pass transmembrane proteins. * In multipass transmembrane proteins, the TMDs may be arranged as a  -sheet in the form of a closed barrel. * Membrane proteins may diffuse laterally within the plane of the lipid bilayer.

25. 1. Single  helix. 2. Multiple  helices. 3.  -barrel (rolled-up  sheet). 4. Anchored with an  helix that partitions into one bilayer leaflet. Figure 10-19 Molecular Biology of the Cell (© Garland Science 2008)

26. 5, 6. Anchored by a lipid attached to the protein. 7, 8. Attached via noncovalent interactions with other proteins; peripheral membrane proteins. Figure 10-19 Molecular Biology of the Cell (© Garland Science 2008)

27. Figure 10-21 Molecular Biology of the Cell (© Garland Science 2008) A segment of a trans- membrane polypeptide chain crossing the lipid bilayer as an  helix.

28. Figure 10-22 Molecular Biology of the Cell (© Garland Science 2008) Using hydropathy plots to predict TMDs

29. Figure 10-27

30. Figure 10-28a Molecular Biology of the Cell (© Garland Science 2008) The carbohydrate layer on the cell surface

31. Figure 10-25 Molecular Biology of the Cell (© Garland Science 2008) Steps in the folding of a multipass membrane protein

32. Solubilizing membrane proteins with a mild detergent Figure 10-30

33. Chapter 11 Membrane Transport of Small Molecules and the Electrical Properties of Membranes

34. Membrane Transport (Ch. 11) * Diffusion across a membrane bilayer is determined by the size and polarity of the solute molecule. * The hydrophobic core of the lipid bilayer serves a barrier to polar molecules. * The barrier function of membranes allows cells to maintain different concentrations of solutes in different compartments. * However, cells must be able to transport specific water-soluble solutes across the membrane bilayer. * Transport across membranes is carried out by specialized transmembrane proteins, known as membrane transport proteins. * Cells can also transport larger molecules, such as proteins, across the membrane bilayer.

35. Figure 11-1/2 (Moles/sec/cm 2 )

36. OUT (mM)IN (mM) Na + 5 - 15 145 K + 140 5 Ca 2+ 10 -4 1 - 2 Cl - 5 - 10 110 Table 11-1

37. Membrane Transport Proteins - Multiple pass transmembrane proteins - Exhibit solute specificity - Two major classes: 1) Transporter proteins - Also called carriers or permeases - Bind to solute and undergo a series of conformation changes that result in the transfer of bound solute across bilayer 2) Channel proteins - Form a hydrophilic pore; do not bind solute - Faster rate of transfer

38. 1) Passive Transport - Also called "Facilitated Diffusion" - Solutes are transferred passively across membrane - Mediated by both carrier & channel proteins 2) Active Transport - Transfer solutes against their electrochemical gradient - Transport is coupled to an expenditure of energy; e.g. ATP hydrolysis - Mediated by carrier proteins Figure 11-4a

39. Forces driving passive transport: For uncharged molecules = concentration gradient For charged = concentration + membrane molecules gradient potential = electrochemical gradient ? + + + - - - -

40. Figure 11-4b Molecular Biology of the Cell (© Garland Science 2008)

41. Three ways of driving active transport. Figure 11-7 Molecular Biology of the Cell (© Garland Science 2008)

42. Types of Carrier Proteins Uniporters Transport a single solute from one side of the membrane to another. Coupled Transporters Transfer of one solute depends on the simul- taneous transport of a second solute. Two types of coupled transport reactions, symport and antiport. Symporters transfer second solute in the same direction as the first. Antiporters transfer second solute in the opposite direction as the first.

43. A model of how a conformational change in a carrier protein could mediate the passive transport of a solute. Figure 11-5 Molecular Biology of the Cell (© Garland Science 2008)

44. Figure 11-9 Molecular Biology of the Cell (© Garland Science 2008) Using the Na + gradient to drive glucose uptake Note: Na + and glucose bind cooperatively

45. Figure 11-11 An asymmetric distribution of carrier proteins underlies transcellular transport in epithelial cells

46. Figure 11-12 Molecular Biology of the Cell (© Garland Science 2008) Three types of ATP-driven pumps

47. Plasma Membrane Na + /K + Pump * Present in the plasma membranes of almost all animal cells. * Functions as an antiporter; Na + is pumped out and K + is pumped in. * Na + gradient is used to regulate cell volume and to drive the transport of sugars and amino acids into the cell. * Almost 33% of the energy required by a typical animal cell is used to drive this pump. * ATP hydrolysis is required for Na + /K + pump action. * This pump can also be run in reverse resulting in the synthesis of ATP. * Pump is electrogenic, as it pumps 3 Na + out for every 2 K + pumped in.

48. Figure 11-12 Molecular Biology of the Cell (© Garland Science 2008) Three types of ATP-driven pumps

49. Figure 11-14 Molecular Biology of the Cell (© Garland Science 2008) Na + -K + Pump

50. Figure 11-15 A model of the pumping cycle of the Na + -K + pump

51. Figure 10-31 Molecular Biology of the Cell (© Garland Science 2008)

52. Osmolarity problem? If the cell does nothing to control osmolarity, the concentration of solutes will be higher inside than outside. - + + + + + + + - - - - - - The concentration of water will be higher outside the cell resulting in a constant influx of water that could lead to cell lysis. Solutions? 1.Control intracellular osmolarity by actively pumping out inorganic ions, such as Na + (animal cells, bacteria). 2.Prevent swelling by possession of a rigid cell wall. At equilibrium, the internal turgor pressure forces out as much water as is entering due to osmolarity differences (plant and yeast cells). 3.Extrude water from special contractile vacuoles (protozoa). Panel 11-1

53. Figure 11-16 Molecular Biology of the Cell (© Garland Science 2008) Responses of human RBC to changes in the osmolarity of the extracellular fluid.

54. ABC Transporters Figure 3-78a MBoC Ed. 5

55. Figure 11-17b Molecular Biology of the Cell (© Garland Science 2008) Multi-drug (MDR) resistance proteins OUT IN

56. The over-expression of multidrug resistance transporter proteins, like MDR1 and MRP1, results in a resistance to chemo- therapeutic agents in many cancer patients. Drug resistance in cancer patients

57. Some ABC transporters act as ion channels Cystic fibrosis (CF) is caused by mutations that inactivate an ABC transporter that functions as a Cl - ion channel in the plasma membrane. In this case, ATP binding and hydrolysis regulates the opening & closing of the channel pore.

58. Ion Channels *Form hydrophilic pores across membranes that allow for passage of specific ions. *Display ion-selectivity. *More efficient than carrier proteins; a single channel can carry >100 million ions per second, a rate 10 5 times greater than that observed with carrier proteins. *Mediate passive transport only. *Transport is regulated by "gates" that open in response to specific stimuli.

59. Figure 11-21 Molecular Biology of the Cell (© Garland Science 2008) Three types of ion channels

60. A schematic representation of an ion channel Figure 11-20 Molecular Biology of the Cell (© Garland Science 2008) How is solute/ion selectivity achieved?

61. Figure 11-23a Figure 11-24 Selectivity filter of bacterial K + channel Ion specificity of channel proteins Na + K+K+

62. K + Leak Channels & Membrane Potential * The membrane potential in animal cells is due primarily to K + leak channels and the K + gradient across the plasma membrane. * K + leak channels are slightly open even in an unstimulated state. * The leakage of K + ions through the leak channels leaves the plasma membrane with a relative negative charge. * The movement of a relatively small number of ions can have a large effect on membrane potential. The ions giving rise to membrane potential lie in a thin (<1 nm) surface layer next to the membrane. * Therefore, almost any change in membrane permeability has an effect on membrane potential. This is the key principle relating the electric excitability of cells to the activity of ion channels.

63. The ionic basis of membrane potential + Figure 11-22

64. Nerve Impulses (Action Potentials) * Neurons utilize ion channels for receiving, conducting and transmitting signals. * The signal carried by neurons is always the same; a change in the electrical potential across the neuronal plasma membrane. * Communication occurs because an electrical disturbance in one part of the cell spreads to other parts. * Signals over a specific threshold result in a self-amplifying response that travels rapidly down the neuron (an action potential). * Membrane potential arises as a result of an electrical charge difference between the two sides of a membrane. * In general, the inside of an animal cell plasma membrane tends to be negative relative to the outside. This results in a resting potential across the plasma membrane of between -20 and -200 mV.

65. Figure 11-28 Molecular Biology of the Cell (© Garland Science 2008) A typical vertebrate neuron

66. Figure 11-21 Molecular Biology of the Cell (© Garland Science 2008) Three types of ion channels

67. Action Potentials * Voltage-gated cation channels are responsible for generating action potentials (AP). * An AP is triggered by a depolarization of the plasma membrane; i.e. by a shift in the membrane potential to a less negative value. * This initial depolarization is generally achieved by the opening of neurotransmitter-gated Na + channels. * This initial depolarization causes voltage-gated Na + channels to open, resulting in a further depolarization of the plasma membrane. * The AP is propagated down the neuron because the local depolarization of the plasma membrane is sufficient to also depolarize neighboring regions of membrane. * The voltage-gated Na + channels possess an automatic inactivating mechanism that causes the channels to rapidly reclose even though the plasma membrane is still depolarized. * These channels remain inactive until several milliseconds after the membrane potential returns to its initial negative value.

68. Figure 11-29 Molecular Biology of the Cell (© Garland Science 2008)

69. + + + - - - - - Closed InactivatedOpen Membrane polarized Membrane depolarized Three states of the voltage-gated Na + channel

70. Figure 11-30a Molecular Biology of the Cell (© Garland Science 2008)

72. Figure 11-31 Molecular Biology of the Cell (© Garland Science 2008) Model for the rapid inactivation of a voltage-gated K + channel

73. Figure 11-29 Triggering the initial depolarization event of the AP Time (milliseconds)

74. Transmitter-gated ion channels * Transmitter-gated ion channels convert chemical signals into electrical ones at chemical synapses. * Neuronal signals are transmitted from cell to cell at specialized sites of contact known as synapses. * Neurotransmitter release is triggered by a change in of electrical potential in the presynaptic cell. * Transmitter crosses synaptic cleft and is bound by transmitter- gated ion channels. * This produces a local change in membrane permeability and hence in membrane potential. If the change is strong enough, nearby voltage-gated cation channels will be opened and an AP initiated.

75. Figure 11-35b Molecular Biology of the Cell (© Garland Science 2008) Chemical Synapse

76. Figure 11-35a Molecular Biology of the Cell (© Garland Science 2008) Transmitter-gated Channels

77. Figure 11-33 Molecular Biology of the Cell (© Garland Science 2008) The technique of patch-clamp recording

78. Membrane Transport - Summary * Transport proteins allow the cell to take up essential nutrients, expel metabolic waste products and regulate intracellular ion concentrations. * Membrane transport proteins allow the cell to maintain different concentrations of a variety of solutes across any given membrane bilayer. * The ionic concentration differences allow cell membranes to store potential energy in the form of electrochemical gradients. * This energy is used to drive various transport processes, control cell volume, convey electrical signals in the nervous system and to produce the majority of the cell's ATP.