1. THIS PART OF THE REVIEW COVERS LECTURES BY DRS MUECKLER, MAHJOUB AND COOPER (TOTAL OF 5 LECTURES) EVERYTHING TESTED ON THE IN-CLASS PORTION OF THE EXAM FOR THE ABOVE LECTURES IS COVERED IN THIS REVIEW. HOWEVER, NOT EVERYTHING COVERED IN THIS REVIEW WILL BE ON THE EXAM. MATERIAL FROM THIS SECTION OF THE REVIEW WILL COUNT FOR 55 OF THE 100 TOTAL POINTS ON THE EXAM MCB EXAM 1 REVIEW : SEPTEMBER 27 2014

2. of The Structure of Biological Membranes

3. Functions of Cellular Membranes 1.Plasma membrane acts as a selectively permeable barrier to the environment Uptake of nutrients Waste disposal Maintains intracellular ionic milieu 2.Plasma membrane facilitates communication With the environment With other cells 3. Intracellular membranes allow compartmentalization and separation of different chemical reaction pathways Increased efficiency through proximity Prevent futile cycling through separation Protein secretion

4. Figure 10-2 Molecular Biology of the Cell (© Garland Science 2008) Structure of Phosphoglycerides All Membrane Lipids are Amphipathic

5. Lipid Bilayer Formation is Driven by the Hydrophobic Effect HE causes hydrophobic surfaces such as fatty acyl chains to aggregate in water Water molecules squeeze hydrophobic molecules into as compact a surface area as possible in order to the minimize the free energy state (G) of the system by maximizing the entropy (S) or degree of disorder of the water molecules  G =  –  S

7. Figure 10-8 Molecular Biology of the Cell (© Garland Science 2008) The Formation of Cell-Like Spherical Water-Filled Bilayers is Energetically Favorable

8. Figure 10-11b Molecular Biology of the Cell (© Garland Science 2008) PhosphoLipid Movements within Bilayers (µM/sec) (10 12 -10 13 /sec) (10 8 -10 9 /sec)

9. Figure 12-57 Molecular Biology of the Cell (© Garland Science 2008) Phosphoglyceride Biosynthesis Occurs at the Cytoplasmic Face of the ER

10. Figure 12-58 Molecular Biology of the Cell (© Garland Science 2008) A “Scramblase” Enzyme Catalyzes Symmetric Growth of Both Leaflets in the ER

11. Figure 10-16 Molecular Biology of the Cell (© Garland Science 2008) The Two Plasma Membrane Leaflets Possess Different Lipid Compositions Enriched in PC, SM, Glycolipids Enriched in PE, PS, PI

12. Figure 12-58 Molecular Biology of the Cell (© Garland Science 2008) A “Flippase” Enzyme promotes Lipid Asymmetry in the Plasma Membrane Flippases are P-type ATPases

13. Figure 10-5 Molecular Biology of the Cell (© Garland Science 2008) C12 How Cholesterol Integrates into a Phospholipid Bilayer 1) INCREASE MEMBRANE FLUIDITY 2) PART OF LIPID RAFTS (SIGNALING FUNCTION)

14. 3 Ways in which Lipids May be Transferred Between Different Intracellular Compartments Vesicle Fusion Direct Protein-Mediated Transfer Soluble Lipid Binding Proteins

15. Figure 3-5 Molecular Biology of the Cell (© Garland Science 2008) Membrane Domains are “Inside-Out” Right-Side Out Soluble Protein

16. Figure 10-29b Molecular Biology of the Cell (© Garland Science 2008) Detergents Exist in Two Different States in Solution (Critical Micelle Concentration) CMC DETERMINANTS 1)CHARGE (IONINC HAS HIGHER CMC THAN NONIONIC) 2)TEMPERATURE 3)CONCENTRATION

17. Detergent Solubilization of Membrane Proteins Desirable for Purification of Integral Membrane Proteins

18. Figure 10-22b Molecular Biology of the Cell (© Garland Science 2008) Transmembrane Domains Can Often be Accurately Identified by Hydrophobicity Analysis CRITERIA: 1)POSITIVE PEAK 2)GREATER THAN 20 AMINO ACIDS IN LENGTH

19. The Signal Hypothesis and the Targeting of Nascent Polypeptides to the Secretory Pathway DR MUECKLER LECTURE 2

20. Intracellular Targeting of Nascent Polypeptides Default targeting occurs to the cytoplasm All other destinations require a targeting sequence Major sorting step occurs at the level of free versus membrane-bound polysomes

21. Figure 12-41a Molecular Biology of the Cell (© Garland Science 2008) Ribosomal Subunits are Shared Between Free and Membrane-Bound Polysomes Targeting information resides in the Nascent polypeptide chain

22. Signal-Mediated Targeting to the RER

23. Properties of Secretory Signal Sequences Hydrophobic Core N Mature Protein 8-12 Residues 15-30 Residues ++ Located at N-terminus 15-30 Residues in length Hydrophobic core of 8-12 residues Often basic residues at N-terminus (Arg, Lys) No sequence similarity cleavage

24. In Vitro Translation of Prolactin mRNA Prolactin is a polypeptide hormone (MW ~ 22 kd) secreted by anterior pituitary 1 2 3 4 5 6 7 8 MW (kd) 25 22 Lanes: 1.Purified prolactin 2.No RM 3.RM 4.No RM /digest with Protease 5.RM /digest with Protease 6.RM /detergent treat and add Protease 7.Prolactin mRNA minus SS + RM /digest with Protease 8.SS-globin mRNA + RM /digest with Protease 18 SDS Gel

25. Figure 12-39b Molecular Biology of the Cell (© Garland Science 2008) Interactions Between SRP and the Signal Sequence and Ribosome

26. Figure 12-44 Molecular Biology of the Cell (© Garland Science 2008) Post-Translational Translocation is Common in Yeast and Bacteria SecA ATPase functions like a piston pushing ~20 aa’s into the channel per cycle

27. The Charge Difference Rule for Multispanning Membrane Proteins NH 2 COOH NH 2 COOH + + – + + NH 2 COOH NH 2 COOH + + + + cytoplasm – – – – – –

28. Figure 12-51 Molecular Biology of the Cell (© Garland Science 2008) N-Linked Oligosaccharides are Added to Nascent Polypeptides in the Lumen of the RER

29. Disulfide Bridges are Formed in the RER by Protein Disulfide Isomerase (PDI)

30. MUECKLER LECTURE 3

31. Mitochondrial Biogenesis Mitochondria contain their own genome and protein synthetic machinery (tRNAs, mRNAs, ribosomes, initiation and elongation factors, etc.) Mitochondria are comprised of hundreds of distinct proteins, only a handful of which are encoded in the mitochondrial genome (varies by species) Most mitochondrial proteins are encoded in nuclear DNA, synthesized in the cytosol, and imported post-translationally into the organelle

32. Protein Import into the Matrix Requires Passage Through Two Separate Membrane Translocons

33. Proteins Traverse the TOM and TIM Translocons in an Unfolded State

34. Figure 12-26 Molecular Biology of the Cell (© Garland Science 2008) Protein Import into the Matrix Requires ATP Hydrolysis and an Intact Proton Gradient Across the Inner Membrane

35. Targeting to the Inner Membrane Occurs Via 3 Distinct Routes Oxa1-MediatedStop-Transfer-MediatedTom70/Tim22/54-Mediated Multi-Pass Proteins Single-Pass Proteins Cytochrome oxidase subunit CoxVa ATP Synthase Subunit 9 ADP/ATP Antiporter

36. Cytochrome B2Cytochrome c Heme Lyase Targeting to the Intermembranous Space Occurs Via Two Distinct Pathways Direct Delivery IM Space Protease

37. Figure 12-27 Molecular Biology of the Cell (© Garland Science 2008) Targeting to the Outer Membrane Via the SAM Protein Complex ( S orting and A ssembly Machinery) (  -Barrell)

38. Figure 12-14 Molecular Biology of the Cell (© Garland Science 2008) Directionality is Conferred on Nuclear Transport by a Gradient of Ran-GDP/GTP Across the Nuclear Envelope

39. Figure 12-15 Molecular Biology of the Cell (© Garland Science 2008) Nuclear Import and Export Operate Via Reciprocal Use of the Ran-GDP/GTP Concentration Gradient

40. Microtubules and their functions in cells. DR. MAHJOUB’S LECTURE

41. Microtubule Structure Cross-section – Hollow tube – 24 nm wide – ~13 protofilaments Helical structure Polar – Plus ends generally distal – Minus ends generally proximal (at MTOC) Composed of Tubulin  Heterodimer

42. Microtubule Structure & Assembly

43. The Master Nucleator γ-TuRC and microtubule nucleation

44. Microtubule Motors Definition – Microtubule-stimulated ATPase – Motility along MT’s Dynein – Moves towards Minus End of MT’s - Retrograde Kinesin – Moves to Plus End of MT’s - Anterograde

45. Microtubules are everywhere… Q: How are heterogeneity and specificity of microtubule function regulated? Different tubulin isoforms A large number of tubulin PTMs Different motors and adaptors Diverse MAPs

46. Organelle Trafficking - ER and Golgi Positioning ER & Golgi: – Golgi near MTOC Minus Ends are at MTOC Golgi Position Requires Dynein – ER Tubular network spread about the cell Kinesin moves the tubules peripherally

47. The centrosome as MTOC 1 centrosome per cell 2 centrioles per centrosome (in interphase) Centriole size is highly conserved throughout evolution: ~250nm diameter, ~500nm length

48. Mechanism of centrosome duplication Q: How can a cell make an exact copy of such an elaborate structure? G1/S SSG2/MS/G2 M/G1 -Templated duplication during the S phase of the cell cycle

49. Centriole structure – establishing 9-fold symmetry Q: How is the 9-fold rotational symmetry of microtubules established during centriole duplication? Sas6 has intrinsic symmetry when forming oligomers

50. Centriole structure – establishing 9-fold symmetry Q: But how come Sas6 doesn’t just form a bunch of rings non-specifically in the cytoplasm? A: - Expression restricted to G1/S phase (temporal regulation) - Recruited to parental centrioles (spatial regulation)…

51. Mitotic Spindle Assembly Centrosome duplicates and is segragated to each side of the nucleus Nuclear envelope breakdown in prophase MT’s rearrange via dynamic instability

52. Spindle microtubules

53. Models for Chromosomes Moving to the Pole Treadmilling? – Depolymerization at pole (MTOC) Depolymerization at Kinetochore? – How to remain bound while end shrinks? Motors at Kinetochore or Pole?

54. Structure of motile cilium: Cross-section

55. Conversion of sliding to bending Q: If all dyneins pull together, how does the axoneme bend back and forth? A: THE ROTATION OF THE CENTRAL PAIR TURNS ON THE DYNEIN ARMS SEQUENTIALLY TO ALLOW BENDING TO OCCUR

56. The primary cilium: non-motile sensory organelle Example: -THE CILIA STICK INTO THE LUMEN OF THE NEPHRON TO SENSE URINE FLOW – MECHANO-SENSATION

57. Fundamental processes in cell biology mediated by primary cilium Extracellular signal Cell division Cell differentiation Cell migration Cell polarity and organization X X X X X

58. Biophysics of Helical Polymers Dr. Cooper

59. Self-Assembly by Proteins - Entropy & the Hydrophobic Effect High Order in Assembled State Implies Lower Entropy, which is Unfavorable ∆G = ∆H - T∆S must be <0 for a Reaction to Occur But ∆H>0, ∆S>>0 ! Higher Entropy => Disorder in Assembled State Ordered Water on Hydrophobic Surface of Protein Subunit is Released

60. Why Use Subunits to Make Large Molecules? Efficient Use of the Genome Error Management Variable Size Disassembly / Reassembly

61. Assembly of Helical Filaments Add & Lose Subunits Only at Ends ON Rate = k + c 1 N OFF Rate = k - N c 1 = Concentration of Monomers N = Concentration of Filament Ends

62. Steady-state Concentrations of Polymer & Monomer [Monomer] [Polymer] [Total] Critical Concentration

63. Critical Concentration and Binding Affinity K a = 1 _c1_c1 K d = [N j+1 ] [N j ] = _c1_c1 _c1_c1

64. Treadmilling Polar Filaments have Two Different Ends Can Have Different Critical Concentrations at the Two Ends Steady State Critical Concentration is an Intermediate Value Net Addition at One End, Net Loss at the Other End

65. How do Cells Regulate the Number and Length of Filaments? Limit Growth – Intrinsic to Protein – Deplete Subunits – Capture by Capping End – Template Create New Filaments – Nucleation - End or Side – Bolus of Subunits - High Concentration

66. Nucleotide Hydrolysis Provides Energy for Dynamic Instability The Basic Facts... – Tubulin Binds GTP or GDP – GTP Tubulin Polymerizes Strongly – GDP Tubulin Polymerizes Poorly – Subunits Exchange w/ Free GTP – GTP on Tubulin Hydrolyzes to GDP over Time after Addition to Microtubule

67. Nucleotide Hydrolysis Provides Energy for Dynamic Instability At Steady State, at any given time... – Most Ends have a GTP “Cap” and Grow Slowly – A Few Ends Lose their GTP Cap Exposing GDP-tubulin subunits so the Microtubule Shrinks Rapidly Occurs In Vitro and In Vivo for Tubulin - Extensive and Relevant

68. Intermediate Filaments Dr. Cooper

69. Intermediate Filament Biochemical Properties In Vitro Very stable. Little subunit exchange. Very strong. Filaments do not break. – MT’s strong but brittle – Actin weak

70. Intermediate Filament Structure & Assembly Key Points: -Monomer and parallel heterodimer have polarity -Antiparallel tetramer stage is where polarity is lost -No directionality for motor proteins to use

71. Regulation of IF Assembly Notoriously Stable – No Nucleotide Filaments Move Little – Precursors Move More Disassemble Somewhat during Mitosis – Phosphorylation by Cyclin-dependent Kinase

72. Vimentin All Cells in Early Development Cage Around Nucleus Interacts with Mt’s Vimentin Knockout Mouse – Initially normal at gross inspection – Cultured cells have altered properties of uncertain significance

73. Desmin Expressed in Muscle Elastic Elements to Prevent Over-stretching Connects / Aligns Z lines Knockout Mouse - Deranged Myofibril Architecture

74. Keratins Expressed in Epithelia Keratin Filaments Connect to Desmosome and Hemidesmosomes Differentiation of Epidermis includes Production of Massive Amounts of Keratin Provides Outer Protection of Skin Composes Hair, Nails, Feathers, etc.

75. Keratin Mutations are Basis for Human Epidermal Diseases Structure/Function Analysis of Keratin Assembly Point Mutation in Terminal Domain Fails to Assemble Mutant is Dominant, even in Low Amounts, in Cultured Cells and Mice

76. Neurofilament Neurofilament H, M, L Copolymer Prevent Axon Breakage Diseases with Clumps of Neurofilaments – Superoxide dismutase model for ALS – Clumps are secondary, not causative

77. Lamins Square Lattice on Inner Surface of Nuclear Membrane Present in Metazoans (Animals, not Plants or unicellular organisms) Mitosis Breakdown – Phosphorylation of A & C by Cyclin-depen Kinase – B remains with Membrane Mutations Cause Accelerated Aging Diseases – Progerias - Dominant Mutations