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  2. 2. Plasma membrane is about 50 atoms thick and serves as a selective barrier.
  3. 3. Membranes include 1. sensors which enable the cell to respond to theenvironment and 2. highly selective channels and pumps. Mechanicalproperties of the membranes are remarkable. Enlarges and changesshape as needed with no loss of integrity.
  4. 4. The lipid bilayer. A. An electron micrograph
  5. 5. The lipids in the cellmembrane areamphipathic.
  6. 6. Phosphatidylcholine is the most common type of phospholipid. Positive negative
  7. 7. Three kinds of membrane lipids, all amphipathic, incudephospholipids, sterols, and glycolipids. Hydrophilic heads
  8. 8. Due to thermal motions, lipid molecules within a monolayer rotate veryrapidly and diffuse rapidly within the fluid membrane. Any drop intemperature decreases the rate of lipid movement, making the lipidbilayer less fluid. This inhibits many functions of the cell’s membranes.All this has beenconfirmed in wholecells.
  9. 9. Viscosity - Fluidity (fluidity = 1/viscosity)Classical Mechanical DefinitionResistance to an isotropic flowDetermines fluid strain rateMembrane Biology DefinitionCommonly defined as the ease of movement of a theoretical particle through the lipid membraneGoverns many physiological and metabolic functions of the cellDetermines mobility of intermembrane proteins
  10. 10. Membrane ViscosityChanges in membrane viscosity are often indicative of intracellular conditionsaffecting functions such as• Carrier mediated Transport• Membrane bound receptors• Membrane bound enzymes
  11. 11. • Membrane fluidity is important to a cell for many reasons. – 1. Enables membrane proteins to diffuse rapidly and interact with one another - crucial in cell signaling etc. – 2. Provides a simple means of distributing membrane lipids and proteins by diffusion from sites of insertion. – 3. Allows membranes to fuse with one another and mix their molecules – 4. Ensures that membrane molecules are distributed evenly between daughter cells.• Remember though, cell has control - cytoskeleton and other interactions can limit the mobility of specific lipids and proteins.
  12. 12. • The fluidity of a lipid bilayer depends on its composition. – As temperature and environment changes, the fluidity of the cell’s membranes must be kept functional. – The closer and more regular the packing of the tails, the more viscous and less fluid the bilayer will be – The length and degree of saturation with hydrogens affect their packing • shorter tails can not interact as much: more fluid • one of the two hydrocarbon tails often has a double bond - unsaturated. This creates a kink - less packing, more fluid.
  13. 13. CLIP
  14. 14. Cholesterol fills in the spaces left by the kinks; stiffens the bilayer andmakes it less fluid and less permeable.
  15. 15. > Tm> Tm
  16. 16. Fluidity of blood cells membranes• changes in membrane fluidity of blood cells have been reported during development and aging and as a result of physiological cell functions.Membrane fluidity changes of blood cells have been described in• thrombocythaemia,• hyperlipidaemia,• hypercholesterolaemia,• hypertension,• diabetes mellitus,• obesity,• septic conditions• allergic and burnt patients• alcoholics,• Alzheimers disease
  17. 17. Current Methods to assess membrane fluidityFRAP (Fluorescence recovery after photobleaching)• Focused laser beam photobleaches area on membranedefects: Induced cross-linking from photo-oxidation May damage functional proteins Error is a function of laser beam radiusFluorescence Anisotropy• Internal rotation changes polarization planeDefects: Rapid photobleachingFilter absorption of signal
  18. 18. Stokes-Einstein equation:the diffusion coefficient, D, for a particle in a free volumedepends on the Boltzmann constant (k), the absolutetemperature (T), the viscosity of the solution (h), and thehydrodynamic radius (R) of the particle (or molecule).
  19. 19. However, lipids and proteins do not all float freely in the membrane.The cell controls the movement of many proteins. Cells have ways ofconfining particular plasma membrane proteins to localizedareas, creating membrane domains which are functionally specialized.Proteins aremoved togetherwhen signaled by Bound by thereceptors like Tethered to the extracellular matrixadhesion cell cortexmolecules. Stopped by diffusion barriors.Held by proteins on another cell
  22. 22. Limiti di intervallo• Solido: r = 1• Liquido a Fluidità infinita: r = 0• 0<r<1
  23. 23. DPH DDTMA-DPH
  24. 24. Fluorescence anisotropy imaging r values are showed in a pseudo-color scale
  25. 25. • SYSTEMIC SCLEROSIS Membrane Meno fluide in SSc
  26. 26. Fluorescence anisotropy for DPH of mononuclear cellsfrom normal controls (NC), IgA nephropathy (IGAN) subjects. nephropathy
  27. 27. Lung cancer
  28. 28. Dot plot of fluidity variable in groups with different stages of the diseasefluidity Sok M. et al.; Ann Thorac Surg 2002;73:1567-1571
  29. 29. Predicted log relative risk (relative to the reference value at the median, for tumors as a function of fluidity , assessed by Cox modeling with restricted cubic splines) Sok M. et al.; Ann Thorac Surg 2002;73:1567-1571
  30. 30. Nanomechanical analysis of cancer cells
  31. 31. the dynamic reorganization of the cytoskeleton has become a specific point ofinterest regarding changes in cell morphology, motility, adhesion and invasion a change in the physical properties, in particular cell elasticity, of tissue cells has been recognized as an indication of disease and has emerged as a marker for cellular phenotypic events associated with cell adhesion and cytoskeletal organization In particular, several studies have shown a reduction in stiffness with increasing metastatic efficiency in human cancer cell lines using several different in vitro biomechanical assays
  32. 32. Labelling for DNA/Ber-EP4/F-actin (Fig. 1c) and DNA/Ber-EP4/Calretinin (Fig. 1d) both showed staining of the small, round cellsfor Ber-EP4 (red), a marker for metastatic adenocarcinoma cells, thusconfirming that the round, balled cells (shown optically in Fig. 1a) wereindeed metastatic adenocarcinoma cells. Arrowheads 1/4 tumour, arrow 1/4 mesothelial cells.
  33. 33. AFM: atomic force microscopeSFM: scanning force microscope AFM AFM probe scans over the laser photodiode surface (in contact) probe piezo- e.g. living cells, chromatin element fibers
  34. 34. Cantilever tip must be positioned on aproper position of the cell body
  35. 35. Data collected from seven different clinical samples (positive for metastatic tumour, n 40; negative for metastatic tumour, n 48) yielded average E values (mean+s.d. of 0.53+0.10 kPa for tumour and 1.97+0.70 kPa for benign mesothelial cells , respectively
  36. 36. tumours controlelasticity
  37. 37. 200nm
  38. 38. 48nm28nm
  39. 39. MOLECULES ENRICHED WITHIN LIPID RAFTS/CAVEOLAECholesterol, sphingolipids, saturated lipids (Palmitoilate)
  40. 40. COINVOLGIMENTO DEI LIPID RAFTSMalattia di AlzheimerInfiammazioneMalattie CardiovascolariDistrofia MuscolareParkinsonLupus eritematosoMalattie da Prioni (encefalopatie spongiformi)TumoriIpertensione
  41. 41. FIG. 8. Signaling through caveolae• Signaling through caveolae. The -adrenergic receptor ( -AR; blue) is a conventional G protein-coupled receptor with seven membrane- spanning domains. When stimulated, the activation of adenylyl cyclase, increases intracellular cAMP concentrations, resulting in the activation of protein kinase A (PKA). On the right, an activated epidermal growth factor receptor (EGF-R) is also shown docking with the caveola, leading to the activation of a proliferative pathway involving several caveolae-associated proteins of the p42/44 mitogen- activated protein kinase cascade (Ras/Raf/MEK/ERK).
  42. 42. Imaging lipid rafts
  43. 43. Imaging lipid rafts : AFM
  44. 44. Imaging lipid rafts : Phase-partitioning probeslissamine rhodamine dipalmitoylphosphatidylethanolamine (rho-DPPE) indocarbocyanine dye DiIPartition in fluid phase (non-rafts)PerylenePartitions in ordered phase (rafts)
  45. 45. Imaging lipid rafts : phase sensitive probesLaurdan is an environmentally sensitive fluorescence probethat exhibits a 50-nm red shift as membranes undergo phasetransition from gel to fluid, due to altered water penetrationinto the lipid bilayer
  46. 46. Ordered Fluid phasephase (non-rafts)(rafts)
  47. 47. Imaging lipid rafts : Fluorescence Excimer formation technique
  48. 48. ExcimerFormationfluorophore-fluorophore interactions
  49. 49. Excimerformationimaging
  50. 50. Imaging lipid rafts : E.M.
  51. 51. Isolation and study of lipid rafts
  52. 52. AB
  53. 53. Electrophoresis/ WB with antibodies Flot-1 (49 kDa) CD55 (70 kDa) Fyn (59 kDa) ALP (38 kDa) Cav-1 (21 kDa) Mit (60 kDa) TfR (85 kDa) GM1 1 2 3 4 5 6 7 8 9 10Gradient distribution of proteins of Caki-1 cells
  54. 54. A new form of mass spectrometry can determine a membrane’s chemical compositionSecondarya resolution of with ion massspectrometry (SIMS) less than 100the sample is bombarded nanometerswith an incident ion ormolecular beam. Thebeam locally vaporizesthe sample into secondarymolecular and atomicions. In time-of-flightSIMS, the incidention beam is pulsed, andthe secondary ion mass-