2. Plasma membrane is about 50 atoms thick and serves as a
selective barrier.
3. Membranes include 1. sensors which enable the cell to respond to the
environment and 2. highly selective channels and pumps. Mechanical
properties of the membranes are remarkable. Enlarges and changes
shape as needed with no loss of integrity.
7. Three kinds of membrane lipids, all amphipathic, incude
phospholipids, sterols, and glycolipids.
Hydrophilic heads
8.
9. Due to thermal motions, lipid molecules within a monolayer rotate very
rapidly and diffuse rapidly within the fluid membrane. Any drop in
temperature decreases the rate of lipid movement, making the lipid
bilayer less fluid. This inhibits many functions of the cell’s membranes.
All this has been
confirmed in whole
cells.
10. Viscosity - Fluidity
(fluidity = 1/viscosity)
Classical Mechanical Definition
Resistance to an isotropic flow
Determines fluid strain rate
Membrane Biology Definition
Commonly defined as the ease of movement of a theoretical particle
through the lipid membrane
Governs many physiological and metabolic functions of the cell
Determines mobility of intermembrane proteins
11. Membrane Viscosity
Changes in membrane viscosity are often
indicative of intracellular conditions
affecting functions such as
• Carrier mediated Transport
• Membrane bound receptors
• Membrane bound enzymes
12. • 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.
13. • 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.
20. 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,
• Alzheimer's disease
21. Current Methods
to assess membrane fluidity
FRAP (Fluorescence recovery after
photobleaching)
• Focused laser beam photobleaches area on
membrane
defects: Induced cross-linking from photo-oxidation
May damage functional proteins
Error is a function of laser beam radius
Fluorescence Anisotropy
• Internal rotation changes polarization plane
Defects: Rapid photobleaching
Filter absorption of signal
22.
23. Stokes-Einstein equation:
the diffusion coefficient, D, for a particle in a free volume
depends on the Boltzmann constant (k), the absolute
temperature (T), the viscosity of the solution (h), and the
hydrodynamic radius (R) of the particle (or molecule).
24.
25. However, lipids and proteins do not all float freely in the membrane.
The cell controls the movement of many proteins. Cells have ways of
confining particular plasma membrane proteins to localized
areas, creating membrane domains which are functionally specialized.
Proteins are
moved together
when signaled by
Bound by the
receptors like Tethered to the extracellular matrix
adhesion cell cortex
molecules.
Stopped by
diffusion
barriors.
Held by proteins on another cell
40. Dot plot of fluidity variable in groups with different stages of the disease
fluidity
Sok M. et al.; Ann Thorac Surg 2002;73:1567-1571
41. 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
43. the dynamic reorganization of the cytoskeleton has become a specific point of
interest 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
44. Labelling for DNA/Ber-EP4/F-actin (Fig. 1c) and DNA/Ber-
EP4/Calretinin (Fig. 1d) both showed staining of the small, round cells
for Ber-EP4 (red), a marker for metastatic adenocarcinoma cells, thus
confirming that the round, balled cells (shown optically in Fig. 1a) were
indeed metastatic adenocarcinoma cells.
Arrowheads 1/4 tumour, arrow 1/4 mesothelial cells.
45. AFM: atomic force microscope
SFM: scanning force microscope AFM
AFM probe scans over the
laser
photodiode surface (in contact)
probe
piezo-
e.g. living cells, chromatin
element
fibers
47. 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
56. COINVOLGIMENTO DEI LIPID RAFTS
Malattia di Alzheimer
Infiammazione
Malattie Cardiovascolari
Distrofia Muscolare
Parkinson
Lupus eritematoso
Malattie da Prioni (encefalopatie spongiformi)
Tumori
Ipertensione
57.
58. 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).
64. Imaging lipid rafts : phase sensitive probes
Laurdan is an environmentally sensitive fluorescence probe
that exhibits a 50-nm red shift as membranes undergo phase
transition from gel to fluid, due to altered water penetration
into the lipid bilayer
76. 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 10
Gradient distribution of proteins of Caki-1 cells
77. A new form of mass
spectrometry can
determine a
membrane’s chemical
composition
Secondarya resolution of
with ion mass
spectrometry (SIMS)
less than 100
the sample is bombarded
nanometers
with an incident ion or
molecular beam. The
beam locally vaporizes
the sample into secondary
molecular and atomic
ions. In time-of-flight
SIMS, the incident
ion beam is pulsed, and
the secondary ion mass-
