CELLULAR NANOTECHNOLOGIES 1

Plasma membrane is about 50 atoms thick and serves as a selective barrier.

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.

The lipid bilayer.

A. An electron micrograph

The lipids in the cell membrane are amphipathic.

Phosphatidylcholine is the most common type of phospholipid.

Positive

negative

Three kinds of membrane lipids, all amphipathic, incude phospholipids, sterols, and glycolipids.

Hydrophilic heads

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.

Viscosity - Fluidity(fluidity = 1/viscosity)

Classical Mechanical Definition Resistance to an isotropic flowDetermines fluid strain rate

Membrane Biology DefinitionCommonly 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

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

• 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.

• 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.

CLIP

Cholesterol fills in the spaces left by the kinks; stiffens the bilayer and makes it less fluid and less permeable.

> Tm

> Tm

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

Current Methodsto assess membrane fluidity

FRAP (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

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).

Stokes-Einstein equation:

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 receptors like adhesion molecules.

Tethered to the cell cortex

Bound by the extracellular matrix

Held by proteins on another cell

Stopped by diffusion barriors.

FLUORESCENCE ANISOTROPY

LIGHT SOURCE

Limiti di intervallo

• Solido: r = 1 • Liquido a Fluidità infinita: r = 0• 0<r<1

DD

TMA-DPH

DPH

Fluorescence anisotropy imaging

r values are showed in a pseudo-color scale

• SYSTEMIC SCLEROSIS

Membrane Meno fluide in SSc

Fluorescence anisotropy for DPH of mononuclear cellsfrom normal controls (NC), IgA nephropathy (IGAN) subjects.

nephropathy

Lung cancer

Sok M. et al.; Ann Thorac Surg 2002;73:1567-1571

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

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)

Nanomechanical analysis of cancer cells

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

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.

AFM: atomic force microscope

AFM probe scans over the surface (in contact)

e.g. living cells, chromatin fibers

laser photodiode

piezo-element

probe

SFM: scanning force microscope AFM

Cantilever tip must be positioned on a proper position of the cell body

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

tumours

control

elasticity

200nm

48nm

28nm

MOLECULES ENRICHED WITHIN LIPID RAFTS/CAVEOLAE

Cholesterol, sphingolipids, saturated lipids

(Palmitoilate)

COINVOLGIMENTO DEI LIPID RAFTS

Malattia di Alzheimer

Infiammazione

Malattie Cardiovascolari

Distrofia Muscolare

Parkinson

Lupus eritematoso

Malattie da Prioni (encefalopatie spongiformi)

Tumori

Ipertensione

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).

Imaging lipid rafts

Imaging lipid rafts : AFM

lissamine rhodamine dipalmitoylphosphatidylethanolamine (rho-DPPE) indocarbocyanine dye DiI Partition in fluid phase (non-rafts)

PerylenePartitions in ordered phase (rafts)

Imaging lipid rafts : Phase-partitioning probes

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

Fluid phase(non-rafts)

Ordered phase (rafts)

Imaging lipid rafts : Fluorescence Excimer formation technique

ExcimerFormationfluorophore-fluorophore interactions

Excimer formation imaging

Imaging lipid rafts : E.M.

Isolation and study of lipid rafts

A

B

CD55 (70 kDa)

Fyn (59 kDa)

Flot-1 (49 kDa)

ALP (38 kDa)

Cav-1 (21 kDa)

TfR (85 kDa)

Mit (60 kDa)

1 2 3 4 5 6 7 8 9 10

GM1

Gradient distribution of proteins of Caki-1 cells

Electrophoresis/ WB with antibodies

A new form of mass spectrometry candetermine a membrane’s chemical compositionwith a resolution of less than 100 nanometers

Secondary ion mass spectrometry (SIMS)the sample is bombarded with an incident ion or molecular beam. The beam locally vaporizesthe sample into secondary molecular and atomic ions. In time-of-flight SIMS, the incidention beam is pulsed, and the secondary ion mass-to-charge ratio (m/z), and hence its identity,is determined by the time it takes to reach the ion detector