Membrane Dynamics PDF

Membrane Dynamics One remarkable feature of all biological membranes is their flexibility—their ability to change shape without losing their integrity and becoming leaky. The basis for this property is the noncovalent interactions among lipids in the bilayer and the motions allowed to indiv...

Membrane Dynamics One remarkable feature of all biological membranes is their flexibility—their ability to change shape without losing their integrity and becoming leaky. The basis for this property is the noncovalent interactions among lipids in the bilayer and the motions allowed to individual lipids because they are not covalently anchored to one another. We turn now to the dynamics of membranes: the motions that occur and the transient structures allowed by these motions. Although the lipid bilayer structure is quite stable, its individual phospholipid and sterol molecules have some freedom of motion… 1  At relatively low temperatures, the lipids in a bilayer form a semisolid gel phase. the bilayer is para-crystalline. At relatively high temperatures, individual hydrocarbon chains of fatty acids are in constant motion produced by rotation about the carbon–carbon bonds of the long acyl side chains. In this liquid-disordered state, or fluid state. At intermediate temperatures, the lipids exist in a liquid-ordered state; there is less thermal motion in the acyl chains of the lipid bilayer, but lateral movement in the plane of the bilayer still takes place. 2 Trans-bilayer Movement of Lipids Requires Catalysis At physiological temperature, trans- bilayer—or “flipflop”— diffusion of a lipid molecule from one leaflet of the bilayer to the other occurs very slowly if at all in most membranes. Trans-bilayer movement requires that a polar or charged head group leave its aqueous environment and move into the hydrophobic interior of the bilayer, a process with a large, positive free-energy change. 3 Lipids and Proteins Diffuse Laterally in the Bilayer This lateral diffusion can be shown experimentally by attaching fluorescent probes to the head groups of lipids and using fluorescence microscopy to follow the probes over time. The rate of fluorescence recovery after photobleaching, or FRAP, is a measure of the rate of lateral diffusion of the lipids. Using the FRAP technique, face and show that movement from one such region to a nearby region is inhibited; lipids behave as though corralled by fences that they can occasionally jump. Many membrane proteins seem to be afloat in a sea of lipids. 4 5  Another technique, single particle tracking, allows one to follow the movement of a single lipid molecule in the plasma membrane on a much shorter time scale. 6  Some membrane proteins associate to form large aggregates (“patches”) on the surface of a cell or organelle in which individual protein molecules do not move relative to one another; for example, acetylcholine receptors (see Fig. below) form dense patches on neuron plasma membranes at synapses. 7 Certain Integral Proteins Mediate Cell-Cell Interactions and Adhesion Several families of integral proteins in the plasma membrane provide specific points of attachment between cells, or between a cell and extracellular matrix proteins. Integrins are heterodimeric proteins (two unlike subunits, and ) anchored to the plasma membrane by a single hydrophobic transmembrane helix in each subunit. Integrins are not merely adhesives; they serve as receptors and signal transducers, conveying information across the plasma membrane in both directions. At least three other families of plasma membrane proteins are also involved in surface adhesion Cadherins undergo homophilic (“with same kind”) interactions with identical cadherins in an adjacent cell. Immunoglobulin-like proteins Selectins 8 9 Solute Transport across Membranes In some cases a membrane protein simply facilitates the diffusion of a solute down its concentration gradient, but transport often occurs against a gradient of concentration, electrical charge, or both, in which case solutes must be “pumped” in a process that requires energy (Figure below). The energy may come directly from ATP hydrolysis or may be supplied in the form of movement of another solute down its electrochemical gradient with enough energy to carry another solute up its gradient. Ions may also move across membranes via ion channels formed by proteins, or they may be carried across by ionophores, small molecules that mask the charge of the ions and allow them to diffuse through the lipid bilayer. 10 11 Passive Transport Is Facilitated by Membrane Proteins When two aqueous compartments containing unequal concentrations of a soluble compound or ion are separated by a permeable divider (membrane), the solute moves by simple diffusion from the region of higher concentration, through the membrane, to the region of lower concentration, until the two compartments have equal solute concentrations (Fig. a below). When ions of opposite charge are separated by a permeable membrane, there is a transmembrane electrical gradient, a membrane potential, Vm (expressed in volts or millivolts). This membrane potential produces a force opposing ion movements that increase Vm and driving ion movements that reduce Vm (Figure b). 12 13  Together, these two factors are referred to as the electrochemical gradient or electrochemical potential. This behavior of solutes is in accord with the second law of thermodynamics: molecules tend to spontaneously assume the distribution of greatest randomness and lowest energy. 14 15 Membrane proteins that speed the movement of a solute across a membrane by facilitating diffusion are called transporters or permeases. There are two very broad categories of transporters: Carriers Carriers bind their substrates with high stereospecificity, catalyze transport at rates well below the limits of free diffusion, and Channels Channels generally allow transmembrane movement at rates several orders of magnitude greater than those typical of carriers, rates approaching the limit of unhindered diffusion. 16 Ion-Selective Channels Allow Rapid Movement of Ions across Membranes Ion-selective channels— provide another mechanism for moving inorganic ions across membranes. Ion channels, together with ion pumps such as the Na+ K+ ATPase, determine a plasma membrane’s permeability to specific ions and regulate the cytosolic concentration of ions and the membrane potential. In neurons, very rapid changes in the activity of ion channels cause the changes in membrane potential (the action potentials) that carry signals from one end of a neuron to the other. In myocytes (muscular cell), rapid opening of Ca2+ channels in the sarcoplasmic reticulum releases the Ca2+ that triggers muscle contraction. 17 18 Basics of ultra-resolution imaging 19 The processes that occur between the absorption and emission of light are usually illustrated by the Jablonski diagram. Jablonski diagrams are often used as the starting point for discussing light absorption and emission. These diagrams are named after Professor Alexander Jablonski A typical Jablonski diagram is shown below. The singlet ground, first, and second electronic states are depicted by S0, S1, and S2, respectively. At each of these electronic energy levels the fluorophores can exist in a number of vibrational energy levels, depicted by 0, 1, 2, etc. Alexander Jablonski (1898–1980) 20  Following light absorption, several processes usually occur. A fluorophore is usually excited to some higher vibrational level of either S1 or S2. Return to the ground state typically occurs to a higher excited vibrational ground state level, which then quickly (10–12 s) reaches thermal equilibrium… 21 22 The Stokes Shift ✓ Examination of the Jablonski diagram reveals that the energy of the emission is typically less than that of absorption. Fluorescence typically occurs at lower energies or longer wavelengths. This phenomenon was first observed by Sir. G. G. Stokes in 1852 at the University of Cambridge. ✓ Energy losses between excitation and emission are observed universally for fluorescent molecules in solution. ✓ One common cause of the Stokes shift is the rapid decay to the lowest vibrational level of S1. Furthermore, fluorophores generally decay to higher vibrational levels of S0, resulting in further loss of excitation energy by thermalization of the excess vibrational energy. ✓ In addition to these effects, fluorophores can display further Stokes shifts due to solvent effects, excited-state reactions, complex formation, and/or energy transfer. 23 Instrumentation for Fluorescence Spectroscopy SPECTROFLUOROMETERS With most spectrofluorometers it is possible to record both absorption (excitation) and emission spectra. An emission spectrum is the wavelength distribution of an emission measured at a single constant excitation wavelength. Conversely, an absorption spectrum is the dependence of emission intensity, measured at a single emission wavelength, upon scanning the excitation wavelength. Such spectra can be presented on either a wavelength scale or a wavenumber scale. Light of a given energy can be described in terms of its wavelength λ, frequency ν, or wavenumber. The usual units for wavelength are nanometers, and wavenumbers are given in units of cm–1. Wavelengths and wavenumbers are easily interconverted by taking the reciprocal of each value. For example, 400 nm corresponds to (400 x 10–7 cm)–1 = 25,000 cm–1. 24 25 Fluorophores A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with several π bonds. Fluorophores or fluorochromes are photoreactive chemicals that absorb and emit energy in a predictable fashion. They re-emit light when excited, making them useful tags for identifying and characterizing cells or molecules in a mixture, visualizing proteins, or quantifying tagged compounds. One of the most popular fluorophores that is used is the Fluorescein. Its application goes from the antibody labeling to the nucleic acids. The most recent generations of fluorophores has been identified as more photostable, brighter and less pH-sensitive, what makes them perform better. Derivates of rhodamine, coumarin and cyanine are examples of other common fluorophores. 26 Fluorescein Rhodamines Cumarin 27 Human epithelial cells using QD conjugates. The colors allowed localization of cellular proteins and substructures: nuclei (cyan), cell proliferation protein Ki-67 (magenta), mitochondria (orange), microtubules (green), and actin filaments (red). Courtesy of Quantum Dot Corp. (Hayward, Calif). 28 KEY PROPERTIES OF FLUOROPHORES ✓ Excitation and Emission Wavelengths ✓ Quantum Yield ✓ Photostability ✓ Fluorescence Lifetime 29 TYPES OF FLUOROPHORES ✓ Organic Dyes ✓ Fluorescent Proteins ✓ Quantum Dots ✓ Fluorescent Nanoparticles 30 APPLICATIONS OF FLUOROPHORES ✓ Fluorescence Microscopy ✓ Flow Cytometry ✓ Molecular Probes and Diagnostic Tools ✓ Immunofluorescence ✓ Live Cell Imaging ✓ In Vivo Imaging 31 32 33 Components in a dividing human cancer cell. DNA, a protein called INCENP and the microtubules 34 3D dual-color super-resolution microscopy with Her2 and Her3 in breast cells, standard dyes: Alexa 488, Alexa 568. 35 36 37  FRET Fluorescence Resonance Energy Transfer 38 Energy Transfer Fluorescence resonance energy transfer (FRET) FRET is an electrodynamic phenomenon that can be explained using classical physics. FRET occurs between a donor (D) molecule in the excited state and an acceptor (A) molecule in the ground state. The donor molecules typically emit at shorter wavelengths that overlap with the absorption spectrum of the acceptor. Energy transfer occurs without the appearance of a photon and is the result of long range dipole–dipole interactions between the donor and acceptor. 39  The rate of energy transfer depends upon the extent of spectral overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor, the quantum yield of the donor, the relative orientation of the donor and acceptor transition dipoles, and the distance between the donor and acceptor molecules. The distance dependence of RET allows measurement of the distances between donors and acceptors. The distance at which RET is 50% efficient is called the Förster distance,1 which is typically in the range of 20 to 60 Å. 40 A) FRET to show protein binding to a specific separate substrate, B) FRET to show protein binding to an attached substrate, C) FRET that occurs when a protein binds to a ligand and undergoes a change in shape, D) FRET that stops when enzymes (in this case a protease) cleave the bond between the fluorophores. Image from Zeiss Fluorescent Protein FRET Biosensors. 41 rate of energy transfer τD is the decay time of the donor in the absence of acceptor R0 is the Förster distance, and r is the donor-to acceptor distance. Hence, the rate of transfer is equal to the decay rate of the donor (1/τD) when the D-to-A distance (r) is equal to the Förster distance (R0), and the transfer efficiency is 50%. 42 Protein-Protein Interactions Because FRET is highly dependent on the distance between the two FPs, it is often used to observe the interactions between two proteins of interest. When the two proteins come close enough together (i.e., through binding), then FRET occurs between the FPs. 43  AFM Atomic Force Microscopy 44 Atomic Force Microscopy to Visualize Membrane Proteins In atomic force microscopy (AFM), the sharp tip of a microscopic probe attached to a flexible cantilever is drawn across an uneven surface such as a membrane. Electrostatic and van der Waals interactions between the tip and the sample produce a force that moves the probe up and down (in the z dimension) as it encounters hills and valleys in the sample A laser beam reflected from the cantilever detects motions of as little as 1 Å. In one type of atomic force microscope, the force on the probe is held constant (relative to a standard force, on the order of piconewtons) by a feedback circuit that causes the platform holding the sample to rise or fall to keep the force constant. 45 46  Single molecules of bacteriorhodopsin in the membranes of the bacterium Halobacterium salinarum are seen as highly regular structures (Fig. 2a). AFM of purified E. coli aquaporin, reconstituted into lipid bilayers and viewed as if from the outside of a cell, shows the fine details of the protein’s periplasmic domains (Fig. 2b). The proton-driven rotor of the chloroplast ATP synthase is composed of many subunits (Fig. 2c) arranged in a circle. 47 Thank you for your attention 48