Chemistry Reference
In-Depth Information
Hence, the residence time of the NTA label can be tuned according to the speci
c
needs from seconds to hours.
7.2.6
Reversible Sequential Labeling (ReSeq)
A novel method for performing reversible sequential (ReSeq)-binding assays on
particular neuroreceptors has recently been developed [37].With this assay, a series of
investigations can be performed on the same cell by repetitively applying speci
c,
fluorescently labeled ligands that have fast association
-
dissociation kinetics. Com-
plete saturation ligand-binding and competition ligand-binding assays have been
obtained on a single cell with excellent accuracy and reproducibility. This new
approach offers several advantages as it (i) substantially reduces the number of cells
needed, (ii) allows the investigation of cell-to-cell variations because extensive data
can be collected from individual cells, and (iii) circumvents problems related to low
expression levels of receptors and photobleaching of
fluorescent ligands, since
measurements can be repetitively performed on the same cell to enhance accuracy.
Moreover, ReSeq-binding assays can be easily automated and implemented in on-
chip analysis, which offers a substantial improvement in reliability, ef
ciency, and
reduction of sample consumption. In a single-molecule imaging experiment,
typically tens of frames are recorded before
fluorophore photobleaching occurs.
Because a reversibly binding
fluorescently-labeled ligand can be washed off
completely and added fresh once again, single-molecule experiments can be repeated
more than 30 times with one cell, and data with excellent statistics can be collected.
7.3
Functional Mobility of Receptors in Cell Membranes
7.3.1
Organization and Dynamics of Cell Membranes
The organization of biological membranes and the lateral diffusion of membrane
proteins are involved in a large number of signaling processes. For example, the
distribution and translational diffusion of membrane receptors regulate the plasticity
and function of neuronal (review: [38]) and immunological synapses [39], growth and
guidance of axons [40] and the gradient sensing of attractant molecules by chemo-
tactic cells [41]. Signal transduction at cellular membranes is often initiated by ligand
binding and followed by the formation of dynamic molecular networks involving the
active receptors. Interactions with structuring elements such as lipid shells and
membrane-associated or cytoskeletal components [42
-
44], or with other membrane
proteins [45, 46], microtubule-associated proteins [47
-
49] or molecules in the
membrane of adjacent cells, inevitably restrict receptor movements, as compared
to Brownian diffusion, and participate in their localization to specialized micro- and
nanometer-sized environments.
