Biology Reference
In-Depth Information
Figure 7.3 Fluorescence speckle microscopy with labelled actin. A newt lung epithelial cell
that was injected with a small amount of X-rhodamine-labelled actin and imaged with a
high magnification, high resolution digital epi-fluorescence microscope system. In the
FSM image, the actin in the lamella and lamellipodium appears as a relatively even
distribution of fluorescent speckles, whose motion, appearance and disappearance can be
tracked over time to reveal molecular dynamics of the actin cytoskeleton. The image on the
right shows the same cell after fixation and staining with alexa-488 phalloidin to reveal in
greater detail the structural organization of the actin. Scale bar is 10 mm
The amount of labelled protein used is generally between 0.1-1% of total
cellular protein (Waterman-Storer and Salmon, 1999). Two incidental
advantages of this technique are, therefore, the low quantities of biological
material required and the lack of overexpression artefacts. An example of
speckles of fluorescently-labelled actin observed in epithelial cells is shown in
Figure 7.3 (Waterman-Storer, unpublished data).
When viewing time-lapse images from FSM, the internal dynamics of
structures such as microtubules, actin stress fibres and the dendritic actin
network in the lamella become apparent (Waterman-Storer and Salmon, 1997;
Waterman-Storer et al., 1999). Speckles can be seen to make up structures and
move in unison in specific identifiable directions. These images can be used to
calculate retrograde flow velocity of filaments and microtubules in different
parts of the cell.
Recent technological advances have allowed progression of the FSM
technique to a level where single fluorophores, and therefore single molecules
of protein, can be detected (Watanabe and Mitchison, 2002). This eliminated
the previous requirement for fluorophores to aggregate although signal
integration during long exposures still ensures that only molecules that are
relatively immobilized, for example by polymerization, can give rise to
speckles. This is an additional advantage of the single-molecule method since
the lifetime of a speckle is a direct indication of the time that the molecule
remains polymerized or otherwise anchored to a quasi-stable structure. In the
pilot
study,
single-molecule speckle fluorescence has
revealed valuable
