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particular PH domain binds specifically to phosphatidylinositol (4,5)-bis-
phosphate (PIP 2 ). Surprisingly, the spatial localization of PIP 2 in mouse
myoblasts appeared to be more stable than expected (Figure 7.2C).
Fluorescence speckle microscopy (FSM)
Conventional fluorescence microscopy works on the basis that a greater
fluorescence signal results in a less noisy image with a higher contrast. While
this is true when looking at stationary structures or fixed cells, it is not always
an advantage when imaging a moving structure or dynamic process. When
studying cytoskeletal proteins, for instance, it is not possible to see the internal
dynamics of a particular structure if it is uniformly labelled due to a high
density of fluorescent molecules. FSM was developed (Waterman-Storer et al.,
1998) in response to a phenomenon observed in an earlier study of
microtubule retrograde flow (Waterman-Storer and Salmon, 1997). Using
FSM, the underlying dynamic organization of labelled proteins can be
observed. Instead of a static homogeneously stained structure, moving
speckles are seen, indicative of molecular translocation. FSM relies on the
incorporation of such small amounts of fluorophore-conjugated protein into
the biopolymers of the cytoskeleton that the fluorescence can only be seen
when chance causes the incorporation of enough labelled molecules in one
small area to register a charge on a CCD camera (Waterman-Storer and
Salmon, 1998). The principle is that only relatively immobile molecules will
show as speckles since freely diffusing molecules move too fast to register
during a long exposure time (*0.5-2 s). Fluorophores can be conjugated
chemically, e.g., AlexaFluor or Cy dyes, or genetically, i.e., GFP and variants.
Figure 7.2 (opposite) Observation of plasma membrane events using TIRF microscopy.
(A) Merrifield, C. et al., unpublished material: A Swiss 3T3 fibroblast expressing (i)
clathrin-DsRed, (ii) dynamin 1-GFP, (iii) and as they appear in an overlay. The lower
panels (iv, v) show a time-resolved montage of an example event (i-ii, white box in upper
panel) showing the transient recruitment of dynamin 1-GFP to a clathrin-coated pit.
Numerals refer to time in seconds relative to the recorded event (t ¼ 0). Scale bars (iii) 5 mm,
(iv) 1 mm. (B) Merrifield, C. et al., unpublished material: A Swiss 3T3 fibroblast stably
expressing GFP-b-actin and transiently expressing clathrin-DsRed: (i) clathrin-DsRed,
taken with epifluorescent illumination, (ii) clathrin-DsRed, taken with TIRF illumination,
(iii) overlay of clathrin-DsRed and GFP-b-actin taken with TIRF illumination. The lower
panels (iv, v, vi) show a time resolved montage of the coated pit indicated in (i-iii, white
square). Numerals refer to time in seconds relative to the recorded event (t ¼ 0). Scale bars
(iii) 5 mm, (iv) 1 mm. (C) Mashanov, G. and Molloy, J., unpublished material: A mouse
myoblast transiently transfected with eGFP fused to the pleckstrin homology domain of
Myosin-X. This image of single fluorophores on the cell membrane was obtained by time-
lapse TIRF microscopy. Note the intensity plots revealing the lifetime of these spots. Scale
bars for space and time are 10 mm and 100 s respectively. (A colour reproduction of this
figure can be found in the colour plate section)
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