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In Xenopus,
mRNAs with proven or probable determinant function become localised
to the vegetal cortex in two phases of oogenesis. Early on, while
the oocyte is small and transparent, a set of RNAs including those
coding for proteins required for germ line development (Xcat2, X-dazl,
Xpat) localises to a specialised macroscopic structure called the
Mitochondrial Cloud (MC).
As part of a collaboration with Mary-Lou King (University
of Miami) we have been studying the mechanism of RNA localisation
to the mitochondrial cloud, by following the behaviour of fluorescently-tagged
Xcat2 and Xdazl mRNA constructs injected into pre-vitellogenic Xenopus
oocytes (Chang
et al, 2004).
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24 hours after injection into Stage I or II oocytes, fluorescent
Xcat2 RNA becomes localised in the Mitochondrial Cloud
(MC)
Tlime lapse movies of the localisation process acquired overnight
suggest that directed transport is not responable, rather
the MC acts as a "sponge" which traps freely diffusing RNA
particles
(see Movie
: 5.8 MB: 1 image every 10 minutes).
The idea that localisation takes place by a "diffusion-entrapment
" mechanism is supported by the results of FRAP (Fluorescence
Recovery After Photobleaching) experiments.
Xcat2 localised in the MC is immobile following photobleaching
(top row of images ), whereas in the cytoplasm it recovers
with typical diffusion kinetics (bottom)
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Movie
(2.1MB) of a FRAP experiment showing recovery in the
cytoplasm but not in the MC |
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Vital labeling
with the Lipophillic dye DiIC16(3) reveals a dense ER network
in the MC
Xcat2 RNA localises progressively within this zone of
concentrated ER (Movie:
5.3 MB)
In peripheral regions, the Xcat2
particles 'dance' with the ER (512 MB). |
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At the onset of vitellogenesis (Stage II) the MC detaches from
the nucleus and merges with the cortex, thus bringing germ plasm with
associated RNAs to the newly-defined vegetal pole. A second group
of RNAs including those encoding Veg-T (which specifies the endoderm
terriory) and Vg1 (a TGFß family mesoderm inducer) then starts
to localise to the vegetal cortex by the microtubule-dependent "late
pathway".
  
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