Agriculture Reference
In-Depth Information
6.2.6
PrABP80 is Poppy Gelsolin
PrABP80 is a Ca
2+
-regulated ABP which may be involved in SI-mediated
actin depolymerization (Huang et al. 2004). PrABP80 was originally iden-
tified by DNase I-Sepharose chromatography during attempts to isolate
native poppy pollen actin. Tandem mass spectrometry sequence analy-
sis and kinetic actin polymerization assays showed that it belongs to the
villin/gelsolin family. Furthermore, the native protein was demonstrated
to have potent Ca
2+
-dependent severing activity in vitro (Huang et al.
2004). As shown in Fig. 6.2, PrABP80 significantly reduced the length of
pre-formed actin filaments after incubation in the presence of 160 µM free
Ca
2+
for 30 min (Fig. 6.2b) compared with incubation in the presence of
15 nM free Ca
2+
(Fig. 6.2a). It was also shown directly with time-lapse
fluorescence imaging that PrABP80 could sever actin filaments in a Ca
2+
-
dependent manner. PrABP80 could, therefore, assist F-actin depolymer-
ization by Ca
2+
-activated severing and the creation of new pointed ends
for depolymerization by profilin. In addition, PrABP80 caps the barbed
end of actin filaments, thereby blocking assembly of the profilin-actin
complex and allowing profilin to function as a simple actin sequestering
protein. The feasibility of such a mechanism was supported by experiments
in which equimolar amounts of profilin were added to pre-existing actin
filaments together with nanomolar amounts of PrABP80. In these assays,
polymer levels were monitored by fluorescence of pyrene-labelled actin in
the fluorimeter. The data from a typical experiment in the presence of 1 µM
free Ca
2+
, which mimics the [Ca
2+
]
i
observed in pollen tubes during the
SIresponse,areshowninFig.6.2c.Theextentandrateofactindepoly-
merization were dramatically increased by substoichiometric amounts of
PrABP80, when compared with profilin alone; moreover, significant de-
polymerization was not observed when free Ca
2+
was reduced to 15 nM
(Fig. 6.2c).
Thus,wehaveidentifiedapotentialmechanismthatlinksSI-induced
[Ca
2+
]
i
changes to actin depolymerization. We propose that PrABP80 func-
tionsatthecentreoftheSIresponsebycreatingnewfilamentpointed
ends for disassembly and by blocking barbed ends from profilin-actin
assembly. To test this molecular model thoroughly, we will need to deter-
mine the cellular concentrations for PrABP80 in pollen, measure its affinity
for plant actin filament ends, and determine the efficiency of severing at
physiological [Ca
2+
]
i
.
The actin depolymerization observed during SI is somewhat unusual,
since the level and extent of depolymerization are far greater than those
required to inhibit growth. Further alterations to the actin cytoskeleton
continue long after the inhibition of tip growth, suggesting that SI-induced
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