20
intrabundle microtubules did not differ significantly from those previously published (Shaw and
499
Lucas, 2011).
500
However, SIM imaging revealed that at the standard time interval used hereby (2.6 s), there were
501
short length transitions resulting in microtubule length changes of ca. 200 nm which were
502
smaller than the resolutions of WF, CLSM, TIRF and SD. Such small length transitions were
503
more frequently observed in intrabundle than in independent microtubules and their occurrence
504
contributed to the higher catastrophe and rescue frequencies which were calculated for
505
intrabundle microtubules.
506
In line with speckled appearance of GFP-TUA6 labeled microtubules in SIM, the kymographic
507
analyses of such labeled microtubules showed alternating bright and dark vertical stripes which
508
remained throughout the entire course of observations. If minor or major translocations of the
509
entire microtubule as frequently observed in plants (this study; e.g., Shaw et al., 2003) were
510
owing to motor-driven gliding then the stripes appearing in kymographs would appear inclined
511
since the stably incorporated GFP-TUA6 speckles would translocate together with the entire
512
microtubule. Since the stripes in SIM kymographs remain vertical (i.e., the GFP-TUA6 speckles
513
remain immotile through time), it can be deduced that such translocations owe to the hybrid
514
treadmilling mechanism described before (Shaw et al., 2003). This mechanism was proven by
515
generating a marker on the wall of growing GFP-TUA6-labeled microtubules by means of
516
photobleaching creating a dark spot, which was translated as a dark stripe on the respective
517
kymograph (Shaw et al., 2003). The photobleaching approach can be applied to a single or few
518
microtubules at a time, thus it is time-demanding to generate the amount of data necessary for
519
quantitative evaluation. On the contrary, application of fluorescence speckle microscopy by
520
means of SIM allows the simultaneous recordings of large populations of microtubules at once
521
because it is a widefield method (Gustaffsson, 2000).
522
Microtubule dynamics in the mpk4 mutant
523
In the
mpk4 mutant, it was found that growth and shrinkage rates were reduced
for both extra-
524
and intrabundle microtubule dynamics. Since these rate reductions occurred in both microtubule
525
classes, it is likely that overabundant but underphosphorylated MAP65-1 in the
mpk4 mutant
526
(Beck et al., 2010) is not related to end-wise microtubule dynamics. This is somewhat expected
527
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21
since at least MAP65-1 does not bind to soluble tubulin dimers (Smertenko et al., 2004) while
528
using in vitro assays, the addition of MAP65-1 is not affecting growth and shrinkage rates
529
(Stoppin-Mellet et al., 2013). Quite contradictory results were obtained from in vivo microtubule
530
dynamic measurements in tobacco BY-2 suspension culture cells stably transformed with
531
proCaMV35S::GFP:AtMAP65-1 construct showing marginal increase of growth rates compared
532
to cells transformed with
proCaMV35S::GFP-MBD (probably insignificant; see Table I from
533
van Damme et al., 2004), but rather steep reduction of depolymerization rates (nearly 50%; see
534
Table I from van Damme et al., 2004).
535
Considering: (a) the inability of MAP65-1 to bind to tubulin dimers and affect their incorporation
536
to microtubule ends (Smertenko et al., 2004, 2008), (b)
the similar
537
polymerization/depolymerization rates measured for extra- and intrabundle microtubules (this
538
study; see also Shaw and Lucas, 2011) and (c) the similar reduction of intra- and extrabundle
539
microtubule dynamic rates in the
mpk4 mutant, it is probable that the overall downregulation of
540
growth and shrinkage
rates observed in the mpk4 mutant is owing to a MAP65-1-independent
541
mechanism.
542
In the
mpk4 mutant, the transition frequencies were also decreased leading to smaller number of
543
both catastrophes and rescues. However, catastrophe and rescue frequencies were similarly
544
reduced for both free and intrabundle microtubules.
In vitro experiments showed that MAP65-1
545
caused decreased catastrophe frequency and amplitude and increased rescue frequency and
546
amplitude (Stoppin-Mellet et al., 2013). These
in vitro frequencies are again contradictory to
in
547
vivo observations (van Damme et al., 2004) showing that in tobacco BY-2 cells heterologously
548
overexpressing AtMAP65-1-GFP fusion protein, catastrophe frequency is comparable to that of
549
GFP-MBD labeled microtubules but rescue frequency is nearly half of the
respective frequency
550
of the GFP-MBD-labeled microtubules.
551
In this context the downregulation of both catastrophe and rescue frequencies in the
mpk4 mutant
552
in combination with the downregulation of polymerization/depolymerization rates, indicate more
553
complex mechanisms of microtubule regulation in the
mpk4 mutant as the simple straightforward
554
involvement of MAP65-1. Elucidation of this mechanism is out of the scope of the present work
555
and deserves further attention. Nevertheless, it was already predicted (Šamajova et al., 2013) that
556
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