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- Microtubule dynamics in the mpk4 mutant
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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
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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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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
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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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