Dynamic interaction of NtMAP65-1a with microtubules in vivo

Abstract

Plant microtubules are intrinsically more dynamic than those from animals. We know little about the dynamics of the interaction of plant microtubule-associated proteins (MAPs) with microtubules. Here, we have used tobacco and Arabidopsis MAPs with relative molecular mass 65 kDa (NtMAP65-1a and AtMAP65-1), to study their interaction with microtubules in vivo. Using fluorescence recovery after photobleaching we report that the turnover of both NtMAP65-1a and AtMAP65-1 bound to microtubules is four- to fivefold faster than microtubule treadmilling (13 seconds compared with 56 seconds, respectively) and that the replacement of NtMAP65-1a on microtubules is by random association rather than by translocation along microtubules. MAP65 will only bind polymerised microtubules and not its component tubulin dimers. The turnover of NtMAP65-1a and AtMAP65-1 on microtubules is similar in the interphase cortical array, the preprophase band and the phragmoplast, strongly suggesting that their role in these arrays is the same. NtMAP65-1a and AtMAP65-1 are not observed to bind microtubules in the metaphase spindle and their rate of recovery is consistent with their cytoplasmic localisation. In addition, the dramatic reappearance of NtMAP65-1a on microtubules at the spindle midzone in anaphase B suggests that NtMAP65-1a is controlled post-translationally. We conclude that the dynamic properties of these MAPs in vivo taken together with the fact that they have been shown not to effect microtubule polymerisation in vitro, makes them ideally suited to a role in crossbridging microtubules that need to retain spatial organisation in rapidly reorganising microtubule arrays.

Control of the AtMAP65-1 interaction with microtubules through the cell cycle

“…However, as the PPB disassembles and the mitotic spindle starts to form, AtMAP65-1 becomes mainly cytoplasmic Van Damme et al, 2004;Chang et al, 2005). As the chromosomes separate AtMAP65-1 is observed to associate with microtubules again in anaphase B, but its localisation is restricted to the midzone of the anaphase spindle and to the phragmoplast midzone during telophase Chang et al, 2005). A similar localisation was reported for the AtMAP65-1 homologue in tobacco NtMAP65-1a (Smertenko et al, 2000) suggesting that both proteins have similar functions (Chang et al, 2005).…”

“…Phragmoplast microtubules depolymerise at the sites where the cell plate has been established and polymerise at the edges of the expanding cell plate to support the centrifugal expansion of the cell plate until it reaches the cell wall of the mother cell . The localisation of AtMAP65-1 at the midzone was detected at the edge of the expanding phragmoplast (Chang et al, 2005) where AtMAP65-1 most probably supports phragmoplast structure until the establishment of a vesicular-tubular membrane network. The role of MAP65 proteins in maintaining the integrity of the phragmoplast was demonstrated in the A. thaliana mutant, pleiade.…”

“…AtMAP65-1 binds to a subset of microtubules in the interphase array and the preprophase band (PPB). However, as the PPB disassembles and the mitotic spindle starts to form, AtMAP65-1 becomes mainly cytoplasmic Van Damme et al, 2004;Chang et al, 2005). As the chromosomes separate AtMAP65-1 is observed to associate with microtubules again in anaphase B, but its localisation is restricted to the midzone of the anaphase spindle and to the phragmoplast midzone during telophase Chang et al, 2005).…”

“…As the cell plate forms the microtubules crossing the division midzone disappear (Segui-Simarro et al, 2004) and MAP65 localisation at the division midzone fades (Chang et al, 2005). Phragmoplast microtubules depolymerise at the sites where the cell plate has been established and polymerise at the edges of the expanding cell plate to support the centrifugal expansion of the cell plate until it reaches the cell wall of the mother cell .…”

Strategies of actin reorganisation in plant cells

“…In all charts showing intensity of GFP, the background values were measured in the area of the cell lacking any visible filaments and subtracted from the plotted signal. The FRAP experiments were performed and quantified as described (Chang et al, 2005). To quantify the thickness of actin bundles, it was assumed that the Lifeact-GFP fluorescence signal level is proportional to the amount of actin located within the filament.…”