xSyntabulin as a candidate interaction partner for xKIF13B

Similar to xKIF13B, xSyntabulin (xSybu) mRNA was also found to be localized to the vegetal pole of Xenopus oocytes (Horvay et al., 2006). Syntabulin (or Syntaxin-1-binding protein) is a microtubule associated protein that serves as an adaptor for conventional kinesin-1 heavy chain (KHC), or KIF5B. In the neurons, this association mediates transport of mitochondria and vesicles containing syntaxin-1 along neuronal axons to the presynaptic membrane in order to form there SNARE core complex (Su et al., 2004; Cai et al., 2005).

Furthermore, the Syntabulin-KIF5B interaction was also shown to mediate the axonal transport of active zone (AZ) precursors generated in the trans-Golgi network to the nascent synapses that is required for presynaptic assembly (Cai et al., 2007). Apart from its role in the nervous system, Syntabulin is also known to regulate the microtubule-dependent transport of the dorsal determinants (DDs) in zebrafish embryos. It is suggested that in zebrafish, Syntabulin links DDs to the maternally expressed kinesin I heavy chain (KIF5B) and mediates their initial vegetal pole localisation and subsequent transport to the prospective dorsal side. This process could link anteroposterior (AP) polarity in oocytes to embryonic dorsoventral (DV) polarity (Nojima et al., 2010).

Since xSybu is vegetally localized in X. laevis oocytes and serves as an adaptor for kinesin motors, its role was analyzed in the context of PGC development and possible interaction with xKIF13B. xSybu mRNA was found to be associated with germ plasm and PGC up to the early tadpole stage, with the exception of the neurula (stage 16-17) (Fig. 31).

Furthermore, expression of xSybu was detected in the dorsal region, probably due its

function in the nervous system, as described for mammalian neurons. Weak expression in the neurula at stage 19 was also observed in the RT-PCR analysis, but no differences between neurula and the tailbud stage 28 were detected in the whole transcriptome analysis. This can be either due to poor handling of the embryos, or a result of a degradation of maternal transcripts prior to the zygotic expression in PGCs. Highest expression of xSybu was observed at stage 37/38. This was mainly mediated by the increased expression in the dorsal region that correlates with the function of xSybu in mature neurons. In contrast, expression of xCentaurin-α1 in the developing neurons was observed to be highest at stage 22 (Fig. 30). RT-PCR analysis revealed presence of two isoforms of xSybu transcript in the embryo, both in dorsal and ventral parts. These isoforms varied in length in approximately 50 base pairs (Fig. 31). The shorter isoform was more uniformly expressed compared to the longer one that was enriched in the ventral part of X. laevis embryos. Expression of xSyntabulin in the PGCs revealed predominant intracellular localization to the specific region of the cell. Similar localization was observed for farnesylated fluorescent proteins, like mRFP, and most likely corresponds to the localization in Golgi apparatus (GA). Function of Syntabulin in the transport of active zone (AZ) precursors generated in the trans-Golgi network to nascent synapses in the neurons supports this assumption (Cai et al., 2007). It was previously demonstrated, that xKIF13B is localized close to the plasma membrane of bleb-like protrusion formed by isolated PGCs (Dzementsei, 2009). However, in contrast to xKIF13B, xSybu was not observed to be localized in this region (Fig. 32). This suggests that xSybu is not involved in the formation of xKIF13B-mediated PIP3 gradient. However, further experiments have to be performed to confirm this statement. To address this issue, co-immunoprecipitation experiments with in vivo and in vitro expressed xKIF13B and xSybu were performed. It seemed that there was no interaction between xKIF13B and xSyntabulin, but the data were inconclusive due to the poor expression of both xKIF13B and xSyntabulin.

In these experiments interaction with the smaller isoform of xSybu was analyzed (data not shown).

Analysis of xSyntabulin function in the PGCs was addressed by morpholino-mediated knock down of xSybu (xSybu MO). Activity and specificity of xSybu MO was verified in vitro (Suppl. Fig. 2). Injections of different concentrations of xSybu MO resulted in the defects in neural tube closer in axis formation at the neurula stage in a concentration-depended manner (Fig. 33). These effects can be explained by the role of xSybu in the transport of dorsal determinant during anterior-posterior axis formation at the early stages of development, described in zebrafish (Nojima et al., 2010). To determine whether xSybu knock-down also affects proliferation of directional migration of PGCs, embryos that survived neurulation were subjected to WMISH with PGC marker Pat and somite marker MyoD. Although average number of PGCs in the xSybu MO-injected embryos was reduced, no significant difference in the number of PGCs was observed in comparison to the control.

From these observations, we conclude that role of xSyntabulin in X. laevis embryogenesis is mainly restricted to the early patterning events. Role of xSybu as a

potential interaction partner for xKIF13B in PGCs is very unlikely, although some additional experiments have to be performed to validate this. Expression in the nervous system also suggests possible role of xSybu in the mature or developing neurons, similar to the mammalian system. Presence of two isoforms detected by the RT-PCR should also be taken into account for the future investigation of xSybu function in X. laevis.