3.3 BucLoc interacts with non-muscle myosin II
3.3.3 Endogenous p-Myl12.2 co-localizes continuously with Buc
As the exosome is involved in RNA degradation it is unclear how it is involved in the localization of Buc protein. Cytoskeletal proteins are more likely to be involved in the dynamic localization of Buc protein. The analyzed cytoskeletal element Sept2 did not show co-localization with BucLoc (Figure 24). Hence, the last candidate among the eight selected proteins, myosin light chain 12, was further analyzed.
Results 3.3.3.1 Non-muscle myosin II components are BucLoc interactors
Myosin light chain 12, genome duplicate 2 (myl12.2) is a regulatory light chain of non-muscle myosin II (NMII) that was identified among the mass spectrometry proteins (Table 12;
Appendix, Table 16). Non-muscle myosins are essential for cytokinesis and are involved in cell adhesion and polarity (Mabuchi and Okuno, 1977; Vicente-Manzanares et al., 2009).
They form a superfamily of ATP-dependent motor proteins, responsible for actin-based motility and are composed of two heavy and two regulatory as well as two essential light chains (Vicente-Manzanares et al., 2009).
Interestingly, Myl12.2 and other proteins of the non-muscle myosin II complex were strongly enriched in the BucLoc Co-IP sample, but not in the Buc-GFP sample (Table 14).
Table 14: Components of non-muscle myosins II complex identified by mass spectrometry analysis. Fold enrichment for Buc11 88-eGFP as well as Buc-eGFP in comparison to eGFP is given for each candidate. Detailed information is presented in the appendix (Table 16).
Fold enrichment in
3.3.3.2 p-Myl12.2 co-localizes with Buc in 2-cell stage embryos
To investigate if non-muscle myosins co-localize with Buc in vivo, a commercial antibody was applied, which was raised against the phosphorylated human homolog of zebrafish Myl12.2. The phosphorylation of regulatory light chains leads to their activation and the formation of a functional NMII complex (Vicente-Manzanares et al., 2009). Previous reports have shown that phosphorylated non-muscle myosin II (p-NMII) co-localizes with germ plasm mRNAs at the cleavage furrow of 2-cell stage zebrafish embryos (Nair et al., 2013).
To investigate if endogenous Buc co-localizes with active phosphorylated non-muscle myosin II regulatory light chain Myl12.2, 2-cell stage wild type embryos were immunostained for Buc as well as p-Myl12.2 and analyzed by confocal fluorescence microscopy. Indeed, 2-cell stage and that active non-muscle myosin II might be involved in this transport.
Results
Figure 26: p-Myl12.2 co-localizes with Buc at the 2-cell stage cleavage furrow. Wild type 2-cell stage embryo immunostained for Buc and p-Myl12.2 and imaged by confocal fluorescence microscopy. Note that single granules are lined up like on a ‘string of pearls’ and together form a rod-like structure (arrowhead). Blastomeres on top of the yolk sac are shown in a lateral view, animal pole to the top. Magnification images of cleavage furrow (A’) are z-stack projections. Scale bars represent 50 µm (A) and 10 µm (A’).
3.3.3.3 p-Myl12.2 permanently co-localizes with Buc during oogenesis and embryogenesis
Localization of phosphorylated non-muscle myosin II in zebrafish was so far described for 2-, 4- and 8-cell stage. In these stages p-NMII localizes in a rod-like structure to the cleavage furrows and in a granular structures to the cortical region of the embryo, similar to Buc-GFP in the transgenic line (Figure 12) (Eno and Pelegri, 2013). If p-Myl12.2 was permanently involved in Buc localization, it would consistently co-localize with Buc.
To test this hypothesis, wild type 256-cell stage embryos and stage IB oocytes were immunostained for Buc as well as p-Myl12.2 and analyzed by confocal fluorescence microscopy. In 256-cell stage embryos, Buc and p-Myl12.2 co-localized in germ plasm aggregates, which is consistent with the identification of Myl12.2 by mass spectrometry analysis (Figure 27A, A’). Furthermore, p-Myl12.2 was localized together with Buc to the Balbiani body already during early stage IB oocytes (Figure 27B). Similarly, in later stage IB oocytes Buc and p-Myl12.2 were both relocated to the cortical region and spread along the cortex of the vegetal pole (Figure 27C). In this stage, p-Myl12.2 was additionally localized in granules within the germinal vesicle.
These results show that p-Myl12.2 consistently co-localizes with Buc from early oogenesis until early embryogenesis. This permanent co-localization supports a role of non-muscle myosin II in Buc localization. Furthermore, this is the first time that a protein directly associated with the cytoskeleton has been described to constantly localize to the germ plasm during zebrafish oogenesis and embryogenesis. Such a permanent protein localization to the germ plasm has so far only been described for Buc (this study).
Results
Figure 27: p-Myl12.2 co-localizes with Buc in oogenesis as well as embryogenesis. Wild type 256-cell stage embryo (A, magnified A’) and stage IB oocytes (B, C) immunostained for Buc and p-Myl12.2 and imaged by confocal fluorescence microscopy. Embryo and oocytes, outlined by a yellow dashed line, are shown in lateral view, animal pole to the top. Scale bars represent 50 µm (A) and 10 µm (A’, B, C).
In summary, the interaction with BucLoc and the localization analysis of non-muscle myosins suggests a role of the NMII-complex, composed of Myh9a, Myl12.2 and Myl6, in the localization of Buc to the germ plasm in zebrafish oogenesis as well as early embryogenesis.
4 Discussion
Buc has been recently identified in zebrafish as the first vertebrate protein that is necessary for germ plasm formation and sufficient for the formation of primordial germ cells (Bontems et al., 2009). Since proper germ plasm localization is a critical step in germ cell development, the localization of the germ plasm regulator Buc is of special interest. Therefore, the aim of this study was to characterize the localization of Buc protein in zebrafish and to analyze the underlying localization mechanism that ensures proper localization of Buc.
Buc was identified in this study as a permanent germ plasm component during zebrafish oogenesis and embryogenesis up to 48 hpf. Buc localization is established by the newly identified BucLoc localization domain in the zebrafish embryo. Using this domain, 213 proteins were identified that specifically interact with the Buc localization domain by Co-IP and subsequent mass spectrometry analysis. Further analysis of the interaction candidates led to the identification of Exocs9 as a germ plasm component in zebrafish oocytes. Moreover, consistent co-localization of Buc with the interacting non-muscle myosin II component Myl12.2 suggests a role of non-muscle myosin II in Buc localization.