Spatial and temporal regulation of cortical proteins on the cell membrane leads to cortical polarization of cells. After cortical polarization, the cell membrane can divide into different domains such as apical domain and basolateral domain. Cortical polarity is crucial for cell differentiation and function. For example, cell polarity is required for the formation of spatially restricted structures, like cell junctions in differentiated cells. Also cell polarity is important for the morphological complexity during embryonic development. In C. elegans embryos, the cell polarization can be found at one-cell stage. With anterior cortical flow created by actomyosin contraction, the cortex of C. elegans embryos separates into anterior side and posterior side. With proliferation and polarization, cells in C. elegans embryos start internalization and migration during gastrulation, embryos separate into ectodermal, endodermal and mesodermal compartments, this is required for organogenesis. The membrane polarization also significantly happens in Drosophila early embryonic development, the cortex of Drosophila embryos differentiates into apical, subapical, lateral, and basal domains during cellularization. Drosophila embryos finish 13 nuclear cycles in about 2h at room temperature, following with embryo cellularization. With the membrane invagination during cellularization, Drosophila embryos divide into more than 6000 cells. Since the polarity of cortical domain is important for embryonic differentiation and development, it is vital to fully understand mechanisms of the cell polarization and functions of different proteins in cell polarization. As the cortex of Drosophila embryos differentiates into four different domains in about 3h, it is a good model to study the cortical polarization in early embryonic development.
The cytoskeleton includes microtubules, microfilaments and intermediate filaments, they are not only providing mechanical support, but are also essential for cortical polarization. Kinesin-1, as a microtubule-dependent motor protein, is required for cargos transport in different cellular processes, such as nuclear positioning, ooplasmic streaming, and cortical polarization. Previous report showed that Kinesin-1 depletion affects the cellularization in Drosophila embryos, the membrane invagination during cellularization is also disrupted in Kinesin-1 RNAi embryos, but mechanisms how Kineisn-1 influences cellularization are not so clear yet. Functions of microtubules and actin network in cell biology and biophysics have been studied for several decades, interactions between microtubules and actin network in core processes have been concerned. However, whether Kinesin-1 depletion affects the polarity of F-actin cap during the syncytial interphase of Drosophila embryos has not been investigated.
To understand how Kinesin-1 regulates the cell polarization during cellularization and how Kinesin-1 influences the organization of F-actin cap during the syncytial interphase, in this study, I utilized Drosophila Kinesin-1 RNAi embryos to checked the localization of cortical components during syncytial stage and cellularization. I also focused on the organization of F-actin cap in Kinesin-1 RNAi embryos. I found that the disruption of cellularization in Kinesin-1 depleted embryos is due to the mislocalization of cortical components, they are stuck at the surface of Kinesin-1 RNAi embryos during cellularization. However, dynamics of GFP-Slam in wild type and Kinesin-1 RNAi embryos are comparable during cellularization, centrosomes and recycling endosomes in Kinesin-1 RNAi embryos are also fine. Although the cortical polarization in Kinesin-1 RNAi embryos is comparable to wild type during the syncytial stage, the localization of Canoe and ELMO/Sponge complex is affected. Furthermore, live images and immunostainings of Capping α (Cpα) indicate that Kinesin-1 is essential for the localization of Cpα at the intercap domain. In Kinesin-1 RNAi embryos, not only the contraction but also the polarity of the F-actin cap are influenced. The accumulation of Cpα at the intercap domain is affected in Kinesin-1 RNAi embryos. Myosin II cannot accumulate to the intercap domain in Kinesin-1 RNAi embryos. By injecting ROCK inhibitor into Drosophila embryos, I found that the disruption of Myosin II affects the polarity of F-actin cap, the distribution of Cpα at the edge of F-actin cap is affected. By inserting the GFP right after the Cpα gene with CRISPR, dynamics of Cpα can be observed. During the interphase, the distribution of Cpα clusters is affected, Cpα clusters are mainly localized to the intercap domain. Dia localizes to the downstream of Kinesin-1, which is also required for the distribution of Cpα clusters at the intercap region. Moreover, I found that Kinesin-1 and plus ends of microtubules are accumulated at the cap domain during the syncytial interphase. I also found that APC2 coprecipitates with Kinesin-1. Although the cellularization is affected in APC2 d40 truncation embryos, the localization of cortical components is comparable, different domains are clearly separated. The disruption of cellularization in APC2 d40 truncation embryos may due to reductions of Slam and Amphiphysin at the basal domain.
I also mapped functions of the slam mRNA sequence for its localization and Slam protein translation. The results indicated that the entire slam mRNA sequence is required for robustly Slam protein expression. Apart from the slam mRNA sequence is essential for Slam protein expression, slam mRNA sequence is also required for the localization of slam mRNA. slam mRNA coding region from 507 nt to 1576 nt has an effect on slam mRNA localization and Slam protein expression. slam mRNA coding sequence from 2818 nt to 3522nt is required for slam mRNA localization.||de