Identification, molecular structure, and analysis of expression of sea urchin unconventional myosins: Characterization of myosin-V in embryogenesis.

摘要

Early sea urchin development requires a dynamic reorganization of both the actin cytoskeleton and cytoskeletal interactions with cellular membranes and may rely upon the activities of multiple myosins. Using RT-PCR with degenerate myosin primers, we identified 11 myosin mRNAs expressed in unfertilized eggs and coelomocytes of the sea urchin Strongylocentrotus purpuratus. Seven of these sea urchin myosins belonged to myosin classes I, II, V, VI, VII, IX and the remaining four might belong to novel classes. Sea urchin myosins-V, -VI, -VII, and amoeboid-type-I were cloned and found to share a high degree of sequence identity with their respective family members from vertebrates and to have class specific structure and domain organization. Analysis of expression of myosin-V, -VI, -VII, and amoeboid-type-I mRNAs during early sea urchin development by RNase protection assays revealed that each myosin mRNA displayed a distinct temporal pattern of expression. Interestingly, the onset of gastrulation appeared to be a pivotal point in modulation of myosin mRNA expression.;We focused on providing a detailed characterization of expression and localization of sea urchin myosin-V in eggs and embryos. To detect myosin-V, polyclonal antibodies specific for sea urchin myosin-V were prepared. Myosin-V from unfertilized eggs interacted with actin filaments in an ATP-sensitive manner. The majority of myosin-V from eggs was present in the soluble, cytosolic pool while a small percentage associated with membrane fractions. Myosin-V protein was maternally stored and the levels of myosin-V remained relatively constant from fertilization through gastrulation. After gastrulation, the amount of myosin-V in embryos decreased and remained at a lower constant level through the pluteus stage. Laser scanning confocal microscopy of immunofluorescently stained embryos showed that myosin-V was expressed in all cell lineages. Myosin-V was localized to small intracellular puncta, interpreted as being vesicular staining, and microtubule-organizing centers. In ectoderm, myosin-V accumulated in the vicinity of the ciliary platform, an actin-rich structure responsible for anchoring/stabilizing individual cilia. Based on these observations, we propose that myosin-V functions in the transport of intracellular organelles and, possibly, organization of microtubule networks, in particular anchoring and/or maintenance of the ciliary platform.