Two regions of the tail are necessary for the isoform-specific functions of nonmuscle myosin IIB.

To function in the cell, nonmuscle myosin II molecules assemble into filaments through their C-terminal tails. Because myosin II isoforms most likely assemble into homo-filaments in vivo, it seems that some self-recognition mechanisms of individual myosin II isoforms should exist. Exogenous expression of myosin IIB rod fragment is thus expected to prevent the function of myosin IIB specifically. We expected to reveal some self-recognition sites of myosin IIB from the phenotype by expressing appropriate myosin IIB rod fragments. We expressed the C-terminal 305-residue rod fragment of the myosin IIB heavy chain (BRF305) in MRC-5 SV1 TG1 cells. As a result, unstable morphology was observed like MHC-IIB(-/-) fibroblasts. This phenotype was not observed in cells expressing BRF305 mutants: 1) with a defect in assembling, 2) lacking N-terminal 57 residues (N-57), or 3) lacking C-terminal 63 residues (C-63). A myosin IIA rod fragment ARF296 corresponding to BRF305 was not effective. However, the chimeric ARF296, in which the N-57 and C-63 of BRF305 were substituted for the corresponding regions of ARF296, acquired the ability to induce unstable morphology. We propose that the N-57 and C-63 of BRF305 are involved in self-recognition when myosin IIB molecules assemble into homo-filament

Ran and Calcineurin Can Participate Collaboratively in the Regulation of Spermatogenesis in Scallop

Calcineurin is a calcium/calmodulin-dependent protein phosphatase that plays important roles in the transduction of calcium signals in a variety of tissues. In addition, calcineurin has been implicated in the process of spermatogenesis. A novel calcineurin-binding protein, CaNBP75, has been identified in scallop testis. The C-terminal region of CaNBP75 is homologous to the C-terminal region of RanBP3, a Ran binding domain-containing protein. A small G-protein Ran has been involved in spermiogenesis by virtue of the fact that its localization in spermatids changes during spermiogenesis. The current study was performed to investigate the functions of Ran and CaNBP75 in the regulation of calcineurin in testis to further understand the basic functions of calcineurin during spermatogenesis. First, cloning and sequencing of a scallop Ran cDNA isolated from testis revealed that scallop Ran is well-conserved at the amino acid level. Secondly, direct binding of Ran to CaNBP75 was demonstrated in an in vitro pull-down assay. Thirdly, analysis of the tissue distribution of Ran, CaNBP75 and calcineurin showed that these proteins are abundantly expressed in testis. Fourthly, comparison of the expression profiles of Ran and CaNBP75 with that of calcineurin in scallop testis during the maturation cycle revealed that Ran and CaNBP75 mRNA levels increase during meiosis and spermiogenesis, similar to calcineurin. Finally, co-immunoprecipitation analysis suggests that Ran, CaNBP75 and calcineurin interact in scallop testis during maturation. These results suggest that Ran, CaNBP75, and calcineurin may act in a coordinated manner to regulate spermatogenesis

Dynamic assembly properties of nonmuscle myosin II isoforms revealed by combination of fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy

Myosin II molecules assemble into filaments through their C-terminal rod region, and are responsible for several cellular motile activities. Three isoforms of nonmuscle myosin II (IIA, IIB and IIC) are expressed in mammalian cells. However, little is known regarding the isoform composition in filaments. To obtain new insight into the assembly properties of myosin II isoforms, especially regarding the isoform composition in filaments, we performed a combination analysis of fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS), which enables us to acquire information on both the interaction and the size of each molecule simultaneously. Using C-terminal rod fragments of IIA and IIB (ARF296 and BRF305) labelled with different fluorescent probes, we demonstrated that hetero-assemblies were formed from a mixture of ARF296 and BRF305, and that dynamic exchange of rod fragments occurred between pre-formed homo-assemblies of each isoform in an isoform-independent manner. We also showed that Mts1 (S100A4) specifically stripped ARF296 away from the hetero-assemblies, and consequently, homo-assemblies of BRF305 were formed. These results suggest that IIA and IIB can form hetero-filaments in an isoform-independent manner, and that a factor like Mts1 can remove one isoform from the hetero-filament, resulting in a formation of homo-filaments consisting of another isoform