Analysis of the molecular dynamics of medaka nuage proteins by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching

The nuage is a unique organelle in animal germ cells that is known as an electron-dense amorphous structure in the perinuclear region. Although the nuage is essential for primordial germ cell (PGC) determination and development, its roles and functions are poorly understood. Herein, we report an analysis of the diffusion properties of the olvas gene product of the medaka fish (Oryzias lapites) in PGCs prepared from embryos, using fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. Olvas-green fluorescent protein (GFP) localized in granules thought to be nuages, and exhibited a constraint movement with two-component diffusion constants of 0.15 and 0.01 microm(2).s(-1). On the other hand, cytosolic Olvas-GFP was also observed to have a diffusion movement of 7.0 microm(2).s(-1). Interestingly, Olvas-GFP could be expressed in HeLa cells, and formed granules that were similar to nuages in medaka PGCs. Olvas-GFP also exhibited a constraint movement in the granules and diffused in the cytosol of HeLa cells, just as in the medaka embryo. The other two gene products, Nanos and Tudor of the medaka, which are known as constituents of the nuage, could also be expressed in HeLa cells and formed granules that colocalized with Olvas-GFP. Nanos-GFP and Tudor-GFP exhibited constraint movement in the granules and diffused in the cytosol of HeLa cells. These results suggest that these granules in the HeLa cell are not simple aggregations or rigid complexes, but dynamic structures consisting of several proteins that shuttle back and forth between the cytosol and the granules.     

Evolutionary history of larval skeletal morphology in sea urchin Echinometridae (Echinoidea: Echinodermata) as deduced from mitochondrial DNA molecular phylogeny

The larval skeletons of sea urchins show considerable morphological diversity, even between closely related species, although the evolutionary history and functional significance of this diversity are poorly understood. To infer the evolutionary history of the skeletal morphology, we focused on echinometrid species for which the morphological variation in larval skeletons had been investigated qualitatively and quantitatively. We reconstructed the phylogenetic relationships among 14 echinometrid species based on mitochondrial ND1 and ND2 genes and mapped the morphological characters onto the resultant trees. The monophyly of each genus in the Echinometridae was well supported by our results, as was the close affinity between Colobocentrotus, Heterocentrotus, and Echinometra. The mapping of the morphological characters of the larval skeletons indicated that the length, direction, and density of spines on the postoral rods was well conserved in each group of Echinometridae and that the abundance of spines and the size and shape of the body skeleton changed relatively frequently and hence were less conserved. In Echinometrid species, morphological variation in relatively unconserved features tends to be associated with latitudinal distributions, rather than phylogenetic relationships, indicating that the morphological diversity of larval skeletons could have been caused by adaptation to the habitat environment. Some morphological differences, however, seem to be nonfunctional and generated by the constraints on larval skeletogenesis. Thus, echinometrid species can be a good model with which to study the evolutionary history from both ecological and developmental standpoints.     

Comparison of energy components of proteins from thermophilic and mesophilic organisms

In order to infer the energetic determinants of thermophilic proteins, molecular mechanics calculations were applied to five proteins from thermophilic eubacteria and their mesophilic homologs. The energy function includes a hydration term as well as the electrostatic contribution from the solvent in addition to the usual conformational energy terms. We calculated energy values for three different states of each protein: the native, near-native, and unfolded structures. The energy difference and its components between pairs of these states were compared. The hypothetical near-native structures have almost the same backbone conformation as the native structure but with largely distorted side-chain packing, thus enabling us to extract the energy components important for stabilizing the native backbone topology itself, irrespective of structural details. It was found that the sum of the electrostatic and hydration energies, although of large positive values, were consistently lower for the thermophilic proteins than for their mesophilic counterparts. This trend was observed in the energy difference not only between the native and unfolded structures, but also between the near-native and unfolded structures. In contrast, the energy components regarding side-chain packing did not show any clear tendency. These results suggest that the thermophilic proteins are stabilized so that the precise packing of the native structure does not significantly affect the stability. Implications of this conclusion are also discussed.     

Microenvironment of endosomal aqueous phase investigated by the mobility of microparticles using fluorescence correlation spectroscopy

Temporal observation of the dynamic behavior of molecules in cells gives information about the physiological environment at the region of interest. Here we report the direct measurement of the mobility of rhodamine-labeled microparticles (14 and 35 nm in diameter) ingested in endosomes of cultured bovine aortic endothelial cells using fluorescence correlation spectroscopy (FCS). The fluctuation of fluorescent signals from microparticles were measured by FCS. Obtained autocorrelation functions (FAFs) were analyzed by the 2-D multicomponent model according to an evaluation procedure we newly developed. It was found that microparticles moved freely in endosomes with average diffusion coefficients of 4.3 x 10(-8) and 2.7 x 10(-8) cm2 s(-1) for 14 and 35 nm, which were 45% slower than in water. This result implies that the endosomal aqueous phase is homogeneous with the viscosity about 2.2 times of water. Our study also proposes the new use of FCS for investigation of the internal space of organelles.     

Role of cysteine residues in the NCKX2 Na+/Ca(2+)-K+ Exchanger: generation of a functional cysteine-free exchanger

Cysteine residues play an important role in many proteins, either in enzymatic activity or by mediating inter- or intramolecular interactions. The Na(+)/Ca(2+)-K(+) exchanger plays a critical role in Ca(2+) homeostasis in retinal rod (NCKX1) and cone (NCKX2) photoreceptors by extruding Ca(2+) that enters rod and cone cells via the cGMP-gated channels. NCKX1 and NCKX2 contain five highly conserved cysteine residues. The objectives of this study were threefold: (1) to examine the importance of cysteine residues in NCKX2 protein function; (2) to examine their role in the interaction between NCKX2 and the CNGA subunit of the cGMP-gated channel; and (3) to generate a functional cysteine-free NCKX2 protein. The latter will facilitate structural studies taking advantage of the unique chemistry of the thiol group following insertion of cysteine residues at specific positions in the cysteine-free background. We generated a cysteine-free NCKX2 mutant protein that showed normal protein synthesis and processing and approximately 50% wild-type cation transport function. Cysteine residues were also not critical for the formation of NCKX2 homo-oligmers or NCKX2 hetero-oligomers with the CNGA subunit of the cGMP-gated channel. Our results appear to rule out a critical importance of an intramolecular disulfide linkage in NCKX2 protein synthesis and folding as had been reported before.     

Eigenvalue analysis of amino acid substitution matrices reveals a sharp transition of the mode of sequence conservation in proteins

The pattern of amino acid substitutions and sequence conservation over many structure-based alignments of protein sequences was analyzed as a function of percentage sequence identity. The statistics of the amino acid substitutions were converted into the form of log-odds amino acid substitution matrices to which eigenvalue decomposition was applied. It was found that the most important component of the substitution matrices exhibited a sharp transition at the sequence identity of 30-35%, which coincides with the twilight zone. Above the transition point, the most dominant component is related to the mutability of amino acids and it acts to disfavor any substitutions, whereas below the transition point, the most dominant component is related to the hydrophobicity of amino acids and substitutions between residues of similar hydrophobic character are positively favored. Implications for protein evolution and sequence analysis are discussed.     

Intracytoplasmic lumina in medullary carcinoma of the thyroid gland. Report of a case with cytologic and immunocytochemical features

BACKGROUND: Intracytoplasmic lumina (ICL) have been observed frequently in breast carcinoma cells. However, they are extremely rare in thyroid gland tumors. We encountered a medullary carcinoma of the thyroid (MCT) with ICL and present a case with cytologic, immunocytochemical and ultrastructural features. CASE: A 15-year-old female was admitted with a left thyroid mass. Ultrasound examination revealed a well-defined tumor in the left lobe of the thyroid. Fine needle aspiration cytology showed mainly dispersed spindle cells with oval nuclei and some polymorphic or triangular tumor cells. The tumor cells containing ICL were noted at high magnification. The ICL contained sparse microvilli and abundant granular material with dense, round bodies on ultrastructural sections. Immunocytochemically, these tumor cells were positive for calcitonin and carcinoembryonic antigen (CEA). Moreover, CEA was recognized in the ICL with immunocytochemical staining. All tumor cells were negative for thyroglobulin. Pathologic examination confirmed the diagnosis of medullary carcinoma of the thyroid gland. CONCLUSION: MCT can include ICL with granular material containing CEA.     

Assembly of retinal rod or cone Na(+)/Ca(2+)-K(+) exchanger oligomers with cGMP-gated channel subunits as probed with heterologously expressed cDNAs

Proper control of intracellular free Ca(2+) is thought to involve subsets of proteins that co-localize to mediate coordinated Ca(2+) entry and Ca(2+) extrusion. The outer segments of vertebrate rod and cone photoreceptors present one example: Ca(2+) influx is exclusively mediated via cGMP-gated channels (CNG), whereas the Na(+)/Ca(2+)-K(+) exchanger (NCKX) is the only Ca(2+) extrusion protein present. In situ, a rod NCKX homodimer and a CNG heterotetramer are thought to be part of a single protein complex. However, NCKX-NCKX and NCKX-CNG interactions have been described so far only in bovine rod outer segment membranes. We have used thiol-specific cross-linking and co-immunoprecipitation to examine NCKX self-assembly and CNG-NCKX co-assembly after heterologous expression of either the rod or cone NCKX/CNG isoforms. Co-immunoprecipitation clearly demonstrated both NCKX homooligomerization and interactions between NCKX and CNG. The NCKX-NCKX and NCKX-CNG interactions were observed for both the rod and the cone isoforms. Thiol-specific cross-linking led to rod NCKX1 dimers and to cone NCKX2 adducts of an apparent molecular weight higher than that predicted for a NCKX2 dimer. The mass of the cross-link product critically depended on the location of the particular cysteine residue used by the cross-linker, and we cannot exclude that NCKX forms a higher oligomer. The NCKX-NCKX and NCKX-CNG interactions were not isoform-specific (i.e., rod NCKX could interact with cone NCKX, rod NCKX could interact with cone CNGA, and vice versa). Deletion of the two large hydrophilic loops from the NCKX protein did not abolish the NCKX oligomerization, suggesting that it is mediated by the highly conserved transmembrane spanning segments.