Regulation of myoblast motility and fusion by the CXCR4-associated sialomucin, CD164

Myoblast fusion is fundamental to the development and regeneration of skeletal muscle. To fuse, myoblasts undergo cell-cell recognition and adhesion and merger of membranes between apposing cells. Cell migration must occur in advance of these events to bring myoblasts into proximity, but the factors that regulate myoblast motility are not fully understood. CD164 is a cell surface sialomucin that is targeted to endosomes and lysosomes via its intracellular region. In hematopoietic progenitor cells, CD164 forms complexes with the motility-stimulating chemokine receptor, CXCR4, in response to the CXCR4 ligand, CXCL12/SDF-1 (Forde, S., Tye, B. J., Newey, S. E., Roubelakis, M., Smythe, J., McGuckin, C. P., Pettengell, R., and Watt, S. M. (2007) Blood 109, 1825-1833). We have previously shown that CD164 stimulates myotube formation in vitro. We report here that CD164 is associated with CXCR4 in C2C12 myoblasts. Cells in which CD164 levels are increased or decreased via overexpression or RNA interference-mediated knockdown, respectively, show enhanced or reduced myotube formation and cell migration, the latter both basally and in response to CXCL12/SDF-1. Furthermore, expression of CD164 cytoplasmic tail mutants that alter the endosome/lysosome targeting sequence and, consequently, the subcellular localization in myoblasts, reveals a similar correlation between cell motility and myotube formation. Finally, Cd164 mRNA is expressed in the dorsal somite (the early myogenic compartment of the mouse embryo) and in premuscle masses. Taken together, these results suggest that CD164 is a regulator of myoblast motility and that this property contributes to its ability to promote myoblast fusion into myotubes.     

Genomic analysis of a murine cell-surface sialomucin, MGC-24/CD164.

MGC-24 is a sialomucin originally found in human gastric carcinoma cells, and in human hematopoietic progenitor cells. In the human, soluble and transmembrane forms of MGC-24 are present, and the transmembrane form has been implicated in adhesion of hematopoietic progenitor cells to marrow stroma cells. In the mouse, we found that only the transmembrane form was expressed in many organs. Northern blotting and in situ hybridization analysis showed that MGC-24 mRNA was widely expressed in various adult and embryonic tissues. The mouse MGC-24 gene, which we isolated, spanned about 12 kb and was comprised of six exons. The transmembrane domain and the cytoplasmic domain were encoded by a single exon; the finding agrees with the absence of an alternatively spliced product of mouse MGC-24. The minimal promoter of mouse MGC-24 was embedded in GC-rich sequences, in which two Sp1 binding motifs were found, but it lacked TATA and CAAT boxes. That the promoter resembles that of house-keeping genes is consistent with the broad expression of mouse MGC-24 mRNA.     

Insulin acts as a myogenic differentiation signal for neural stem cells with multilineage differentiation potential

Reports of non-neural differentiation of neural stem cells (NSCs) have been challenged by alternative explanations for expanded differentiation potentials. In an attempt to demonstrate the plasticity of NSC, neurospheres were generated from single retrovirally labeled embryonic cortical precursors. In a defined serum-free insulin-containing media, 40% of the neurospheres contained both myogenic and neurogenic differentiated progeny. The number of NSCs displaying multilineage differentiation potential declines through gestation but does exist in the adult animal. In this system, insulin appears to function as a survival and dose-dependent myogenic differentiation signal for multilineage NSCs (MLNSC). MLNSC-derived cardiomyocytes contract synchronously, respond to sympathetic and parasympathetic stimulation, and regenerate injured heart tissues. These studies provide support for the hypothesis that MLNSCs exist throughout the lifetime of the animal, and potentially provide a population of stem cells for cell-based regenerative medicine strategies inside and outside of the nervous system.     

The ratio of splicing variants of MGC-24/CD164, a sialomucin, correlates with the metastatic potential of colorectal carcinomas

MGC-24/CD164 is a sialomucin expressed in many normal and cancerous tissues. In humans, soluble and transmembrane forms of MGC-24 are produced by alternative splicing. The total MGC-24 RNA level was found to be lower in human colorectal carcinomas as compared with the adjacent normal mucosal tissues. Lower MGC-24 mRNA levels in colon carcinomas and in the adjacent normal mucosa epithelium correlate with lymphatic vessel invasion by the carcinoma. The ratio of the soluble form to the transmembrane form of the mRNA in colorectal carcinomas was determined by ribonuclease protection assay. Higher ratios were correlated with less venous invasion and less remote metastasis, which became evident during postoperative observation.     

Identification of a role for actin in translational fidelity in yeast

Numerous studies have suggested a role for actin in translation, but the molecular details of this role are unknown. To elucidate the function(s) of actin in translation, we have studied 25 isogenic, conditional yeast actin mutants. Strikingly, analysis of these mutants indicates that none of those tested have conditional growth defects caused by reduced rates of protein synthesis; and analysis of latrunculin A-treated wild-type cells indicates that even complete disruption of the actin cytoskeleton has no significant effect on the rate of translation. However, analysis of the effect of the 25 actin mutations on fidelity and sensitivity to translation inhibitors identified two mutations ( act1-2 and act1-122) that cause a significant reduction in the fidelity of translation, as assayed by nonsense suppression, and several mutants that are sensitive to paromomycin, which affects translational fidelity. Translation elongation factor 1A (eEF1A) also has a role in fidelity, and in the presence of excess eEF1A four of the mutants ( act1-2, act1-20, act1-120, and act1-125) are even more sensitive to paromomycin, while one mutant ( act1-122) becomes less sensitive. Together, these findings suggest that actin may not be important for the rate of translation, but may have a critical role in ensuring translational fidelity.     

Cell-surface MuSK self-association: a crucial role for the putative signal sequence

The receptor tyrosine kinase MuSK plays a crucial role-both as a signaling molecule and structurally-in the process of clustering nicotinic acetylcholine receptors at the neuromuscular junction. Immunofluorescence microscopy of transiently transfected fibroblasts has been used to visualize the cell-surface distribution of MuSK, which is found in discrete, punctate clusters. This distribution does not result from targeting of MuSK to identified plasma membrane subdomains, and MuSK's association with itself is specific, as MuSK clusters at the cell surface are segregated from clusters of other cotransfected receptor tyrosine kinases. A mutational analysis, using coexpressed pairs of MuSK mutants and chimeras, demonstrates that the putative signal peptide is both necessary and sufficient for association with MuSK. Removal of the intracellular domain or most of the extracellular domain, or replacement of the transmembrane domain, does not abolish MuSK self-association. The N-terminus of the MuSK protein, however, is sufficient to recruit another receptor tyrosine kinase to MuSK clusters. Quantitation and statistical analysis of the amount of colocalization between coexpressed MuSK mutants and chimeras confirm these results.     

Induction of myogenic differentiation by an expression vector encoding the DNA methyltransferase cDNA sequence in the antisense orientation.

To test the hypothesis that DNA methylation controls the state of differentiation of a mammalian cell, we transfected the stable mesenchymal line 10T1/2 with an expression vector encoding sequences from the DNA methyltransferase (DNA MeTase) cDNA in the antisense orientation. 10T1/2 cells transfected with the antisense construct (pZ alpha M), but not with the vector alone, exhibit morphological changes, convert into multinucleated tubular cells, and express the skeletal myosin heavy chain protein. The conversion to myogenic phenotype is a late event and is dependent on the number of replication events that the cell has undergone, suggesting that induction of myogenesis is a multistep process. Demethylation of sequences that are not involved in the myogenic process is detected at early passages, while demethylation and expression of the MyoD gene is a late event. This report establishes for the first time that demethylation is a very early event in commitment to myogenic differentiation, while demethylation and expression of MyoD is a late event. We suggest that other genes serve as the initial targets for demethylation and commitment of mesenchymal cells to myogenesis. The cell lines described in this report can serve as an important system for identifying these genes.     

Construction of yeast secretion vectors designed for production of mature proteins using the signal sequence of yeast invertase

Summary Two kinds of yeast secretion vectors were constructed by site-directed mutagenesis of the invertase signal sequence and ligation of synthetic oligonucleotides coding appropriate signals. Each has a cloning site for a foreign gene preceded by a sequence encoding either the signal peptide cleavage site or a Lys-Arg sequence which is a cleavage site for the product of the KEX2 gene. Both vectors were able to direct the expression and secretion of mouse amylase. One of them has a SalI site within the signal sequence, and an attempt to clone sequences enhancing secretion of amylase with this vector is reported.     

Identification of a novel functional specificity signal within the GPI anchor signal sequence of carcinoembryonic antigen

Exchanging the glycophosphatidylinositol (GPI) anchor signal sequence of neural cell adhesion molecule (NCAM) for the signal sequence of carcinoembryonic antigen (CEA) generates a mature protein with NCAM external domains but CEA-like tumorigenic activity. We hypothesized that this resulted from the presence of a functional specificity signal within this sequence and generated CEA/NCAM chimeras to identify this signal. Replacing the residues (GLSAG) 6-10 amino acids downstream of the CEA anchor addition site with the corresponding NCAM residues resulted in GPI-anchored proteins lacking the CEA-like biological functions of integrin modulation and differentiation blockage. Transferring this region from CEA into NCAM in conjunction with the upstream proline (PGLSAG) was sufficient to specify the addition of the CEA anchor. Therefore, this study identifies a novel specificity signal consisting of six amino acids located within the GPI anchor attachment signal, which is necessary and sufficient to specify the addition of a particular functional GPI anchor and, thereby, the ultimate function of the mature protein.     

Haemonchus contortus: evaluation of two signal sequence trapping systems for detection of secreted molecules

Given that signal sequences between secreted proteins of different species can be interchanged, it is reasonable to expect that both mammalian and yeast signal sequence trapping (SST) systems would secrete Haemonchus contortus proteins with similar efficiency and quality. To determine if H. contortus cDNAs that contain a signal sequence could re-establish secretion of a reporter protein, mammalian and yeast SST vectors were designed, 10 H. contortus genes selected, and their respective cDNAs cloned into these two SST vectors. The selected molecules included genes known to code for excretory/secretory or membrane-bound proteins as potential test 'positives', and genes known to code for non-secreted proteins as test 'negatives'. While differentiation between secretion and non-secretion was evident in both systems, the results indicated greater efficiency was achieved when the mammalian system was used. Therefore, mammalian SST using COS cells would be a more useful tool to screen H. contortus cDNA libraries for potential secreted and type-1 integral membrane proteins than yeast SST.     

The role of a conserved dodecamer sequence in yeast mitochondrial gene expression

All mRNAs on the yeast mitochondrial genome terminate at a conserved dodecamer sequence 5'-AAUAAUAUUCUU-3'. We have characterized two mutants with altered dodecamers. One contains a deletion of the dodecamer at the end of the var1 gene, and the other contains two adjacent transversions in the dodecamer at the end of the reading frame of fit1, a gene within the omega+ allele of the 21S rRNA gene. In each mutant, expression of the respective gene is blocked completely. A dominant nuclear suppressor, SUV3-1, was isolated that suppresses the var1 deletion but is without effect on the fit1 dodecamer mutations. Unexpectedly, however, we found that SUV3-1 blocks expression of the wild-type fit1 allele by blocking processing at its dodecamer. SUV3-1 has pleiotropic effects on mitochondrial gene expression, affecting RNA processing, RNA stability, and translation. Our results suggest that RNA metabolism and translation may be part of a multicomponent complex within mitochondria.     

Identification and purification of a protein that binds the yeast ARS consensus sequence

We have identified a yeast protein that binds specifically to the ARS consensus sequence. By two-step chromatography we have purified the factor to apparent homogeneity as a single polypeptide of 67 kd. The purified ARS consensus-binding protein (ACBP) recognizes the ARS consensus of the four genomic ARS elements tested, binding preferentially to the T-rich single strand. Point mutations in the consensus significantly reduce the affinity of the single-strand binding. At the histone H4 ARS, ACBP recognizes both the perfect ARS consensus and a 9/11 match 3' of it. These two binding sites correlate with the boundaries of the minimal functional H4 ARS element. A similar configuration of binding sites is found at ARS1. We propose a model implicating this factor in an early step of the initiation of DNA replication.     

Identification of a peptide sequence involved in association of subunits of yeast hexokinases

The chemistry of the proteolytic conversion of the native yeast hexokinases P-I and P-II to the respective modified forms S-I and S-II was studied in detail. The conversion of P-I to S-I was found to involve the removal of one six and one five residue peptide from P-I; these peptides were isolated and sequenced, and a comparison with the partial sequence of native P-I demonstrated that they were cleaved from the amino terminal end. Since results indicated that exactly the same peptides were cleaved from P-II during conversion to S-II, it is concluded that the first 11 amino acids in P-I and P-II have the same sequence. That sequence is: val · his · leu · gly · pro · lys · lys · pro · gln · ala · arg The basicity of these peptides was reflected in the decrease in isolectric point observed when a P-form is converted to an S-form. The peptides are clearly involved in the association of the subunits of yeast hexokinase, since their removal greatly weakens the tendency of the subunits to dimerize.     

Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells.

The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide.     

Identification of genes encoding mouse oocyte secretory and transmembrane proteins by a signal sequence trap

The oocyte plays a key role in follicular development. At all stages of follicular development, oocytes interact with surrounding granulosa cells and promote their differentiation into the types of cells that support further oocyte growth and developmental competence. These interactions suggest the existence of an oocyte-granulosa cell regulatory loop that includes both secreted proteins and cell surface receptors on both cell types. Factors involved in the regulatory loop will therefore contain a signal sequence, which can be used to identify them through a signal sequence trap (SST). A screen of an oocyte SST library identified three classes of oocyte-expressed sequences: known mouse genes, sequences homologous to known mammalian genes, and novel sequences of unknown function. Many of the recovered genes may have roles in the oocyte-granulosa cell regulatory loop. For several of the known mouse genes, new roles in follicular development are implied by identification of their expression, for the first time, in the oocyte. The future characterization of novel sequences may lead to the identification of novel proteins participating in the regulatory loop.     

The role of the DAP12 signal in mouse myeloid differentiation

DAP12 is a recently cloned, immunoreceptor tyrosine-based activation motif-bearing transmembrane adapter molecule that is associated with the NK-activating receptors. Previous reports showed that the DAP12 message could be detected not only in NK cells but also in granulocytes, monocytes, dendritic cells, and macrophages. In this study we found a significant level of DAP12 protein expression in macrophage-related cell lines and organs. Additionally, we observed increased expression of DAP12 after LPS-induced differentiation of M1 cells into macrophages. To examine the role of DAP12 in the myeloid cell lineage, we established M1 FLAG-DAP12 transfectants (FDAP-M1) and demonstrated the marked morphological changes in FDAP-M1 cells caused by signaling through DAP12. Cell surface phenotypic analysis showed up-regulation of macrophage markers CD11b, 2.4G2, and adhesion molecule B7-2. Additionally, after stimulation through DAP12, phosphorylated FLAG -DAP12 could be immunoprecipitated using anti-phosphotyrosine mAbs. Collectively, these findings indicate that direct DAP12 signaling has an important role in macrophage differentiation.     

Nucleosome positioning on yeast plasmids is determined only by the internal signal of DNA sequence occupied by the nucleosome

The possible role of border factors in determining the nucleosome positioning on a DNA sequence was investigated. To this end a family of recombinant plasmids based on Gal10Cyc1 promoter and neomycin phosphotransferase gene NPTII were created. A DNA sequence adjoining the GalCyc promoter was varied in these plasmids. Three nearly equally represented nucleosome positions on the GalCyc promoter were found. In the basal plasmid an FRT sequence adjoins the GalCyc promoter at the right. It contains an internal signal of multiple positioning. Its replacement with different DNA sequences does not affect nucleosome positioning on the GalCyc promoter. The nucleosome positioning on the GalCyc promoter does not depend on nucleosome positioning (or its absence) on adjoining sequences. The same is true for nucleosome positioning on FRT sequence. It was found also that nucleosomes' positioning on the NPTII gene and their mutual disposition, namely the spacing between neighboring nucleosomes (linker length) are determined by the location of positioning signals only. Generally the nucleosome positioning in our experimental model is determined solely by internal DNA sequence occupied by nucleosome. On the other hand, the action of this internal positioning signal does not extend to neighboring DNA sequences.