Cell surface exposure of phosphatidylserine (PS) is shown to be part of normal physiology of skeletal muscle development and to mediate myotube formation. A transient exposure of PS was observed on mouse embryonic myotubes at E13, at a stage of development when primary myotubes are formed. The study of this process in cell cultures of differentiating C2C12 and H9C2 myoblasts also reveals a transient expression of PS at the cell surface. This exposure of PS locates mainly at cell-cell contact areas and takes place at a stage when the structural organization of the sarcomeric protein titin is initiated, prior to actual fusion of individual myoblast into multinucleated myotubes. Myotube formation in vitro can be inhibited by the PS binding protein annexin V, in contrast to its mutant M1234, which lacks the ability to bind to PS. Although apoptotic myoblasts also expose PS, differentiating muscle cells show neither loss of mitochondrial membrane potential nor detectable levels of active caspase-3 protein. Moreover, myotube formation and exposure of PS cannot be blocked by the caspase inhibitor zVAD(OMe)-fmk. Our findings indicate that different mechanisms regulate PS exposure during apoptosis and muscle cell differentiation, and that surface exposed PS plays a crucial role in the process of myotube formation.

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The expression efficiency of both cassettes was tested in two pUC-based expression vectors, that are otherwise comparable. In both constructs the respective polyadenylation sequences are fused to the coding sequences of the chloramphenicol acetyltransferase (CAT) gene. The expression of CAT activity was assayed when driven by a weak or by a strong promoter. As weak promoters the first 161 (PstI) or 525 bp (DraI) (4) nucleotides upstream of the transcription initiation site of the rat carbamoylphosphate synthase (CPS) gene were used, while the Rous sarcoma virus long terminal repeat (RSV-LTR) was used as a strong promoter. After CsCl purification, 50 Ag of these constructs was transfected into 1 x 107 FTO2B rat hepatoma cells as described (5). As an internal control 1 /tg pRSVluc was included in each transfection. Two days after electrotransfection, the cells were harvested in ice-cold PBS supplemented with 2 mM EDTA pH 8.0, and lysed in 0.1 % Triton X-100 in 100 mM potassium phosphate buffer pH 7.6. In each lysate the protein concentration (BCA protein reagent kit, Pierce), luciferase (6) and chloramphenicol acetyl transferase activity (7) was determined.Both vectors expressed no detectable CAT activity, when promoter sequences were absent. The activities of the weak promoters when expressed from the vector containing the shorter polyadenylation sequence are below the limit of detection of the assay, whereas when expressed in combination with the longer polyadenylation sequence a low but reproducible activity was detected (data not shown). When expression of the CPS promoter constructs was hormonally stimulated (10-7 M dexamethasone, 10-4 M dibutyryl cAMP and 10-3 M 3-isobutyl-1-methylxanthine), CAT expression became measurable even with the shorter polyadenylation sequence, but calculated activities were more than 100-fold lower than those obtained with the longer sequences for polyadenylation (Table 1). When CAT activity was driven by the strong viral RSV-LTR promoter, a 50-fold enhancement of gene expression was found with the longer polyadenylation sequence compared to the shorter (Table 1). A quantitatively comparable enhancement of expression of these constructs was observed in rat 5123 hepatoma cells, in rat Rat-I fibroblasts and in mouse NIH 3T3 fibroblasts. The shorter polyadenylation sequence in the late orientation has been shown to be more efficient than in the early orientation (10). If the shorter sequences for polyadenylation in the early orientation is extended up to the EcoRI site, gene expression proves to be at least equally if not more efficient than the short one in the late orientation.A convenient cloning vector should contain only the essential sequences necessary for high expression. The plasmid containing the RSV-LTR promoter upstream of the CAT gene and the longer polyadenylation sequence downstream of the CAT gene was used to identify the shortest sequence having the full capacity. The supercoiled plasmid was linearized with restriction enzymes BamHI, ApaI, PstI and NdeI to create seq...

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