Abstract.We have previously shown that a fluorescent derivative of ceramide, N-(e-7-nitrobenz-2-oxa-l,3- diazol-4-yl-aminocaproyl)-D-erythro-sphingosine (C6-NBD-Cer), vitally stains the Golgi apparatus of cells (Lipsky, N. G., and R. E. Pagano. 1985. Science (Wash. DC). 228:745-747). In the present paper we demonstrate that C6-NBD-Cer also accumulates at the Golgi apparatus of fixed cells and we explore the mechanism by which this occurs. When human skin fibroblasts were fixed with glutaraldehyde and then incubated with C6-NBD-Cer at 2°C, the fluorescent lipid spontaneously transferred into the cells, labeling the Golgi apparatus as well as other intracellular membranes. Subsequent incubations with defatted BSA at 24°C removed excess C6-NBD-Cer from the cells such that fluorescence was then detected only at the Golgi apparatus. Similar results were obtained using other cell types. A method for visualizing the fluorescent lipid at the electron microscopic level, based on the photoconversion of a fluorescent marker to a diaminobenzidine product (Sandell, J. H., and R. H. Masland. 1988. J. Histochem. Cytochem.36:555-559), is described and evidence is presented that C6-NBD-Cer was localized to the trans cisternae of the Golgi apparatus. While accumulation occurred in cells fixed in various ways, it was inhibited when fixation protocols that extract or modify cellular lipids were used. In addition, Filipin, which forms complexes with cellular cholesterol, labeled the Golgi apparatus of fixed cells and inhibited accumulation of C6-NBD-Cer at the Golgi apparatus. These results are discussed in terms of a simple model based on the physical properties of C6-NBD-Cer and its interactions with endogenous lipids of the Golgi apparatus. Possible implications of these findings for metabolism and transport of (fluorescent) sphingolipids in vivo are also presented.

We used null mutations of Drosophila actin and myosin genes to investigate two aspects of myofibril assembly.First, we eliminated all actin or myosin in flight muscles to evaluate contributions of thick and thin filaments to sarcomere formation. Results demonstrate that thick and thin filament arrays can assemble independently but that both are essential for sarcomeric order and periodicity. Second, we examined how filament stoichiometry affects myofibril assembly. We find that heterozygotes for actin (Act88F) or myosin heavy chain (Mhc36B) null alleles have complex myofibrillar defects, whereas Mhc36B-/+ ; Act88F-/+ double heterozygotes have nearly normal myofibrils. These results imply that most defects observed in single heterozygotes are due to filament imbalances, not deficits, and suggest that thick and thin filament interactions regulate myofibrillar growth and alignment.

The N-terminal caveolin binding motif (CBM) in Na/K-ATPase (NKA) α1 subunit is essential for cell signaling and somitogenesis in animals. To further investigate the molecular mechanism, we have generated CBM mutant human induced pluripotent stem cells (iPSCs) through CRISPR/Cas9 genome editing and examined their ability to differentiate into skeletal muscle (Skm) cells. Compared to the parental wild type human iPSCs, the CBM mutant cells lost their ability of Skm differentiation, which was evidenced by the absence of spontaneous cell contraction, marker gene expression, and subcellular myofiber banding structures in the final differentiated iSkm (induced Skm) cells. Another NKA functional mutant, A420P, which lacks NKA/Src signaling function, did not produce a similar defect. Indeed, A420P mutant iPSCs retained intact pluripotency and ability of Skm differentiation. Mechanistically, the myogenic transcription factor MYOD was greatly suppressed by the CBM mutation. Overexpression of a mouse Myod cDNA through lentiviral delivery restored the CBM mutant cells’ ability to differentiate into Skm. Upstream of MYOD, Wnt signaling was demonstrated from the TOPFlash assay to have a similar inhibition. This effect on Wnt activity was further confirmed functionally by defective induction of the presomitic mesoderm marker genes BRACHYURY (T) and MESOGENIN1 (MSGN1) by Wnt3a ligand or the GSK3 inhibitor/Wnt pathway activator CHIR. Further investigation through immunofluorescence imaging and cell fractionation revealed a shifted membrane localization of β-catenin in CBM mutant iPSCs, revealing a novel molecular component of NKA-Wnt regulation. This study sheds light on a genetic regulation of myogenesis through the CBM of NKA and control of Wnt/β-catenin signaling.

A family of muscle-specific helix-loop-helix transcription factors (myoD, myogenin, myf-5 and MRF4) has been implicated in the control of vertebrate skeletal myogenesis. Searches for homologues of this family in Caenorhabditis elegans identified a single family member, hlh-1, which is expressed in striated muscles and their clonal precursors. We have isolated a null allele of hlh-1 following chemical mutagenesis. Animals homozygous for the null mutation produce contractile body-wall muscles, although muscle contractions are weak and coordination is defective. In addition to the evident muscle defects, mutant animals fail to complete embryonic elongation and die as larvae or young adults. Ultrastructural analysis of the mutant muscle reveals an apparently normal local lattice of thick and thin filaments, with more global defects in sarcomere organization and muscle cell placement. Mosaic studies using the point mutation and an extrachromosomal transgene indicate that the requirement for hlh-1 is fully zygotic, with no maternal hlh-1 requirement for either muscle development or viability.