Oxygen-Glucose Deprivation Decreases the Motility and Length of Axonal Mitochondria in Cultured Dorsal Root Ganglion Cells of Rats.

Controlling axonal mitochondria is important for maintaining normal function of the neural network. Oxygen-glucose deprivation (OGD), a model used for mimicking ischemia, eventually induces neuronal cell death similar to axonal degeneration. Axonal mitochondria are disrupted during OGD-induced neural degeneration; however, the mechanism underlying mitochondrial dysfunction has not been completely understood. We focused on the dynamics of mitochondria in axons exposed to OGD; we observed that the number of motile mitochondria significantly reduced in 1 h following OGD exposure. In our observation, the decreased length of stationary mitochondria was affected by the following factors: first, the halt of motile mitochondria; second, the fission of longer stationary mitochondria; and third, a transformation from tubular to spherical shape in OGD-exposed axons. Motile mitochondria reduction preceded stationary mitochondria fragmentation in OGD exposure; these conditions induced the decrease of stationary mitochondria in three different ways. Our results suggest that mitochondrial morphological changes precede the axonal degeneration while ischemia-induced neurodegeneration.

Universal phase behaviors of intracellular lipid droplets.

Lipid droplets are cytoplasmic microscale organelles involved in energy homeostasis and handling of cellular lipids and proteins. The core structure is mainly composed of two kinds of neutral lipids, triglycerides and cholesteryl esters, which are coated by a phospholipid monolayer and proteins. Despite the liquid crystalline nature of cholesteryl esters, the connection between the lipid composition and physical states is poorly understood. Here, we present a universal intracellular phase diagram of lipid droplets, semiquantitatively consistent with the in vitro phase diagram, and reveal that cholesterol esters cause the liquid-liquid crystal phase transition under near-physiological conditions. We moreover combine in vivo and in vitro studies, together with the theory of confined liquid crystals, to suggest that the radial molecular alignments in the liquid crystallized lipid droplets are caused by an anchoring force at the droplet surface. Our findings on the phase transition of lipid droplets and resulting molecular organization contribute to a better understanding of their biological functions and diseases.

Syntaxin 17 promotes lipid droplet formation by regulating the distribution of acyl-CoA synthetase 3.

Lipid droplets (LDs) are ubiquitous organelles that contain neutral lipids and are surrounded by a phospholipid monolayer. How proteins specifically localize to the phospholipid monolayer of the LD surface has been a matter of extensive investigations. In the present study, we show that syntaxin 17 (Stx17), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein whose expression in the liver is regulated by diet, participates in LD biogenesis by regulating the distribution of acyl-CoA synthetase (ACSL)3, a key enzyme for LD biogenesis that redistributes from the endoplasmic reticulum (ER) to LDs during LD formation. Stx17 interacts with ACSL3, but not with LD formation-unrelated ACSL1 or ACSL4, through its SNARE domain. The interaction occurs at the ER-mitochondria interface and depends on the active site occupancy of ACSL3. Depletion of Stx17 impairs ACSL3 redistribution to nascent LDs. The defect in LD maturation due to Stx17 knockdown can be compensated for by ACSL3 overexpression, suggesting that Stx17 increases the efficiency of ACSL3 redistribution to LDs. Moreover, we show that the interaction between Stx17 and ACSL3 during LD maturation may be regulated by synaptosomal-associated protein of 23 kDa.

Linkage between lipid droplet formation and nuclear deformation in HeLa human cervical cancer cells.

Because lipid droplets (LDs) and the nucleus are cellular organelles that regulate seemingly very different biochemical processes, very little attention has been focused on their possible interplay. Here, we report a correlation between nuclear morphology and cytoplasmic LD formation in HeLa human cervical cells. When the cells were treated with oleic acid (OA), LDs were formed in the cytoplasm, but not in the nucleoplasm. Interestingly, cells harboring OA-induced cytoplasmic LDs showed deformity of the nucleus, particularly at the nuclear rim. Conversely, when alteration from a single spherical nuclear shape to a multinucleated form was enforced by coadministration of paclitaxel and reversine, a significant amount of LDs was detected in the cytoplasm of the multinucleated cells. These two distinct pharmacological culture conditions not only allow analysis of the previously underappreciated organelle relationship, but also provide insights into the mutual affectability of LD formation and nuclear deformation.

The Lipid Droplet and the Endoplasmic Reticulum.

Lipid droplets (LDs) are often found adjacent to the endoplasmic reticulum (ER). The ER-LD association may appear morphologically similar to the prototypical membrane contact sites found between the ER and other organelles, but the functional relationship between the ER and LDs is unique in that highly hydrophobic lipid esters are transported between them. This transportation is thought to occur through some form of membrane continuity, but its details are yet to be defined. Lipin, seipin, and FIT proteins, which are located at the ER-LD interface, may be involved in the lipid ester transport and probably play important roles for functional connectivity of the two organelles. More recently, LDs in the nucleus were found to be closely adhered to the inner nuclear membrane, representing a specialized form of the ER-LD association. In this article, we will give an overview of the ER-LD association, which is still filled with many unanswered questions.

Alleviation of seipinopathy-related ER stress by triglyceride storage.

Mutations affecting the N-glycosylation site in Berardinelli-Seip lipodystrophy (BSCL)-associated gene BSCL2/seipin lead to a dominantly inherited spastic paraplegia termed seipinopathy. While the loss of function of seipin leads to severe congenital lipodystrophy, the effects of seipin N-glycosylation mutations on lipid balance in the nervous system are unknown. In this study, we show that expression of seipin N-glycosylation mutant N88S led to decreased triglyceride (TG) content in astrocytoma and motor neuron cell lines. This was corrected by supplementation with exogenous oleic acid. Upon oleic acid loading, seipin N88S protein was relocated from the endoplasmic reticulum (ER) to the surface of lipid droplets and this was paralleled by alleviation of ER stress induced by the mutant protein. This effect was not limited to seipin N88S, as oleic acid loading also reduced tunicamycin-induced ER stress in motor neuron cells. Furthermore, both seipin N88S and tunicamycin-induced ER stress were decreased by inhibiting lipolysis, suggesting that lipid droplets protected neuronal cells from ER stress. In developing zebrafish larvae, seipin N88S expression led to TG imbalance and reduced spontaneous free swimming. Importantly, supplementation with exogenous oleic acid reduced ER stress in the zebrafish head and increased fish motility. We propose that the decreased TG content contributes to the pathology induced by seipin N88S, and that rescuing TG levels may provide a novel therapeutic strategy in seipinopathy.

Inhibition of Niemann-Pick-type C1-like1 by ezetimibe activates autophagy in human hepatocytes and reduces mutant α1-antitrypsin Z deposition.

UNLABELLED: Autophagy can degrade aggregate-prone proteins, but excessive autophagy can have adverse effects. It would be beneficial if autophagy could be enhanced in a cell type-specific manner, but this has been difficult because the basic mechanism of autophagy is common. In the present study we found that inhibition of Niemann-Pick-type C1-like 1 (NPC1L1) by ezetimibe activates autophagy only in hepatocytes and small intestinal epithelia, but not in other cells. Ezetimibe induced accumulation of free cholesterol in the late endosome/lysosome and increased partitioning of a Ragulator component, LAMTOR1, in rafts. The latter change led to down-regulation of mammalian target of rapamycin (mTOR)C1 activity by decreasing mTOR recruitment to the late endosome/lysosome and activated autophagy. A primary effect of ezetimibe was found to be a decrease of free cholesterol in the plasma membrane, because all the results caused by ezetimibe were suppressed by supplementation of cholesterol as a methyl-ß-cyclodextrin complex. By enhancing autophagy in human primary hepatocytes with ezetimibe, insoluble mutant α1-antitrypsin Z was reduced significantly. CONCLUSION: Inhibition of NPC1L1 by ezetimibe activates autophagy in human hepatocytes by modulating cholesterol homeostasis. Ezetimibe may be used to ameliorate liver degeneration in α1-antitrypsin deficiency.

Fish oil-derived n-3 polyunsaturated fatty acids downregulate aquaporin 9 protein expression of liver and white adipose tissues in diabetic KK mice and 3T3-L1 adipocytes.

Aquaporin 9 (AQP9) is an integral membrane protein that facilitates glycerol transport in hepatocytes and adipocytes. Glycerol is necessary as a substrate for gluconeogenesis in the physiological fasted state, suggesting that inhibiting AQP9 function may be beneficial for treating type 2 diabetes associated with fasting hyperglycemia. The n-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are rich in fish oil and lower the risk of metabolic syndrome; however, the effects of EPA and DHA on AQP9 expression in obese and type 2 diabetes are unclear. The KK mouse is an animal model of obesity and type 2 diabetes because of the polymorphisms on leptin receptor gene, which results in a part of cause for obese and diabetic conditions. In this study, we determined the effect of fish oil-derived n-3 PUFA on AQP9 protein expression in the liver and white adipose tissue (WAT) of KK mice and mouse 3T3-L1 adipocytes. The expression of AQP9 protein in the liver, epididymal WAT, and inguinal WAT were markedly decreased following fish oil administration. We also demonstrated that n-3 PUFAs, such as DHA, and to a lesser extent EPA, downregulated AQP9 protein expression in 3T3-L1 adipocytes. Our results suggest that fish oil-derived n-3 PUFAs may regulate the protein expressions of AQP9 in glycerol metabolism-related organs in KK mice and 3T3-L1 adipocytes.

Stress-strain relationship of individual hamstring muscles: A human cadaver study.

The incidence of hamstring muscle strain varies among muscles, suggesting that the mechanical stresses associated with elongation may differ among muscles. However, the passive mechanical properties of whole human muscles have rarely been directly measured and clarified. This study aimed to clarify the stress-strain relationship of the hamstring muscles using a soft-embalmed Thiel cadaver. The long heads of the biceps femoris (BFlh), semimembranosus (SM), and semitendinosus (ST) muscles were dissected from eight cadavers. The proximal and distal hamstring tendons were affixed to the mechanical testing machine. Slack length was defined as the muscle length at the initial loading point detected upon the application of a tensile load. Muscle length was measured using a tape measure, and the anatomical cross-sectional area (ACSA) of the muscle was measured at the proximal and distal sites using B-mode ultrasonography. In the loading protocol, the muscle was elongated from its slack length to a maximum of 8% strain at an average rate of 0.83 L0/s, and the amount of displacement and tensile load were measured for each muscle. Further, the strain (%, displacement/slack muscle length) and stress (kPa, tensile load/ACSA) were calculated to evaluate the mechanical properties. Two-way repeated-measures analysis of variance (ANOVA) was used to compare stress changes with increasing muscle strain. A significant interaction between the muscle and strain factors was observed with respect to stress. Post-hoc tests revealed higher stresses in the BFlh and SM than in ST after 3% strain (P < 0.01). However, no significant differences were observed between the BFlh and SM groups. At 8% strain, the BFlh, SM, and ST exhibited stresses of 63.7 ± 12.1, 53.7 ± 23.2, and 21.0 ± 11.9 kPa, respectively. The results indicate that the stress changes associated with muscle strain differed among muscles. In particular, the stress applied to the three muscles at the same strain was found to be higher in the BFlh and SM. Thus, these findings suggest that increased mechanical stress during elongation may contribute to the frequent occurrence of muscle strain in BFlh and SM.

The interplay between the epithelial permeability barrier, cell migration and mitochondrial metabolism of growth factors and their inhibitors in a human endometrial carcinoma cell line.

It is known that there are abnormalities of tight junction functions, cell migration and mitochondrial metabolism in human endometriosis and endometrial carcinoma. In this study, we investigated the effects of growth factors and their inhibitors on the epithelial permeability barrier, cell migration and mitochondrial metabolism in 2D and 2.5D cultures of human endometrioid endometrial carcinoma Sawano cells. We also investigated the changes of bicellular and tricellular tight junction molecules and ciliogenesis induced by these inhibitors. The growth factors TGF-ß and EGF affected the epithelial permeability barrier, cell migration and expression of bicellular and tricellular tight junction molecules in 2D and 2.5D cultures of Sawano cells. EW-7197 (a TGF-ß receptor inhibitor), AG1478 (an EGFR inhibitor) and SP600125 (a JNK inhibitor) affected the epithelial permeability barrier, cell migration and mitochondrial metabolism and prevented the changes induced by TGF-ß and EGF in 2D and 2.5D cultures. EW-7197 and AG1478 induced ciliogenesis in 2.5D cultures. In conclusion, TGF-ß and EGF promoted the malignancy of endometrial cancer via interplay among the epithelial permeability barrier, cell migration and mitochondrial metabolism. EW-7197 and AG1478 may be useful as novel therapeutic treatments options for endometrial cancer.

Role of ORPs in sterol transport from plasma membrane to ER and lipid droplets in mammalian cells.

In this study, we investigated the mechanisms of sterol transport from the plasma membrane (PM) to the endoplasmic reticulum (ER) and lipid droplets (LDs) in HeLa cells. By overexpressing all mammalian oxysterol-binding protein-related proteins (ORPs), we found that especially ORP1S and ORP2 enhanced PM-to-LD sterol transport. This reflected the stimulation of transport from the PM to the ER, rather than from the ER to LDs. Double knockdown of ORP1S and ORP2 inhibited sterol transport from the PM to the ER and LDs, suggesting a physiological role for these ORPs in the process. A two phenylalanines in an acidic tract (FFAT) motif in ORPs that mediates interaction with VAMP-associated proteins (VAPs) in the ER was not necessary for the enhancement of sterol transport by ORPs. However, VAP-A and VAP-B silencing slowed down PM-to-LD sterol transport. This was accompanied by enhanced degradation of ORP2 and decreased levels of several FFAT motif-containing ORPs, suggesting a role for VAPs in sterol transport by stabilization of ORPs.

ORP10, a cholesterol binding protein associated with microtubules, regulates apolipoprotein B-100 secretion.

ORP10/OSBPL10 is a member of the oxysterol-binding protein family, and genetic variation in OSBPL10 is associated with dyslipidemias and peripheral artery disease. In this study we investigated the ligand binding properties of ORP10 in vitro as well as its localization and function in human HuH7 hepatocytes. The pleckstrin homology (PH) domain of ORP10 selectively interacts with phosphatidylinositol-4-phosphate, while the C-terminal ligand binding domain binds cholesterol and several acidic phospholipids. Full-length ORP10 decorates microtubules (MT), while the ORP10 N-terminal fragment (aa 1-318) localizes at Golgi membranes. Removal of the C-terminal aa 712-764 of ORP10 containing a predicted coiled-coil segment abolishes the MT association, but allows partial Golgi targeting. A PH domain-GFP fusion protein is distributed mainly in the cytosol and the plasma membrane, indicating that the Golgi affinity of ORP10 involves other determinants in addition to the PH domain. HuH7 cells expressing ORP10-specific shRNA display increased accumulation of apolipoprotein B-100 (apoB-100), but not of albumin, in culture medium, and contain reduced levels of intracellular apoB-100. Pulse-chase analysis of cellular [(35)S]apoB-100 demonstrates enhanced apoB-100 secretion by cells expressing ORP10-specific shRNA. The apoB-100 secretion phenotype is replicated in HepG2 cells transduced with the ORP10 shRNA lentiviruses. As a conclusion, the present study dissects the determinants of ORP10 association with MT and the Golgi complex and provides evidence for a specific role of this protein in ß-lipoprotein secretion by human hepatocytes.

Modified thoracoabdominal nerve block through perichondrial approach (M-TAPA): an anatomical study to evaluate the spread of dye after a simulated injection in soft embalmed Thiel cadavers.

BACKGROUND AND OBJECTIVES: There is still no consensus on the analgesic range and mechanisms of action of modified thoracoabdominal nerve block through perichondrial approach (M-TAPA). This cadaveric study aimed to determine the spread of an injectate following simulated M-TAPA. METHODS: Simulated M-TAPA injections (n=8) were administered on both sides of soft embalmed Thiel cadavers with 25 mL of a saline-soluble dye. Anatomic dissection was performed to document staining (deeply, faintly, or not stained) of the anterior cutaneous branches of the thoracoabdominal nerves and determine the extent of the injectate spread of the dye to the intercostal space in the thoracic cage following a simulated M-TAPA. RESULTS: The median (IQR) dermatome of the stained segmental nerve was T10 (T8-T11) and the median (IQR) number of stained segmental nerves was 3 (4-2). The T9, T10 and T11 segmental nerves were stained in 75%, 100% and 62.5% of simulated M-TAPA, respectively. Conversely, the T8 segmental nerve was stained in only 25% of simulated M-TAPA. No injectate spread of dye to the intercostal space in the thoracic cage was observed in eight simulated injections of M-TAPA. CONCLUSION: Our findings suggest that M-TAPA most likely involves the T9, T10 and T11 segmental nerves and that the local anesthetic may not spread to the intercostal space in the thoracic cage in M-TAPA. Further studies are required to confirm the precise mechanism of action and efficacy of M-TAPA in a large sample of human participants.

I-gel Plus acts as a superior conduit for fiberoptic intubation than standard i-gel.

The supraglottic airway (SGA) is widely used. I-gel Plus is a next-generation i-gel with some improvements, including facilitation of fiberoptic tracheal intubation (FOI). To compare the performance of i-gel Plus and standard i-gel as conduits for FOI, a Thiel-embalmed cadaveric study was conducted. Twenty-two anesthesiologists were enrolled as operators in Experiment 1. The i-gel Plus and standard i-gel were inserted into one cadaver, and the FOI was performed through each SGA. The primary outcome was time required for FOI. The secondary outcomes were the number of attempts and visual analog scale (VAS) score for difficulty in FOI. Moreover, fiberoptic views of the vocal cords in each SGA were assessed by an attending anesthesiologist using nine cadavers in Experiment 2. The percentage of glottic opening (POGO) score without fiberscope tip upward flexion and upward angle of the fiberscope tip to obtain a 100% POGO score were evaluated as secondary outcomes. The time for FOI through i-gel Plus was significantly shorter than that through standard i-gel (median (IQR), i-gel Plus: 30.3 (25.4-39.0) s, vs standard i-gel: 54.7 (29.6-135.0) s; median of differences, 24.4 s; adjusted 95% confidence interval, 3.0-105.7; adjusted P = 0.040). Although the number of attempts for successful FOI was not significantly different, the VAS score for difficulty in the i-gel Plus group was significantly lower (easier) than that in the standard i-gel group. Moreover, i-gel Plus required a significantly smaller upward angle of the fiberscope tip to obtain a 100% POGO score. FOI can be performed more easily using i-gel Plus than using standard i-gel because of the improved fiberoptic visibility of vocal cords.

Effects of superficial tissue and intermuscular connections on rectus femoris muscle shear modulus heterogeneity.

INTRODUCTION: Intramuscular heterogeneity exists in the shear modulus of the rectus femoris (RF) muscle. However, the underlying heterogeneity mechanisms are not entirely understood. Previous research has reported that detachment of superficial tissues reduces the shear modulus by 50%. The aim of this study was to examine the effects of the skin, deep fascia, and intermuscular connections on the shear modulus of the RF at multiple sites. MATERIALS AND METHODS: Eleven donors were fixed using the Thiel method. Measurements were performed at 0°, 60°, and 120° knee flexion in a neutral hip position. Tissue processing was performed under four conditions: superficial tissue (CONT), skin off (SKIN), deep fascia detachment (FASC), and intermuscular connections detachment (ALL). The shear modulus at the proximal, central, and distal regions were measured using ultrasound shear wave elastography. The study was approved by the Sapporo Medical University Ethical Committee. RESULTS: Three-way ANOVA revealed no significant interaction between treatment, site, and angle (P = 0.156), treatment and angle (P = 0.067), or site and angle (P = 0.441). There was a significant effect of treatment (P < 0.001), site (P = 0.010), and angle (P < 0.001) and interaction between treatment and site (P < 0.001). The proximal shear modulus was greater than the central for CONT. There were no significant differences between the measurement sites for SKIN. The distal shear modulus was greater than the proximal for FASC. The distal shear modulus was also greater than the proximal and central for ALL. CONCLUSIONS: Intramuscular regional differences that influence superficial tissue and intermuscular connections of RF elasticity heterogeneity were observed.

Relationship between shear elastic modulus and passive muscle force in human hamstring muscles using a Thiel soft-embalmed cadaver.

PURPOSE: Assessing muscle flexibility and architecture is important for hamstring strain injury (HSI) prevention. We investigated the relationship between shear modulus and passive force in hamstring muscles at different sites and the effect of muscle architecture on the slope of the shear modulus-passive force using shear wave elastography (SWE). METHODS: The biceps femoris long head (BFlh), semitendinosus (ST), and semimembranosus (SM) muscles were dissected from nine Thiel-embalmed cadavers and fixed to a custom-made mechanical testing machine. Calibrated weights (0-1800 g) were applied gradually in 150-g increments. The shear modulus and anatomical cross-sectional area (ACSA) were measured at proximal, central, and distal points using SWE. The muscle mass and length were measured before the loading test. The shear modulus-passive load relationship of each tested muscle region was analyzed by fitting a least-squares regression line. The increase in shear modulus slope per unit load was calculated and compared between the muscles before and after normalization by the muscle mass, length, and ACSA. RESULTS: The shear modulus and passive force for all hamstring muscles in each region showed a statistically significant linear correlation. Furthermore, the increase in shear modulus slope was greater for BFlh and ST than for SM (P < 0.05), but after normalization by the muscle length and ACSA, there were no significant differences among the muscles. CONCLUSION: The local mechanical properties of individual hamstring muscles can be indirectly estimated using SWE, and the slope of increase in shear modulus reflects characteristics of the muscle architecture.

Oleic acid-bound FABP7 drives glioma cell proliferation through regulation of nuclear lipid droplet formation.

Fatty acid-binding protein 7 (FABP7), one of the fatty acid (FA) chaperones involved in the modulation of intracellular FA metabolism, is highly expressed in glioblastoma, and its expression is associated with decreased patients' prognosis. Previously, we demonstrated that FABP7 requires its binding partner to exert its function and that a mutation in the FA-binding site of FABP7 affects tumour biology. Here, we explored the role of FA ligand binding for FABP7 function in tumour proliferation and examined the mechanism of FABP7 and ligand interaction in tumour biology. We discovered that among several FA treatment, oleic acid (OA) boosted cell proliferation of FABP7-expressing cells. In turn, OA increased FABP7 nuclear localization, and the accumulation of FABP7-OA complex in the nucleus induced the formation of nuclear lipid droplet (nLD), as well as an increase in colocalization of nLD with promyelocytic leukaemia (PML) nuclear bodies. Furthermore, OA increased mRNA levels of proliferation-related genes in FABP7-expressing cells through histone acetylation. Interestingly, these OA-boosted functions were abrogated in FABP7-knockout cells and mutant FABP7-overexpressing cells. Thus, our findings suggest that FABP7-OA intracellular interaction may modulate nLD formation and the epigenetic status thereby enhancing transcription of proliferation-regulating genes, ultimately driving tumour cell proliferation.

Distinct features of two lipid droplets types in cell nuclei from patients with liver diseases.

Lipid droplets (LDs) have been observed in the nuclei of hepatocytes; however, their significance in liver disease remains unresolved. Our purpose was to explore the pathophysiological features of intranuclear LDs in liver diseases. We included 80 patients who underwent liver biopsies; the specimens were dissected and fixed for electron microscopy analysis. Depending on the presence of adjacent cytoplasmic invagination of the nuclear membrane, LDs in the nuclei were classified into two types: nucleoplasmic LDs (nLDs) and cytoplasmic LD invagination with nucleoplasmic reticulum (cLDs in NR). nLDs were found in 69% liver samples and cLDs in NR were found in 32%; no correlation was observed between the frequencies of the two LD types. nLDs were frequently found in hepatocytes of patients with nonalcoholic steatohepatitis, whereas cLDs in NR were absent from the livers of such patients. Further, cLDs in NR were often found in hepatocytes of patients with lower plasma cholesterol level. This indicates that nLDs do not directly reflect cytoplasmic lipid accumulation and that formation of cLDs in NR is inversely correlated to the secretion of very low-density lipoproteins. Positive correlations were found between the frequencies of nLDs and endoplasmic reticulum (ER) luminal expansion, suggesting that nLDs are formed in the nucleus upon ER stress. This study unveiled the presence of two distinct nuclear LDs in various liver diseases.

Novel cell-based system to assay cell-cell fusion during myotube formation.

A live assay tool has been established to uncover the precise molecular mechanisms underlying complex cell fusion events in myoblasts. The novel cell-based assay, HiMy (HiBiT-based myoblast fusion), utilizes a recently developed split-luciferase technology. The assay successfully detected cell fusion in differentiating C2C12 myoblast cultures. This allowed us to measure mixing of the cytoplasm, which occurred several hours after the initiation of C2C12 differentiation. Unlike what was reported earlier, the fusion was detected a few hours after the initiation of differentiation. Thus, this assay is sensitive enough to monitor fusion events before they become detectable using conventional methods. Furthermore, a panel of laboratory compounds, including a variety of inhibitors of cellular enzymes or activities, were assayed using the HiMy assay. Lovastatin, a cholesterol biogenesis inhibitor, decreased HiMy activity by approximately 50%. In contrast, mevalonolactone, a precursor for cholesterol synthesis, increased fusion activity. These results confirmed the previous finding that the amount of cellular cholesterol positively correlates with the rate of myoblast fusion during myogenesis. These results indicate that the novel cell fusion assay is a quick, accurate, and robust method to monitor intercellular fusion events.