肌肉發育起源於胚胎前驅細胞，隨著其肌細胞增長、分化，最終肌細胞在融合作用下，形成具有功能的多核肌小管，接著形成肌肉組織。對於肌肉失養症或肌肉萎縮症而言，肌肉組織的修復高度倚賴肌原母細胞的分化與融合以形成肌小管，儘管我們對於肌肉轉錄調控的機制已有諸多了解，然而如何控制肌肉細胞間的融合作用則有待進一步釐清。在肌細胞融合前，我們發現有顯著一部分肌原母細胞表面，動態地突出「泡狀結構」，不同於一般在細胞膜上發現的小泡，百分之七十五的初代細胞培養中也會有這些泡狀結構出現，並且在四小時內，這些初代細胞會與周圍的細胞行融合作用，我們便假設這些泡狀結構對於細胞融合是必要的，並且其為細胞融合處建立基礎，因此，我們檢視這些泡狀結構的動態表現以及參與其中的組成分子，泡狀結構從原始細胞膜突出至縮回原處的平均時間約七十秒，並且在肌肉分化過程中，該結構會持續約一小時，研究資料顯示該結構中富含有肌肉黏著分子(M-cadherin)、β-catenin、肌動蛋白 (actin) 以及磷脂質，其中包含磷酯肌醇二磷酸 (PIP2)、磷酯肌醇三磷酸 (PIP3) 與磷脂絲胺酸 (PS)，這些分子對於肌肉細胞融合都是十分重要，抑制肌動蛋白、肌球蛋白 (myosin II) 與增加細胞質中的鈣離子含量，會中斷泡狀結構的出現，其中伴隨著磷脂質耗竭。除此之外，我們首次展示在肌肉細胞融合中，磷脂絲胺酸會在細胞內膜積聚，並且主導肌細胞融合的發生。總結以上，在肌肉發育過程中，我們的實驗結果發現並描述在肌肉細胞表面上的泡狀結構動態，並且其中包含肌肉黏著分子、肌動蛋白與磷脂質參
與在肌肉細胞融合之過程。

Myogenesis begins with specification of embryonic progenitors, followed by proliferation and differentiation of myoblasts, ultimately fusion to form functional multi-nucleated myotubes and then muscles. For muscular dystrophy/atrophy, recovery of muscle tissues highly depends on the differentiation and fusion of myoblasts to generate myofibers. The transcriptional regulation of myogenic program is well-studied; however, the control of fusion events remains to be elucidated. Prior to myoblast fusion, we observed significant numbers of dynamic “bubble-like” structures protruding from the surface of myoblasts, distinct from the cellular blebbing. More than 75% of the primary myoblasts generated bubble-like structures, and then they actively fused to other surrounding myoblasts mainly within 4 hours. We hypothesized that these bubble-like structures are required for fusion and may set the stage for fusion sites. We thus examined the dynamics and components of these bubble-like structures. The average turnover period of the individual bubble appeared and disappeared from the original membrane range was estimated to be around 70 seconds. The emergence of bubble-like structures would last for about 1 hour during myogenesis. Our data suggest that these bubble-like structures were enriched with M-cadherin, β-catenin, actin as well as the phospholipids, including phosphatidylinositol-(4,5)-bisphosphate (PIP2), phosphatidylinositol-(3,4,5)-trisphosphate (PIP3) and phosphatidylserine (PS), which are critical for the myoblasts fusion. Inhibition of actin, myosin II as well as enhancement of calcium influx would interrupt the formation of bubble-like structures with the depletion of phospholipids from the plasma membrane. Moreover, for the first time, we discovered that the accumulation of PS on the inner leaflet of plasma membrane was required to initiate myoblast fusion. In summary, our results so far characterized the dynamics of the bubble-like structures at the surface of myoblasts and suggest the involvement of M-cadherin, actin and phospholipids in the fusion process during myogenesis.

Contents (1)
Abstract (4)
摘要 (7)
Acknowledgements (8)
Introduction (10)
Myogenesis (10)
Rhabdomyosarcoma (13)
Dynamics of Cell Plasma Membrane (15)
Distribution and Functions of Phospholipids in Cell Membrane (18)
Material and methods (22)
Reagents (22)
Cell culture (22)
Plasmids, transfection and viral infection (24)
Fluorescence microscopy and time-lapse imaging (25)
Statistical analysis (26)
Results (27)
Bubble-like structures during myogenesis (27)
The appearance of bubble-like structures is distinct from blebbing-mediated migration (28)
Characteristics of bubble-like structures (30)
Actin – membrane interactions during the formation of bubble-like structures (31)
PIP2, PIP3 and PS are enriched in the bubble-like structures during myogenesis (36)
Manipulation of PIP2, PIP3 and PS within bubble-like structures (39)
PS is enriched at the fusion site (40)
Bubble-like structures were also observed on rhabdomyosarcoma cell membrane. (43)
Discussion (45)
Figures (49)
Figure 1. Bubble-like structures formed during myogenesis. (50)
Figure 2. Orientation of myoblast fusion. (51)
Figure 3. The appearance of bubble-like structures is distinct from blebbing-mediated migration in differentiating myoblasts. (53)
Figure 4. Characteristics of bubble-like structure dynamics. (55)
Figure 5. Bubble-like structures formed with the enrichment of M-cadherin and β-catenin on C2C12 cell plasma membrane. (56)
Figure 6. Actin cytoskeleton interacted with plasma membrane during the formation of bubble-like structures in C2C12 cells. (57)
Figure 7. Treatment of actin inhibitors increased the turnover time of bubble-like structures. (58)
Figure 8. Whole cell analysis processed with ADAPT. (60)
Figure 9. Effects of myosin II inhibitor on actin and PIP2 during bubble-like structure formation and fusion. (61)
Figure 10. Distribution of PIP2, PIP3 and PS during myogenesis. (63)
Figure 11. PIP2, PIP3 and PS are enriched within different microdomains of bubble-like structures. (65)
Figure 12. Detection of PS via Annexin V staining. (66)
Figure 13. Detection of PS with the treatment of TopFluor-PS. (68)
Figure 14. Calcium-induced redistribution of F-actin, PIP2, PIP3 and PS. (70)
Figure 15. Effects of calcium influx during bubble-like structure formation and fusion. (72)
Figure 16. PS is enriched at the fusion contact site. (73)
Figure 17. Bubble-like structures were also observed on rhabdomyosarcoma cell membrane. (75)
Figure 18. Membrane remodeling and redistribution of PIP2, PIP3 and PS during myogenesis. (76)
Tables (77)
Table 1. List of antibodies dilution for immunofluorescence staining (77)
Table 2. List of primers for cloning (77)
Table 3. Comparison of myoblast bubbling and blebbing (78)
Reference (79)

1. Messina, G., and Cossu, G. (2009) The origin of embryonic and fetal myoblasts: a role of Pax3 and Pax7. Genes & development 23, 902-905
2. Tajbakhsh, S. (2009) Skeletal muscle stem cells in developmental versus regenerative myogenesis. Journal of internal medicine 266, 372-389
3. Buckingham, M. (2006) Myogenic progenitor cells and skeletal myogenesis in vertebrates. Current opinion in genetics & development 16, 525-532
4. Buckingham, M., and Rigby, P. W. (2014) Gene regulatory networks and transcriptional mechanisms that control myogenesis. Developmental cell 28, 225-238
5. Shi, X., and Garry, D. J. (2006) Muscle stem cells in development, regeneration, and disease. Genes & development 20, 1692-1708
6. Haslett, J. N., Sanoudou, D., Kho, A. T., Bennett, R. R., Greenberg, S. A., Kohane, I. S., Beggs, A. H., and Kunkel, L. M. (2002) Gene expression comparison of biopsies from Duchenne muscular dystrophy (DMD) and normal skeletal muscle. Proceedings of the National Academy of Sciences of the United States of America 99, 15000-15005
7. McGreevy, J. W., Hakim, C. H., McIntosh, M. A., and Duan, D. (2015) Animal models of Duchenne muscular dystrophy: from basic mechanisms to gene therapy. Disease models & mechanisms 8, 195-213
8. Knight, J. D., and Kothary, R. (2011) The myogenic kinome: protein kinases critical to mammalian skeletal myogenesis. Skeletal muscle 1, 29
9. Bentzinger, C. F., Wang, Y. X., and Rudnicki, M. A. (2012) Building muscle: molecular regulation of myogenesis. Cold Spring Harbor perspectives in biology 4
10. Moncaut, N., Rigby, P. W., and Carvajal, J. J. (2013) Dial M(RF) for myogenesis. The FEBS journal 280, 3980-3990
11. Kim, J. H., Jin, P., Duan, R., and Chen, E. H. (2015) Mechanisms of myoblast fusion during muscle development. Current opinion in genetics & development 32, 162-170
12. Kim, J. H., Ren, Y., Ng, W. P., Li, S., Son, S., Kee, Y. S., Zhang, S., Zhang, G., Fletcher, D. A., Robinson, D. N., and Chen, E. H. (2015) Mechanical tension drives cell membrane fusion. Developmental cell 32, 561-573
13. Strunkelnberg, M., Bonengel, B., Moda, L. M., Hertenstein, A., de Couet, H. G., Ramos, R. G., and Fischbach, K. F. (2001) rst and its paralogue kirre act redundantly during embryonic muscle development in Drosophila. Development 128, 4229-4239
14. Ruiz-Gomez, M., Coutts, N., Price, A., Taylor, M. V., and Bate, M. (2000) Drosophila dumbfounded: a myoblast attractant essential for fusion. Cell 102, 189-198
15. Bour, B. A., Chakravarti, M., West, J. M., and Abmayr, S. M. (2000) Drosophila SNS, a member of the immunoglobulin superfamily that is essential for myoblast fusion. Genes & development 14, 1498-1511
16. Artero, R. D., Castanon, I., and Baylies, M. K. (2001) The immunoglobulin-like protein Hibris functions as a dose-dependent regulator of myoblast fusion and is differentially controlled by Ras and Notch signaling. Development 128, 4251-4264
17. Beckett, K., and Baylies, M. K. (2007) 3D analysis of founder cell and fusion competent myoblast arrangements outlines a new model of myoblast fusion. Developmental biology 309, 113-125
18. Bogdan, S., and Klambt, C. (2003) Kette regulates actin dynamics and genetically interacts with Wave and Wasp. Development 130, 4427-4437
19. Luo, L., Liao, Y. J., Jan, L. Y., and Jan, Y. N. (1994) Distinct morphogenetic functions of similar small GTPases: Drosophila Drac1 is involved in axonal outgrowth and myoblast fusion. Genes & development 8, 1787-1802
20. Cifuentes-Diaz, C., Nicolet, M., Alameddine, H., Goudou, D., Dehaupas, M., Rieger, F., and Mege, R. M. (1995) M-cadherin localization in developing adult and regenerating mouse skeletal muscle: possible involvement in secondary myogenesis. Mechanisms of development 50, 85-97
21. Volonte, D., Peoples, A. J., and Galbiati, F. (2003) Modulation of myoblast fusion by caveolin-3 in dystrophic skeletal muscle cells: implications for Duchenne muscular dystrophy and limb-girdle muscular dystrophy-1C. Molecular biology of the cell 14, 4075-4088
22. Posey, A. D., Jr., Demonbreun, A., and McNally, E. M. (2011) Ferlin proteins in myoblast fusion and muscle growth. Current topics in developmental biology 96, 203-230
23. Sohn, R. L., Huang, P., Kawahara, G., Mitchell, M., Guyon, J., Kalluri, R., Kunkel, L. M., and Gussoni, E. (2009) A role for nephrin, a renal protein, in vertebrate skeletal muscle cell fusion. Proceedings of the National Academy of Sciences of the United States of America 106, 9274-9279
24. Millay, D. P., O'Rourke, J. R., Sutherland, L. B., Bezprozvannaya, S., Shelton, J. M., Bassel-Duby, R., and Olson, E. N. (2013) Myomaker is a membrane activator of myoblast fusion and muscle formation. Nature 499, 301-305
25. Bi, P., Ramirez-Martinez, A., Li, H., Cannavino, J., McAnally, J. R., Shelton, J. M., Sanchez-Ortiz, E., Bassel-Duby, R., and Olson, E. N. (2017) Control of muscle formation by the fusogenic micropeptide myomixer. Science 356, 323-327
26. Zhang, Q., Vashisht, A. A., O'Rourke, J., Corbel, S. Y., Moran, R., Romero, A., Miraglia, L., Zhang, J., Durrant, E., Schmedt, C., Sampath, S. C., and Sampath, S. C. (2017) The microprotein Minion controls cell fusion and muscle formation. Nature communications 8, 15664
27. Aguilar, P. S., Baylies, M. K., Fleissner, A., Helming, L., Inoue, N., Podbilewicz, B., Wang, H., and Wong, M. (2013) Genetic basis of cell-cell fusion mechanisms. Trends in genetics : TIG 29, 427-437
28. El Demellawy, D., McGowan-Jordan, J., de Nanassy, J., Chernetsova, E., and Nasr, A. (2017) Update on molecular findings in rhabdomyosarcoma. Pathology 49, 238-246
29. Tiffin, N., Williams, R. D., Shipley, J., and Pritchard-Jones, K. (2003) PAX7 expression in embryonal rhabdomyosarcoma suggests an origin in muscle satellite cells. British journal of cancer 89, 327-332
30. Fletcher, C. D., Gustafson, P., Rydholm, A., Willen, H., and Akerman, M. (2001) Clinicopathologic re-evaluation of 100 malignant fibrous histiocytomas: prognostic relevance of subclassification. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 19, 3045-3050
31. Zambo, I., and Vesely, K. (2014) [WHO classification of tumours of soft tissue and bone 2013: the main changes compared to the 3rd edition]. Ceskoslovenska patologie 50, 64-70
32. Ognjanovic, S., Linabery, A. M., Charbonneau, B., and Ross, J. A. (2009) Trends in childhood rhabdomyosarcoma incidence and survival in the United States, 1975-2005. Cancer 115, 4218-4226
33. Marshall, A. D., and Grosveld, G. C. (2012) Alveolar rhabdomyosarcoma - The molecular drivers of PAX3/7-FOXO1-induced tumorigenesis. Skeletal muscle 2, 25
34. Missiaglia, E., Williamson, D., Chisholm, J., Wirapati, P., Pierron, G., Petel, F., Concordet, J. P., Thway, K., Oberlin, O., Pritchard-Jones, K., Delattre, O., Delorenzi, M., and Shipley, J. (2012) PAX3/FOXO1 fusion gene status is the key prognostic molecular marker in rhabdomyosarcoma and significantly improves current risk stratification. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 30, 1670-1677
35. Monti, E., and Fanzani, A. (2016) Uncovering metabolism in rhabdomyosarcoma. Cell cycle 15, 184-195
36. Chhabra, E. S., and Higgs, H. N. (2007) The many faces of actin: matching assembly factors with cellular structures. Nature cell biology 9, 1110-1121
37. Burnette, D. T., Manley, S., Sengupta, P., Sougrat, R., Davidson, M. W., Kachar, B., and Lippincott-Schwartz, J. (2011) A role for actin arcs in the leading-edge advance of migrating cells. Nature cell biology 13, 371-381
38. Giannone, G., Dubin-Thaler, B. J., Rossier, O., Cai, Y., Chaga, O., Jiang, G., Beaver, W., Dobereiner, H. G., Freund, Y., Borisy, G., and Sheetz, M. P. (2007) Lamellipodial actin mechanically links myosin activity with adhesion-site formation. Cell 128, 561-575
39. Charras, G. T. (2008) A short history of blebbing. Journal of microscopy 231, 466-478
40. Croft, D. R., Coleman, M. L., Li, S., Robertson, D., Sullivan, T., Stewart, C. L., and Olson, M. F. (2005) Actin-myosin-based contraction is responsible for apoptotic nuclear disintegration. The Journal of cell biology 168, 245-255
41. Charras, G., and Paluch, E. (2008) Blebs lead the way: how to migrate without lamellipodia. Nature reviews. Molecular cell biology 9, 730-736
42. Paluch, E. K., and Raz, E. (2013) The role and regulation of blebs in cell migration. Current opinion in cell biology 25, 582-590
43. Paluch, E., van der Gucht, J., and Sykes, C. (2006) Cracking up: symmetry breaking in cellular systems. The Journal of cell biology 175, 687-692
44. Charras, G. T., Hu, C. K., Coughlin, M., and Mitchison, T. J. (2006) Reassembly of contractile actin cortex in cell blebs. The Journal of cell biology 175, 477-490
45. Totsukawa, G., Wu, Y., Sasaki, Y., Hartshorne, D. J., Yamakita, Y., Yamashiro, S., and Matsumura, F. (2004) Distinct roles of MLCK and ROCK in the regulation of membrane protrusions and focal adhesion dynamics during cell migration of fibroblasts. The Journal of cell biology 164, 427-439
46. Rossy, J., Gutjahr, M. C., Blaser, N., Schlicht, D., and Niggli, V. (2007) Ezrin/moesin in motile Walker 256 carcinosarcoma cells: signal-dependent relocalization and role in migration. Experimental cell research 313, 1106-1120
47. McClatchey, A. I. (2014) ERM proteins at a glance. Journal of cell science 127, 3199-3204
48. Fehon, R. G., McClatchey, A. I., and Bretscher, A. (2010) Organizing the cell cortex: the role of ERM proteins. Nature reviews. Molecular cell biology 11, 276-287
49. Tyson, R. A., Zatulovskiy, E., Kay, R. R., and Bretschneider, T. (2014) How blebs and pseudopods cooperate during chemotaxis. Proceedings of the National Academy of Sciences of the United States of America 111, 11703-11708
50. Friedl, P., and Wolf, K. (2003) Tumour-cell invasion and migration: diversity and escape mechanisms. Nature reviews. Cancer 3, 362-374
51. Otto, A., Collins-Hooper, H., Patel, A., Dash, P. R., and Patel, K. (2011) Adult skeletal muscle stem cell migration is mediated by a blebbing/amoeboid mechanism. Rejuvenation research 14, 249-260
52. Krahn, M. P., and Wodarz, A. (2012) Phosphoinositide lipids and cell polarity: linking the plasma membrane to the cytocortex. Essays in biochemistry 53, 15-27
53. Vance, J. E. (2008) Phosphatidylserine and phosphatidylethanolamine in mammalian cells: two metabolically related aminophospholipids. Journal of lipid research 49, 1377-1387
54. Murphy, E. J., Anderson, D. K., and Horrocks, L. A. (1993) Phospholipid and phospholipid fatty acid composition of mixed murine spinal cord neuronal cultures. Journal of neuroscience research 34, 472-477
55. Leventis, P. A., and Grinstein, S. (2010) The distribution and function of phosphatidylserine in cellular membranes. Annual review of biophysics 39, 407-427
56. McMahon, H. T., and Boucrot, E. (2015) Membrane curvature at a glance. Journal of cell science 128, 1065-1070
57. Czech, M. P. (2000) PIP2 and PIP3: complex roles at the cell surface. Cell 100, 603-606
58. Raucher, D., Stauffer, T., Chen, W., Shen, K., Guo, S., York, J. D., Sheetz, M. P., and Meyer, T. (2000) Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion. Cell 100, 221-228
59. Wennstrom, S., Hawkins, P., Cooke, F., Hara, K., Yonezawa, K., Kasuga, M., Jackson, T., Claesson-Welsh, L., and Stephens, L. (1994) Activation of phosphoinositide 3-kinase is required for PDGF-stimulated membrane ruffling. Current biology : CB 4, 385-393
60. Funamoto, S., Milan, K., Meili, R., and Firtel, R. A. (2001) Role of phosphatidylinositol 3' kinase and a downstream pleckstrin homology domain-containing protein in controlling chemotaxis in dictyostelium. The Journal of cell biology 153, 795-810
61. Insall, R. H., and Weiner, O. D. (2001) PIP3, PIP2, and cell movement--similar messages, different meanings? Developmental cell 1, 743-747
62. Kay, J. G., Koivusalo, M., Ma, X., Wohland, T., and Grinstein, S. (2012) Phosphatidylserine dynamics in cellular membranes. Molecular biology of the cell 23, 2198-2212
63. Helming, L., Winter, J., and Gordon, S. (2009) The scavenger receptor CD36 plays a role in cytokine-induced macrophage fusion. Journal of cell science 122, 453-459
64. Marty, N. J., Holman, C. L., Abdullah, N., and Johnson, C. P. (2013) The C2 domains of otoferlin, dysferlin, and myoferlin alter the packing of lipid bilayers. Biochemistry 52, 5585-5592
65. Blondelle, J., Ohno, Y., Gache, V., Guyot, S., Storck, S., Blanchard-Gutton, N., Barthelemy, I., Walmsley, G., Rahier, A., Gadin, S., Maurer, M., Guillaud, L., Prola, A., Ferry, A., Aubin-Houzelstein, G., Demarquoy, J., Relaix, F., Piercy, R. J., Blot, S., Kihara, A., Tiret, L., and Pilot-Storck, F. (2015) HACD1, a regulator of membrane composition and fluidity, promotes myoblast fusion and skeletal muscle growth. Journal of molecular cell biology 7, 429-440
66. Bach, A. S., Enjalbert, S., Comunale, F., Bodin, S., Vitale, N., Charrasse, S., and Gauthier-Rouviere, C. (2010) ADP-ribosylation factor 6 regulates mammalian myoblast fusion through phospholipase D1 and phosphatidylinositol 4,5-bisphosphate signaling pathways. Molecular biology of the cell 21, 2412-2424
67. Bothe, I., Deng, S., and Baylies, M. (2014) PI(4,5)P2 regulates myoblast fusion through Arp2/3 regulator localization at the fusion site. Development 141, 2289-2301
68. Oikawa, T., Oyama, M., Kozuka-Hata, H., Uehara, S., Udagawa, N., Saya, H., and Matsuo, K. (2012) Tks5-dependent formation of circumferential podosomes/invadopodia mediates cell-cell fusion. The Journal of cell biology 197, 553-568
69. Balagopalan, L., Chen, M. H., Geisbrecht, E. R., and Abmayr, S. M. (2006) The CDM superfamily protein MBC directs myoblast fusion through a mechanism that requires phosphatidylinositol 3,4,5-triphosphate binding but is independent of direct interaction with DCrk. Molecular and cellular biology 26, 9442-9455
70. Ijuin, T., and Takenawa, T. (2012) Role of phosphatidylinositol 3,4,5-trisphosphate (PIP3) 5-phosphatase skeletal muscle- and kidney-enriched inositol polyphosphate phosphatase (SKIP) in myoblast differentiation. The Journal of biological chemistry 287, 31330-31341
71. Jeong, J., and Conboy, I. M. (2011) Phosphatidylserine directly and positively regulates fusion of myoblasts into myotubes. Biochemical and biophysical research communications 414, 9-13
72. Hochreiter-Hufford, A. E., Lee, C. S., Kinchen, J. M., Sokolowski, J. D., Arandjelovic, S., Call, J. A., Klibanov, A. L., Yan, Z., Mandell, J. W., and Ravichandran, K. S. (2013) Phosphatidylserine receptor BAI1 and apoptotic cells as new promoters of myoblast fusion. Nature 497, 263-267
73. van den Eijnde, S. M., van den Hoff, M. J., Reutelingsperger, C. P., van Heerde, W. L., Henfling, M. E., Vermeij-Keers, C., Schutte, B., Borgers, M., and Ramaekers, F. C. (2001) Transient expression of phosphatidylserine at cell-cell contact areas is required for myotube formation. Journal of cell science 114, 3631-3642
74. Hirama, T., Das, R., Yang, Y., Ferguson, C., Won, A., Yip, C. M., Kay, J. G., Grinstein, S., Parton, R. G., and Fairn, G. D. (2017) Phosphatidylserine dictates the assembly and dynamics of caveolae in the plasma membrane. The Journal of biological chemistry
75. Bondesen, B. A., Mills, S. T., Kegley, K. M., and Pavlath, G. K. (2004) The COX-2 pathway is essential during early stages of skeletal muscle regeneration. American journal of physiology. Cell physiology 287, C475-483
76. Barry, D. J., Durkin, C. H., Abella, J. V., and Way, M. (2015) Open source software for quantification of cell migration, protrusions, and fluorescence intensities. The Journal of cell biology 209, 163-180
77. Charrasse, S., Comunale, F., Fortier, M., Portales-Casamar, E., Debant, A., and Gauthier-Rouviere, C. (2007) M-cadherin activates Rac1 GTPase through the Rho-GEF trio during myoblast fusion. Molecular biology of the cell 18, 1734-1743
78. Hsiao, S. P., and Chen, S. L. (2010) Myogenic regulatory factors regulate M-cadherin expression by targeting its proximal promoter elements. The Biochemical journal 428, 223-233
79. Yaffe, D., and Saxel, O. (1977) Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature 270, 725-727
80. Sahai, E., and Marshall, C. J. (2003) Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nature cell biology 5, 711-719
81. Jones, A. E., Price, F. D., Le Grand, F., Soleimani, V. D., Dick, S. A., Megeney, L. A., and Rudnicki, M. A. (2015) Wnt/beta-catenin controls follistatin signalling to regulate satellite cell myogenic potential. Skeletal muscle 5, 14
82. Sens, K. L., Zhang, S., Jin, P., Duan, R., Zhang, G., Luo, F., Parachini, L., and Chen, E. H. (2010) An invasive podosome-like structure promotes fusion pore formation during myoblast fusion. The Journal of cell biology 191, 1013-1027
83. Stadler, B., Blattler, T. M., and Franco-Obregon, A. (2010) Time-lapse imaging of in vitro myogenesis using atomic force microscopy. Journal of microscopy 237, 63-69
84. Riedl, J., Crevenna, A. H., Kessenbrock, K., Yu, J. H., Neukirchen, D., Bista, M., Bradke, F., Jenne, D., Holak, T. A., Werb, Z., Sixt, M., and Wedlich-Soldner, R. (2008) Lifeact: a versatile marker to visualize F-actin. Nature methods 5, 605-607
85. Morton, W. M., Ayscough, K. R., and McLaughlin, P. J. (2000) Latrunculin alters the actin-monomer subunit interface to prevent polymerization. Nature cell biology 2, 376-378
86. Cooper, J. A. (1987) Effects of cytochalasin and phalloidin on actin. The Journal of cell biology 105, 1473-1478
87. Constantin, B., Imbert, N., Besse, C., Cognard, C., and Raymond, G. (1995) Cultured rat skeletal muscle cells treated with cytochalasin exhibit normal dystrophin expression and intracellular free calcium control. Biology of the cell 85, 125-135
88. Dhawan, J., and Helfman, D. M. (2004) Modulation of acto-myosin contractility in skeletal muscle myoblasts uncouples growth arrest from differentiation. Journal of cell science 117, 3735-3748
89. Nowak, S. J., Nahirney, P. C., Hadjantonakis, A. K., and Baylies, M. K. (2009) Nap1-mediated actin remodeling is essential for mammalian myoblast fusion. Journal of cell science 122, 3282-3293
90. O'Connell, C. B., Tyska, M. J., and Mooseker, M. S. (2007) Myosin at work: motor adaptations for a variety of cellular functions. Biochimica et biophysica acta 1773, 615-630
91. Yoshida, K., and Soldati, T. (2006) Dissection of amoeboid movement into two mechanically distinct modes. Journal of cell science 119, 3833-3844
92. Ivanov, A. I., Hopkins, A. M., Brown, G. T., Gerner-Smidt, K., Babbin, B. A., Parkos, C. A., and Nusrat, A. (2008) Myosin II regulates the shape of three-dimensional intestinal epithelial cysts. Journal of cell science 121, 1803-1814
93. Kovacs, M., Toth, J., Hetenyi, C., Malnasi-Csizmadia, A., and Sellers, J. R. (2004) Mechanism of blebbistatin inhibition of myosin II. The Journal of biological chemistry 279, 35557-35563
94. Yamashiro, S., Mizuno, H., Smith, M. B., Ryan, G. L., Kiuchi, T., Vavylonis, D., and Watanabe, N. (2014) New single-molecule speckle microscopy reveals modification of the retrograde actin flow by focal adhesions at nanometer scales. Molecular biology of the cell 25, 1010-1024
95. Holz, R. W., Hlubek, M. D., Sorensen, S. D., Fisher, S. K., Balla, T., Ozaki, S., Prestwich, G. D., Stuenkel, E. L., and Bittner, M. A. (2000) A pleckstrin homology domain specific for phosphatidylinositol 4, 5-bisphosphate (PtdIns-4,5-P2) and fused to green fluorescent protein identifies plasma membrane PtdIns-4,5-P2 as being important in exocytosis. The Journal of biological chemistry 275, 17878-17885
96. Lacalle, R. A., Peregil, R. M., Albar, J. P., Merino, E., Martinez, A. C., Merida, I., and Manes, S. (2007) Type I phosphatidylinositol 4-phosphate 5-kinase controls neutrophil polarity and directional movement. The Journal of cell biology 179, 1539-1553
97. Lokuta, M. A., Senetar, M. A., Bennin, D. A., Nuzzi, P. A., Chan, K. T., Ott, V. L., and Huttenlocher, A. (2007) Type Igamma PIP kinase is a novel uropod component that regulates rear retraction during neutrophil chemotaxis. Molecular biology of the cell 18, 5069-5080
98. Saarikangas, J., Zhao, H., and Lappalainen, P. (2010) Regulation of the actin cytoskeleton-plasma membrane interplay by phosphoinositides. Physiological reviews 90, 259-289
99. Nakanishi, K., Kakiguchi, K., Yonemura, S., Nakano, A., and Morishima, N. (2015) Transient Ca2+ depletion from the endoplasmic reticulum is critical for skeletal myoblast differentiation. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 29, 2137-2149
100. Varnai, P., and Balla, T. (1998) Visualization of phosphoinositides that bind pleckstrin homology domains: calcium- and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools. The Journal of cell biology 143, 501-510
101. Tapscott, S. J., Thayer, M. J., and Weintraub, H. (1993) Deficiency in rhabdomyosarcomas of a factor required for MyoD activity and myogenesis. Science 259, 1450-1453
102. Fackler, O. T., and Grosse, R. (2008) Cell motility through plasma membrane blebbing. The Journal of cell biology 181, 879-884
103. Welf, E. S., Driscoll, M. K., Dean, K. M., Schafer, C., Chu, J., Davidson, M. W., Lin, M. Z., Danuser, G., and Fiolka, R. (2016) Quantitative Multiscale Cell Imaging in Controlled 3D Microenvironments. Developmental cell 36, 462-475
104. Callahan, M. K., Williamson, P., and Schlegel, R. A. (2000) Surface expression of phosphatidylserine on macrophages is required for phagocytosis of apoptotic thymocytes. Cell death and differentiation 7, 645-653
105. Helming, L., and Gordon, S. (2009) Molecular mediators of macrophage fusion. Trends in cell biology 19, 514-522
106. Ramachandran, S., Kumar, P. B., and Laradji, M. (2008) Lipid flip-flop driven mechanical and morphological changes in model membranes. The Journal of chemical physics 129, 125104
107. Fang, M., Rivas, M. P., and Bankaitis, V. A. (1998) The contribution of lipids and lipid metabolism to cellular functions of the Golgi complex. Biochimica et biophysica acta 1404, 85-100
108. Rambold, A. S., Cohen, S., and Lippincott-Schwartz, J. (2015) Fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics. Developmental cell 32, 678-692
109. Sharom, F. J. (2011) Flipping and flopping--lipids on the move. IUBMB life 63, 736-746
110. Andersen, J. P., Vestergaard, A. L., Mikkelsen, S. A., Mogensen, L. S., Chalat, M., and Molday, R. S. (2016) P4-ATPases as Phospholipid Flippases-Structure, Function, and Enigmas. Frontiers in physiology 7, 275
111. Lin, Y. C., Niewiadomski, P., Lin, B., Nakamura, H., Phua, S. C., Jiao, J., Levchenko, A., Inoue, T., Rohatgi, R., and Inoue, T. (2013) Chemically inducible diffusion trap at cilia reveals molecular sieve-like barrier. Nature chemical biology 9, 437-443
112. Miyamoto, T., DeRose, R., Suarez, A., Ueno, T., Chen, M., Sun, T. P., Wolfgang, M. J., Mukherjee, C., Meyers, D. J., and Inoue, T. (2012) Rapid and orthogonal logic gating with a gibberellin-induced dimerization system. Nature chemical biology 8, 465-470
113. Rudge, S. A., and Wakelam, M. J. (2014) SnapShot: Lipid kinase and phosphatase reaction pathways. Cell 156, 376-376 e371
114. Teng, S., Stegner, D., Chen, Q., Hongu, T., Hasegawa, H., Chen, L., Kanaho, Y., Nieswandt, B., Frohman, M. A., and Huang, P. (2015) Phospholipase D1 facilitates second-phase myoblast fusion and skeletal muscle regeneration. Molecular biology of the cell 26, 506-517
115. Sekiya, T., Takenawa, T., and Nozawa, Y. (1984) Reorganization of membrane cholesterol during membrane fusion in myogenesis in vitro: a study using the filipin-cholesterol complex. Cell structure and function 9, 143-155
116. Nakanishi, M., Hirayama, E., and Kim, J. (2001) Characterisation of myogenic cell membrane: II. Dynamic changes in membrane lipids during the differentiation of mouse C2 myoblast cells. Cell biology international 25, 971-979
117. Nagata, Y., Kobayashi, H., Umeda, M., Ohta, N., Kawashima, S., Zammit, P. S., and Matsuda, R. (2006) Sphingomyelin levels in the plasma membrane correlate with the activation state of muscle satellite cells. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 54, 375-384
118. Nagata, Y., Partridge, T. A., Matsuda, R., and Zammit, P. S. (2006) Entry of muscle satellite cells into the cell cycle requires sphingolipid signaling. The Journal of cell biology 174, 245-253
119. Meacci, E., Nuti, F., Donati, C., Cencetti, F., Farnararo, M., and Bruni, P. (2008) Sphingosine kinase activity is required for myogenic differentiation of C2C12 myoblasts. Journal of cellular physiology 214, 210-220
120. Maekawa, M., and Fairn, G. D. (2015) Complementary probes reveal that phosphatidylserine is required for the proper transbilayer distribution of cholesterol. Journal of cell science 128, 1422-1433
121. Makino, A., Abe, M., Ishitsuka, R., Murate, M., Kishimoto, T., Sakai, S., Hullin-Matsuda, F., Shimada, Y., Inaba, T., Miyatake, H., Tanaka, H., Kurahashi, A., Pack, C. G., Kasai, R. S., Kubo, S., Schieber, N. L., Dohmae, N., Tochio, N., Hagiwara, K., Sasaki, Y., Aida, Y., Fujimori, F., Kigawa, T., Nishibori, K., Parton, R. G., Kusumi, A., Sako, Y., Anderluh, G., Yamashita, M., Kobayashi, T., Greimel, P., and Kobayashi, T. (2017) A novel sphingomyelin/cholesterol domain-specific probe reveals the dynamics of the membrane domains during virus release and in Niemann-Pick type C. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 31, 1301-1322
122. Li, H. G., Wang, Q., Li, H. M., Kumar, S., Parker, C., Slevin, M., and Kumar, P. (2007) PAX3 and PAX3-FKHR promote rhabdomyosarcoma cell survival through downregulation of PTEN. Cancer letters 253, 215-223
123. Zhu, B., Zhang, M., Williams, E. M., Keller, C., Mansoor, A., and Davie, J. K. (2016) TBX2 represses PTEN in rhabdomyosarcoma and skeletal muscle. Oncogene 35, 4212-4224