A novel interaction of CLN3 with nonmuscle myosin-IIB and defects in cell motility of Cln3(-/-) cells.
Juvenile neuronal ceroid lipofuscinosis (JNCL) is a pediatric lysosomal storage disorder characterized by accumulation of autofluorescent storage material and neurodegeneration, which result from mutations in CLN3. The function of CLN3, a lysosomal membrane protein, is currently unknown. We report that CLN3 interacts with cytoskeleton-associated nonmuscle myosin-IIB. Both CLN3 and myosin-IIB are ubiquitously expressed, yet mutations in either produce dramatic consequences in the CNS such as neurodegeneration in JNCL patients and Cln3(-/-) mouse models, or developmental deficiencies in Myh10(-/-) mice, respectively. A scratch assay revealed a migration defect associated with Cln3(-/-) cells. Inhibition of nonmuscle myosin-II with blebbistatin in WT cells resulted in a phenotype that mimics the Cln3(-/-) migration defect. Moreover, inhibiting lysosome function by treating cells with chloroquine exacerbated the migration defect in Cln3(-/-). Cln3(-/-) cells traversing a transwell filter under gradient trophic factor conditions displayed altered migration, further linking lysosomal function and cell migration. The myosin-IIB distribution in Cln3(-/-) cells is elongated, indicating a cytoskeleton defect caused by the loss of CLN3. In summary, cells lacking CLN3 have defects that suggest altered myosin-IIB activity, supporting a functional and physical interaction between CLN3 and myosin-IIB. We propose that the migration defect in Cln3(-/-) results, in part, from the loss of the CLN3-myosin-IIB interaction.
Getty
Amanda L
AL
Center for Neural Development and Disease, Aab Institute of Biomedical Sciences, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Benedict
Jared W
JW
Pearce
David A
DA
D000818:Animals; D054443:Cell Migration Assays; D002465:Cell Movement / Q000235:genetics*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000378:metabolism / Q000502:physiology; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D041681:NIH 3T3 Cells; D009472:Neuronal Ceroid-Lipofuscinoses / Q000235:genetics / Q000378:metabolism; D024501:Nonmuscle Myosin Type IIB / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000502:physiology
C102229:CLN3 protein, mouse; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; D024501:Nonmuscle Myosin Type IIB; C103815:nonmuscle myosin type IIB heavy chain; D018995:Myosin Heavy Chains