A novel interaction of CLN3 with nonmuscle myosin-IIB and defects in cell motility of Cln3−/− cells

Abstract

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 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 the migration defect in Cln3−/− results, in part, from the loss of the CLN3-myosin-IIB interaction.

CLN3 Deficient Cells Display Defects in the ARF1-Cdc42 Pathway and Actin-Dependent Events

“…Consistent with these phenotypes, CLN3 has been reported to associate directly or indirectly with the actin regulatory proteins myosin-IIb, fodrin, and hook1 [15], [19], [30]. Our data implicate the small GTPase Cdc42 as a common link.…”

The juvenile Batten disease protein, CLN3, and its role in regulating anterograde and retrograde post-Golgi trafficking

“…Luiro and colleagues reported that endogenous CLN3 in neurons is enriched in synaptosomal fractions isolated from nerve terminals [66], and alterations in neurotransmitter and receptor levels and in activation of receptors at the synapse in Cln3 genetic mouse models have been documented [67–69]. CLN3 function near the plasma membrane may involve interaction with components that regulate the actin cytoskeleton (β-fodrin and nonmuscle myosin-IIB) [70,71], with lipid rafts that are enriched in cholesterol and sphingolipids such as galactosylceramide [59,72], and/or with ion channels or their accessory proteins (Na + /K + ATPase, calsenilin/KChip3) (Figure 3) [70,73]. These interactions may mediate directed trafficking of membrane proteins and lipids, particularly sphingolipids and cholesterol from lipid raft microdomains, which may have immediate and downstream effects on cell survival and ion homeostasis [59,72–74].…”

Neuronal ceroid lipofuscinosis protein CLN3 interacts with motor proteins and modifies location of late endosomal compartments

“…An interaction between Btn1p and Sdo1p, Saccharomyces cerevisiae orthologues of CLN3 and Shwachmann-Bodian-Diamond syndrome protein (SBDS), respectively, was suggested to regulate yeast vacuolar homeostasis through a ribosome maturation pathway [22]. Recently, loss of the novel CLN3-myosin-IIB interaction was proposed to result in cell migration defects of Cln3-deficient mouse fibroblasts [23].…”