Plasma membrane localization of MLC1 regulates cellular morphology and motility

Junmo Hwang,Hyun-Ho Lim,Hung M Vu,Min-Sik Kim

doi:10.1186/s13041-019-0540-6

Abstract

BackgroundMegalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare form of infantile-onset leukodystrophy. The disorder is caused primarily by mutations of MLC1 that leads to a series of phenotypic outcomes including vacuolation of myelin and astrocytes, subcortical cysts, brain edema, and macrocephaly. Recent studies have indicated that functional interactions among MLC1, GlialCAM, and ClC-2 channels play key roles in the regulation of neuronal, glial and vascular homeostasis. However, the physiological role of MLC1 in cellular homeostatic communication remains poorly understood. In the present study, we investigated the cellular function of MLC1 and its effects on cell–cell interactions.MethodsMLC1-dependent cellular morphology and motility were analyzed by using confocal and live cell imaging technique. Biochemical approaches such as immunoblotting, co-immunoprecipitation, and surface biotinylation were conducted to support data.ResultsWe found that the altered MLC1 expression and localization led to a great alteration in cellular morphology and motility through actin remodeling. MLC1 overexpression induced filopodia formation and suppressed motility. And, MLC1 proteins expressed in patient-derived MLC1 mutants resulted in trapping in the ER although no changes in morphology or motility were observed. Interestingly knockdown of Mlc1 induced Arp3-Cortactin interaction, lamellipodia formation, and increased the membrane ruffling of the astrocytes. These data indicate that subcellular localization of expressed MLC1 at the plasma membrane is critical for changes in actin dynamics through ARP2/3 complex. Thus, our results suggest that misallocation of pathogenic mutant MLC1 may disturbs the stable cell-cell communication and the homeostatic regulation of astrocytes in patients with MLC.

Highlights

Patients with Megalencephalic leukoencephalopathy with subcortical cysts (MLC) exhibit abnormalities in brain ion and water homeostasis, resulting in diffuse cerebral white matter signaling on spectroscopic MRI, abnormal swelling of the white matter, and macrocephaly [1,2,3]
Phalloidin staining indicates that the MLC1-green fluorescent protein (GFP) expression induces the structural changes of filamentous actin (F-actin): Filopodia formation was increased, but lamellipodia structures were diminished (Fig. 1a, b)
To eliminate the possibility that the morphological changes were caused by a large GFP-tag itself or perturbation of any cell signaling by the protein tagging at the C-terminal end of MLC1, we examined the effect of MLC1 with a relatively small tag (Myc) on the putative loop between predicted transmembrane domain 3 and 4

Summary

Results

MLC1 regulates morphological changes via remodeling of the actin cytoskeleton To investigate the functional role of MLC1, we firstly analyzed the effect of MLC1 over-expression in the heterologous expression system (COS-7 cells). Unlike to the wildtype MLC1, the cells expressing patient-derived mutants failed to exhibit morphological changes in the plasma membrane: Lamellipodia remained intact, and a dense actin network was observed (Fig. 2a). The severity of deceasing filopodia numbers is somewhat correlated with the degree of decreasing surface expression levels of MLC1 proteins (Fig. 2b, e) These results suggest that the localization of MLC1 at the plasma membrane may be linked to the filopodia formation via actin remodeling. Filopodia formation was increased at the cell-cell contact point, and interactions remained stable for the remainder of the imaging period (at 480 min, stabilizing stage) (Fig. 4f and Additional file 9: Video S1) These results indicate that the translocation of MLC1 stabilizes cell-cell contact by decreasing in cell motility. Immunofluorescence staining showed that knockdown of Mlc repressed filopodia formation but induced lamellipodia formation as indicated by Phalloidin staining pattern (A)

Introduction

Stabilizing

Methods