Willin/FRMD6 Influences Mechanical Phenotype and Neuronal Differentiation in Mammalian Cells by Regulating ERK1/2 Activity

Abstract

Willin/FRMD6 is part of a family of proteins with a 4.1 ezrin-radixin-moesin (FERM) domain. It has been identified as an upstream activator of the Hippo pathway and, when aberrant in its expression, is associated with human diseases and disorders. Even though Willin/FRMD6 was originally discovered in the rat sciatic nerve, most studies have focused on its functional roles in cells outside of the nervous system, where Willin/FRMD6 is involved in the formation of apical junctional cell-cell complexes and in regulating cell migration. Here, we investigate the biochemical and biophysical role of Willin/FRMD6 in neuronal cells, employing the commonly used SH-SY5Y neuronal model cell system and combining biochemical measurements with Elastic Resonator Interference Stress Micropscopy (ERISM). We present the first direct evidence that Willin/FRMD6 expression influences both the cell mechanical phenotype and neuronal differentiation. By investigating cells with increased and decreased Willin/FRMD6 expression levels, we show that Willin/FRMD6 not only affects proliferation and migration capacity of cells but also leads to changes in cell morphology and an enhanced formation of neurite-like membrane extensions. These changes were accompanied by alterations of biophysical parameters such as cell force, the organization of actin stress fibers and the formation of focal adhesions. At the biochemical level, changes in Willin/FRMD6 expression inversely affected the activity of the extracellular signal-regulated kinases (ERK) pathway and downstream transcriptional factor NeuroD1, which seems to prime SH-SY5Y cells for retinoic acid (RA)-induced neuronal differentiation.

Impaired SNF2L Chromatin Remodeling Prolongs Accessibility at Promoters Enriched for Fos/Jun Binding Sites and Delays Granule Neuron Differentiation

“…Our finding that this was specific to the Ex6DEL cultures may simply reflect the delayed differentiation of these cultures. Several of these studies have also linked MAPK-ERK1/2 activation directly to Neurod1 activity which we mentioned above was upregulated ( Petersen et al, 2002 ; Kronenberg et al, 2020 ). One study of particular interest demonstrated that Wnt3 promoted GNP differentiation through a non-canonical Wnt signaling pathway that activated the MAPK-ERK1/2 pathway instead of the β-catenin canonical pathway ( Anne et al, 2013 ).…”

“…When we compared expression profiles at 3 DIV we observed activation of the ERK1/2 pathway which we confirmed by immunoblots of the protein extracts from the cultures. Multiple studies have previously shown that a wide range of soluble factors (Glucose, IGF-1, Wnt3) can promote cerebellar granule differentiation through MAPK-ERK1/2 activation ( Torres-Aleman et al, 1998 ; Petersen et al, 2002 ; Anne et al, 2013 ; Kronenberg et al, 2020 ). Our finding that this was specific to the Ex6DEL cultures may simply reflect the delayed differentiation of these cultures.…”

Willin/FRMD6: A Multi-Functional Neuronal Protein Associated with Alzheimer’s Disease

“…In aging cardiac microvascular endothelial cells (CMECs), Willin/FRMD6 facilitates BDNF-induced migration, as knockdown of Willin/FRMD6 abrogates hallmarks of BDNF-induced migration such as increased actin polarity, stress fiber polymerization, and pseudopod migration [ 30 ]. In contrast, Willin/FRMD6 overexpression in neuronal SH-SY5Y cells results in significantly decreased proliferation and migration, but without changes in actin arrangement or cell morphology [ 73 ].…”

“…In contrast to the results in fibroblasts, MCF10A, HeLa, and PC cells, Willin/FRMD6 knockdown in aged CMECS results in increased transcription of MST1/2, LATS1/2, and YAP [ 30 ]. In neuronal SH-SY5Y cells, Willin/FRMD6 knockdown leads to decreased nuclear TAZ and decreased YAP expression [ 73 ]. Willin/FRMD6 overexpression in SH-SY5Y cells results in significantly increased nuclear TAZ [ 73 ].…”

Real-time imaging of cellular forces using optical interference

“…We recently introduced an interference-based approach, elastic resonator interference stress microscopy (ERISM), which offers reference-free, high-precision force measurements with µm lateral resolution and low probe-light intensity 17 , 18 . However, the use of ERISM has been limited to studying phenomena on the second timescale and longer 19 , 20 , 21 , as each ERISM measurement requires the acquisition of at least 50 images at different illumination wavelengths which limits the acquisition speed to 0.4 fps; this is followed by data analysis that takes ~10 s per image.…”