Cell migration is important for development and its aberrant regulation contributes to many diseases. The Scar/WAVE complex is essential for Arp2/3 mediated lamellipodia formation during mesenchymal cell migration and several coinciding signals activate it. However, so far, no direct negative regulators are known. We have identified Nance-Horan Syndrome-like 1 protein (NHSL1) as a novel, direct binding partner of the Scar/WAVE complex, which co-localise at protruding lamellipodia. This interaction is mediated by the Abi SH3 domain and two binding sites in NHSL1. Furthermore, active Rac binds to NHSL1 at two regions that mediate leading edge targeting of NHSL1 suggesting that Rac recruits NHSL1. Surprisingly, NHSL1 inhibits cell migration through its interaction with the Scar/WAVE complex. Mechanistically, NHSL1 may reduce cell migration efficiency by impeding Arp2/3 activity, as measured in cells using a novel Arp2/3 FRET-FLIM biosensor, resulting in reduced F-actin content of lamellipodia, and consequently impairing the stability of lamellipodia protrusions.Scar/WAVE complex 6 . Lpd functions to promote cell migration via the Scar/WAVE complex 6,7 , which is consistent with a positive role for the Scar/WAVE complex in enhancing migration [8][9][10][11] . Several signals including active Rac, tyrosine phosphorylation and binding to phosphoinositides are known to activate the Scar/WAVE complex 2 .However, so far, it is not known how the Scar/WAVE complex is directly inhibited at the leading edge.Here, we identify NHSL1 (Nance-Horan Syndrome-like 1) protein as a negative regulator of cell migration and we found that this is mediated by its interaction with the Scar/WAVE complex. NHSL1 belongs to the poorly investigated Nance-Horan Syndrome protein family along with Nance-Horan Syndrome (NHS) and NHSL2 proteins. Mutations in the NHS gene cause Nance-Horan syndrome, which is characterised by dental abnormalities, developmental delay, and congenital cataracts [12][13][14] . We show that NHSL1 directly binds to the Scar/WAVE complex and co-localises with it at the very edge of protruding lamellipodia. We found that active Rac binds to NHSL1 at two regions that mediate leading edge targeting of NHSL1 suggesting that Rac recruits NHSL1. The negative regulatory function of NHSL1 in cell migration may be due to its role in lamellipodia since we found that it reduces lamellipodia stability. NHSL1 acts to reduce Arp2/3 activity, which is consistent with our finding that NHSL1 reduces F-actin content of lamellipodia via its interaction with the Scar/WAVE complex. Taken together, our data suggest that NHSL1 negatively regulates the Scar/WAVE complex, and hence reduces Arp2/3 activity, to control lamellipodia stability and consequently cell migration efficiency. Results NHSL1 localises to the very edge of lamellipodiaThe Nance-Horan Syndrome (NHS) protein family consists of the Nance-Horan Syndrome (NHS) protein, Nance-Horan Syndrome-like 1 (NHSL1) protein and Nance-Horan Syndrome-like 2 (NHSL2) protein [12][13][14][15][16] . E...

Cell migration is important for development and its aberrant regulation contributes to many diseases. The Scar/WAVE complex is essential for Arp2/3 mediated lamellipodia formation during mesenchymal cell migration and several coinciding signals activate it. However, so far, no direct negative regulators are known. Here we identify Nance-Horan Syndrome-like 1 protein (NHSL1) as a direct binding partner of the Scar/WAVE complex, which co-localise at protruding lamellipodia. This interaction is mediated by the Abi SH3 domain and two binding sites in NHSL1. Furthermore, active Rac binds to NHSL1 at two regions that mediate leading edge targeting of NHSL1. Surprisingly, NHSL1 inhibits cell migration through its interaction with the Scar/WAVE complex. Mechanistically, NHSL1 may reduce cell migration efficiency by impeding Arp2/3 activity, as measured in cells using a Arp2/3 FRET-FLIM biosensor, resulting in reduced F-actin density of lamellipodia, and consequently impairing the stability of lamellipodia protrusions.

Autophagy is essential for neuronal development and its deregulation contributes to neurodegenerative diseases. NDR1 and NDR2 are highly conserved kinases, implicated in neuronal development, mitochondrial health and autophagy, but how they affect mammalian brain development in vivo is not known. Using single and doubleNdr1/2knockout mouse models, we show that only dual loss ofNdr1/2in neurons causes neurodegeneration. This phenotype was present when NDR kinases were deleted both during embryonic development, as well as in adult mice. Proteomic and phosphoproteomic comparisons betweenNdr1/2knockout and control brains revealed novel kinase substrates and indicated that endocytosis is significantly affected in the absence of NDR1/2. We validated the endocytic protein Raph1/Lpd1, as a novel NDR1/2 substrate, and showed that both NDR1/2 and Raph1 are critical for endocytosis and membrane recycling. In NDR1/2 knockout brains, we observed prominent accumulation of transferrin receptor, p62 and ubiquitinated proteins, indicative of a major impairment of protein homeostasis. Furthermore, the levels of LC3-positive autophagosomes were reduced in knockout neurons, implying that reduced autophagy efficiency mediates p62 accumulation and neurotoxicity. Mechanistically, pronounced mislocalisation of the transmembrane autophagy protein ATG9A at the neuronal periphery, impaired axonal ATG9A trafficking and increased ATG9A surface levels further confirm defects in membrane trafficking, and could underlie the impairment in autophagy. We provide novel insight into the roles of NDR1/2 kinases in maintaining neuronal health.

NDR1 and NDR2 are highly conserved kinases that regulate neuronal development, mitochondrial health and autophagy, but their roles in the mammalian brain in vivo are unexplored. Using constitutive Ndr1 and neuron-specific Ndr2 knockout mice we show that dual loss of Ndr1/2 in neurons causes neurodegeneration. Phosphoproteomic comparisons between Ndr1/2 knockout and control brains revealed novel kinase substrates and highlighted changes in endocytosis in the absence of NDR1/2. The endocytic protein Raph1/Lpd1 is a new NDR1/2 substrate and both NDR1/2 and Raph1 are critical for endocytosis and membrane recycling. NDR1/2 knockout brains exhibited a prominent accumulation of transferrin receptor, p62 and ubiquitinated proteins, indicative of a major impairment in protein homeostasis. Furthermore, autophagosome levels were reduced in knockout neurons, implying that reduced autophagy efficiency mediates p62 accumulation. Mechanistically, pronounced mislocalisation of the transmembrane autophagy protein ATG9A at the neuronal periphery, impaired axonal ATG9A trafficking and increased ATG9A surface levels further confirm defects in membrane trafficking and could underlie the impairment in autophagy. We provide novel insight into the roles of NDR1/2 kinases in maintaining neuronal health.