Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

Abstract

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...

PPP2R1A Regulates Migration Persistence through the WAVE Shell Complex

“…Yet the extracts prepared from NHSL1-depleted cells and those prepared from cells simultaneously depleted of PPP2R1A and NHSL1 were as competent as wild type extracts in supporting actin polymerization. Both PPP2R1A and NHSL1 were detected associated with GTP-bound RAC1 Q61L (Fig.S4), in line with the fact that both CYFIP1 and NHSL1 harbor binding sites for active RAC1 7,16 . Together, these results show that PPP2R1A requires NHSL1 to regulate cell migration and actin polymerization.…”

“…Upon transient transfection of 293T cells with GFP fusion proteins of NHSL1 fragments, we found that the 4 th fragment at the C-terminus of NHSL1 retrieved a large amount of PPP2R1A upon GFP immunoprecipitation (Fig.6a). The 2 nd fragment of NHSL1 that contains the two previously reported binding sites for the SH3 domain of ABI1 16 also retrieved PPP2R1A, but to a much lesser extent than the 4 th fragment. The AlphaFold2 software predicted with high confidence three motifs in fragment 4 that interact with the PPP2R1A scaffold or the PPP2R5D regulatory subunit retrieved in the WSC TAP (Fig.6b).…”

“…NHSL1 was previously described as a negative regulator of cell migration 16 . It was thus surprising to find PPP2R1A and NHSL1 biochemically associated.…”

OpenCell: proteome-scale endogenous tagging enables the cartography of human cellular organization

“…Thus, NHSL1, NHSL2 and KIAA1522 also act to regulate actin assembly. A recent mechanistic study supports this hypothesis: NHSL1 localizes at the cell's leading edge and directly binds SCAR/WAVE to negatively regulate its activity, reducing F-actin content in lamellipodia and inhibiting cell migration (41). The authors identified NHSL1's SCAR/WAVE binding sites, and we find these sequences to be conserved in NSHL2 and KIA1522 (Fig.…”

OpenCell: Endogenous tagging for the cartography of human cellular organization

“…Thus, NHSL1, NHSL2, and KIAA1522 might also act to regulate actin assembly. A recent mechanistic study supports this hypothesis: NHSL1 localizes at the cell’s leading edge and directly binds SCAR/WAVE to negatively regulate its activity, reducing F-actin content in lamellipodia and inhibiting cell migration ( 41 ). The authors identified NHSL1’s SCAR/WAVE binding sites, and we found these sequences to be conserved in NSHL2 and KIA1522 (Fig.…”