SummaryThe basement membrane (BM) is a thin layer of extracellular matrix (ECM) beneath nearly all epithelial cell types that is critical for cellular and tissue function. It is composed of numerous components conserved among all bilaterians [1]; however, it is unknown how all of these components are generated and subsequently constructed to form a fully mature BM in the living animal. Although BM formation is thought to simply involve a process of self-assembly [2], this concept suffers from a number of logistical issues when considering its construction in vivo. First, incorporation of BM components appears to be hierarchical [3, 4, 5], yet it is unclear whether their production during embryogenesis must also be regulated in a temporal fashion. Second, many BM proteins are produced not only by the cells residing on the BM but also by surrounding cell types [6, 7, 8, 9], and it is unclear how large, possibly insoluble protein complexes [10] are delivered into the matrix. Here we exploit our ability to live image and genetically dissect de novo BM formation during Drosophila development. This reveals that there is a temporal hierarchy of BM protein production that is essential for proper component incorporation. Furthermore, we show that BM components require secretion by migrating macrophages (hemocytes) during their developmental dispersal, which is critical for embryogenesis. Indeed, hemocyte migration is essential to deliver a subset of ECM components evenly throughout the embryo. This reveals that de novo BM construction requires a combination of both production and distribution logistics allowing for the timely delivery of core components.

Cell migration is hypothesised to involve a cycle of behaviours beginning with leading edge extension. However, recent evidence suggests that the leading edge may be dispensable for migration, raising the question of what actually controls cell directionality. Here we exploit the embryonic migration of Drosophila macrophages to bridge the different temporal scales of the behaviours controlling motility. This reveals that edge fluctuations during random motility are impersistent and weakly correlated with motion. In contrast, flow of the actin network behind the leading edge is highly persistent. Quantification of actin flow structure during migration reveals a stable organisation and asymmetry in the cell-wide flowfield that strongly correlates with cell Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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.