Highlights d Labeled ECM in fly embryos can be examined from initiation to homeostasis d Quantifying ECM levels to homeostasis allows for modeling of basal turnover rate d Embryonic ECM has a half-life of 10 h, which was confirmed by pulse-chase analysis d Inhibiting MMPs or ECM interactions alters the basal turnover rate

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:

Measuring the organization of the cellular cytoskeleton and the surrounding extracellular matrix (ECM) is currently of wide interest as changes in both local and global alignment can highlight alterations in cellular functions and material properties of the extracellular environment. Different approaches have been developed to quantify these structures, typically based on fiber segmentation or on matrix representation and transformation of the image, each with its own advantages and disadvantages. Here we present AFT − Alignment by Fourier Transform, a workflow to quantify the alignment of fibrillar features in microscopy images exploiting 2D Fast Fourier Transforms (FFT). Using pre-existing datasets of cell and ECM images, we demonstrate our approach and compare and contrast this workflow with two other well-known ImageJ algorithms to quantify image feature alignment. These comparisons reveal that AFT has a number of advantages due to its grid-based FFT approach. 1) Flexibility in defining the window and neighborhood sizes allows for performing a parameter search to determine an optimal length scale to carry out alignment metrics. This approach can thus easily accommodate different image resolutions and biological systems. 2) The length scale of decay in alignment can be extracted by comparing neighborhood sizes, revealing the overall distance that features remain anisotropic. 3) The approach is ambivalent to the signal source, thus making it applicable for a wide range of imaging modalities and is dependent on fewer input parameters than segmentation methods. 4) Finally, compared to segmentation methods, this algorithm is computationally inexpensive, as high-resolution images can be evaluated in less than a second on a standard desktop computer. This makes it feasible to screen numerous experimental perturbations or examine large images over long length scales. Implementation is made available in both MATLAB and Python for wider accessibility, with example datasets for single images and batch processing. Additionally, we include an approach to automatically search parameters for optimum window and neighborhood sizes, as well as to measure the decay in alignment over progressively increasing length scales.

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.

The cell/microenvironment interface is the starting point of integrin-mediated mechanotransduction, but many details of mechanotransductive signal integration remain elusive due to the complexity of the involved (extra)cellular structures, such as the glycocalyx. We used nano-bio-interfaces reproducing the complex nanotopographical features of the extracellular matrix to analyse the glycocalyx impact on PC12 cell mechanosensing at the nanoscale (e.g., by force spectroscopy with functionalised probes). Our data demonstrates that the glycocalyx configuration affects spatio-temporal nanotopography-sensitive mechanotransductive events at the cell/microenvironment interface. Opposing effects of major glycocalyx removal were observed, when comparing flat and specific nanotopographical conditions. The excessive retrograde actin flow speed and force loading are strongly reduced on certain nanotopographies upon strong reduction of the native glycocalyx, while on the flat substrate we observe the opposite trend. Our results highlight the importance of the glycocalyx configuration in a molecular clutch force loading-dependent cellular mechanism for mechanosensing of microenvironmental nanotopographical features. Graphical Abstract

The hyaluronic acid component of the glycocalyx plays a role in cell mechanotransduction by selectively transmitting mechanical signals to the cell cytoskeleton or to the cell membrane. The aim of this study was to evaluate the mechanical link between the hyaluronic acid molecule and the cell cytoskeleton by means of atomic force microscopy single molecule force spectroscopy. Hyaluronic acid molecules on live cells were targeted with probes coated with hyaluronic acid binding protein. Two different types of events were observed when the detachment of the target molecule from the probe occurred, suggesting the presence of cytoskeleton- and membrane-anchored molecules. Membrane-anchored molecules facilitated the formation of tethers when pulled. About 15% of the tested hyaluronic acid molecules were shown to be anchored to the cytoskeleton. When multiple molecules bonded to the probe, specific detachment patterns were observed, suggesting that a cytoskeletal bond needed to be broken to improve the ability to pull tethers from the cell membrane. This likely resulted in the formation of tethering structures maintaining a cytoskeletal core similar to the ones observed for cells over-expressing HA synthases. The different observed rupture events were associated with separate mechanotransductive mechanisms in an analogous manner to that previously proposed for the endothelial glycocalyx. Single cytoskeleton anchored rupture events represent HA molecules linked to the cytoskeleton and therefore transmitting mechanical stimuli into the inner cell compartments. Single membrane tethers would conversely represent the glycocalyx molecules connected to areas of the membrane where an abundance of signalling molecules reside.

BackgroundThe relationship between extracranial venous system abnormalities and central nervous system disorders has been recently theorized. In this paper we delve into this hypothesis by modeling the venous drainage in brain and spinal column areas and simulating the intracranial flow changes due to extracranial morphological stenoses.MethodsA lumped parameter model of the cerebro-spinal venous drainage was created based on anatomical knowledge and vessels diameters and lengths taken from literature. Each vein was modeled as a hydraulic resistance, calculated through Poiseuille’s law. The inputs of the model were arterial flow rates of the intracranial, vertebral and lumbar districts. The effects of the obstruction of the main venous outflows were simulated. A database comprising 112 Multiple Sclerosis patients (Male/Female = 42/70; median age ± standard deviation = 43.7 ± 10.5 years) was retrospectively analyzed.ResultsThe flow rate of the main veins estimated with the model was similar to the measures of 21 healthy controls (Male/Female = 10/11; mean age ± standard deviation = 31 ± 11 years), obtained with a 1.5 T Magnetic Resonance scanner. The intracranial reflux topography predicted with the model in cases of internal jugular vein diameter reduction was similar to those observed in the patients with internal jugular vein obstacles.ConclusionsThe proposed model can predict physiological and pathological behaviors with good fidelity. Despite the simplifications introduced in cerebrospinal venous circulation modeling, the key anatomical feature of the lumped parameter model allowed for a detailed analysis of the consequences of extracranial venous impairments on intracranial pressure and hemodynamics.Electronic supplementary materialThe online version of this article (doi:10.1186/s12883-015-0352-y) contains supplementary material, which is available to authorized users.