Netrin-1-Regulated Distribution of UNC5B and DCC in Live Cells Revealed by TICCS.

Netrins are secreted proteins that direct cell migration and adhesion during development. Netrin-1 binds its receptors deleted in colorectal cancer (DCC) and the UNC5 homologs (UNC5A-D) to activate downstream signaling that ultimately directs cytoskeletal reorganization. To investigate how netrin-1 regulates the dynamic distribution of DCC and UNC5 homologs, we applied fluorescence confocal and total internal reflection fluorescence microscopy, and sliding window temporal image cross correlation spectroscopy, to measure time profiles of the plasma membrane distribution, aggregation state, and interaction fractions of fluorescently tagged netrin receptors expressed in HEK293T cells. Our measurements reveal changes in receptor aggregation that are consistent with netrin-1-induced recruitment of DCC-enhanced green fluorescent protein (EGFP) from intracellular vesicles to the plasma membrane. Netrin-1 also induced colocalization of coexpressed full-length DCC-EGFP with DCC-T-mCherry, a putative DCC dominant negative that replaces the DCC intracellular domain with mCherry, consistent with netrin-1-induced receptor oligomerization, but with no change in aggregation state with time, providing evidence that signaling via the DCC intracellular domain triggers DCC recruitment to the plasma membrane. UNC5B expressed alone was also recruited by netrin-1 to the plasma membrane. Coexpressed DCC and UNC5 homologs are proposed to form a heteromeric netrin-receptor complex to mediate a chemorepellent response. Application of temporal image cross correlation spectroscopy to image series of cells coexpressing UNC5B-mCherry and DCC-EGFP revealed a netrin-1-induced increase in colocalization, with both receptors recruited to the plasma membrane from preexisting clusters, consistent with vesicular recruitment and receptor heterooligomerization. Plasma membrane recruitment of DCC or UNC5B was blocked by application of the netrin-1 VI-V peptide, which fails to activate chemoattraction, or by pharmacological block of Src family kinase signaling, consistent with receptor recruitment requiring netrin-1-activated signaling. Our findings reveal a mechanism activated by netrin-1 that recruits DCC and UNC5B to the plasma membrane.

Independent synchronized control and visualization of interactions between living cells and organisms.

To investigate the early stages of cell-cell interactions occurring between living biological samples, imaging methods with appropriate spatiotemporal resolution are required. Among the techniques currently available, those based on optical trapping are promising. Methods to image trapped objects, however, in general suffer from a lack of three-dimensional resolution, due to technical constraints. Here, we have developed an original setup comprising two independent modules: holographic optical tweezers, which offer a versatile and precise way to move multiple objects simultaneously but independently, and a confocal microscope that provides fast three-dimensional image acquisition. The optical decoupling of these two modules through the same objective gives users the possibility to easily investigate very early steps in biological interactions. We illustrate the potential of this setup with an analysis of infection by the fungus Drechmeria coniospora of different developmental stages of Caenorhabditis elegans. This has allowed us to identify specific areas on the nematode's surface where fungal spores adhere preferentially. We also quantified this adhesion process for different mutant nematode strains, and thereby derive insights into the host factors that mediate fungal spore adhesion.

Low-cost multimodal light sheet microscopy for optically cleared tissues and living specimens.

Light sheet microscopy techniques have expanded with designs to address many new applications. Due to rapid advancements in computing power, camera/detector technologies, and tissue clearing techniques, light sheet methods are becoming increasingly popular for biomedical imaging applications at the cellular and tissue levels. Light sheet imaging modalities couple rapid imaging rates, low-levels of phototoxicity, and excellent signal to noise ratios, contributing to their popularity for experimental biology. However, the current major limitation of light sheet microscopy arises from optical aberrations, with the main drawback being the defocusing introduced by refractive index variations that accompany clearing techniques. Here, we propose an inexpensive and easy to build light sheet based instrumentation to overcome this limitation by optomechanically decoupling the sample scanning movement from the detection step. Our solution is relatively simple to implement and also provides increased modularity by using a swappable excitation arm. This expands the range of samples we can image on a single system, from high resolution for single cells at ? m spatial resolution, to tissues with mm spatial resolution. We demonstrate our approach, using the system to image iDISCO cleared embryos and sciatic nerves, and provide the full three-dimensional reconstruction of these objects in minutes.

Physiological epidermal growth factor concentrations activate high affinity receptors to elicit calcium oscillations.

Signaling mediated by the epidermal growth factor (EGF) is crucial in tissue development, homeostasis and tumorigenesis. EGF is mitogenic at picomolar concentrations and is known to bind its receptor on high affinity binding sites depending of the oligomerization state of the receptor (monomer or dimer). In spite of these observations, the cellular response induced by EGF has been mainly characterized for nanomolar concentrations of the growth factor, and a clear definition of the cellular response to circulating (picomolar) concentrations is still lacking. We investigated Ca2+ signaling, an early event in EGF responses, in response to picomolar doses in COS-7 cells where the monomer/dimer equilibrium is unaltered by the synthesis of exogenous EGFR. Using the fluo5F Ca2+ indicator, we found that picomolar concentrations of EGF induced in 50% of the cells a robust oscillatory Ca2+ signal quantitatively similar to the Ca2+ signal induced by nanomolar concentrations. However, responses to nanomolar and picomolar concentrations differed in their underlying mechanisms as the picomolar EGF response involved essentially plasma membrane Ca2+ channels that are not activated by internal Ca2+ store depletion, while the nanomolar EGF response involved internal Ca2+ release. Moreover, while the picomolar EGF response was modulated by charybdotoxin-sensitive K+ channels, the nanomolar response was insensitive to the blockade of these ion channels.

Probing the plasma membrane organization in living cells by spot variation fluorescence correlation spectroscopy.

While intrinsic Brownian agitation within a lipid bilayer does homogenize the molecular distribution, the extremely diverse composition of the plasma membrane, in contrast, favors the development of inhomogeneity due to the propensity of such a system to minimize its total free energy. Precisely, deciphering such inhomogeneous organization with appropriate spatiotemporal resolution remains, however, a challenge. In accordance with its ability to accurately measure diffusion parameters, fluorescence correlation spectroscopy (FCS) has been developed in association with innovative experimental strategies to monitor modes of molecular lateral confinement within the plasma membrane of living cells. Here, we describe a method, namely spot variation FCS (svFCS), to decipher the dynamics of the plasma membrane organization. The method is based on questioning the relationship between the diffusion time τ(d) and the squared waist of observation w(2). Theoretical models have been developed to predict how geometrical constraints such as the presence of adjacent or isolated domains affect the svFCS observations. These investigations have allowed significant progress in the characterization of cell membrane lateral organization at the suboptical level, and have provided, for instance, compelling evidence for the in vivo existence of raft nanodomains.

Mapping molecular diffusion in the plasma membrane by Multiple-Target Tracing (MTT).

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT). Single-molecule videomicroscopy, offering millisecond and nanometric resolution, allows a detailed representation of membrane organization by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions. We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis. Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution. The nanometric accuracy obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods such as STORM, PALM, RESOLFT or STED, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.