Victor Racine

The extracellular matrix guides the orientation of the cell division axis

The cell division axis determines the future positions of daughter cells and is therefore critical for cell fate. The positioning of the division axis has been mostly studied in systems such as embryos or yeasts, in which cell shape is well defined. In these cases, cell shape anisotropy and cell polarity affect spindle orientation. It remains unclear whether cell geometry or cortical cues are determinants for spindle orientation in mammalian cultured cells. The cell environment is composed of an extracellular matrix (ECM), which is connected to the intracellular actin cytoskeleton via transmembrane proteins. We used micro-contact printing to control the spatial distribution of the ECM on the substrate and demonstrated that it has a role in determining the orientation of the division axis of HeLa cells. On the basis of our analysis of the average distributions of actin-binding proteins in interphase and mitosis, we propose that the ECM controls the location of actin dynamics at the membrane, and thus the segregation of cortical components in interphase. This segregation is further maintained on the cortex of mitotic cells and used for spindle orientation.

Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity

Control of the establishment of cell polarity is an essential function in tissue morphogenesis and renewal that depends on spatial cues provided by the extracellular environment. The molecular role of cell–cell or cell–extracellular matrix (ECM) contacts on the establishment of cell polarity has been well characterized. It has been hypothesized that the geometry of the cell adhesive microenvironment was directing cell surface polarization and internal organization. To define how the extracellular environment affects cell polarity, we analyzed the organization of individual cells plated on defined micropatterned substrates imposing cells to spread on various combinations of adhesive and nonadhesive areas. The reproducible normalization effect on overall cell compartmentalization enabled quantification of the spatial organization of the actin network and associated proteins, the spatial distribution of microtubules, and the positioning of nucleus, centrosome, and Golgi apparatus. By using specific micropatterns and statistical analysis of cell compartment positions, we demonstrated that ECM geometry determines the orientation of cell polarity axes. The nucleus–centrosome orientations were reproducibly directed toward cell adhesive edges. The anisotropy of the cell cortex in response to the adhesive conditions did not affect the centrosome positioning at the cell centroid. Based on the quantification of microtubule plus end distribution we propose a working model that accounts for that observation. We conclude that, in addition to molecular composition and mechanical properties, ECM geometry plays a key role in developmental processes.

Experimental and theoretical study of mitotic spindle orientation

The architecture and adhesiveness of a cell microenvironment is a critical factor for the regulation of spindle orientation in vivo. Using a combination of theory and experiments, we have investigated spindle orientation in HeLa (human) cells. Here we show that spindle orientation can be understood as the result of the action of cortical force generators, which interact with spindle microtubules and are activated by cortical cues. We develop a simple physical description of this spindle mechanics, which allows us to calculate angular profiles of the torque acting on the spindle, as well as the angular distribution of spindle orientations. Our model accounts for the preferred spindle orientation and the shape of the full angular distribution of spindle orientations observed in a large variety of different cellular microenvironment geometries. It also correctly describes asymmetric spindle orientations, which are observed for certain distributions of cortical cues. We conclude that, on the basis of a few simple assumptions, we can provide a quantitative description of the spindle orientation of adherent cells.

Activity-Dependent Tuning of Inhibitory Neurotransmission Based on GABAAR Diffusion Dynamics

An activity-dependent change in synaptic efficacy is a central tenet in learning, memory, and pathological states of neuronal excitability. The lateral diffusion dynamics of neurotransmitter receptors are one of the important parameters regulating synaptic efficacy. We report here that neuronal activity modifies diffusion properties of type-A GABA receptors (GABA(A)R) in cultured hippocampal neurons: enhanced excitatory synaptic activity decreases the cluster size of GABA(A)Rs and reduces GABAergic mIPSC. Single-particle tracking of the GABA(A)R gamma2 subunit labeled with quantum dots reveals that the diffusion coefficient and the synaptic confinement domain size of GABA(A)R increases in parallel with neuronal activity, depending on Ca(2+) influx and calcineurin activity. These results indicate that GABA(A)R diffusion dynamics are directly linked to rapid and plastic modifications of inhibitory synaptic transmission in response to changes in intracellular Ca(2+) concentration. This transient activity-dependent reduction of inhibition would favor the onset of LTP during conditioning.

The human Nup107–160 nuclear pore subcomplex contributes to proper kinetochore functions

We previously demonstrated that a fraction of the human Nup107–160 nuclear pore subcomplex is recruited to kinetochores at the onset of mitosis. However, the molecular determinants for its kinetochore targeting and the functional significance of this localization were not investigated. Here, we show that the Nup107–160 complex interacts with CENP‐F, but that CENP‐F only moderately contributes to its targeting to kinetochores. In addition, we show that the recruitment of the Nup107–160 complex to kinetochores mainly depends on the Ndc80 complex. We further demonstrate that efficient depletion of the Nup107–160 complex from kinetochores, achieved either by combining siRNAs targeting several of its subunits excluding Seh1, or by depleting Seh1 alone, induces a mitotic delay. Further analysis of Seh1‐depleted cells revealed impaired chromosome congression, reduced kinetochore tension and kinetochore–microtubule attachment defects. Finally, we show that the presence of the Nup107–160 complex at kinetochores is required for the recruitment of Crm1 and RanGAP1–RanBP2 to these structures. Together, our data thus provide the first molecular clues underlying the function of the human Nup107–160 complex at kinetochores.

Surface Trafficking of Neurotransmitter Receptor: Comparison between Single-Molecule/Quantum Dot Strategies

The cellular traffic of neurotransmitter receptors has captured a lot of attention over the last decade, mostly because synaptic receptor number is adjusted during synaptic development and plasticity. Although each neurotransmitter receptor family has its own trafficking characteristics, two main

Visualization and quantification of vesicle trafficking on a three-dimensional cytoskeleton network in living cells

Recent progress in biology and microscopy has made it possible to acquire multidimensional data on rapid cellular activities. Unfortunately, the data analysis needed to describe the observed biological process still remains a major bottleneck. We here present a novel method of studying membrane trafficking by monitoring vesicular structures moving along a three‐dimensional cytoskeleton network. It allows the dynamics of such structures to be qualitatively and quantitatively investigated. Our tracking method uses kymogram analysis to extract the consistent part of the temporal information and to allow the meaningful representation of vesicle dynamics. A fully automatic extension of this method, together with a statistical tool for dynamic comparisons, allows the precise analysis and comparison of overall speed distributions and directions. It can handle typical complex situations, such as a dense set of vesicles moving at various velocities, fusing and dissociating with each other or with other cell compartments. The overall approach has been characterized and validated on synthetic data. We chose the Rab6A protein, a GTPase involved in the regulation of intracellular membrane trafficking, as a molecular model. The application of our method to GFP‐Rab6A stable cells acquired using fast four‐dimensional deconvolution video‐microscopy gives considerable cellular dynamic information unreachable using other techniques.

Pom1 kinase links division plane position to cell polarity by regulating Mid1p cortical distribution

In fission yeast, Mid1p, a major determinant for division plane position, defines a medial cortical compartment where it recruits myosin II at the onset of mitosis to initiate contractile ring assembly. How Mid1p is restricted to the medial cortex is unknown. We report here that in a pom1 polarity mutant, which displays a monopolar growth pattern, Mid1p distribution expands towards the non-growing cell tip, uncoupling Mid1p localization from nuclear position. This accounts for the displacement of the contractile ring during mitosis. By contrast, Mid1p localization is normal in a bud6Δ strain, indicating that Mid1p misdistribution is not a general consequence of monopolar growth. We conclude that Pom1 kinase acts as a negative regulator of Mid1p distribution, excluding Mid1p from non-growing ends, whereas a Pom1-independent mechanism prevents Mid1p association with growing ends. Our work therefore provides evidence that cell polarity regulators influence the distribution of Mid1p, linking division plane position to cell polarity.

Wavelet analysis for single molecule localization microscopy

Localization of single molecules in microscopy images is a key step in quantitative single particle data analysis. Among them, single molecule based super-resolution optical microscopy techniques require high localization accuracy as well as computation of large data sets in the order of 10(5) single molecule detections to reconstruct a single image. We hereby present an algorithm based on image wavelet segmentation and single particle centroid determination, and compare its performance with the commonly used gaussian fitting of the point spread function. We performed realistic simulations at different signal-to-noise ratios and particle densities and show that the calculation time using the wavelet approach can be more than one order of magnitude faster than that of gaussian fitting without a significant degradation of the localization accuracy, from 1 nm to 4 nm in our range of study. We propose a simulation-based estimate of the resolution of an experimental single molecule acquisition.

Myosin Va Mediates Docking of Secretory Granules at the Plasma Membrane

Myosin Va (MyoVa) is a prime candidate for controlling actin-based organelle motion in neurons and neuroendocrine cells. Its function in secretory granule (SG) trafficking was investigated in enterochromaffin cells by wide-field and total internal reflection fluorescence microscopy. The distribution of endogenous MyoVa partially overlapped with SGs and microtubules. Impairing MyoVa function by means of a truncated construct (MyoVa tail) or RNA interference prevented the formation of SG-rich regions at the cell periphery and reduced SG density in the subplasmalemmal region. Individual SG trajectories were tracked to analyze SG mobility. A wide distribution of their diffusion coefficient, Dxy, was observed. Almost immobile SGs (Dxy < 5 × 10−4 μm2 · s−1) were considered as docked at the plasma membrane based on two properties: (1) SGs that undergo exocytosis have a Dxy below this threshold value for at least 2 s before fusion; (2) a negative autocorrelation of the vertical motion was found in subtrajectories with a Dxy below the threshold. Using this criterion of docking, we found that the main effect of MyoVa inhibition was to reduce the number of docked granules, leading to reduced secretory responses. Surprisingly, this reduction was not attributable to a decreased transport of SGs toward release sites. In contrast, MyoVa silencing reduced the occurrence of long-lasting, but not short-lasting, docking periods. We thus propose that, despite its known motor activity, MyoVa directly mediates stable attachment of SGs at the plasma membrane.