Weimiao Yu

Quantitative neurite outgrowth measurement based on image segmentation with topological dependence

The study of neuronal morphology and neurite outgrowth has been enhanced by the combination of imaging informatics and high content screening, in which thousands of images are acquired using robotic fluorescent microscopy. To understand the process of neurite outgrowth in the context of neuroregeneration, we used mouse neuroblastoma N1E115 as our model neuronal cell. Six‐thousand cellular images of four different culture conditions were acquired with two‐channel widefield fluorescent microscopy. We developed a software package called NeuronCyto. It is a fully automatic solution for neurite length measurement and complexity analysis. A novel approach based on topological analysis is presented to segment cells. The detected nuclei were used as references to initialize the level set function. Merging and splitting of cells segments were prevented using dynamic watershed lines based on the constraint of topological dependence. A tracing algorithm was developed to automatically trace neurites and measure their lengths quantitatively on a cell‐by‐cell basis. NeuronCyto analyzes three important biologically relevant features, which are the length, branching complexity, and number of neurites. The application of NeuronCyto on the experiments of Toca‐1 and serum starvation show that the transfection of Toca‐1 cDNA induces longer neurites with more complexities than serum starvation.

Evolving generalized Voronoi diagrams for accurate cellular image segmentation

Analyzing cellular morphologies on a cell‐by‐cell basis is vital for drug discovery, cell biology, and many other biological studies. Interactions between cells in their culture environments cause cells to touch each other in acquired microscopy images. Because of this phenomenon, cell segmentation is a challenging task, especially when the cells are of similar brightness and of highly variable shapes. The concept of topological dependence and the maximum common boundary (MCB) algorithm are presented in our previous work (Yu et al., Cytometry Part A 2009;75A:289–297). However, the MCB algorithm suffers a few shortcomings, such as low computational efficiency and difficulties in generalizing to higher dimensions. To overcome these limitations, we present the evolving generalized Voronoi diagram (EGVD) algorithm. Utilizing image intensity and geometric information, EGVD preserves topological dependence easily in both 2D and 3D images, such that touching cells can be segmented satisfactorily. A systematic comparison with other methods demonstrates that EGVD is accurate and much more efficient.

Collective Cell Motility Promotes Chemotactic Prowess and Resistance to Chemorepulsion

Collective cell migration is a widespread biological phenomenon, whereby groups of highly coordinated, adherent cells move in a polarized fashion. This migration mode is a hallmark of tissue morphogenesis during development and repair and of solid tumor dissemination. In addition to circulating as solitary cells, lymphoid malignancies can assemble into tissues as multicellular aggregates. Whether malignant lymphocytes are capable of coordinating their motility in the context of chemokine gradients is, however, unknown. Here, we show that, upon exposure to CCL19 or CXCL12 gradients, malignant B and T lymphocytes assemble into clusters that migrate directionally and display a wider chemotactic sensitivity than individual cells. Physical modeling recapitulates cluster motility statistics and shows that intracluster cell cohesion results in noise reduction and enhanced directionality. Quantitative image analysis reveals that cluster migration runs are periodically interrupted by transitory rotation and random phases that favor leader cell turnover. Additionally, internalization of CCR7 in leader cells is accompanied by protrusion retraction, loss of polarity, and the ensuing replacement by new leader cells. These mechanisms ensure sustained forward migration and resistance to chemorepulsion, a behavior of individual cells exposed to steep CCL19 gradients that depends on CCR7 endocytosis. Thus, coordinated cluster dynamics confer distinct chemotactic properties, highlighting unexpected features of lymphoid cell migration.

Positive and Negative Regulation of Gli Activity by Kif7 in the Zebrafish Embryo

Loss of function mutations of Kif7, the vertebrate orthologue of the Drosophila Hh pathway component Costal2, cause defects in the limbs and neural tubes of mice, attributable to ectopic expression of Hh target genes. While this implies a functional conservation of Cos2 and Kif7 between flies and vertebrates, the association of Kif7 with the primary cilium, an organelle absent from most Drosophila cells, suggests their mechanisms of action may have diverged. Here, using mutant alleles induced by Zinc Finger Nuclease-mediated targeted mutagenesis, we show that in zebrafish, Kif7 acts principally to suppress the activity of the Gli1 transcription factor. Notably, we find that endogenous Kif7 protein accumulates not only in the primary cilium, as previously observed in mammalian cells, but also in cytoplasmic puncta that disperse in response to Hh pathway activation. Moreover, we show that Drosophila Costal2 can substitute for Kif7, suggesting a conserved mode of action of the two proteins. We show that Kif7 interacts with both Gli1 and Gli2a and suggest that it functions to sequester Gli proteins in the cytoplasm, in a manner analogous to the regulation of Ci by Cos2 in Drosophila. We also show that zebrafish Kif7 potentiates Gli2a activity by promoting its dissociation from the Suppressor of Fused (Sufu) protein and present evidence that it mediates a Smo dependent modification of the full length form of Gli2a. Surprisingly, the function of Kif7 in the zebrafish embryo appears restricted principally to mesodermal derivatives, its inactivation having little effect on neural tube patterning, even when Sufu protein levels are depleted. Remarkably, zebrafish lacking all Kif7 function are viable, in contrast to the peri-natal lethality of mouse kif7 mutants but similar to some Acrocallosal or Joubert syndrome patients who are homozygous for loss of function KIF7 alleles.

Endocytic reawakening of motility in jammed epithelia

Dynamics of epithelial monolayers has recently been interpreted in terms of a jamming or rigidity transition. How cells control such phase transitions is, however, unknown. Here we show that RAB5A, a key endocytic protein, is sufficient to induce large-scale, coordinated motility over tens of cells and ballistic motion in otherwise kinetically-arrested monolayers. This is linked to increased traction forces and to the extension of cell protrusions, which align with local velocity. Molecularly, impairing endocytosis, macropinocytosis or increasing fluid efflux abrogates RAB5A-induced collective motility. A simple model based on mechanical junctional tension and an active cell reorientation mechanism for the velocity of self-propelled cells identifies regimes of monolayer dynamics that explain endocytic reawakening of locomotion in terms of a combination of large-scale directed migration and local unjamming. These changes in multicellular dynamics enable collectives to migrate under physical constraints and may be exploited by tumors for interstitial dissemination.

Nuclear import of a lipid-modified transcription factor: mobilization of NFAT5 isoform a by osmotic stress.

Lipid-modified transcription factors (TFs) are biomolecular oddities since their reduced mobility and membrane attachment appear to contradict nuclear import required for their gene-regulatory function. NFAT5 isoform a (selected from an in silico screen for predicted lipid-modified TFs) is shown to contribute about half of all endogenous expression of human NFAT5 isoforms in the isotonic state. Wild-type NFAT5a protein is indeed myristoylated and palmitoylated on its transport to the plasmalemma via the endoplasmic reticulum and the Golgi. In contrast, its lipid anchor-deficient mutants as well as isoforms NFAT5b/c are diffusely localized in the cytoplasm without preference to vesicular structures. Quantitative/live microscopy shows the plasmamembrane-bound fraction of NFAT5a moving into the nucleus upon osmotic stress despite the lipid anchoring. The mobilization mechanism is not based on proteolytic processing of the lipid-anchored N-terminus but appears to involve reversible palmitoylation. Thus, NFAT5a is an example of TFs immobilized with lipid anchors at cyotoplasmic membranes in the resting state and that, nevertheless, can translocate into the nucleus upon signal induction.

Level Set Segmentation of Cellular Images Based on Topological Dependence

Segmentation of cellular images presents a challenging task for computer vision, especially when the cells of irregular shapes clump together. Level set methods can segment cells with irregular shapes when signal-to-noise ratio is low, however they could not effectively segment cells that are clumping together. We perform topological analysis on the zero level sets to enable effective segmentation of clumped cells. Geometrical shapes and intensities are important information for segmentation of cells. We assimilated them in our approach and hence we are able to gain from the advantages of level sets while circumventing its shortcoming. Validation on a data set of 4916 neural cells shows that our method is 93.3 ±0.6% accurate.

Cortical forces and CDC-42 control clustering of PAR proteins for Caenorhabditis elegans embryonic polarization

Cell polarization enables zygotes to acquire spatial asymmetry, which in turn patterns cellular and tissue axes during development. Local modification in the actomyosin cytoskeleton mediates spatial segregation of partitioning-defective (PAR) proteins at the cortex, but how mechanical changes in the cytoskeleton are transmitted to PAR proteins remains elusive. Here we uncover a role of actomyosin contractility in the remodelling of PAR proteins through cortical clustering. During embryonic polarization in Caenorhabditis elegans, actomyosin contractility and the resultant cortical tension stimulate clustering of PAR-3 at the cortex. Clustering of atypical protein kinase C (aPKC) is supported by PAR-3 clusters and is antagonized by activation of CDC-42. Cortical clustering is associated with retardation of PAR protein exchange at the cortex and with effective entrainment of advective cortical flows. Our findings delineate how cytoskeleton contractility couples the cortical clustering and long-range displacement of PAR proteins during polarization. The principles described here would apply to other pattern formation processes that rely on local modification of cortical actomyosin and PAR proteins.

Quantitative 3D analysis of complex single border cell behaviors in coordinated collective cell migration

Understanding the mechanisms of collective cell migration is crucial for cancer metastasis, wound healing and many developmental processes. Imaging a migrating cluster in vivo is feasible, but the quantification of individual cell behaviours remains challenging. We have developed an image analysis toolkit, CCMToolKit, to quantify the Drosophila border cell system. In addition to chaotic motion, previous studies reported that the migrating cells are able to migrate in a highly coordinated pattern. We quantify the rotating and running migration modes in 3D while also observing a range of intermediate behaviours. Running mode is driven by cluster external protrusions. Rotating mode is associated with cluster internal cell extensions that could not be easily characterized. Although the cluster moves slower while rotating, individual cells retain their mobility and are in fact slightly more active than in running mode. We also show that individual cells may exchange positions during migration.

Steering cell migration by alternating blebs and actin-rich protrusions

BackgroundHigh directional persistence is often assumed to enhance the efficiency of chemotactic migration. Yet, cells in vivo usually display meandering trajectories with relatively low directional persistence, and the control and function of directional persistence during cell migration in three-dimensional environments are poorly understood.ResultsHere, we use mesendoderm progenitors migrating during zebrafish gastrulation as a model system to investigate the control of directional persistence during migration in vivo. We show that progenitor cells alternate persistent run phases with tumble phases that result in cell reorientation. Runs are characterized by the formation of directed actin-rich protrusions and tumbles by enhanced blebbing. Increasing the proportion of actin-rich protrusions or blebs leads to longer or shorter run phases, respectively. Importantly, both reducing and increasing run phases result in larger spatial dispersion of the cells, indicative of reduced migration precision. A physical model quantitatively recapitulating the migratory behavior of mesendoderm progenitors indicates that the ratio of tumbling to run times, and thus the specific degree of directional persistence of migration, are critical for optimizing migration precision.ConclusionsTogether, our experiments and model provide mechanistic insight into the control of migration directionality for cells moving in three-dimensional environments that combine different protrusion types, whereby the proportion of blebs to actin-rich protrusions determines the directional persistence and precision of movement by regulating the ratio of tumbling to run times.