Sarah E. Smith

Differentiation-Defective Stem Cells Outcompete Normal Stem Cells for Niche Occupancy in the Drosophila Ovary

Rapid progress has recently been made regarding how the niche controls stem cell function, but little is yet known about how stem cells in the same niche interact with one another. In this study, we show that differentiation-defective Drosophila ovarian germline stem cells (GSCs) can outcompete normal ones for niche occupancy in a cadherin-dependent manner. The differentiation-defective bam or bgcn mutant GSCs invade the niche space of neighboring wild-type GSCs and gradually push them out of the niche by upregulating E-cadherin expression. Furthermore, the bam/bgcn-mediated GSC competition requires E-cadherin and normal GSC division, but not the self-renewal-promoting BMP niche signal, while different E-cadherin levels can sufficiently stimulate GSC competition. Therefore, we propose that GSCs have a competitive relationship for niche occupancy, which may serve as a quality control mechanism to ensure that accidentally differentiated stem cells are rapidly removed from the niche and replaced by functional ones.

Symmetry Breaking in the Life Cycle of the Budding Yeast

The budding yeast Saccharomyces cerevisiae has been an invaluable model system for the study of the establishment of cellular asymmetry and growth polarity in response to specific physiological cues. A large body of experimental observations has shown that yeast cells are able to break symmetry and establish polarity through two coupled and partially redundant intrinsic mechanisms, even in the absence of any pre-existing external asymmetry. One of these mechanisms is dependent upon interplay between the actin cytoskeleton and the Rho family GTPase Cdc42, whereas the other relies on a Cdc42 GTPase signaling network. Integral to these mechanisms appear to be positive feedback loops capable of amplifying small and stochastic asymmetries. Spatial cues, such as bud scars and pheromone gradients, orient cell polarity by modulating the regulation of the Cdc42 GTPase cycle, thereby biasing the site of asymmetry amplification.

Arrestin 3 Mediates Endocytosis of CCR7 following Ligation of CCL19 but Not CCL21

Internalization of ligand bound G protein-coupled receptors, an important cellular function that mediates receptor desensitization, takes place via distinct pathways, which are often unique for each receptor. The C-C chemokine receptor (CCR7) G protein-coupled receptor is expressed on naive T cells, dendritic cells, and NK cells and has two endogenous ligands, CCL19 and CCL21. Following binding of CCL21, 21 ± 4% of CCR7 is internalized in the HuT 78 human T cell lymphoma line, while 76 ± 8% of CCR7 is internalized upon binding to CCL19. To determine whether arrestins mediated differential internalization of CCR7/CCL19 vs CCR7/CCL21, we used small interfering RNA (siRNA) to knock down expression of arrestin 2 or arrestin 3 in HuT 78 cells. Independent of arrestin 2 or arrestin 3 expression, CCR7/CCL21 internalized. In contrast, following depletion of arrestin 3, CCR7/CCL19 failed to internalize. To examine the consequence of complete loss of both arrestin 2 and arrestin 3 on CCL19/CCR7 internalization, we examined CCR7 internalization in arrestin 2−/−/arrestin 3−/− murine embryonic fibroblasts. Only reconstitution with arrestin 3-GFP but not arrestin 2-GFP rescued internalization of CCR7/CCL19. Loss of arrestin 2 or arrestin 3 blocked migration to CCL19 but had no effect on migration to CCL21. Using immunofluorescence microscopy, we found that arrestins do not cluster at the membrane with CCR7 following ligand binding but cap with CCR7 during receptor internalization. These are the first studies that define a role for arrestin 3 in the internalization of a chemokine receptor following binding of one but not both endogenous ligands.

Neural Crest Invasion is a Spatially-Ordered Progression Into the Head with Higher Cell Proliferation at the Migratory Front as Revealed by the Photoactivatable Protein, KikGR

Neural crest cell (NCC) invasion is a complex sculpting of individual cells into organized migratory streams that lead to organ development along the vertebrate axis. Key to our understanding of how molecular mechanisms modulate the NCC migratory pattern is information about cell behaviors, yet it has been challenging to selectively mark and analyze migratory NCCs in a living embryo. Here, we apply an innovative in vivo strategy to investigate chick NCC behaviors within the rhombomere 4 (r4) migratory stream by combining photoactivation of KikGR and confocal time-lapse analysis of H2B-mRFP1 transfected NCCs. We find that the spatial order of r4 NCC emergence translates into a distal-to-proximal invasion of the 2nd branchial arch. Lead and trailing NCCs display similar average cell speeds and directionalities. Surprisingly, we find that lead NCCs proliferate along the migratory route and grow to outnumber trailing NCCs by nearly 3 to 1. A simple, cell-based computational model reproduces the r4 NCC migratory pattern and predicts the invasion order can be disrupted by slower, less directional lead cells or by environmental noise. Our results suggest a model in which NCC behaviors maintain a spatially-ordered invasion of the branchial arches with differences in cell proliferation between the migratory front and trailing NCCs.

Non-uniform membrane diffusion enables steady-state cell polarization via vesicular trafficking

Actin-based vesicular trafficking of Cdc42, leading to a polarized concentration of the GTPase, has been implicated in cell polarization, but it was recently debated whether this mechanism allows stable maintenance of cell polarity. Here we show that endocytosis and exocytosis are spatially segregated in the polar plasma membrane, with sites of exocytosis correlating with microdomains of higher concentration and slower diffusion of Cdc42 compared with surrounding regions. Numerical simulations using experimentally obtained diffusion coefficients and trafficking geometry revealed that non-uniform membrane diffusion of Cdc42 in fact enables temporally sustained cell polarity. We show further that phosphatidylserine, a phospholipid recently found to be crucial for cell polarity, is enriched in Cdc42 microdomains. Weakening a potential interaction between phosphatidylserine and Cdc42 enhances Cdc42 diffusion in the microdomains but impedes the strength of polarization. These findings demonstrate a critical role for membrane microdomains in vesicular trafficking-mediated cell polarity.

Independence of symmetry breaking on Bem1-mediated autocatalytic activation of Cdc42

Rather than acting directly on Cdc42, Bem1 works in concert with the Cdc42 binding partner Rdi1 to relocalize Cdc42 to the cytosol during symmetry breaking in the absence of an intact actin cytoskeleton.

Structured illumination with particle averaging reveals novel roles for yeast centrosome components during duplication

Duplication of the yeast centrosome (called the spindle pole body, SPB) is thought to occur through a series of discrete steps that culminate in insertion of the new SPB into the nuclear envelope (NE). To better understand this process, we developed a novel two-color structured illumination microscopy with single-particle averaging (SPA-SIM) approach to study the localization of all 18 SPB components during duplication using endogenously expressed fluorescent protein derivatives. The increased resolution and quantitative intensity information obtained using this method allowed us to demonstrate that SPB duplication begins by formation of an asymmetric Sfi1 filament at mitotic exit followed by Mps1-dependent assembly of a Spc29- and Spc42-dependent complex at its tip. Our observation that proteins involved in membrane insertion, such as Mps2, Bbp1, and Ndc1, also accumulate at the new SPB early in duplication suggests that SPB assembly and NE insertion are coupled events during SPB formation in wild-type cells. DOI: http://dx.doi.org/10.7554/eLife.08586.001

Analysis of membrane proteins localizing to the inner nuclear envelope in living cells

Few tools exist to examine the inner nuclear membrane (INM) in living cells, and the INM-specific proteome is poorly characterized. Smoyer et al. combine a split-GFP screen and fluorescence correlation spectroscopy analysis to identify membrane proteins that access the INM and study INM-specific interactions.

Nanoscale architecture of the Schizosaccharomyces pombe contractile ring

The contractile ring is a complex molecular apparatus which physically divides many eukaryotic cells. Despite knowledge of its protein composition, the molecular architecture of the ring is not known. Here we have applied super-resolution microscopy and FRET to determine the nanoscale spatial organization of Schizosaccharomyces pombe contractile ring components relative to the plasma membrane. Similar to other membrane-tethered actin structures, we find proteins localize in specific layers relative to the membrane. The most membrane-proximal layer (0–80 nm) is composed of membrane-binding scaffolds, formin, and the tail of the essential myosin-II. An intermediate layer (80–160 nm) consists of a network of cytokinesis accessory proteins as well as multiple signaling components which influence cell division. Farthest from the membrane (160–350 nm) we find F-actin, the motor domains of myosins, and a major F-actin crosslinker. Circumferentially within the ring, multiple proteins proximal to the membrane form clusters of different sizes, while components farther from the membrane are uniformly distributed. This comprehensive organizational map provides a framework for understanding contractile ring function.

Aneuploidy as a cause of impaired chromatin silencing and mating-type specification in budding yeast

Aneuploidy and epigenetic alterations have long been associated with carcinogenesis, but it was unknown whether aneuploidy could disrupt the epigenetic states required for cellular differentiation. In this study, we found that ~3% of random aneuploid karyotypes in yeast disrupt the stable inheritance of silenced chromatin during cell proliferation. Karyotype analysis revealed that this phenotype was significantly correlated with gains of chromosomes III and X. Chromosome X disomy alone was sufficient to disrupt chromatin silencing and yeast mating-type identity as indicated by a lack of growth response to pheromone. The silencing defect was not limited to cryptic mating type loci and was associated with broad changes in histone modifications and chromatin localization of Sir2 histone deacetylase. The chromatin-silencing defect of disome X can be partially recapitulated by an extra copy of several genes on chromosome X. These results suggest that aneuploidy can directly cause epigenetic instability and disrupt cellular differentiation.