Stephanie Pellegrin

Actin stress fibres.

Animal cell movement is effected through a combination of protrusive and contractile events. Non-muscle cells contain stress fibres – bundles of actomyosin that are the major mediators of cell contraction and that can be compared to the highly organised actomyosin arrays of muscle cells. Recent studies have defined regulatory mechanisms that control stress fibre formation, placing the ROCK protein kinase at the centre of a complex signalling network controlling actomyosin contractility and stress fibre assembly. As we uncover the details of stress fibre construction, it is becoming clear that different categories of stress fibres exist. Some of these structures are less suited for cell motility and more suited to static contraction. In keeping with this, many specialised contractile cell types use stress fibres to remodel tissues and extracellular matrix.

The majority of the in vitro erythroid expansion potential resides in CD34- cells, outweighing the contribution of CD34+ cells and significantly increasing the erythroblast yield from peripheral blood samples

The study of human erythropoiesis in health and disease requires a robust culture system that consistently and reliably generates large numbers of immature erythroblasts that can be induced to differentiate synchronously. We describe a culture method modified from Leberbauer et al. (2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34+ cells. This pure population of immature erythroblasts can be expanded to obtain 4×108 erythroblasts from 1×108 PBMC after 13–14 days in culture. Upon synchronized differentiation, high levels of enucleation (80–90%) and low levels of cell death (<10%) are achieved. We compared the yield of erythroblasts obtained from PBMC, CD34+ cells or PBMC depleted of CD34+ cells and show that CD34− cells represent the most significant early erythroid progenitor population. This culture system may be particularly useful for investigating the pathophysiology of anemic patients where only small blood volumes are available.

Synergistic roles for the Map and Tir effector molecules in mediating uptake of enteropathogenic Escherichia coli (EPEC) into non-phagocytic cells.

Enteropathogenic Escherichia coli (EPEC) are a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens. Enteropathogenic Escherichia coli encode a type III secretion system (TTSS) to transfer effector proteins into host cells, a process which is essential for virulence. In addition to generation of A/E lesions, the TTSS is also implicated in the ability of EPEC to invade cultured cells but the effector proteins responsible for promoting invasion have not been identified. In this paper we confirm the requirement of TTSS in EPEC invasion and demonstrate important roles for the Map and Tir effector molecules. Whereas in trans expression of Tir in the tir mutant restored invasion to wild‐type levels, similar complementation of the map mutation by in trans expression of Map results in a hyperinvasive phenotype. The Map effector protein has two distinct functions within host cells, mediating Cdc42‐dependent filopodia formation and targeting mitochondria to elicit dysfunction. The former function appears to be related to Maps ability to promote invasion as this was inhibited by interference with Cdc42 signalling. Conversely, Map targeting to mitochondria is not necessary for invasion. Promotion of EPEC invasion by Tir appears to involve interaction with intimin but is independent of pedestal formation, and intimin–Tir interaction is neither necessary nor sufficient for invasion. Comparison of the invasiveness of strains lacking Tir and/or Map with wild‐type or mutant strains expressing the effectors in trans provides evidence that Map and Tir stimulate invasion by synergistic mechanisms. This synergism, which is in stark contrast to the antagonistic actions of Map and Tir in regulating filopodia and pedestal formation, further illustrates the complex interplay between EPEC effectors.

Critical band 3 multiprotein complex interactions establish early during human erythropoiesis

Band 3, the major anion transport protein of human erythrocytes, forms the core of a multiprotein complex in the erythrocyte membrane. Here we studied the spatiotemporal mechanisms of band 3 multiprotein complex assembly during erythropoiesis. Significant pools of intracellular band 3 and Rh-associated glycoprotein (RhAG) were found in the basophilic erythroblast. These intracellular pools decreased in the polychromatic erythroblast, whereas surface expression increased and were lowest in the orthochromatic erythroblast and reticulocytes. Protease treatment of intact cells to remove extracellular epitopes recognized by antibodies to band 3 and RhAG was used to study surface delivery kinetics and intracellular complex composition from the proerythroblast stage to the enucleated reticulocyte. Newly synthesized band 3 and protein 4.2 interact initially in the early stages of the secretory pathway and are found associated at the plasma membrane from the basophilic stage of erythropoiesis. Although we could successfully coimmunoprecipitate Rh with RhAG from plasma membrane pools at a similar stage, no intracellular interaction between these proteins was detectable. Knockdown of RhAG during early erythropoiesis was accompanied by a concomitant drop in membrane expression of Rh polypeptides. These data are consistent with assembly of major components of the band 3 macrocomplex at an early stage during erythropoiesis.

The small GTPase Rif is an alternative trigger for the formation of actin stress fibers in epithelial cells

Actin stress fibers are fundamental components of the actin cytoskeleton that produce contractile force in non-muscle cells. The formation of stress fibers is controlled by the small GTPase RhoA and two highly related proteins, RhoB and RhoC. Together, this subgroup of actin-regulatory proteins represents the canonical pathway of stress-fiber formation. Here, we show that the Rif GTPase is an alternative trigger of stress-fiber formation in epithelial cells. Rif is distantly related to RhoA; however, we show that the two proteins share a common downstream partner in stress-fiber formation – the Diaphanous-related formin mDia1. Rif-induced stress fibers also depend on the activity of the ROCK protein kinase. Unlike RhoA, Rif does not raise ROCK activity in cells, instead Rif appears to regulate the localization of myosin light chain phosphorylation. This study establishes Rif as a general regulator of Diaphanous-related formins and shows how non-classical Rho family members can access classical Rho pathways to create new signaling interfaces in cytoskeletal regulation.

Investigating the key membrane protein changes during in vitro erythropoiesis of protein 4.2 (−) cells (mutations Chartres 1 and 2)

Background Protein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47, CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex. Design and Methods We used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis. Results We report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47, higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(−) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression, arguing against a direct reciprocal relationship. Conclusions We have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated, in part, by increased CD44-cytoskeleton binding.

Characteristic phenotypes associated with congenital dyserythropoietic anemia (type II) manifest at different stages of erythropoiesis.

Congenital dyserythropoietic anemia type II is an autosomally recessive form of hereditary anemia caused by SEC23B gene mutations. Patients exhibit characteristic phenotypes including multinucleate erythroblasts, erythrocytes with hypoglycosylated membrane proteins and an apparent double plasma membrane. Despite ubiquitous expression of SEC23B, the effects of mutations in this gene are confined to the erythroid lineage and the basis of this erythroid specificity remains to be defined. In addition, little is known regarding the stage at which the disparate phenotypes of this disease manifest during erythropoiesis. We employ an in vitro culture system to monitor the appearance of the defining phenotypes associated with congenital dyserythropoietic anemia type II during terminal differentiation of erythroblasts derived from small volumes of patient peripheral blood. Membrane protein hypoglycosylation was detected by the basophilic stage, preceding the onset of multinuclearity in orthochromatic erythroblasts that occurs coincident with the loss of secretory pathway proteins including SEC23A during erythropoiesis. Endoplasmic reticulum remnants were observed in nascent reticulocytes of both diseased and healthy donor cultures but were lost upon further maturation of normal reticulocytes, implicating a defect of ER clearance during reticulocyte maturation in congenital dyserythropoietic anemia type II. We also demonstrate distinct isoform and species-specific expression profiles of SEC23 during terminal erythroid differentiation and identify a prolonged expression of SEC23A in murine erythropoiesis compared to humans. We propose that SEC23A is able to compensate for the absence of SEC23B in mouse erythroblasts, providing a basis for the absence of phenotype within the erythroid lineage of a recently described SEC23B knockout mouse.

Rho GTPase Activation Assays

The Rho GTPase family of signaling proteins controls a wide range of highly dynamic cellular processes. Activation of Rho GTPases can be investigated and quantified in cell extracts using so‐called pull‐down assays. Proteins that bind specifically to the activated form of the Rho GTPase are used to capture it onto a bead support. Western blotting of the captured samples with specific antibodies then allows for quantification of the level of Rho GTPase activation in the sample. This unit describes the techniques for preparing the reagents required for assays of RhoA, Rac, and Cdc42 and gives practical tips for the successful application of the assay in a range of situations. Curr. Protoc. Cell Biol. 38:14.8.1‐14.8.19.

Scanning electron microscopy of cell surface morphology.

The surface of metazoan cells is a landscape not clearly visualized by light microscopy. Many cells elaborate protrusive structures such as microvilli, filopodia, lamellipodia, and surface ruffles that play important roles in the interaction between the cell and its environment. The high resolution of scanning electron microscopy makes it an ideal technique for studies of the cell surface; however, preservation of fine surface structure can be problematic. Here we highlight the critical factors in sample preparation to ensure optimal high‐resolution imaging of the surface of mammalian cells and tissues. Curr. Protoc. Cell Biol. 37:4.17.1‐4.17.13.

Severe Ankyrin-R deficiency results in impaired surface retention and lysosomal degradation of RhAG in human erythroblasts

Ankyrin-R provides a key link between band 3 and the spectrin cytoskeleton that helps to maintain the highly specialized erythrocyte biconcave shape. Ankyrin deficiency results in fragile spherocytic erythrocytes with reduced band 3 and protein 4.2 expression. We use in vitro differentiation of erythroblasts transduced with shRNAs targeting ANK1 to generate erythroblasts and reticulocytes with a novel ankyrin-R ‘near null’ human phenotype with less than 5% of normal ankyrin expression. Using this model, we demonstrate that absence of ankyrin negatively impacts the reticulocyte expression of a variety of proteins, including band 3, glycophorin A, spectrin, adducin and, more strikingly, protein 4.2, CD44, CD47 and Rh/RhAG. Loss of band 3, which fails to form tetrameric complexes in the absence of ankyrin, alongside GPA, occurs due to reduced retention within the reticulocyte membrane during erythroblast enucleation. However, loss of RhAG is temporally and mechanistically distinct, occurring predominantly as a result of instability at the plasma membrane and lysosomal degradation prior to enucleation. Loss of Rh/RhAG was identified as common to erythrocytes with naturally occurring ankyrin deficiency and demonstrated to occur prior to enucleation in cultures of erythroblasts from a hereditary spherocytosis patient with severe ankyrin deficiency but not in those exhibiting milder reductions in expression. The identification of prominently reduced surface expression of Rh/RhAG in combination with direct evaluation of ankyrin expression using flow cytometry provides an efficient and rapid approach for the categorization of hereditary spherocytosis arising from ankyrin deficiency.