Correction: The Endocytic Adaptor Eps15 Controls Marginal Zone B Cell Numbers.

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220⁺ bone marrow cells, CD19⁻ thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis

Loss of the Actin Remodeler Eps8 Causes Intestinal Defects and Improved Metabolic Status in Mice

In a variety of organisms, including mammals, caloric restriction improves metabolic status and lowers the incidence of chronic-degenerative diseases, ultimately leading to increased lifespan. Here we show that knockout mice for Eps8, a regulator of actin dynamics, display reduced body weight, partial resistance to age- or diet-induced obesity, and overall improved metabolic status. Alteration in the liver gene expression profile, in behavior and metabolism point to a calorie restriction-like phenotype in Eps8 knockout mice. Additionally, and consistent with a calorie restricted metabolism, Eps8 knockout mice show increased lifespan. The metabolic alterations in Eps8 knockout mice correlated with a significant reduction in intestinal fat absorption presumably caused by a 25% reduction in intestinal microvilli length. Our findings implicate actin dynamics as a novel variable in the determination of longevity. Additionally, our observations suggest that subtle differences in energy balance can, over time, significantly affect bodyweight and metabolic status in mice

cAMP and in vivo hypoxia induce tob, ifr1, and fos expression in erythroid cells of the chick embryo

During avian embryonic development, terminal erythroid differentiation occurs in the circulation. Some of the key events, such as the induction of erythroid 2,3-bisphosphoglycerate (2,3-BPG), carbonic anhydrase (CAII), and pyrimidine 5'-nucleotidase (P5N) synthesis are oxygen dependent (Baumann R, Haller EA, Schöning U, and Weber M, Dev Biol 116: 548-551, 1986; Dragon S and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 280: R870-R878, 2001; Dragon S, Carey C, Martin K, and Baumann R, J Exp Biol 202: 2787-2795, 1999; Dragon S, Glombitza S, Götz R, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 271: R982-R989, 1996; Dragon S, Hille R, Götz R, and Baumann R, Blood 91: 3052-3058, 1998; Million D, Zillner P, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 261: R1188-R1196, 1991) in an indirect way: hypoxia stimulates the release of norepinephrine (NE)/adenosine into the circulation (Dragon et al., J Exp Biol 202: 2787-2795, 1999; Dragon et al., Am J Physiol Regulatory Integrative Comp Physiol 271: R982-R989, 1996). This leads via erythroid beta-adrenergic/adenosine A(2) receptor activation to a cAMP signal inducing several proteins in a transcription-dependent manner (Dragon et al., Am J Physiol Regulatory Integrative Comp Physiol 271: R982-R989, 1996; Dragon et al., Blood 91: 3052-3058, 1998; Glombitza S, Dragon S, Berghammer M, Pannermayr M, and Baumann R, Am J Physiol Regulatory Integrative Comp Physiol 271: R973-R981, 1996). To understand how the cAMP-dependent processes are initiated, we screened an erythroid cDNA library for cAMP-regulated genes. We detected three genes that were strongly upregulated (>5-fold) by cAMP in definitive and primitive red blood cells. They are homologous to the mammalian Tob, Ifr1, and Fos proteins. In addition, the genes are induced in the intact embryo during short-term hypoxia. Because the genes are regulators of proliferation and differentiation in other cell types, we suggest that cAMP might promote general differentiating processes in erythroid cells, thereby allowing adaptive modulation of the latest steps of erythroid differentiation during developmental hypoxia

The eps8 Family of Proteins Links Growth Factor Stimulation to Actin Reorganization Generating Functional Redundancy in the Ras/Rac Pathway

Sos-1, a guanine nucleotide exchange factor (GEF), eps8 and Abi1, two signaling proteins, and the lipid kinase phosphoinositide 3-kinase (PI3-K), assemble in a multimolecular complex required for Rac activation leading to actin cytoskeletal remodeling. Consistently, eps8 –/– fibroblasts fail to form membrane ruffles in response to growth factor stimulation. Surprisingly, eps8 null mice are healthy, fertile, and display no overt phenotype, suggesting the existence of functional redundancy within this pathway. Here, we describe the identification and characterization of a family of eps8-related proteins, comprising three novel gene products, named eps8L1, eps8L2, and eps8L3. Eps8Ls display collinear topology and 27–42% identity to eps8. Similarly to eps8, eps8Ls interact with Abi1 and Sos-1; however, only eps8L1 and eps8L2 activate the Rac-GEF activity of Sos-1, and bind to actin in vivo. Consistently, eps8L1 and eps8L2, but not eps8L3, localize to PDGF-induced, F-actin–rich ruffles and restore receptor tyrosine kinase (RTK)-mediated actin remodeling when expressed in eps8 –/– fibroblasts. Thus, the eps8Ls define a novel family of proteins responsible for functional redundancy in the RTK-activated signaling pathway leading to actin remodeling. Finally, the patterns of expression of eps8 and eps8L2 in mice are remarkably overlapping, thus providing a likely explanation for the lack of overt phenotype in eps8 null mice

Increased Ethanol Resistance and Consumption in Eps8 Knockout Mice Correlates with Altered Actin Dynamics

Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-Daspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol

Eps8 regulates hair bundle length and functional maturation of mammalian auditory hair cells

Hair cells of the mammalian cochlea are specialized for the dynamic coding of sound stimuli. The transduction of sound waves into electrical signals depends upon mechanosensitive hair bundles that project from the cell's apical surface. Each stereocilium within a hair bundle is composed of uniformly polarized and tightly packed actin filaments. Several stereociliary proteins have been shown to be associated with hair bundle development and function and are known to cause deafness in mice and humans when mutated. The growth of the stereociliar actin core is dynamically regulated at the actin filament barbed ends in the stereociliary tip. We show that Eps8, a protein with actin binding, bundling, and barbed-end capping activities in other systems, is a novel component of the hair bundle. Eps8 is localized predominantly at the tip of the stereocilia and is essential for their normal elongation and function. Moreover, we have found that Eps8 knockout mice are profoundly deaf and that IHCs, but not OHCs, fail to mature into fully functional sensory receptors. We propose that Eps8 directly regulates stereocilia growth in hair cells and also plays a crucial role in the physiological maturation of mammalian cochlear IHCs. Together, our results indicate that Eps8 is critical in coordinating the development and functionality of mammalian auditory hair cells

The endocytic adaptor Eps15 controls marginal zone B cell numbers.

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220⁺ bone marrow cells, CD19⁻ thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis

Eps15-KO mice show increased marginal zone B cell numbers.

<p><b>A.</b> Dot plots of bone marrow cells from 4-month-old Eps15-WT and Eps15-KO mice stained for B220 and IgM to identify pre−/pro-B cells (B220⁺IgM⁻), immature (B220⁺IgM⁺) and mature (B220^highIgM⁺) B cells. Pre−/pro-B cells were further analyzed for expression of CD43 to identify pro- (CD43⁺) and pre- (CD43⁻) B cells. <b>B.–C.</b> Dot plots depicting the total number of mature (B220⁺⁺), immature (Imm.) and pre−/pro- and B cells (<b>B</b>) and pre- and pro-B cells (<b>C</b>) in the bone marrow of Eps15-WT (WT, white symbols, n = 5) and Eps15-KO (KO, black symbols, n = 6) mice. <b>D.</b> Top panel: dot plot of thymocytes from 2-month Eps15-WT and Eps15-KO mice stained for CD19 and TCRβ to identify B and T cells, respectively. Middle panel: dot plots for thymocytes from 4-month-old Eps15-WT and Eps15-KO mice staind for CD4 and CD8 to identify thymocyte subpopulations. Bottom panel: the CD4/CD8 double negative population (DN) was further analyzed for immature thymocytes using CD44 and CD25. <b>E.–G.</b> Dot plots depicting the total number of the following cell populations in the thymi of Eps15-WT (WT, white symbols) and Eps15-KO (KO, black symbols) mice: (<b>E</b>) TCRβ⁺ and CD19⁺ cells (n = 3), (<b>F</b>) DN (double negative, CD4⁻CD8⁻), DP (double positive, CD4⁺CD8⁺), CD4⁺ and CD8⁺ (n = 4) and (<b>G</b>) of DN1 (CD44⁺CD25⁻), DN2 (CD44⁺CD25⁺), DN3 (CD44⁻CD25⁻) cells (n = 4). <b>H</b>. Dot plot of peritoneal B cells from 2–4 month old Eps15-WT and Eps15-KO mice stained for CD19 and B220 gated for CD19⁺B220^low B1 and CD19⁺B220^high B2 B cells. B1 B cells were further stained and gated for CD5 to identify CD5⁺ B1a and CD5⁻ B1b B cells. <b>I.–J.</b> Dot plots depicting the percentage of B1 and B2 (I) and B1a and B1b (J) B cells in the peritoneum of Eps15-WT (WT, white symbols) and Eps15-KO (KO, black symbols) mice (n = 8). <b>K.</b> Dot plot of splenocytes from Eps15-WT and Eps15-KO mice stained for CD19 and gated for CD21 and CD23 to identify Fo B (CD19⁺/CD21⁺/CD23⁺) and MZ B (CD19⁺/CD21⁺/CD23⁻) cells. <b>L.</b> Dot plots depicting the total number of CD19+, Fo B and MZ B cells in the spleens of Eps15-WT (WT, white symbols) and Eps15-KO (KO, black symbols) mice (n = 5). Values are depicted as mean±standard error mean. Statistical significance was assessed using Student’s t-test and significant differences are indicated as * = p<0.05.</p

FACS analysis of the cellular populations present in the spleen three months after competitive bone marrow transplantation.

<p>Distribution of CD45.1+ and CD45.2+ cells in the spleen of recipient mice 3 months after bone marrow transplantation. CD45.1+ WT and CD45.2+ WT or CD45.2+ KO donor cells were mixed at a 1∶1 ratio prior to injection into recipient CD45.1+ mice. The percentage of total cells gated and the percentage of CD45.1+ or CD45.2+ cells for any given gate are shown. Significance was assessed using Student’s t-test and p-values are indicated as *p<0.05, **p<0.01, ***p<0.001.</p