Diana M. Gilligan

Characterization of glycolytic enzyme interactions with murine erythrocyte membranes in wild-type and membrane protein knockout mice

Previous research has shown that glycolytic enzymes (GEs) exist as multienzyme complexes on the inner surface of human erythrocyte membranes. Because GE binding sites have been mapped to sequences on the membrane protein, band 3, that are not conserved in other mammalian homologs, the question arose whether GEs can organize into complexes on other mammalian erythrocyte membranes. To address this, murine erythrocytes were stained with antibodies to glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, lactate dehydrogenase, and pyruvate kinase and analyzed by confocal microscopy. GEs were found to localize to the membrane in oxygenated erythrocytes but redistributed to the cytoplasm upon deoxygenation, as seen in human erythrocytes. To identify membrane proteins involved in GE assembly, erythrocytes from mice lacking each of the major erythrocyte membrane proteins were examined for GE localization. GEs from band 3 knockout mice were not membrane associated but distributed throughout the cytoplasm, regardless of erythrocyte oxygenation state. In contrast, erythrocytes from mice lacking alpha-spectrin, ankyrin, protein 4.2, protein 4.1, beta-adducin, or dematin headpiece exhibited GEs bound to the membrane. These data suggest that oxygenation-dependent assembly of GEs on the membrane could be a general phenomenon of mammalian erythrocytes and that stability of these interactions depends primarily on band 3.

Impaired Synaptic Plasticity and Learning in Mice Lacking β-Adducin, an Actin-Regulating Protein

The adducin family of proteins interacts with the actin cytoskeleton and the plasma membrane in a calcium- and cAMP-dependent manner. Thus, adducins may be involved in changes in cytoskeletal organization resulting from synaptic stimulation. β-Adducin knock-out mice were examined in physiological and behavioral paradigms related to synaptic plasticity to elucidate the role the adducin family plays in processes underlying learning and memory. In situ hybridization for α- and β-adducin demonstrates that these mRNAs are found throughout the brain, with high levels of expression in the hippocampus. Schaffer collateral-CA1 tetanic long-term potentiation decayed rapidly in acute hippocampal slices from β-adducin knock-out mice, although baseline spine morphology and postsynaptic density were normal. Interestingly, the input-output relationship was significantly increased in hippocampal slices from β-adducin knock-out mice. Furthermore, β-adducin knock-out mice were impaired in performance of fear conditioning and the water maze paradigm. The current results indicate that β-adducin may play an important role in the cellular mechanisms underlying activity-dependent synaptic plasticity associated with learning and memory.

Targeted deletion of α-adducin results in absent β- and γ-adducin, compensated hemolytic anemia, and lethal hydrocephalus in mice

In the red blood cell (RBC), adducin is present primarily as tetramers of alpha- and beta-subunits at spectrin-actin junctions, or junctional complexes. Mouse RBCs also contain small amounts of gamma-adducin. Platelets contain alpha- and gamma-adducin only. Adducin functions as a barbed-end actin capping protein to regulate actin filament length and recruits spectrin to the ends of actin filaments. To further define adducins role in vivo, we generated alpha-adducin knockout mice. alpha-Adducin is absent in all tissues examined in homozygous null mice. In RBCs, beta- and gamma-adducin are also absent, indicating that alpha-adducin is the limiting subunit in tetramer formation at the spectrin-actin junction. Similarly, gamma-adducin is absent in alpha-null platelets. alpha-Adducin-null mice display compensated hemolytic anemia with features characteristic of RBCs in hereditary spherocytosis (HS), including spherocytes with significant loss of surface area, decreased mean corpuscular volume (MCV), cell dehydration, and increased osmotic fragility. Platelets maintain their normal discoid shape, and bleeding times are normal. alpha-Adducin-null mice show growth retardation at birth and throughout adulthood. Approximately 50% develop lethal communicating hydrocephalus with striking dilation of the lateral, third, and fourth ventricles. These data indicate that adducin plays a role in RBC membrane stability and in cerebrospinal fluid homeostasis.

Combined deletion of mouse dematin-headpiece and β-adducin exerts a novel effect on the spectrin-actin junctions leading to erythrocyte fragility and hemolytic anemia

Dematin and adducin are actin-binding proteins of the erythrocyte “junctional complex.” Individually, they exert modest effects on erythrocyte shape and membrane stability, and their homologues are expressed widely in non-erythroid cells. Here we report generation and characterization of double knock-out mice lacking β-adducin and the headpiece domain of dematin. The combined mutations result in altered erythrocyte morphology, increased membrane instability, and severe hemolysis. Peripheral blood analysis shows evidence of severe hemolytic anemia with reduced number of erythrocytes/hematocrit/hemoglobin and an ∼12-fold increase in the number of circulating reticulocytes. The presence of a variety of misshapen and fragmented erythrocytes correlates with increased osmotic fragility and reduced in vivo life span. Despite the apparently normal protein composition of the mutant erythrocyte membrane, the retention of the spectrin-actin complex in the membrane under low ionic strength conditions is significantly reduced by the double mutation. Atomic force microscopy reveals an increase in grain size and a decrease in filament number of the mutant membrane cytoskeleton, although the volume parameter is similar to wild type erythrocytes. Aggregated, disassembled, and irregular features are visualized in the mutant membrane, consistent with the presence of large protein aggregates. Importantly, purified dematin binds to the stripped inside-out vesicles in a saturable manner, and dematin-membrane binding is abolished upon pretreatment of membrane vesicles with trypsin. Together, these results reveal an essential role of dematin and adducin in the maintenance of erythrocyte shape and membrane stability, and they suggest that the dematin-membrane interaction could link the junctional complex to the plasma membrane in erythroid cells.

In vivo inactivation of MASTL kinase results in thrombocytopenia.

OBJECTIVE A missense mutation in the microtubule-associated serine/threonine-like kinase gene (MASTL, FLJ14813) on human chromosome 10 was previously linked to a novel form of autosomal dominant inherited thrombocytopenia in a single pedigree. The mutation results in an amino acid change from glutamic acid at position 167 to aspartic acid and segregates perfectly with thrombocytopenic individuals within this extended family. The phenotype is characterized by mild thrombocytopenia with an average platelet count of 60,000 platelets per microliter of blood. We wanted to determine the expression and localization of MASTL, as well as its role in developing thrombocytes using an in vivo model system. MATERIALS AND METHODS Northern blot analysis allowed us to examine expression patterns. Morpholino knockdown assays in zebrafish (Danio rerio) were employed to determine in vivo contribution to thrombocyte development. Transient expression in baby hamster kidney cells resulted in localization of both the wild-type and E167D mutant forms of MASTL kinase to the nucleus. RESULTS Northern blot analysis indicates that MASTL messenger RNA is restricted in its expression to hematopoietic and cancer cell lines. A transient knockdown of MASTL in zebrafish results in deficiency of circulating thrombocytes. Transient expression of recombinant MASTL kinase in vitro demonstrates localization to the nucleus. CONCLUSIONS Functional studies presented here demonstrate a direct relationship between transient knockdown of MASTL kinase gene expression and reduction of circulating thrombocytes in zebrafish. This transient knockdown of MASTL in zebrafish correlates with a decrease in the expression of the thrombopoietin receptor, c-mpl, and the CD41 platelet adhesion protein, GpIIb, but has no effect on essential housekeeping zebrafish gene, EF1alpha.

Dynamin 3 participates in the growth and development of megakaryocytes

High-density oligonucleotide microarrays were used to compare gene expression profiles from uncultured CD34+/CD38lo cells and culture-derived megakaryocytes (MKs). As previously published, three replicate microarray data sets from three different sources of organ donor marrow were analyzed using the software program Rosetta Resolver. After setting a stringent p value of <or=0.001 with a fold change cutoff of three or more in expression level, dynamin 3 (DNM3) was identified to be differentially expressed during the course of MK development with a mean fold-change of 8.2+/-2.1 (mean+/-standard deviation). DNM3 is a member of a family of mechanochemical enzymes (DNM1, DNM2, and DNM3) known for their participation in membrane dynamics by hydrolyzing nucleotides to link cellular membranes to the actin cytoskeleton. Real-time quantitative polymerase chain reaction confirmed that DNM3 increased by 20.7-+/-3.4-fold (n=4, p=0.09) during megakaryocytopoiesis and Western blot analysis showed that DNM3 protein was expressed in human MKs. Confocal microscopy revealed that DNM3 was distributed diffusely throughout the cytoplasm of MKs with a punctate appearance in proplatelet processes. Immunogold electron microscopy also showed that DNM3 is widely distributed in the cytoplasm of MKs, with no apparent localization to specific organelles. The open reading frame of DNM3 was cloned from culture-derived human MKs and determined to be 100% identical to the protein encoded by the DNM3 transcript variant ENST00000367731 published in the Ensemble database. Overexpression of DNM3 in umbilical cord blood CD34+ cells resulted in an increase in total nucleated cells, an amplification of total colony-forming cells and colony-forming unit-megakaryocytes, and a concomitant increase in the expression of nuclear factor erythroid 2 (NF-E2) and beta-tubulin. Together these findings provide the first evidence that a member of the dynamin family of mechanochemical enzymes is present in human MKs and indicate that DNM3 is an excellent candidate for playing an important role in mediating cytoskeleton and membrane changes that occur during MK/platelet development.

The mouse adducin gene family: alternative splicing and chromosomal localization.

Abstract. Mouse cDNA sequences encoding α, β, and γ adducins were cloned from a mouse reticulocyte cDNA library. The purified clones contain alternatively spliced exons from all three adducin genes. In the case of α and β, the inclusion of the alternatively spliced exons results in truncated polypeptide isoforms (called α-2 and β-2). The mouse predicted amino acid sequences are compared with published rat and human sequences. For completion of this comparison, cDNA encoding the rat β-1 carboxy terminus was cloned by PCR. The carboxy terminal region containing MARCKS homology, calmodulin-binding region-2, and spectrin-actin-binding site, is conserved among α-1, β-1, and γ-1 isoforms in mouse, rat, and humans. We also report here the localization of the gene encoding γ adducin (Add3) to murine Chr 19, in a region that shows conserved synteny with human Chr 10.

Distinct functional effects for dynamin 3 during megakaryocytopoiesis.

Dynamin 3 (DNM3) is a member of a family of motor proteins that participate in a number of membrane rearrangements such as cytokinesis, budding of transport vesicles, phagocytosis, and cell motility. Recently, DNM3 was implicated as having a role in megakaryocyte (MK) development. To further investigate the functional role of DNM3 during megakaryocytopoiesis, we introduced sequence-specific short hairpin RNAs (shRNAs) into developing MKs. The results showed that knockdown of DNM3 inhibited a stage of MK development that involved progenitor amplification. This was evident by significant decreases in the number of colony forming unit-megakaryocytes, the total number of nucleated cells, and the number of CD41(+) and CD61(+) MKs produced in culture. Using a styrl membrane dye to quantify the demarcation membrane system (DMS) of terminally differentiated MKs, we found that DNM3 co-localized with the DMS and that DNM3 lentiviral shRNAs precluded the formation of the DMS. Knockdown of dynamin 3 in murine MKs also caused a decrease in the number of morphologically large MKs and the overall size of large MKs was decreased relative to controls. MK protein lysates were used in overlay blots to show that both DNM3 and actin bind to nonmuscle myosin IIA (MYH9). Consistent with these observations, immunofluorescence studies of MKs and proplatelet processes showed co-localization of DNM3 with MYH9. Overall, these studies demonstrate that DNM3 not only participates in MK progenitor amplification, but is also involved in cytoplasmic enlargement and the formation of the DMS.

Effect of recombinant human megakaryocyte growth and development factor coupled with polyethylene glycol on the platelet storage lesion

BACKGROUND: Platelet production is regulated by a thrombopoietic growth factor (Mpl ligand).The receptor for this platelet growth factor (Mpl) is expressed on the platelet surface membrane. A recombinant thrombopoietic cytokine, recombinant human megakaryocyte growth and development factor coupled with polyethylene glycol (PEG‐rHuMGDF), was added to apheresis platelets in vitro to determine whether Mpl ligand–receptor binding produced any beneficial or adverse effect on the development of the platelet storage lesion during 5 days of storage.

Exclusion of the stomatin, α-adducin and β-adducin loci in a large kindred with dehydrated hereditary stomatocytosis

Defects in stomatin, α‐and β‐adducin have been implicated in erythrocyte disorders of cation permeability. We performed linkage analysis of the genetic loci for these proteins in a large kindred with xerocytosis (dehydrated hereditary stomatocytosis). Using polymerase chain reaction‐based genotyping techniques, all three loci are excluded as disease gene candidates. Am. J. Hematol. 60:72–74, 1999.