Search

The spectrin membrane skeleton controls the disposition of selected membrane channels, receptors, and transporters. In the brain [beta]III spectrin binds directly to the excitatory amino acid transporter (EAAT4), the glutamate receptor delta, and other proteins. Mutations in [beta]III spectrin link strongly to human spinocerebellar ataxia type 5 (SCA5), correlating with alterations in EAAT4. We have explored the mechanistic basis of this phenotype by targeted gene disruption of Spnb3. Mice lacking intact [beta]III spectrin develop normally. By 6 months they display a mild nonprogressive ataxia. By 1 year most [Spnb3.sup.-/-] animals develop a myoclonic seizure disorder with significant reductions of EAAT4, EAAT1, GluR[delta], IP3R, and NCAM140. Other synaptic proteins are normal. The cerebellum displays increased dark Purkinje cells (PC), a thin molecular layer, fewer synapses, a loss of dendritic spines, and a 2-fold expansion of the PC dendrite diameter. Membrane and expanded Golgi profiles fill the PC dendrite and soma, and both regions accumulate EAAT4. Correlating with the seizure disorder are enhanced hippocampal levels of neuropeptide Y and EAAT3 and increased calpain proteolysis of [alpha]II spectrin. It appears that [beta]III spectrin disruption impairs synaptogenesis by disturbing the intracellular pathways selectively regulating protein trafficking to the synapse. The mislocalization of these proteins secondarily disrupts glutamate transport dynamics, leading to seizures, neuronal damage, and compensatory changes in EAAT3 and neuropeptide Y. cytoskeleton | membrane | spinocerebellar ataxia type 5 | excitatory amino acid transporter 4 | Purkinje doi/10.1073/pnas.1001522107

Author(s): Adriana Donovan [1]; Alison Brownlie [1, 2]; Yi Zhou [1]; Jennifer Shepard [1]; Stephen J. Pratt [3]; John Moynihan [1]; Barry H. Paw [1]; Anna Drejer [1]; Bruce Barut [...]

The relationship between the lateral diffusion rate of the band 3 molecule and the spectrin content of the human red blood cell membrane was investigated in patients with severe autosomal recessive hereditary spherocytosis and selective spectrin deficiency. Results showed that the rotational mobility of band 3 in hereditary spherocytosis is identical to that in normal red blood cells. It was therefore concluded that the lateral mobility of band 3 is inhibited by steric interactions with the membrane skeleton.

Spectrin is an important structural component of the plasma membrane skeleton. Heretofore-unidentified isoforms of spectrin also associate with Golgi and other organelles. We have discovered another member of the [Beta]-spectrin gene family by homology searches of the GenBank databases and by 5[prime] rapid amplification of cDNA ends of human brain cDNAs. Collectively, 7,938 nucleotides of contiguous clones are predicted to encode a 271,294-Da protein, called [Beta]III spectrin, with conserved actin-, protein 4.1-, and ankyrin-binding domains, membrane association domains 1 and 2, a spectrin dimer self-association site, and a pleckstrinhomology domain. [Beta]III spectrin transcripts are concentrated in the brain and present in the kidneys, liver, and testes and the prostate, pituitary, adrenal, and salivary glands. All of the tested tissues contain major 9.0-kb and minor 11.3-kb transcripts. The human [Beta]III spectrin gene (SPTBN2) maps to chromosome 11q13 and the mouse gene (Spnb3) maps to a syntenic region close to the centromere on chromosome 19. Indirect immunofluorescence studies of cultured cells using antisera specific to human [Beta]III spectrin reveal a Golgi-associated and punctate cytoplasmic vesicle-like distribution, suggesting that [Beta]III spectrin associates with intracellular organelles. This distribution overlaps that of several Golgi and vesicle markers, including mannosidase II, p58, transGolgi network (TGN)38, and [Beta]-COP and is distinct from the endoplasmic reticulum markers calnexin and Bip. Liver Golgi membranes and other vesicular compartment markers cosediment in vitro with [Beta]III spectrin. [Beta]III spectrin thus constitutes a major component of the Golgi and vesicular membrane skeletons.

Proximal tubule (PT) fragments were purified to 100% from the renal cortex to determine the epithelial ankyrin (Ank3) spliceoforms found in the mouse PT cells. Distal tubule fragments had a substantial amount of Ank3(sub 200/215) but no Ank3(sub 170) or Ank3(sub 120). Isolated PT fragments had all four spliceoforms but radically reduced Ank3(sub 200/215). These larger spliceoforms bind Na-K-ATPase in diverse cell types.

Ankyrin-3, also known as ankyrin G, was cloned and characterized. Ankyrin-3 was expressed in mouse macrophages as two small isoforms of 120 and 100 kD that lack NH2 repeats. Sequence analysis of Ank3 cDNA clones showed that its spectrin-binding and regulatory domains are identical to those in kidney ankyrin-3. Dark appearance of ankyrin-3-positive vesicles on phase contrast microscopy suggested that they are lysosomes.

The function of the red blood cell anion exchanger, AE1, in in vivo RBC development is investigated by gene targeting. Specifically, null mutation in mice is carried out by targeted gene disruption in embryonic stem cells. The hematological effects of AE1 deletion do not affect fetal loss of homozygotes. Instances of survival to adulthood and fertility are even reported. Results suggest that AE1 is vital to membrane lipid stability but its plasma membrane localization is not required for either terminal RBC differentiation or membrane skeleton assembly.

The ability of kidney nephrons to maintain ionic homeostasis depends on the subcellular distribution and activity of the sodium-ion/potassium-ion/adenosinetriphosphatase (NA+/K+/ATPase) along the nephron. Immunofluorescence microscopy shows that the subcellular and segmental distributions of Na+/K+/ATPase paralleled the distributions of the membrane cytoskeleton proteins fodrin and ankyrin along the nephron, but not the distributions of the tight junction protein ZO-1 or E-cadherin, indicating selective location of Na+/K+/ATPase in the basolateral plasma membrane.

Hereditary spherocytosis is passed as an autosomal (not sex-linked) dominant disorder and typically occurs in individuals of northern European heritage. The disorder affects red blood cells, which are marked by a pathologic fragility that leads to their rupture and to decreased oxygen-carrying ability in the blood. Consequently, anemia is a common condition among patients with hereditary spherocytosis. Microscopic examination reveals that a significant number of these red blood cells have a spheroidal shape, rather than the normal biconcave (dumbbell-shaped) morphology. Previous studies on the biochemical deficit of the spherocytic cells have associated the condition with various gene products that are involved in the structure of the red blood cell membrane. The present study evaluates a family of individuals with dominant hereditary spherocytosis; use of a new molecular DNA probe revealed a disorder of the gene for the membrane protein ankyrin. However, the primary defect of the ankyrin gene in this family remains unidentified. (Consumer Summary produced by Reliance Medical Information, Inc.)

Hereditary spherocytosis (HS) is characterized by the appearance of red blood cells which are globular, rather than with the normal indentation which is seen as a pale center under the microscope. This condition is the most common form of hereditary hemolytic anemia. Some patients have a mutation in the protein spectrin, which is found in large quantities just inside the membrane of red blood cells. However, the majority of patients with hereditary spherocytosis have no such mutation. Furthermore, the alpha and beta chains of spectrin have their genes on chromosomes 1 and 14, respectively, while the gene for HS seems to map to chromosome 8. Researchers have now cloned the gene for ankyrin, another important red cell protein, and found it to be located on chromosome 8, precisely at 8p11.2. Additionally, in two unrelated patients with HS, a deletion has been identified in this chromosomal region. Affected red cells were demonstrated to be ankyrin-deficient. While the location of the HS gene is not mapped with sufficient precision to say that it is indeed the anykrin gene, the results suggest that a defect or deficiency in ankyrin may be responsible for hereditary spherocytosis. (Consumer Summary produced by Reliance Medical Information, Inc.)

The relationship of microcytic anemia in mk mice and the functional properties of the nuclear DNA-binding protein NF-E2 was investigated. NF-E2 mediates the strong erythroid-enhancing activity of the alpha- and beta-globin locus control region, and is involved in the synthesis of porphobilinogen deaminase and ferrochelatase, two key enzymes in heme biosynthesis. The results showed that the mk mutation affects the gene encoding the p45 polypeptide component of NF-E2. Microcytic anemia in mk mice is thus due to impaired regulation of globin synthesis and iron metabolism arising from impaired NF-E2 function.

The ternary complex of spectrin, F-actin and protein 4.1R binds the N-terminal region of the spectrin chain containing two calponin homology domains CH1 and CH2. It is observed that the formation and binding of spectrin-actin-4.1R ternary complex is inhibited by the polypeptide 1-301 and this binding acts like a regulatory switch in the cell.