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N V Hayes

Publications and source records attributed to N V Hayes.

15 recordsLinked to original sources
PubMed2000

Identification of a novel C-terminal variant of beta II spectrin: two isoforms of beta II spectrin have distinct intracellular locations and activities.

It is established that variations in the structure and activities of betaI spectrin are mediated by differential mRNA splicing. The two betaI spectrin splice forms so far identified have either long or short C-terminal regions. Are analogous mechanisms likely to mediate regulation of betaII spectrins? Thus far, only a long form of betaII spectrin is reported in the literature. Five human expressed sequence tags indicated the existence of a short splice variant of betaII spectrin. The occurrence and DNA sequence of the short C-terminal variant was confirmed by analysis of human and rat cDNA. The novel variant lacks a pleckstrin homology domain, and has 28 C-terminal residues not present in the previously recognized longer form. Transcripts of the short C-terminal variant (7.5 and 7. 0 kb) were most abundant in tissues originating from muscle and nervous system. Antibodies raised to a unique sequence of short C-terminal variant recognized 240 kDa polypeptides in cardiac and skeletal muscle and in nervous tissue; in cerebellum and forebrain, additional 270 kDa polypeptides were detected. In rat heart and skeletal muscle, both long and short C-terminal forms of betaII spectrin localized in the region of the Z line. The central region of the sarcomere, coincident with the M line, was selectively labeled with antibodies to the short C-terminal form. In cerebellum, the short form was not detectable in parallel fibers, structures in which the long form was readily detected. In cultured cerebellar granule neurons, the long form was dominant in neurites, with the short form being most abundant in cell bodies. In vitro, the short form was found to lack the binding activity for the axonal protein fodaxin, which characterizes the C-terminal region of the long form. Subcellular fractionation of brain revealed that the short form was scarcely detectable in post-synaptic density preparations, in which the long form was readily detected. We conclude that variation in the structure of the C-terminal regions of betaII spectrin isoforms correlates with their differential intracellular targeting.

Alternative Splicing↗
PubMed1997

Definition of a sequence unique in beta II spectrin required for its axon-specific interaction with fodaxin (A60).

Spectrin isotypes segregate in neurons and are differentially distributed between axons and somatodendritic compartments. Their functions in those compartments are likely to be mediated by proteins that interact selectively with one or other isotype. Fodaxin (an axon-specific protein previously termed A60) colocalizes in CNS neurons with axonal spectrin and in vitro binds brain spectrin (a mixture of alpha I, beta I, and beta II polypeptides) but not erythrocyte spectrin (alpha I and beta I). Because alpha II and beta II spectrin polypeptides are enriched in axons, we investigated a possible binding of fodaxin to the types of spectrin found in axons. Fodaxin did not bind to isolated brain alpha chains. Bacterially expressed C-terminal segments 18-19 of beta II spectrin bound to fodaxin and inhibited the binding of fodaxin to whole brain spectrin. By contrast, recombinant segments 18-19 of the somatodendritic beta I sigma 2 spectrin showed no interaction with fodaxin. Within beta II, fodaxin binding activity was localized to residues 2,087-2,198, which are unique to beta II and link between the end of segment 18 and the pleckstrin homology domain in segment 19. The divergent regions of sequence in segments 19 of beta II and beta I sigma 2 are candidates to mediate the isotype-specific functions of spectrin. Fodaxin is the first protein to be described that discriminates between the unique regions of beta spectrin isoforms.

Animals↗
PubMed1995

A60, an axonal membrane-skeletal spectrin-binding protein.

A60 is a 60 kDa protein which is associated with the axonal cortical cytoskeleton in adult central nervous system neurons. It was originally defined by the use of a monoclonal antibody which showed that it was closely associated with the cytoplasic face of axonal plasma membranes. A60 appears to be neuron-specific and biochemical studies show that it is tightly bound to brain membranes. Affinity chromatography has revealed that A60 interacts with brain spectrin but not with erythrocyte spectrin. As erythrocyte spectrin is closely related to the isotype of spectrin that is localized in dendrites this raises the possibility that A60 is restricted to axons by interaction with the isotype of spectrin that is found in axons. During post-natal cerebellar development (days 1-13) A60 is initially located in the perikarya of precursor Purkinje cells and is then localized in the initial dendrites of these cells as well as in the white matter. In contrast, in the adult cerebellum the location of A60 is exclusively axonal. These data indicate that A60 has a spectrin-binding activity in the adult axonal membrane skeleton which is only required after initial axon growth has occurred. A60 is transiently expressed during embryonic and post-natal development of rat dorsal root ganglia (DRG). It is located in the large light DRG cells but is essentially absent from the small dark DRG cells.

Aging↗
PubMed1995

Cytoplasmic chaperonin complexes enter neurites developing in vitro and differ in subunit composition within single cells.

Chaperonins containing t-complex polypeptide-1 (CCT) are cytosolic molecular chaperone particles implicated especially in the biogenesis of cytoskeletal proteins by promoting the correct folding of the major ubiquitous cytoskeletal components, tubulin and actin. We have purified cytosolic chaperonins from the ND7/23 cell line, determined their subunit composition and examined changes in the intracellular locations of their components during differentiation of ND7/23 cells to a neuronal phenotype by using immunocytochemistry and immunoblots. Chaperonins containing the CCT alpha (TCP1) subunit enter neuritic processes and are particularly noticeable at the leading edge of growth cone-like structures where they co-localise with actin. Chaperonins containing three other components (CCT beta, epsilon and gamma), however, remain predominantly restricted to perikaryal cytoplasm. These findings suggest a heterogeneous population of chaperonin particles within single differentiated ND7/23 cells and this may reflect specialisation of chaperonin function in different cytoplasmic compartments of a neurone. Further, since ribosomes do not enter neurites while CCT alpha-containing chaperonins do, the latter may play roles, subsequent to translation, which influence cytoskeletal elaboration during neuritogenesis.

Actins↗
PubMed1994

Axonal membrane-skeletal protein A60: association with a brain spectrin-binding activity and entry into cerebellar axons at a stage after the initiation of axonal growth.

A60 is a 60-kDa component of the axonal cortical cytoskeleton in CNS neurones. It appears to be neurone specific and is tightly bound to brain membranes. In this study the cytoskeletal activities and developmental expression of A60 in rat cerebellum have been examined using the monoclonal antibody DR1. A60 in a partially purified soluble extract of brain membranes interacts selectively with brain but not erythrocyte spectrin. Because erythrocyte spectrin is more closely related to the dendritic form of spectrin than the axonal form, this raises the possibility that A60 localises in axons by interaction with the axonal form of spectrin only. A60 is not found in rat cerebellum before the day of birth. However, during postnatal development of the cerebellum (days 1-13) DR1 reactivity appears progressively. On postnatal day 1, a small population of cells in the mantle layer (presumptive Purkinje cells) is DR1 positive. There is no DR1 reactivity found in Purkinje cell axons during their initial phase of growth. By postnatal day 7, Purkinje cell bodies, initial dendritic segments, and the cerebellar white matter are all positive. This pattern of labelling is strengthened up until postnatal day 13. By contrast, in adult rat cerebellum, the location of A60 has changed so that it is most concentrated in axons, and dendritic staining is lost. These data indicate that A60 is a spectrin-binding component of the adult axonal membrane skeleton, the presence of which is only required in axons after the initial phase of growth.

Aging↗
PubMed1991

Site specificity in the interactions of synapsin 1 with tubulin.

Synapsin 1 is one of a family of phosphoproteins located on small synaptic vesicles (SSV) in the presynaptic terminal, and probably plays a critical role in the process of neuronal exocytosis by providing regulated linkages between SSV and the cytoskeleton. Two forms of synapsin 1 are produced from a single gene by differential mRNA splicing: 1a, 706 amino acid residues, and 1b, 670 residues. Synapsin 1 has two structural domains, a globular N-terminal head domain and an elongated tail domain. Electron microscopy of nerve terminals in situ and reconstitution studies in vitro indicates that synapsin 1 can interact with microtubules, microfilaments and brain spectrin. In vitro, synapsin 1 can bundle microtubules. This could either occur by synapsin 1 being at least bivalent for microtubules, or by univalent synapsin 1 molecules aggregating to form complexes that are more than univalent. To resolve this question, we have taken the approach of preparing defined fragments of synapsin 1 from each structural domain and analysing them for tubulin-binding activity. Our results show that there are tubulin-binding sites in both head and tail domains. We conclude that synapsin 1 monomers should be able to cross-link microtubules.

Amino Acid Sequence↗
PubMed1991

Selective Ca2(+)-dependent interaction of calmodulin with the head domain of synapsin 1.

The calcium-dependent regulatory protein calmodulin is a critical element in the machinery regulating exocytosis at nerve terminals. Okabe & Sobue [(1987) FEBS Lett. 213, 184-188] showed that calmodulin interacts with one of the proteins intimately connected with the neuronal exocytotic process, i.e. synapsin 1. We have investigated the site at which calmodulin interacts with synapsin 1. We find that it is possible to generate chemically cross-linked Ca2(+)-dependent complexes between synapsin 1 and calmodulin in vitro, and have used covalent cross-linking in conjunction with calmodulin affinity chromatography to identify fragments of synapsin 1 that interact with calmodulin. Ca2(+)-dependent calmodulin binding is restricted to the 'head' domain (residues 1-453 in bovine synapsin 1). Within this domain the binding site is located in a unique 11 kDa Staphylococcus aureus V8 proteinase generated fragment. This fragment does not contain the site for cyclic-AMP-dependent phosphorylation and therefore does not represent the N-terminus of the protein.

Animals↗
PubMed1991

p103 and A60: novel proteins of the neuronal membrane-associated cytoskeleton.

Associated with the neuronal plasma membrane are cytoskeletal proteins which probably control the specialization of the membrane into axonal and dendritic domains. Specialized isoforms of the proteins spectrin and ankyrin are located in each region and provide molecular mechanisms for locating specific transmembrane proteins at required points. However, spectrin and ankyrin were defined by extensions of the model for the erythrocyte membrane, an analogy unlikely to provide a complete account of the neuronal membrane skeleton. We have defined two new proteins of the neuronal membrane skeleton, designated p103 and A60. p103 is enriched in post-synaptic densities and binds with high affinity to integral membrane proteins--we suggest that it may have a role in linking the cytoskeleton to synaptic glycoproteins. A60 is a 60 kDa axonal protein, which appears to form a lining to the axolemma. It is almost exclusively axonal, although some neurons (such as Purkinje cells) appear to contain it in the cell body and initial dendrite segment. A60 binds both ankyrin and neurofilaments, and may have a role in transmitting information critical to axonal morphology to the membrane.

Ankyrins↗
PubMed1991

Purification and properties of p103, a novel 103-kDa component of postsynaptic densities.

A 103-kDa protein present in membrane cytoskeletal preparations from bovine brain has been identified. We have purified this protein to greater than 95% homogeneity using gel filtration and ion-exchange chromatography. This protein, p103, is an asymmetric dimer in dilute solution and has two major variants that can be distinguished by isoelectric focussing, pI 5.60 and 5.75. Using subcellular fractionation, it is most enriched in postsynaptic densities. Immunolocalization with anti-p103-specific antibodies reveals that it is confined to the dendrites and perikarya; it is apparently absent from spinal cord axons. It coextracts from brain membrane-skeletal preparations with brain spectrin and actin, but in vitro, it does not interact with them.

Animals↗