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M B Willard

Publications and source records attributed to M B Willard.

16 recordsLinked to original sources
PubMed2005

Molecular differentiation between chromosomally defined incipient species of Anopheles funestus.

Anopheles funestus Giles is one of the most important vectors of malaria in sub-Saharan Africa. The population structure of this mosquito in Burkina Faso, West Africa based on chromosomal inversion data led to the description of two chromosomal forms, Kiribina and Folonzo. Because both forms co-occur in the same locales yet differ significantly, both in the frequency of inverted arrangements on chromosome arms 3R and 2R and in vectorial capacity, they were hypothesized to be emerging species with at least partial barriers to gene flow. This hypothesis would be strengthened by molecular evidence of differentiation between Kiribina and Folonzo at loci outside chromosomal inversions. We surveyed molecular variation in sympatric populations of the two forms using sequences from the mitochondrial ND5 gene and genotypes at sixteen microsatellite loci distributed across the genome. Both classes of marker revealed slight but significant differentiation between the two forms (mtDNA F(ST) = 0.023, P < 0.001; microsatellite F(ST) = 0.004, P < 0.001; R(st) = 0.009, P = 0.002). Locus-by-locus analysis of the microsatellite data showed that significant differentiation was not genome-wide, but could be attributed to five loci on chromosome 3R (F(ST) = 0.010, P < 0.001; R(st) = 0.016, P = 0.002). Importantly, three of these loci are outside of, and in linkage equilibrium with, chromosomal inversions, suggesting that differentiation between chromosomal forms extends beyond the inversions themselves. The slight overall degree of differentiation indicated by both marker classes is likely an underestimate because of recent population expansion inferred for both Folonzo and Kiribina. The molecular evidence from this study is consistent with the hypothesis of incipient speciation between Kiribina and Folonzo.

Animals↗
PubMed1992

Evidence for unequal crossing over in the evolution of the neurofilament polypeptide H.

Two allelic forms of the rabbit neurofilament protein H, designated H1 and H2, differ by approximately 6% in their electrophoretic mobilities. We have used techniques of peptide and nucleic acid analysis to determine that this difference is located in the central portion of the COOH-terminal tail, a region of the H protein that can project from the filament, and may form cross-bridges. This region comprises tandem amino acid motifs containing the sequence KSP, in which the serine residues are sites of phosphorylation. The sequence of this repetitive region of H2 is 132 nucleotides (44 amino acids) shorter than H1 and differs significantly in the arrangement of the repeated motifs. The difference suggests that both forms have evolved from a common ancestor over the past several million years by the process of unequal crossing over and that this process figured importantly in generating the repetitive region. In addition, the results indicate that this region is functionally redundant and that the length and structure of the tail of rabbit H resemble H from other mammalian species more closely than was suggested by a previously reported cDNA, on account of a single nucleotide difference.

Alleles↗
PubMed1992

GAP-43, a protein associated with axon growth, is phosphorylated at three sites in cultured neurons and rat brain.

GAP-43 is a neuronal calmodulin-binding phosphoprotein that is concentrated in growth cones and presynaptic terminals. By sequencing tryptic and endoproteinase Asp-N phosphopeptides and directly determining the release of radioactive phosphate, we have identified three sites (serines 41 and 96 and threonine 172) that are phosphorylated, both in cultured neurons and in neonatal rat brain. These three sites account for most of the 32PO4 that was incorporated into GAP-43 in cultured neurons; 8-15% of each site was occupied with phosphate in GAP-43 isolated from neonatal rat brain. Phosphorylation of serine 41 in cultured neurons was stimulated by phorbol ester, indicating that it is the only site phosphorylated by protein kinase C. The resemblance of the sequence surrounding the other two sites suggests that they may be substrates for the same protein kinase. None of the sites phosphorylated by casein kinase II in vitro was phosphorylated in living cells or in neonatal rat brain. These results show that GAP-43 is a substrate for at least one protein kinase in addition to protein kinase C in living cells and brain.

Amino Acid Sequence↗
PubMed1992

Identification of six phosphorylation sites in the COOH-terminal tail region of the rat neurofilament protein M.

The COOH-terminal tail domain of the neurofilament polypeptide M from rat nervous tissue contains approximately six molecules of phosphate. We report here that protein kinases in a crude cytoskeleton preparation of rat nervous tissue phosphorylated a set of tryptic peptides of M similar (but not identical) to those phosphorylated by living dorsal root ganglion cells in culture. Using these phosphopeptides as markers, we purified these same peptides from rat spinal cord and identified six specific phosphorylation sites in M by enzymatic and chemical criteria. These sites, serines 502, 506, 536, 606, 608, and 666, are all located in the COOH-terminal tail domain. Four are embedded in the repeated motif KSP whereas two are within variants of this motif, KSD and ESP. All of the sites that were preceded by lysine were resistant to alkaline phosphatase prior to modification of the lysine with citraconic anhydride. The identification of these sites should aid in investigations of the function of the phosphorylation of this protein and provides criteria for identifying the relevant kinases.

Amino Acid Sequence↗
PubMed1992

Does GAP-43 support axon growth by increasing the axonal transport velocity of calmodulin?

GAP-43 is a neuronal protein whose synthesis is elevated during developmental and regenerative axon growth. We propose that one consequence of this increased synthesis may be the delivery of calmodulin-like proteins to the distal portions of the growing axon at an increased velocity; this is because calmodulin, which is transported slowly in mature intact axons, can bind to GAP-43, which is transported rapidly. The release of calmodulin from GAP-43 would be regulated by phosphorylation by protein kinase C. Such a rapid carrier function could be important for allowing certain recently synthesized slowly transported proteins to reach the moving growth cone in time to support its function. This hypothetical carrier mechanism is consistent with the phosphorylation pattern, calmodulin binding, transport velocity, and growth-association of GAP-43, and suggests an explanation for the specific importance of newly synthesized GAP-43 in supporting axon growth.

Animals↗
PubMed1991

Production of the neuronal growth-associated protein GAP-43 in a bacterial expression system.

GAP-43, a major protein of neuronal growth cones and certain presynaptic terminals, is a candidate for important functions in both axon growth and synaptic plasticity. To facilitate studies that may elucidate these functions, we have efficiently generated large quantities of GAP-43 by introducing a GAP-43 cDNA into a bacterial expression system driven by T7-RNA polymerase. Two constructs were expressed in this system: one (pT7Ava-GAP) produces a fusion protein in which the first 16 amino acids of GAP-43 are replaced by 11 amino acids of the phage T7 capsid protein; the other (pT7FL-GAP) produces full length GAP-43. After the bacteria were lysed, both products were soluble, and could be efficiently purified by HPLC chromatography on a C4 reversed-phase column. One liter of bacterial culture yielded 50 mg of purified fusion protein or 10 mg of complete GAP-43. When it was incubated with protein kinase C, the fusion protein was phosphorylated at the same single site (serine 41) that is phosphorylated in cultured neurons. The ability to produce large quantities of GAP-43 by this procedure should expedite future studies investigating its structure, posttranslational modification, and function.

Amino Acid Sequence↗
PubMed1991

Sequence of the rabbit neurofilament protein L.

In the course of screening a rabbit brain cDNA library with a probe for the H neurofilament protein, we identified a neurofilament L-cDNA. Its nucleotide sequence is 88% identical to that of human, indicating that L is highly conserved among species. The similarities between the sequences of L from rabbit and mouse suggest that the species-specific accumulation of neurofilaments that occurs in rabbit during aluminum intoxication is not a consequence of the primary structure of L.

Amino Acid Sequence↗
PubMed1991

Expression of the growth-associated protein GAP-43 in adult rat retinal ganglion cells following axon injury.

We have studied the expression of the growth-associated protein GAP-43 after injury to the axons of adult rat retinal ganglion cells (CNS neurons that do not normally regenerate injured axons). Both the biosynthetic labeling of GAP-43 and the GAP-43 immunoreactivity of the retina increased after axotomy, but only when the injury was within 3 mm of the eye. These results suggest the following conclusions: First, axon injury is sufficient to alter GAP-43 expression in CNS neurons, even in the absence of regeneration. Second, mechanisms that regulate GAP-43 expression are sensitive to the length of uninterrupted axon remaining after injury. Finally, the conditions that favor increased GAP-43 are similar to those that favor regrowth of injured CNS axons into grafts of peripheral nerve, suggesting that GAP-43 induction is accompanied by an increased potential of injured CNS neurons to regenerate.

Animals↗
PubMed1991

The major 35S-methionine-labeled rapidly transported protein (superprotein) is identical to SNAP-25, a protein of synaptic terminals.

Superprotein is a rapidly axonally transported protein that is conspicuously labeled with 35S-methionine supplied to the cell bodies of retinal ganglion cells. Superprotein candidates are apparent among the rapidly transported proteins of many neurons from the CNS and PNS, including cranial, sympathetic, sensory, and motor neurons from mammals, fish, and amphibians. To determine the identity of Superprotein, we purified it from rabbit visual system and spinal cord and determined the amino acid sequence of seven of its tryptic peptides. The sequence shows that Superprotein is SNAP-25, a protein recently predicted from a cDNA sequence; SNAP-25 has been reported to be concentrated in the synaptic terminals of a selected population of CNS neurons. We measured the amount of radioactivity associated with Superprotein in tissue containing axons (optic tract) and synaptic terminals (superior collicules) of rabbit retinal ganglion cells. Labeled Superprotein disappeared from the superior colliculus more rapidly than another protein (synapsin I-like protein) that is concentrated in synaptic terminals. These results serve to unite the observations on the synthesis, distribution, metabolism, and axonal transport of Superprotein with observations of SNAP-25 and its mRNA.

Animals↗
PubMed1989

Characterization of the interaction between calpactin I and fodrin (non-erythroid spectrin).

Calpactin I, a calcium-binding protein associated with the membrane cytoskeleton, has been reported to bind to a calcium-dependent manner to fodrin, to certain phospholipids, and to F-actin. We have investigated the interaction between calpactin I and fodrin. Using a gel filtration assay, we observed one or more calpactin I molecules were bound calcium-dependently only at high concentrations of calpactin (greater than 1 microM), indicating that the interaction is of only moderate affinity. At higher concentrations of calpactin I, the calpactin coprecipitated with fodrin in a calcium-dependent manner. The molar ratio of calpactin to fodrin tetramer in the precipitate was greater than 25:1, indicating that the calpactin binds to a large number of sites. Moreover, the monomeric form of calpactin I (p36), which did not induce precipitation of fodrin, showed no evidence of saturation in its binding to fodrin even when more than 30 mol of p36 were bound per mole of fodrin tetramer. Several proteins other than fodrin, including clathrin, alpha-actinin, and neurofilament-H, also interacted calcium-dependently with calpactin I in the gel filtration assay. These results demonstrate that the interaction between calpactin and fodrin is not of high affinity, is not readily saturated, and is not specific for fodrin. Our results suggest that calpactin's interaction with fodrin is a particular example of a calcium-dependent, but promiscuous, binding of calpactin to proteins.

Animals↗
PubMed1987

Posttranslational modification of neurofilament polypeptides in rabbit retina.

Three polypeptides that compose neurofilaments, designated H, M, and L, are synthesized in the cell bodies of neurons and subsequently conveyed down their axons by the process of slow axonal transport. The axonal form of H, which is a component of the cross bridges between the neurofilaments, is antigenically different from the form in the cell bodies and dendrites. To understand how this special form of H is directed to the axon, and more generally how intracellular differentiation is established and maintained by the selective delivery of different molecular species to different compartments of a cell, we have studied the events that occur immediately after the synthesis of the three neurofilament polypeptides in the retinas of rabbits. We observed that H and M are synthesized in the retina as precursor polypeptides, EH and EM, that migrate markedly faster on SDS polyacrylamide gels than their mature axonal forms. The maturation of these precursors requires more than one day and appears to involve their phosphorylation. Only the electrophoretically mature forms appear in the axons of the retinal ganglion cells in the optic nerve. We consider the following interpretation of these observations. Shortly after they are translated in the cell body, the neurofilament polypeptides become phosphorylated at multiple sites. However, only after they have moved a distance of several hundred micrometers down the axon, H and M are phosphorylated at additional sites, causing their conformation or binding properties to change. This change, which is reflected in the reduction of their electrophoretic mobility and the appearance of new antigenic determinants, may function to alter the H-mediated crossbridges and produces the morphological and structural properties of the neurofilament lattice that is characteristic of axons.

Animals↗
PubMed1986

Growth-associated protein, GAP-43, a polypeptide that is induced when neurons extend axons, is a component of growth cones and corresponds to pp46, a major polypeptide of a subcellular fraction enriched in growth cones.

Growth-associated protein, GAP-43, is a polypeptide that is induced in neurons when they grow axons. We show by means of subcellular fractionation and immunohistochemical localization that GAP-43 is a component of neuronal growth cones as well as growing neurites; it is similar to a major phosphoprotein, pp46, of a growth cone-enriched subcellular fraction. These conclusions are consistent with the possibility that the induction of GAP-43/pp46 is an important event in the establishment of a productive growth state in which a neuron is competent to extend an axon.

Animals↗
PubMed1984

Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton.

Neurofilaments in the axons of mammalian spinal cord neurons are extensively cross-linked; consequently, the filaments and their cross-bridges compose a three-dimensional lattice. We have used antibody decoration in situ combined with tissue preparation by the quick-freeze, deep-etch technique to locate three neurofilament polypeptides (195, 145, and 73 Kd) within this lattice. When antibodies against each polypeptide were incubated with detergent-extracted, formaldehyde-fixed samples of rabbit spinal cord, each antibody assumed a characteristic distribution: anti-73-Kd decorated the neurofilament core uniformly, but not the cross-bridges; anti-145-Kd also decorated the core, but less uniformly; sometimes the anti-145-Kd antibodies were located over the bases of cross-bridges. In contrast, anti-195-Kd primarily decorated the cross-bridges between the neurofilaments. These observations show that the 73-Kd polypeptide is a component of the central core of neurofilaments, and that the 195-Kd polypeptide is a component of the inter-neurofilamentous cross-bridges. It is consistent with this conclusion that we found few cross-bridges between neurofilaments in the optic nerves of neonatal rabbits during a developmental period when the ratio of 195 to 73 or 145-Kd polypeptides is much lower than in adults. The ratio of 195-Kd polypeptide to the other two neurofilament polypeptides also appeared much lower in the cell bodies and dendrites than in axons of adult spinal cord neurons, when the dispositions of the three polypeptides were studied by immunofluorescence experiments. The cell bodies apparently contain neurofilaments composed primarily of 145- and 73-Kd polypeptides, because we observed antibody decoration of individual neurofilaments in the cell bodies with anti-73- and -145-Kd, but not with anti-195-Kd. We conclude that the 195-Kd polypeptide participates in a cross-linking function, and that this function is, at least in certain neurons, most prevalent in the mature axon.

Aging↗
PubMed1977

The intra-axonal transport of polypeptide H: evidence for a fifth (very slow) group of transported proteins in the retinal ganglion cells of the rabbit.

We have determined that a genetically polymorphic polypeptide (H, molecular weight approximately equal to 195,000) of the rabbit nervous system is transported down the retinal ganglion cell axons at a velocity of 0.7-1.1 mm/day. The H-polypeptide and probably at least two additional polypeptides (molecular weights approximately 145,000 and 73,000) therefore compose a group of intra-axonally transported proteins which moves more slowly than the 4 groups previously described in these neurons. The polypeptides of this fifth group are similar in molecular weight to certain polypeptides transported slowly in other mammalian neurons.

Animals↗
PubMed1976

Genetically determined protein polymorphism in the rabbit nervous system.

One of the polypeptides (H1) of the rabbit nervous system occurs in an altered form (H2) in some rabbits. The electrophoretic mobility of H2 on sodium dodecyl sulfate-polyacrylamide gels is about 6% greater than that of H1, suggesting that the two polypeptides differ in molecular weight by about 10,000. The alteration is genetically determined since (i) rabbit phenotypes corresponding to all possible genotypes (H1H1, H1H2, H2H2) were present in an outbred population of rabbits, (ii) the frequency of the phenotype corresponding to the heterozygous genotype (H1H2) was smaller in partially inbred rabbit populations than in outbred populations, and (iii) all of the individuals examined from two partially inbred strains (WH/J and X/J) were of the phenotype that would be expected if they were homozygous (H2H2) for the rare allele of the gene. Preliminary evidence indicates that this polymorphic polypeptide is most abundant in the white matter of the nervous systems of several mammalian species, and is distributed with buffers of low ionic strength and centrifuged at 100,000 Xg.

Albinism↗