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1.
Dev Dyn ; 248(4): 296-305, 2019 04.
Article in English | MEDLINE | ID: mdl-30682232

ABSTRACT

BACKGROUND: The mammalian guanine deaminase (GDA), called cypin, is important for proper neural development, by regulating dendritic arborization through modulation of microtubule (MT) dynamics. Additionally, cypin can promote MT assembly in vitro. However, it has never been tested whether cypin (or other GDA orthologs) binds to MTs or modulates MT dynamics. Here, we address these questions and characterize Xenopus laevis GDA (Gda) for the first time during embryonic development. RESULTS: We find that exogenously expressed human cypin and Gda both display a cytosolic distribution in primary embryonic cells. Furthermore, while expression of human cypin can promote MT polymerization, Xenopus Gda has no effect. Additionally, we find that the tubulin-binding collapsin response mediator protein (CRMP) homology domain is only partially conserved between cypin and Gda. This likely explains the divergence in function, as we discovered that the cypin region containing the CRMP homology and PDZ-binding domain is necessary for regulating MT dynamics. Finally, we observed that gda is strongly expressed in the kidneys during late embryonic development, although it does not appear to be critical for kidney development. CONCLUSIONS: Together, these results suggest that GDA has diverged in function between mammals and amphibians, and that mammalian GDA plays an indirect role in regulating MT dynamics. Developmental Dynamics 248:296-305, 2019. © 2019 Wiley Periodicals, Inc.


Subject(s)
Guanine Deaminase/physiology , Kidney/enzymology , Xenopus Proteins/physiology , Xenopus laevis/embryology , Animals , Embryo, Nonmammalian/enzymology , Guanine Deaminase/metabolism , Humans , Kidney/embryology , Microtubules/metabolism , Xenopus Proteins/metabolism , Xenopus laevis/metabolism
2.
Nat Neurosci ; 7(2): 145-52, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14730308

ABSTRACT

Dendrite branching has an important role in normal brain function. Here we report that overexpression of cypin, a protein that has guanine deaminase activity and is expressed in developing processes in rat hippocampal neurons, results in increased dendrite branching in primary culture. Mutant cypin proteins that lack guanine deaminase activity act in a dominant-negative manner when expressed in primary neurons. Furthermore, we knocked down cypin protein levels using a new strategy: expressing a 5' end-mutated U1 small nuclear RNA (snRNA) to inhibit maturation of cypin mRNA. Neurons that express this mutant snRNA show little or no detectable cypin protein and fewer dendrites than normal. In addition, we found that cypin binds directly to tubulin heterodimers and promotes microtubule polymerization. Thus, our results demonstrate a new pathway by which dendrite patterning is regulated, and we also introduce a new method for decreasing endogenous protein expression in neurons.


Subject(s)
Body Patterning/physiology , Carrier Proteins/physiology , Dendrites/physiology , Guanine Deaminase/physiology , Hippocampus/embryology , Microtubules/physiology , Animals , Cells, Cultured , Embryo, Mammalian , Immunohistochemistry , Mutation , RNA, Small Nuclear/genetics , Rats , Transfection
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