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Teneurin-1 is expressed in interconnected regions of the developing brain and is processed in vivo.

Kenzelmann D, Chiquet-Ehrismann R, Leachman NT, Tucker RP - BMC Dev. Biol. (2008)

Bottom Line: Moreover we found complementary patterns of teneurin-1 and-2 expression in many parts of the brain, including the retina, optic tectum, olfactory bulb, and cerebellum as well as in brain nuclei involved in processing of sensory information.Finally, the teneurin-1 intracellular domain was found to contain a nuclear localization signal, which is required for nuclear localization in transfected cells.Our data support the hypothesis that teneurins can be proteolytically processed leading to the release of the intracellular domain and its translocation to the nucleus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Friedrich Miescher Institute, Novartis Research Foundation, Maulbeerstr. 66, 4057 Basel, Switzerland. daniela.kenzelmann@fmi.ch

ABSTRACT

Background: Teneurins are a unique family of transmembrane proteins conserved from C. elegans and D. melanogaster to mammals. In vertebrates there are four paralogs (teneurin-1 to -4), all of which are expressed prominently in the developing central nervous system.

Results: Analysis of teneurin-1 expression in the developing chick brain by in situ hybridization and immunohistochemistry defined a unique, distinct expression pattern in interconnected regions of the brain. Moreover we found complementary patterns of teneurin-1 and-2 expression in many parts of the brain, including the retina, optic tectum, olfactory bulb, and cerebellum as well as in brain nuclei involved in processing of sensory information. Based on these expression patterns, we suspect a role for teneurins in neuronal connectivity. In contrast to the cell-surface staining of the antibody against the extracellular domain, an antibody recognizing the intracellular domain revealed nuclear staining in subpopulations of neurons and in undifferentiated mesenchyme. Western blot analysis of brain lysates showed the presence of N-terminal fragments of teneurin-1 containing the intracellular domain indicating that proteolytic processing occurs. Finally, the teneurin-1 intracellular domain was found to contain a nuclear localization signal, which is required for nuclear localization in transfected cells.

Conclusion: Teneurin-1 and -2 are expressed by distinct interconnected populations of neurons in the developing central nervous system. Our data support the hypothesis that teneurins can be proteolytically processed leading to the release of the intracellular domain and its translocation to the nucleus.

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The teneurin-1 intracellular domain contains a nuclear localization signal. The amino acid sequence representing a putative nuclear localization signal (NLS) which was mutagenized to alanines (A). Western blot analysis of total lysates, cytoplasmic and nuclear fractions of COS-7 cells mock transfected, transfected with wild type ICD (ICD-WT) or transfected with the mutated NLS (NLS-Mut) constructs (B). Blots were probed with the anti-ICD, an antibody to the cytoplasmic protein vinculin, or an antibody to the nuclear protein Oct-1. Western blot quantification and calculation of the nuclear-to-cytoplasmic ICD ratio (C). Immunofluorescence staining of transfected COS-7 cells (D-G) and quantification of nuclear localization by cell counting (H). Both western blot analysis and immunofluorescence show that the overexpressed wild-type ICD is localized in the nucleus, whereas the NLS-Mut construct is predominantly found in the cytoplasm.
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Figure 5: The teneurin-1 intracellular domain contains a nuclear localization signal. The amino acid sequence representing a putative nuclear localization signal (NLS) which was mutagenized to alanines (A). Western blot analysis of total lysates, cytoplasmic and nuclear fractions of COS-7 cells mock transfected, transfected with wild type ICD (ICD-WT) or transfected with the mutated NLS (NLS-Mut) constructs (B). Blots were probed with the anti-ICD, an antibody to the cytoplasmic protein vinculin, or an antibody to the nuclear protein Oct-1. Western blot quantification and calculation of the nuclear-to-cytoplasmic ICD ratio (C). Immunofluorescence staining of transfected COS-7 cells (D-G) and quantification of nuclear localization by cell counting (H). Both western blot analysis and immunofluorescence show that the overexpressed wild-type ICD is localized in the nucleus, whereas the NLS-Mut construct is predominantly found in the cytoplasm.

Mentions: The ICD of teneurin-1 contains the amino acid sequence arginine-lysine-arginine-lysine at position 62 to 65 from the N-terminus. This resembles a classical nuclear localization signal (NLS), which directs the nuclear import of proteins. This putative NLS is conserved in chicken, mouse and human teneurin-1. To test whether it is functional, the four basic amino acids were replaced by alanines in the construct named NLS-mut (Fig. 5A). COS-7 cells were transfected either with a construct encoding the ICD of teneurin-1 (ICD-wt) or the NLS-mut construct. Western blot analysis of cytoplasmic and nuclear extracts of the transfected cells showed decreased nuclear accumulation of the NLS-mut construct compared to the ICD-wt construct (Fig. 5B). Quantification of the nuclear-to-cytoplasmic ratio showed that ICD-wt accumulates in the nucleus, which is not the case for NLS-mut (Fig. 5C). The ICD-wt protein is detected in the nucleus by immunofluorescence (Fig. 5D, E). However, nuclear localization of the ICD was abolished if the NLS is mutated (Fig. 5F, G). More than 80% of the cells expressing the wt-construct exhibited nuclear staining, whereas this was only observed in 20% of the cells expressing the NLS-mut version (Fig. 5H). In addition to the strongly decreased nuclear localization, these cells also exhibited a more pronounced localization of the teneurin-1 ICD to the cell membrane and cell processes.


Teneurin-1 is expressed in interconnected regions of the developing brain and is processed in vivo.

Kenzelmann D, Chiquet-Ehrismann R, Leachman NT, Tucker RP - BMC Dev. Biol. (2008)

The teneurin-1 intracellular domain contains a nuclear localization signal. The amino acid sequence representing a putative nuclear localization signal (NLS) which was mutagenized to alanines (A). Western blot analysis of total lysates, cytoplasmic and nuclear fractions of COS-7 cells mock transfected, transfected with wild type ICD (ICD-WT) or transfected with the mutated NLS (NLS-Mut) constructs (B). Blots were probed with the anti-ICD, an antibody to the cytoplasmic protein vinculin, or an antibody to the nuclear protein Oct-1. Western blot quantification and calculation of the nuclear-to-cytoplasmic ICD ratio (C). Immunofluorescence staining of transfected COS-7 cells (D-G) and quantification of nuclear localization by cell counting (H). Both western blot analysis and immunofluorescence show that the overexpressed wild-type ICD is localized in the nucleus, whereas the NLS-Mut construct is predominantly found in the cytoplasm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2289808&req=5

Figure 5: The teneurin-1 intracellular domain contains a nuclear localization signal. The amino acid sequence representing a putative nuclear localization signal (NLS) which was mutagenized to alanines (A). Western blot analysis of total lysates, cytoplasmic and nuclear fractions of COS-7 cells mock transfected, transfected with wild type ICD (ICD-WT) or transfected with the mutated NLS (NLS-Mut) constructs (B). Blots were probed with the anti-ICD, an antibody to the cytoplasmic protein vinculin, or an antibody to the nuclear protein Oct-1. Western blot quantification and calculation of the nuclear-to-cytoplasmic ICD ratio (C). Immunofluorescence staining of transfected COS-7 cells (D-G) and quantification of nuclear localization by cell counting (H). Both western blot analysis and immunofluorescence show that the overexpressed wild-type ICD is localized in the nucleus, whereas the NLS-Mut construct is predominantly found in the cytoplasm.
Mentions: The ICD of teneurin-1 contains the amino acid sequence arginine-lysine-arginine-lysine at position 62 to 65 from the N-terminus. This resembles a classical nuclear localization signal (NLS), which directs the nuclear import of proteins. This putative NLS is conserved in chicken, mouse and human teneurin-1. To test whether it is functional, the four basic amino acids were replaced by alanines in the construct named NLS-mut (Fig. 5A). COS-7 cells were transfected either with a construct encoding the ICD of teneurin-1 (ICD-wt) or the NLS-mut construct. Western blot analysis of cytoplasmic and nuclear extracts of the transfected cells showed decreased nuclear accumulation of the NLS-mut construct compared to the ICD-wt construct (Fig. 5B). Quantification of the nuclear-to-cytoplasmic ratio showed that ICD-wt accumulates in the nucleus, which is not the case for NLS-mut (Fig. 5C). The ICD-wt protein is detected in the nucleus by immunofluorescence (Fig. 5D, E). However, nuclear localization of the ICD was abolished if the NLS is mutated (Fig. 5F, G). More than 80% of the cells expressing the wt-construct exhibited nuclear staining, whereas this was only observed in 20% of the cells expressing the NLS-mut version (Fig. 5H). In addition to the strongly decreased nuclear localization, these cells also exhibited a more pronounced localization of the teneurin-1 ICD to the cell membrane and cell processes.

Bottom Line: Moreover we found complementary patterns of teneurin-1 and-2 expression in many parts of the brain, including the retina, optic tectum, olfactory bulb, and cerebellum as well as in brain nuclei involved in processing of sensory information.Finally, the teneurin-1 intracellular domain was found to contain a nuclear localization signal, which is required for nuclear localization in transfected cells.Our data support the hypothesis that teneurins can be proteolytically processed leading to the release of the intracellular domain and its translocation to the nucleus.

View Article: PubMed Central - HTML - PubMed

Affiliation: Friedrich Miescher Institute, Novartis Research Foundation, Maulbeerstr. 66, 4057 Basel, Switzerland. daniela.kenzelmann@fmi.ch

ABSTRACT

Background: Teneurins are a unique family of transmembrane proteins conserved from C. elegans and D. melanogaster to mammals. In vertebrates there are four paralogs (teneurin-1 to -4), all of which are expressed prominently in the developing central nervous system.

Results: Analysis of teneurin-1 expression in the developing chick brain by in situ hybridization and immunohistochemistry defined a unique, distinct expression pattern in interconnected regions of the brain. Moreover we found complementary patterns of teneurin-1 and-2 expression in many parts of the brain, including the retina, optic tectum, olfactory bulb, and cerebellum as well as in brain nuclei involved in processing of sensory information. Based on these expression patterns, we suspect a role for teneurins in neuronal connectivity. In contrast to the cell-surface staining of the antibody against the extracellular domain, an antibody recognizing the intracellular domain revealed nuclear staining in subpopulations of neurons and in undifferentiated mesenchyme. Western blot analysis of brain lysates showed the presence of N-terminal fragments of teneurin-1 containing the intracellular domain indicating that proteolytic processing occurs. Finally, the teneurin-1 intracellular domain was found to contain a nuclear localization signal, which is required for nuclear localization in transfected cells.

Conclusion: Teneurin-1 and -2 are expressed by distinct interconnected populations of neurons in the developing central nervous system. Our data support the hypothesis that teneurins can be proteolytically processed leading to the release of the intracellular domain and its translocation to the nucleus.

Show MeSH