Limits...
Expression and localization of FRMD7 in human fetal brain, and a role for F-actin.

Pu J, Li Y, Liu Z, Yan Y, Tian J, Chen S, Zhang B - Mol. Vis. (2011)

Bottom Line: The COOH-terminus of FRMD7 was found to play a key role in the subcellular localization of FRMD7 in mouse neuroblastoma cells (Neuro-2a) and human embryonic kidney 293 cells (HEK293T).While no differences in the co-localization of F-actin between the wild-type and missense mutation-type (c.781C>G and c.886G>C) proteins was observed, an additional mutant, c.1003C>T, which results in a COOH-terminally truncated protein, exhibited a nuclear localization pattern which did not co-localize with the cytoplasmic distribution of F-actin.The results of the present study indicate that FRMD7 may play an important role in the brainstem in the early stages of development of the human fetal brain, and provides clues for the mechanism of mutation FRMD7, which may be involved in influencing F-actin dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China.

ABSTRACT

Purpose: FERM domain containing 7 (FRMD7) is a member of the four-point-one, ezrin, radixin, moesin (FERM) family of proteins, and has been reported to cause X-linked idiopathic congenital nystagmus (ICN), a disease which affects ocular motor control. There have been over 30 mutations reported for FRMD7, however, their role in the pathogenesis of ICN remains unclear. The purpose of this study is to perform the expression distributes of protein FRMD7 from human fetal brain during development and to understand the relationship with cytoskeletal protein F-actin between wild-type and mutation-type FRMD7.

Methods: Expression of protein FRMD7 from developing human fetal brain was tested by immunohistochemistry. Enhanced green fluorescent protein (EGFP)-tagged recombinant plasmids DNA encoding the normal or mutant FRMD7 were used to transiently transfect the mouse neuroblastoma cells (Neuro-2a) and human embryonic kidney 293 cells (HEK293T). Further, confocal microscopic analysis was used to determine the subcellular localization of the fusion proteins. To visualize F-actin, fixed HEK293T cells were stained with rhodamine-phalloidin.

Results: We show that expression of FRMD7 was mainly detected in the brainstem (a region associated with ocular motor control), while limited level was observed in the cortex. The COOH-terminus of FRMD7 was found to play a key role in the subcellular localization of FRMD7 in mouse neuroblastoma cells (Neuro-2a) and human embryonic kidney 293 cells (HEK293T). While no differences in the co-localization of F-actin between the wild-type and missense mutation-type (c.781C>G and c.886G>C) proteins was observed, an additional mutant, c.1003C>T, which results in a COOH-terminally truncated protein, exhibited a nuclear localization pattern which did not co-localize with the cytoplasmic distribution of F-actin.

Conclusions: The results of the present study indicate that FRMD7 may play an important role in the brainstem in the early stages of development of the human fetal brain, and provides clues for the mechanism of mutation FRMD7, which may be involved in influencing F-actin dynamics.

Show MeSH

Related in: MedlinePlus

Co-localization of F-actin and FRMD7. HEK293T cells transiently transfected with EGFP-tagged wild-type FRMD7 and FRMD7 mutants, c.781C>G and c.886G>C, (shown in green) were stained with TRITC-conjugated rhodamine–phalloidin fluorescein (red). All three fusion proteins exhibited a diffuse localization pattern in the cytoplasm, and also in actin-rich regions of the HEK293T cells (arrows). However, HEK293T cells expressing the FRMD7 mutant, c.1003C>T, fused to EGFP, primarily exhibited localization of the fusion protein to the nucleus and did not co-localize with F-actin (merge). Scale bars: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3049738&req=5

f3: Co-localization of F-actin and FRMD7. HEK293T cells transiently transfected with EGFP-tagged wild-type FRMD7 and FRMD7 mutants, c.781C>G and c.886G>C, (shown in green) were stained with TRITC-conjugated rhodamine–phalloidin fluorescein (red). All three fusion proteins exhibited a diffuse localization pattern in the cytoplasm, and also in actin-rich regions of the HEK293T cells (arrows). However, HEK293T cells expressing the FRMD7 mutant, c.1003C>T, fused to EGFP, primarily exhibited localization of the fusion protein to the nucleus and did not co-localize with F-actin (merge). Scale bars: 20 μm.

Mentions: It is hypothesized that FRMD7 regulates neuronal development by influencing F-actin dynamics. Therefore, EGFP-tagged mutated versions of FRMD7 were transiently transfected into HEK293T cells and these cells were stained with TRITC rhodamine–phalloidin to detect F-actin. In these experiments, both wild-type FRMD7 and c.781C>G and c.886G>C FRMD7 mutants exhibited a diffuse distribution in the cytoplasm, and also in actin-rich regions. However, another FRMD7 mutant, c.1003C>T, exhibited changes in its subcellular distribution pattern and did not co-localize with F-actin (Figure 3).


Expression and localization of FRMD7 in human fetal brain, and a role for F-actin.

Pu J, Li Y, Liu Z, Yan Y, Tian J, Chen S, Zhang B - Mol. Vis. (2011)

Co-localization of F-actin and FRMD7. HEK293T cells transiently transfected with EGFP-tagged wild-type FRMD7 and FRMD7 mutants, c.781C>G and c.886G>C, (shown in green) were stained with TRITC-conjugated rhodamine–phalloidin fluorescein (red). All three fusion proteins exhibited a diffuse localization pattern in the cytoplasm, and also in actin-rich regions of the HEK293T cells (arrows). However, HEK293T cells expressing the FRMD7 mutant, c.1003C>T, fused to EGFP, primarily exhibited localization of the fusion protein to the nucleus and did not co-localize with F-actin (merge). Scale bars: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3049738&req=5

f3: Co-localization of F-actin and FRMD7. HEK293T cells transiently transfected with EGFP-tagged wild-type FRMD7 and FRMD7 mutants, c.781C>G and c.886G>C, (shown in green) were stained with TRITC-conjugated rhodamine–phalloidin fluorescein (red). All three fusion proteins exhibited a diffuse localization pattern in the cytoplasm, and also in actin-rich regions of the HEK293T cells (arrows). However, HEK293T cells expressing the FRMD7 mutant, c.1003C>T, fused to EGFP, primarily exhibited localization of the fusion protein to the nucleus and did not co-localize with F-actin (merge). Scale bars: 20 μm.
Mentions: It is hypothesized that FRMD7 regulates neuronal development by influencing F-actin dynamics. Therefore, EGFP-tagged mutated versions of FRMD7 were transiently transfected into HEK293T cells and these cells were stained with TRITC rhodamine–phalloidin to detect F-actin. In these experiments, both wild-type FRMD7 and c.781C>G and c.886G>C FRMD7 mutants exhibited a diffuse distribution in the cytoplasm, and also in actin-rich regions. However, another FRMD7 mutant, c.1003C>T, exhibited changes in its subcellular distribution pattern and did not co-localize with F-actin (Figure 3).

Bottom Line: The COOH-terminus of FRMD7 was found to play a key role in the subcellular localization of FRMD7 in mouse neuroblastoma cells (Neuro-2a) and human embryonic kidney 293 cells (HEK293T).While no differences in the co-localization of F-actin between the wild-type and missense mutation-type (c.781C>G and c.886G>C) proteins was observed, an additional mutant, c.1003C>T, which results in a COOH-terminally truncated protein, exhibited a nuclear localization pattern which did not co-localize with the cytoplasmic distribution of F-actin.The results of the present study indicate that FRMD7 may play an important role in the brainstem in the early stages of development of the human fetal brain, and provides clues for the mechanism of mutation FRMD7, which may be involved in influencing F-actin dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology (Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education), 2nd Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China.

ABSTRACT

Purpose: FERM domain containing 7 (FRMD7) is a member of the four-point-one, ezrin, radixin, moesin (FERM) family of proteins, and has been reported to cause X-linked idiopathic congenital nystagmus (ICN), a disease which affects ocular motor control. There have been over 30 mutations reported for FRMD7, however, their role in the pathogenesis of ICN remains unclear. The purpose of this study is to perform the expression distributes of protein FRMD7 from human fetal brain during development and to understand the relationship with cytoskeletal protein F-actin between wild-type and mutation-type FRMD7.

Methods: Expression of protein FRMD7 from developing human fetal brain was tested by immunohistochemistry. Enhanced green fluorescent protein (EGFP)-tagged recombinant plasmids DNA encoding the normal or mutant FRMD7 were used to transiently transfect the mouse neuroblastoma cells (Neuro-2a) and human embryonic kidney 293 cells (HEK293T). Further, confocal microscopic analysis was used to determine the subcellular localization of the fusion proteins. To visualize F-actin, fixed HEK293T cells were stained with rhodamine-phalloidin.

Results: We show that expression of FRMD7 was mainly detected in the brainstem (a region associated with ocular motor control), while limited level was observed in the cortex. The COOH-terminus of FRMD7 was found to play a key role in the subcellular localization of FRMD7 in mouse neuroblastoma cells (Neuro-2a) and human embryonic kidney 293 cells (HEK293T). While no differences in the co-localization of F-actin between the wild-type and missense mutation-type (c.781C>G and c.886G>C) proteins was observed, an additional mutant, c.1003C>T, which results in a COOH-terminally truncated protein, exhibited a nuclear localization pattern which did not co-localize with the cytoplasmic distribution of F-actin.

Conclusions: The results of the present study indicate that FRMD7 may play an important role in the brainstem in the early stages of development of the human fetal brain, and provides clues for the mechanism of mutation FRMD7, which may be involved in influencing F-actin dynamics.

Show MeSH
Related in: MedlinePlus