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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.

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Subcellular localization of full-length FRMD7 versus various domains of FRMD7. HEK293T cells transiently transfected with EGFP-fusion proteins of FRMD7 (green) were stained with DAPI (blue) and imaged. Full-length FRMD7 was diffusely localized to the cytoplasm, while the NH2-terminal FERM domain (aa 1–279), and the FERM domain plus the FA domain, primarily localized to the nucleus (arrows). Constructs containing the COOH-terminal region of FRMD7 (e.g., △FERM and △FERM+FA) exhibited an expression pattern similar to that of full-length FRMD7, however, the expression level was decreased and some aggregates of these truncated proteins were detected in the cytoplasm. The results obtained from Neuro-2a cells (not shown) was the same as HEK293T cells. FERM: four-point-one, ezrin, radixin, moesin; FERM+FA: FERM domain and FERM Adjacent domain; △FERM: truncated NH2-terminal FERM domain; △FERM+FA; truncated NH2-terminal FERM domain and FERM Adjacent domain. Scale bars: 20 μm.
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f2: Subcellular localization of full-length FRMD7 versus various domains of FRMD7. HEK293T cells transiently transfected with EGFP-fusion proteins of FRMD7 (green) were stained with DAPI (blue) and imaged. Full-length FRMD7 was diffusely localized to the cytoplasm, while the NH2-terminal FERM domain (aa 1–279), and the FERM domain plus the FA domain, primarily localized to the nucleus (arrows). Constructs containing the COOH-terminal region of FRMD7 (e.g., △FERM and △FERM+FA) exhibited an expression pattern similar to that of full-length FRMD7, however, the expression level was decreased and some aggregates of these truncated proteins were detected in the cytoplasm. The results obtained from Neuro-2a cells (not shown) was the same as HEK293T cells. FERM: four-point-one, ezrin, radixin, moesin; FERM+FA: FERM domain and FERM Adjacent domain; △FERM: truncated NH2-terminal FERM domain; △FERM+FA; truncated NH2-terminal FERM domain and FERM Adjacent domain. Scale bars: 20 μm.

Mentions: To better understand the function of FRMD7, HEK293T and Neuro-2a cells were transiently transfected with various EGFP-tagged fragments of FRMD7. In these experiments, full-length FRMD7 was found to be uniformly distributed in the cytoplasm. In contrast, when various regions of FRMD7 were overexpressed, only the COOH-terminal regions of FRMD7 (△FERM and △FERM+FA) resulted in a distribution pattern that was similar to that of full-length FRMD7. However, for these modified FRMD7 proteins, the expression levels were lower, and some aggregates were observed in the cytoplasm. For FRMD7 constructs containing only the NH2-terminal FERM domain (aa 1–279) of FRMD7, or a combination of the FERM domain and the FA domain (adjacent to the FERM domain; aa 294–336), a predominantly nuclear distribution was observed (Figure 2).


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)

Subcellular localization of full-length FRMD7 versus various domains of FRMD7. HEK293T cells transiently transfected with EGFP-fusion proteins of FRMD7 (green) were stained with DAPI (blue) and imaged. Full-length FRMD7 was diffusely localized to the cytoplasm, while the NH2-terminal FERM domain (aa 1–279), and the FERM domain plus the FA domain, primarily localized to the nucleus (arrows). Constructs containing the COOH-terminal region of FRMD7 (e.g., △FERM and △FERM+FA) exhibited an expression pattern similar to that of full-length FRMD7, however, the expression level was decreased and some aggregates of these truncated proteins were detected in the cytoplasm. The results obtained from Neuro-2a cells (not shown) was the same as HEK293T cells. FERM: four-point-one, ezrin, radixin, moesin; FERM+FA: FERM domain and FERM Adjacent domain; △FERM: truncated NH2-terminal FERM domain; △FERM+FA; truncated NH2-terminal FERM domain and FERM Adjacent domain. Scale bars: 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Subcellular localization of full-length FRMD7 versus various domains of FRMD7. HEK293T cells transiently transfected with EGFP-fusion proteins of FRMD7 (green) were stained with DAPI (blue) and imaged. Full-length FRMD7 was diffusely localized to the cytoplasm, while the NH2-terminal FERM domain (aa 1–279), and the FERM domain plus the FA domain, primarily localized to the nucleus (arrows). Constructs containing the COOH-terminal region of FRMD7 (e.g., △FERM and △FERM+FA) exhibited an expression pattern similar to that of full-length FRMD7, however, the expression level was decreased and some aggregates of these truncated proteins were detected in the cytoplasm. The results obtained from Neuro-2a cells (not shown) was the same as HEK293T cells. FERM: four-point-one, ezrin, radixin, moesin; FERM+FA: FERM domain and FERM Adjacent domain; △FERM: truncated NH2-terminal FERM domain; △FERM+FA; truncated NH2-terminal FERM domain and FERM Adjacent domain. Scale bars: 20 μm.
Mentions: To better understand the function of FRMD7, HEK293T and Neuro-2a cells were transiently transfected with various EGFP-tagged fragments of FRMD7. In these experiments, full-length FRMD7 was found to be uniformly distributed in the cytoplasm. In contrast, when various regions of FRMD7 were overexpressed, only the COOH-terminal regions of FRMD7 (△FERM and △FERM+FA) resulted in a distribution pattern that was similar to that of full-length FRMD7. However, for these modified FRMD7 proteins, the expression levels were lower, and some aggregates were observed in the cytoplasm. For FRMD7 constructs containing only the NH2-terminal FERM domain (aa 1–279) of FRMD7, or a combination of the FERM domain and the FA domain (adjacent to the FERM domain; aa 294–336), a predominantly nuclear distribution was observed (Figure 2).

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