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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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Related in: MedlinePlus

Expression of FRMD7 protein. A-F: FRMD7 protein expression in the brainstem of developing human fetal brain. Sections from human fetal brainstem tissue were examined by immunohistochemistry with anti-FRMD7 antibody. Positive staining (brown) was showed in the cytoplasm (arrows), and strong immunoreactivity was detected at 16–17 weeks post-conception (wpc) (A: 100×; D: 400×), while lower levels of FRMD7 immunoreactivity were observed at 21 wpc (B: 100×, E: 400×) and 25 wpc (C: 100×; F: 400×). Scale bars: (A, B, and C; 400 μm), (D, E, and F; 100 μm). G-L: Immunohistochemical staining of protein FRMD7 in the cortex of human fetal brain tissue using anti-FRMD7 antibody. Limited expression (arrow) was manifested in the cortex plate at 16–17 weeks post-conception (wpc; G: 100×; J: 400×), and at 21 wpc (H: 100×, K: 400×) and 25 wpc (I: 100×; L: 400×) there were little positive staining detected. Scale bars: (G, H, and I; 400 μm), (J, K, and L; 100 μm).
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f1: Expression of FRMD7 protein. A-F: FRMD7 protein expression in the brainstem of developing human fetal brain. Sections from human fetal brainstem tissue were examined by immunohistochemistry with anti-FRMD7 antibody. Positive staining (brown) was showed in the cytoplasm (arrows), and strong immunoreactivity was detected at 16–17 weeks post-conception (wpc) (A: 100×; D: 400×), while lower levels of FRMD7 immunoreactivity were observed at 21 wpc (B: 100×, E: 400×) and 25 wpc (C: 100×; F: 400×). Scale bars: (A, B, and C; 400 μm), (D, E, and F; 100 μm). G-L: Immunohistochemical staining of protein FRMD7 in the cortex of human fetal brain tissue using anti-FRMD7 antibody. Limited expression (arrow) was manifested in the cortex plate at 16–17 weeks post-conception (wpc; G: 100×; J: 400×), and at 21 wpc (H: 100×, K: 400×) and 25 wpc (I: 100×; L: 400×) there were little positive staining detected. Scale bars: (G, H, and I; 400 μm), (J, K, and L; 100 μm).

Mentions: Expression of the nystagmus-related FRMD7 protein was examined during three stages of human fetal development (16–17, 21, and 25 wpc) and in five different regions (cortex, brainstem, hippocampus, cerebellum, and diencephalons) for each stage. At 16–17 wpc, FRMD7 was observed to be highly expressed in the pons, medulla oblongata, and midbrain (brainstem; Figure 1A-F), which are important regions associated with ocular motor control. FRMD7 was also observed in the cerebellar region and diencephalons at 16–17 wpc. During the later stages of fetal development (i.e., 21 and 25 wpc), overall expression of FRMD7 was found to significantly decrease in the brainstem, and from the cortex, limited expression was manifested in the cortex plate (Figure 1G-L).


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)

Expression of FRMD7 protein. A-F: FRMD7 protein expression in the brainstem of developing human fetal brain. Sections from human fetal brainstem tissue were examined by immunohistochemistry with anti-FRMD7 antibody. Positive staining (brown) was showed in the cytoplasm (arrows), and strong immunoreactivity was detected at 16–17 weeks post-conception (wpc) (A: 100×; D: 400×), while lower levels of FRMD7 immunoreactivity were observed at 21 wpc (B: 100×, E: 400×) and 25 wpc (C: 100×; F: 400×). Scale bars: (A, B, and C; 400 μm), (D, E, and F; 100 μm). G-L: Immunohistochemical staining of protein FRMD7 in the cortex of human fetal brain tissue using anti-FRMD7 antibody. Limited expression (arrow) was manifested in the cortex plate at 16–17 weeks post-conception (wpc; G: 100×; J: 400×), and at 21 wpc (H: 100×, K: 400×) and 25 wpc (I: 100×; L: 400×) there were little positive staining detected. Scale bars: (G, H, and I; 400 μm), (J, K, and L; 100 μm).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Expression of FRMD7 protein. A-F: FRMD7 protein expression in the brainstem of developing human fetal brain. Sections from human fetal brainstem tissue were examined by immunohistochemistry with anti-FRMD7 antibody. Positive staining (brown) was showed in the cytoplasm (arrows), and strong immunoreactivity was detected at 16–17 weeks post-conception (wpc) (A: 100×; D: 400×), while lower levels of FRMD7 immunoreactivity were observed at 21 wpc (B: 100×, E: 400×) and 25 wpc (C: 100×; F: 400×). Scale bars: (A, B, and C; 400 μm), (D, E, and F; 100 μm). G-L: Immunohistochemical staining of protein FRMD7 in the cortex of human fetal brain tissue using anti-FRMD7 antibody. Limited expression (arrow) was manifested in the cortex plate at 16–17 weeks post-conception (wpc; G: 100×; J: 400×), and at 21 wpc (H: 100×, K: 400×) and 25 wpc (I: 100×; L: 400×) there were little positive staining detected. Scale bars: (G, H, and I; 400 μm), (J, K, and L; 100 μm).
Mentions: Expression of the nystagmus-related FRMD7 protein was examined during three stages of human fetal development (16–17, 21, and 25 wpc) and in five different regions (cortex, brainstem, hippocampus, cerebellum, and diencephalons) for each stage. At 16–17 wpc, FRMD7 was observed to be highly expressed in the pons, medulla oblongata, and midbrain (brainstem; Figure 1A-F), which are important regions associated with ocular motor control. FRMD7 was also observed in the cerebellar region and diencephalons at 16–17 wpc. During the later stages of fetal development (i.e., 21 and 25 wpc), overall expression of FRMD7 was found to significantly decrease in the brainstem, and from the cortex, limited expression was manifested in the cortex plate (Figure 1G-L).

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