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EHBP-1 functions with RAB-10 during endocytic recycling in Caenorhabditis elegans.

Shi A, Chen CC, Banerjee R, Glodowski D, Audhya A, Rongo C, Grant BD - Mol. Biol. Cell (2010)

Bottom Line: A similar role was found for mammalian Rab10 in MDCK cells, suggesting that a conserved mechanism regulates these related pathways in metazoans.We also show that loss of EHBP-1 disrupts transport of membrane proteins to the plasma membrane of the nonpolarized germline cells, a defect that can be phenocopied by codepletion of RAB-10 and its closest paralog RAB-8.These results indicate that RAB-10 and EHBP-1 function together in many cell types and suggests that there are differences in the level of redundancy among Rab family members in polarized versus nonpolarized cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA.

ABSTRACT
Caenorhabditis elegans RAB-10 functions in endocytic recycling in polarized cells, regulating basolateral cargo transport in the intestinal epithelia and postsynaptic cargo transport in interneurons. A similar role was found for mammalian Rab10 in MDCK cells, suggesting that a conserved mechanism regulates these related pathways in metazoans. In a yeast two-hybrid screen for binding partners of RAB-10 we identified EHBP-1, a calponin homology domain (CH) protein, whose mammalian homolog Ehbp1 was previously shown to function during endocytic transport of GLUT4 in adipocytes. In vivo we find that EHBP-1-GFP colocalizes with RFP-RAB-10 on endosomal structures of the intestine and interneurons and that ehbp-1 loss-of-function mutants share with rab-10 mutants specific endosome morphology and cargo localization defects. We also show that loss of EHBP-1 disrupts transport of membrane proteins to the plasma membrane of the nonpolarized germline cells, a defect that can be phenocopied by codepletion of RAB-10 and its closest paralog RAB-8. These results indicate that RAB-10 and EHBP-1 function together in many cell types and suggests that there are differences in the level of redundancy among Rab family members in polarized versus nonpolarized cells.

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(A–E) Nomarski images of the gonads in wild-type, rab-10(q373), rab-8(RNAi), rab-10(q373);rab-8(RNAi), and ehbp-1(tm2523) animals. Developmental defects of gonads were found in rab-10(q373);rab-8(RNAi) animals and ehbp-1(tm2523) mutants (D and E). Arrows indicate the normal large squared-off oocyte cells in the proximal gonad region (A–C) and the lack of the characteristic row of oocyte cells in the proximal gonad regions (D and E). Scale bar, 10 μm. (F–H‴) Depletion of EHBP-1 or codepletion of RAB-8 and -10 result in a similar defect in germline secretion. GFP-SNB-1 localized mostly to the plasma membrane of developing compartments in the germline and colocalized with mCherry-PH at steady state (F–F″). Line-scan analysis showed that fluorescence intensity of GFP-SNB-1 and mCherry-PH peaked at identical points (F‴). RNAi depletion of RAB-8 and -10 caused a defect in the localization of GFP-SNB-1 at the plasma membrane (G–G‴). RNAi depletion of EHBP-1 caused a similar defect in GFP-SNB-1 delivery to the plasma membrane (H–H‴).
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Figure 8: (A–E) Nomarski images of the gonads in wild-type, rab-10(q373), rab-8(RNAi), rab-10(q373);rab-8(RNAi), and ehbp-1(tm2523) animals. Developmental defects of gonads were found in rab-10(q373);rab-8(RNAi) animals and ehbp-1(tm2523) mutants (D and E). Arrows indicate the normal large squared-off oocyte cells in the proximal gonad region (A–C) and the lack of the characteristic row of oocyte cells in the proximal gonad regions (D and E). Scale bar, 10 μm. (F–H‴) Depletion of EHBP-1 or codepletion of RAB-8 and -10 result in a similar defect in germline secretion. GFP-SNB-1 localized mostly to the plasma membrane of developing compartments in the germline and colocalized with mCherry-PH at steady state (F–F″). Line-scan analysis showed that fluorescence intensity of GFP-SNB-1 and mCherry-PH peaked at identical points (F‴). RNAi depletion of RAB-8 and -10 caused a defect in the localization of GFP-SNB-1 at the plasma membrane (G–G‴). RNAi depletion of EHBP-1 caused a similar defect in GFP-SNB-1 delivery to the plasma membrane (H–H‴).

Mentions: As mentioned earlier, we found that adult ehbp-1(tm2523) mutant animals are completely sterile (0 eggs produced, n = 18 adult hermaphrodites). The sterile adults have abnormally small gonads and completely lack the characteristic row of large squared-off oocyte cells in the proximal gonad region (Figure 8, A and E, arrows). This suggests a defect in oocyte growth and/or differentiation in these animals. Although such a phenotype might be explained by severe membrane-trafficking defects in the germline cells, this phenotype cannot be completely explained simply by loss of RAB-10 or -8 function, because neither the rab-10 nor -8 mutant is sterile; however, each single mutant does display a reduced brood size. We measured rab-10 mutant brood size as 162 ± 10 (n = 18) and rab-8 mutant brood size as 152 ± 4 (n = 18), compared with wild-type N2 controls that displayed a brood size of 280 ± 12 (n = 18).


EHBP-1 functions with RAB-10 during endocytic recycling in Caenorhabditis elegans.

Shi A, Chen CC, Banerjee R, Glodowski D, Audhya A, Rongo C, Grant BD - Mol. Biol. Cell (2010)

(A–E) Nomarski images of the gonads in wild-type, rab-10(q373), rab-8(RNAi), rab-10(q373);rab-8(RNAi), and ehbp-1(tm2523) animals. Developmental defects of gonads were found in rab-10(q373);rab-8(RNAi) animals and ehbp-1(tm2523) mutants (D and E). Arrows indicate the normal large squared-off oocyte cells in the proximal gonad region (A–C) and the lack of the characteristic row of oocyte cells in the proximal gonad regions (D and E). Scale bar, 10 μm. (F–H‴) Depletion of EHBP-1 or codepletion of RAB-8 and -10 result in a similar defect in germline secretion. GFP-SNB-1 localized mostly to the plasma membrane of developing compartments in the germline and colocalized with mCherry-PH at steady state (F–F″). Line-scan analysis showed that fluorescence intensity of GFP-SNB-1 and mCherry-PH peaked at identical points (F‴). RNAi depletion of RAB-8 and -10 caused a defect in the localization of GFP-SNB-1 at the plasma membrane (G–G‴). RNAi depletion of EHBP-1 caused a similar defect in GFP-SNB-1 delivery to the plasma membrane (H–H‴).
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Figure 8: (A–E) Nomarski images of the gonads in wild-type, rab-10(q373), rab-8(RNAi), rab-10(q373);rab-8(RNAi), and ehbp-1(tm2523) animals. Developmental defects of gonads were found in rab-10(q373);rab-8(RNAi) animals and ehbp-1(tm2523) mutants (D and E). Arrows indicate the normal large squared-off oocyte cells in the proximal gonad region (A–C) and the lack of the characteristic row of oocyte cells in the proximal gonad regions (D and E). Scale bar, 10 μm. (F–H‴) Depletion of EHBP-1 or codepletion of RAB-8 and -10 result in a similar defect in germline secretion. GFP-SNB-1 localized mostly to the plasma membrane of developing compartments in the germline and colocalized with mCherry-PH at steady state (F–F″). Line-scan analysis showed that fluorescence intensity of GFP-SNB-1 and mCherry-PH peaked at identical points (F‴). RNAi depletion of RAB-8 and -10 caused a defect in the localization of GFP-SNB-1 at the plasma membrane (G–G‴). RNAi depletion of EHBP-1 caused a similar defect in GFP-SNB-1 delivery to the plasma membrane (H–H‴).
Mentions: As mentioned earlier, we found that adult ehbp-1(tm2523) mutant animals are completely sterile (0 eggs produced, n = 18 adult hermaphrodites). The sterile adults have abnormally small gonads and completely lack the characteristic row of large squared-off oocyte cells in the proximal gonad region (Figure 8, A and E, arrows). This suggests a defect in oocyte growth and/or differentiation in these animals. Although such a phenotype might be explained by severe membrane-trafficking defects in the germline cells, this phenotype cannot be completely explained simply by loss of RAB-10 or -8 function, because neither the rab-10 nor -8 mutant is sterile; however, each single mutant does display a reduced brood size. We measured rab-10 mutant brood size as 162 ± 10 (n = 18) and rab-8 mutant brood size as 152 ± 4 (n = 18), compared with wild-type N2 controls that displayed a brood size of 280 ± 12 (n = 18).

Bottom Line: A similar role was found for mammalian Rab10 in MDCK cells, suggesting that a conserved mechanism regulates these related pathways in metazoans.We also show that loss of EHBP-1 disrupts transport of membrane proteins to the plasma membrane of the nonpolarized germline cells, a defect that can be phenocopied by codepletion of RAB-10 and its closest paralog RAB-8.These results indicate that RAB-10 and EHBP-1 function together in many cell types and suggests that there are differences in the level of redundancy among Rab family members in polarized versus nonpolarized cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA.

ABSTRACT
Caenorhabditis elegans RAB-10 functions in endocytic recycling in polarized cells, regulating basolateral cargo transport in the intestinal epithelia and postsynaptic cargo transport in interneurons. A similar role was found for mammalian Rab10 in MDCK cells, suggesting that a conserved mechanism regulates these related pathways in metazoans. In a yeast two-hybrid screen for binding partners of RAB-10 we identified EHBP-1, a calponin homology domain (CH) protein, whose mammalian homolog Ehbp1 was previously shown to function during endocytic transport of GLUT4 in adipocytes. In vivo we find that EHBP-1-GFP colocalizes with RFP-RAB-10 on endosomal structures of the intestine and interneurons and that ehbp-1 loss-of-function mutants share with rab-10 mutants specific endosome morphology and cargo localization defects. We also show that loss of EHBP-1 disrupts transport of membrane proteins to the plasma membrane of the nonpolarized germline cells, a defect that can be phenocopied by codepletion of RAB-10 and its closest paralog RAB-8. These results indicate that RAB-10 and EHBP-1 function together in many cell types and suggests that there are differences in the level of redundancy among Rab family members in polarized versus nonpolarized cells.

Show MeSH
Related in: MedlinePlus