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The Arp2/3 complex has essential roles in vesicle trafficking and transcytosis in the mammalian small intestine.

Zhou K, Sumigray KD, Lechler T - Mol. Biol. Cell (2015)

Bottom Line: We found that in the absence of ArpC3, enterocytes had defects in the organization of the endolysosomal system.These defects were physiologically relevant, as transcytosis of IgG was disrupted, lipid absorption was perturbed, and neonatal mice died within days of birth.These data highlight the important roles of the Arp2/3 complex in vesicle trafficking in enterocytes and suggest that defects in cytoplasmic F-actin assembly by the Arp2/3 complex, rather than cortical pools, underlie many of the phenotypes seen in the mutant small intestine.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology and Department of Cell Biology, Duke University Medical Center, Durham, NC 27710.

No MeSH data available.


Related in: MedlinePlus

Normal cortical F-actin, polarity, and adhesion in ArpC3 cKO mice. (A–B′) F-actin staining of WT (A) and ArpC3 cKO (B) intestinal sections. A′ and B′ are brightness enhanced to visualize the low levels of lateral F-actin. (C) Quantitation of fluorescence intensity of phalloidin staining of F-actin. (D, E) Transmission electron micrographs of the brush borders of WT and ArpC3 cKO intestine. Scale bar, 2 10 μm. (F, G) Transmission electron micrographs of the apical junctions of WT and ArpC3 cKO intestine. (H, I) Tight junction (ZO-1, green) and adherens junctions (E-cadherin, red) staining in WT and ArpC3 cKO intestine. Scale bar, 10 μm. Dashed line indicates the basement membrane. (J, K) Occludin (red) localization in WT and ArpC3 cKO intestine. (L–O) Whole-mount intestines stained for ZO1. L–N represent the predominant phenotype, and O represents the status of ZO1 in a smaller number of mutant villi. (P, Q) Ezrin (green) and E-cadherin (red) staining of WT and mutant intestine. (R, S) WT and mutant intestinal sections were stained for the trans-Golgi network protein, Grasp64 (red) and F-actin (green). (T, U) Staining for the terminal web and brush border marker myosin 1D (red) in WT and ArpC3 cKO intestine. Scale bars, 10 μm.
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Figure 2: Normal cortical F-actin, polarity, and adhesion in ArpC3 cKO mice. (A–B′) F-actin staining of WT (A) and ArpC3 cKO (B) intestinal sections. A′ and B′ are brightness enhanced to visualize the low levels of lateral F-actin. (C) Quantitation of fluorescence intensity of phalloidin staining of F-actin. (D, E) Transmission electron micrographs of the brush borders of WT and ArpC3 cKO intestine. Scale bar, 2 10 μm. (F, G) Transmission electron micrographs of the apical junctions of WT and ArpC3 cKO intestine. (H, I) Tight junction (ZO-1, green) and adherens junctions (E-cadherin, red) staining in WT and ArpC3 cKO intestine. Scale bar, 10 μm. Dashed line indicates the basement membrane. (J, K) Occludin (red) localization in WT and ArpC3 cKO intestine. (L–O) Whole-mount intestines stained for ZO1. L–N represent the predominant phenotype, and O represents the status of ZO1 in a smaller number of mutant villi. (P, Q) Ezrin (green) and E-cadherin (red) staining of WT and mutant intestine. (R, S) WT and mutant intestinal sections were stained for the trans-Golgi network protein, Grasp64 (red) and F-actin (green). (T, U) Staining for the terminal web and brush border marker myosin 1D (red) in WT and ArpC3 cKO intestine. Scale bars, 10 μm.

Mentions: In the C. elegans intestine, RNAi knockdown or mutation of Arp2/3 complex components or its activators alters filamentous actin (F-actin) organization (Bernadskaya et al., 2011). To determine whether F-actin organization was notably disrupted in the mutant, we stained intestinal sections with fluorescent phalloidin. Surprisingly, we did not observe gross changes in cortical F-actin in the mutant intestine (Figure 2, A and B). Phalloidin strongly labeled the brush border in both wild-type and ArpC3 cKO mice, and quantitation of fluorescence intensity demonstrated that the ArpC3 cKO was not significantly different from the wild type (Figure 2C). Whereas the brush border has the highest concentration of F-actin, the basolateral membrane also has an enrichment. There was no difference in intensity or organization of F-actin at these lateral sites when longer exposures were used (Figure 2, A′, B′, and C). Consistent with these data, electron microscopy of ultrathin sections revealed that microvilli were normal in appearance (Figure 2, D and E). Other F-actin structures within the cytoplasm and fine details of cortical actin organization cannot be discerned by traditional light microscopy techniques. That said, our data demonstrate that ArpC3 is not required for the generation of the bulk of cortical F-actin within the enterocyte.


The Arp2/3 complex has essential roles in vesicle trafficking and transcytosis in the mammalian small intestine.

Zhou K, Sumigray KD, Lechler T - Mol. Biol. Cell (2015)

Normal cortical F-actin, polarity, and adhesion in ArpC3 cKO mice. (A–B′) F-actin staining of WT (A) and ArpC3 cKO (B) intestinal sections. A′ and B′ are brightness enhanced to visualize the low levels of lateral F-actin. (C) Quantitation of fluorescence intensity of phalloidin staining of F-actin. (D, E) Transmission electron micrographs of the brush borders of WT and ArpC3 cKO intestine. Scale bar, 2 10 μm. (F, G) Transmission electron micrographs of the apical junctions of WT and ArpC3 cKO intestine. (H, I) Tight junction (ZO-1, green) and adherens junctions (E-cadherin, red) staining in WT and ArpC3 cKO intestine. Scale bar, 10 μm. Dashed line indicates the basement membrane. (J, K) Occludin (red) localization in WT and ArpC3 cKO intestine. (L–O) Whole-mount intestines stained for ZO1. L–N represent the predominant phenotype, and O represents the status of ZO1 in a smaller number of mutant villi. (P, Q) Ezrin (green) and E-cadherin (red) staining of WT and mutant intestine. (R, S) WT and mutant intestinal sections were stained for the trans-Golgi network protein, Grasp64 (red) and F-actin (green). (T, U) Staining for the terminal web and brush border marker myosin 1D (red) in WT and ArpC3 cKO intestine. Scale bars, 10 μm.
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Figure 2: Normal cortical F-actin, polarity, and adhesion in ArpC3 cKO mice. (A–B′) F-actin staining of WT (A) and ArpC3 cKO (B) intestinal sections. A′ and B′ are brightness enhanced to visualize the low levels of lateral F-actin. (C) Quantitation of fluorescence intensity of phalloidin staining of F-actin. (D, E) Transmission electron micrographs of the brush borders of WT and ArpC3 cKO intestine. Scale bar, 2 10 μm. (F, G) Transmission electron micrographs of the apical junctions of WT and ArpC3 cKO intestine. (H, I) Tight junction (ZO-1, green) and adherens junctions (E-cadherin, red) staining in WT and ArpC3 cKO intestine. Scale bar, 10 μm. Dashed line indicates the basement membrane. (J, K) Occludin (red) localization in WT and ArpC3 cKO intestine. (L–O) Whole-mount intestines stained for ZO1. L–N represent the predominant phenotype, and O represents the status of ZO1 in a smaller number of mutant villi. (P, Q) Ezrin (green) and E-cadherin (red) staining of WT and mutant intestine. (R, S) WT and mutant intestinal sections were stained for the trans-Golgi network protein, Grasp64 (red) and F-actin (green). (T, U) Staining for the terminal web and brush border marker myosin 1D (red) in WT and ArpC3 cKO intestine. Scale bars, 10 μm.
Mentions: In the C. elegans intestine, RNAi knockdown or mutation of Arp2/3 complex components or its activators alters filamentous actin (F-actin) organization (Bernadskaya et al., 2011). To determine whether F-actin organization was notably disrupted in the mutant, we stained intestinal sections with fluorescent phalloidin. Surprisingly, we did not observe gross changes in cortical F-actin in the mutant intestine (Figure 2, A and B). Phalloidin strongly labeled the brush border in both wild-type and ArpC3 cKO mice, and quantitation of fluorescence intensity demonstrated that the ArpC3 cKO was not significantly different from the wild type (Figure 2C). Whereas the brush border has the highest concentration of F-actin, the basolateral membrane also has an enrichment. There was no difference in intensity or organization of F-actin at these lateral sites when longer exposures were used (Figure 2, A′, B′, and C). Consistent with these data, electron microscopy of ultrathin sections revealed that microvilli were normal in appearance (Figure 2, D and E). Other F-actin structures within the cytoplasm and fine details of cortical actin organization cannot be discerned by traditional light microscopy techniques. That said, our data demonstrate that ArpC3 is not required for the generation of the bulk of cortical F-actin within the enterocyte.

Bottom Line: We found that in the absence of ArpC3, enterocytes had defects in the organization of the endolysosomal system.These defects were physiologically relevant, as transcytosis of IgG was disrupted, lipid absorption was perturbed, and neonatal mice died within days of birth.These data highlight the important roles of the Arp2/3 complex in vesicle trafficking in enterocytes and suggest that defects in cytoplasmic F-actin assembly by the Arp2/3 complex, rather than cortical pools, underlie many of the phenotypes seen in the mutant small intestine.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology and Department of Cell Biology, Duke University Medical Center, Durham, NC 27710.

No MeSH data available.


Related in: MedlinePlus