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SNX16 negatively regulates the migration and tumorigenesis of MCF-7 cells.

Zhang L, Qin D, Hao C, Shu X, Pei D - Cell Regen (Lond) (2013)

Bottom Line: Ectopic expression of SNX16 reduces the migration and the tumor formation activity of MCF-7 cells.Our results indicate that, in addition to the PI3P, there is a SNX23- and microtubule-dependent cargo transport pathway required for the proper subcellular distribution of SNX16.SNX16 plays a negative regulatory role during cell migration and tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, 510530 China.

ABSTRACT

Background: Sorting nexins are a large family of proteins that are associated with various components of the endosome system and they play many roles in processes such as endocytosis, intracellular protein trafficking and cell signaling. The subcellular distribution patterns of many of them remain controversial and their in vivo functions have not been characterized yet.

Results: We investigated the subcellular distribution and function of SNX16 in this study. SNX16 is detected on Rab5-positive endosomes localized adjacent to focal adhesions at cell cortex. Inhibition of SNX23, polymerization of microtubule filaments as well as the PI3-kinase all disrupt the cell cortex distribution of SNX16. Ectopic expression of SNX16 reduces the migration and the tumor formation activity of MCF-7 cells.

Conclusion: Our results indicate that, in addition to the PI3P, there is a SNX23- and microtubule-dependent cargo transport pathway required for the proper subcellular distribution of SNX16. SNX16 plays a negative regulatory role during cell migration and tumorigenesis.

No MeSH data available.


Related in: MedlinePlus

SNX23 and PI3-kinase are required for the cell cortex distribution of SNX16. (A) SNX16 co-localizes with SNX23 to cell cortex. MCF-7 cells were transfected with the Flag-tagged SNX16 and the GFP-tagged SNX23 and the subcellular distribution of them determined as described above. (B) Efficiency of siRNAs to SNX23 as determined by real-time RT-PCR. (C) Down-regulation of SNX23 by siRNA disrupts the cell cortex distribution of SNX16. (D) The effects of small chemical inhibitors on the subcellular distribution of SNX16. Cells expressing SNX16-GFP were treated with the indicated inhibitors and the subcellular distribution of SNX16 determined as described above. Inhibition the polymerization of microtubules by colchicine or inhibition the activity of PI3-kinase by wortmannin abolishes the cell cortex distribution of SNX16. Inhibition of actin filaments (cytochalasin B) or mTOR (rapamycin) does not affect the cell cortex distribution of SNX16. Treatment of cells with monensin, staurosporine or okadaic acid has no effect on the cell cortex distribution of SNX16.
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Fig2: SNX23 and PI3-kinase are required for the cell cortex distribution of SNX16. (A) SNX16 co-localizes with SNX23 to cell cortex. MCF-7 cells were transfected with the Flag-tagged SNX16 and the GFP-tagged SNX23 and the subcellular distribution of them determined as described above. (B) Efficiency of siRNAs to SNX23 as determined by real-time RT-PCR. (C) Down-regulation of SNX23 by siRNA disrupts the cell cortex distribution of SNX16. (D) The effects of small chemical inhibitors on the subcellular distribution of SNX16. Cells expressing SNX16-GFP were treated with the indicated inhibitors and the subcellular distribution of SNX16 determined as described above. Inhibition the polymerization of microtubules by colchicine or inhibition the activity of PI3-kinase by wortmannin abolishes the cell cortex distribution of SNX16. Inhibition of actin filaments (cytochalasin B) or mTOR (rapamycin) does not affect the cell cortex distribution of SNX16. Treatment of cells with monensin, staurosporine or okadaic acid has no effect on the cell cortex distribution of SNX16.

Mentions: SNX23/KIF16B is a kinesin family protein that can regulate the microtubule-based peripheral transport of early endosomes. It is reported to co-localize with early endosome marker EEA1 at the cell cortex in Hela cells [30]. This distribution pattern of SNX23 is similar to what we observed for SNX16 here, so we compared the subcellular distribution patterns of SNX16 and SNX23. We co-transfected SNX16 and 23 into the MCF-7 cells and found that they co-localize with each other at cell cortex (Figure 2A). Since SNX23 is a motor protein that can regulate the cell peripheral transport of early endosomes, we determined whether the SNX23 transport pathway is required for the cell cortex distribution of SNX16. We knocked-down SNX23 by siRNAs then determined the subcellular distribution pattern of SNX16. Our siRNAs effectively down-regulate the mRNA level of SNX23 (Figure 2B) and we found that down-regulation of SNX23 abolishes the peripheral distribution of SNX16. In fact, the majority of SNX16 vesicles are now detected at the perinuclear regions (Figure 2C, similar result was observed for siSNX23-2). The microtubule filaments are required for the SNX23-mediated cargo transport [30], so we investigated whether the microtubules are involved in the trafficking of SNX16 vesicles. Pretreatment of MCF-7 cells with colchicine, an inhibitor of microtubule polymerization, disrupts the cortex localization of SNX16 vesicles. On the other hand, inhibition of the actin filaments by cytochalasin B does not affect the cell cortex distribution of SNX16 (Figure 2D). So, the SNX23- and microtubule-dependent transport route is required for the cell cortex distribution of SNX16 vesicles.Figure 2


SNX16 negatively regulates the migration and tumorigenesis of MCF-7 cells.

Zhang L, Qin D, Hao C, Shu X, Pei D - Cell Regen (Lond) (2013)

SNX23 and PI3-kinase are required for the cell cortex distribution of SNX16. (A) SNX16 co-localizes with SNX23 to cell cortex. MCF-7 cells were transfected with the Flag-tagged SNX16 and the GFP-tagged SNX23 and the subcellular distribution of them determined as described above. (B) Efficiency of siRNAs to SNX23 as determined by real-time RT-PCR. (C) Down-regulation of SNX23 by siRNA disrupts the cell cortex distribution of SNX16. (D) The effects of small chemical inhibitors on the subcellular distribution of SNX16. Cells expressing SNX16-GFP were treated with the indicated inhibitors and the subcellular distribution of SNX16 determined as described above. Inhibition the polymerization of microtubules by colchicine or inhibition the activity of PI3-kinase by wortmannin abolishes the cell cortex distribution of SNX16. Inhibition of actin filaments (cytochalasin B) or mTOR (rapamycin) does not affect the cell cortex distribution of SNX16. Treatment of cells with monensin, staurosporine or okadaic acid has no effect on the cell cortex distribution of SNX16.
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Fig2: SNX23 and PI3-kinase are required for the cell cortex distribution of SNX16. (A) SNX16 co-localizes with SNX23 to cell cortex. MCF-7 cells were transfected with the Flag-tagged SNX16 and the GFP-tagged SNX23 and the subcellular distribution of them determined as described above. (B) Efficiency of siRNAs to SNX23 as determined by real-time RT-PCR. (C) Down-regulation of SNX23 by siRNA disrupts the cell cortex distribution of SNX16. (D) The effects of small chemical inhibitors on the subcellular distribution of SNX16. Cells expressing SNX16-GFP were treated with the indicated inhibitors and the subcellular distribution of SNX16 determined as described above. Inhibition the polymerization of microtubules by colchicine or inhibition the activity of PI3-kinase by wortmannin abolishes the cell cortex distribution of SNX16. Inhibition of actin filaments (cytochalasin B) or mTOR (rapamycin) does not affect the cell cortex distribution of SNX16. Treatment of cells with monensin, staurosporine or okadaic acid has no effect on the cell cortex distribution of SNX16.
Mentions: SNX23/KIF16B is a kinesin family protein that can regulate the microtubule-based peripheral transport of early endosomes. It is reported to co-localize with early endosome marker EEA1 at the cell cortex in Hela cells [30]. This distribution pattern of SNX23 is similar to what we observed for SNX16 here, so we compared the subcellular distribution patterns of SNX16 and SNX23. We co-transfected SNX16 and 23 into the MCF-7 cells and found that they co-localize with each other at cell cortex (Figure 2A). Since SNX23 is a motor protein that can regulate the cell peripheral transport of early endosomes, we determined whether the SNX23 transport pathway is required for the cell cortex distribution of SNX16. We knocked-down SNX23 by siRNAs then determined the subcellular distribution pattern of SNX16. Our siRNAs effectively down-regulate the mRNA level of SNX23 (Figure 2B) and we found that down-regulation of SNX23 abolishes the peripheral distribution of SNX16. In fact, the majority of SNX16 vesicles are now detected at the perinuclear regions (Figure 2C, similar result was observed for siSNX23-2). The microtubule filaments are required for the SNX23-mediated cargo transport [30], so we investigated whether the microtubules are involved in the trafficking of SNX16 vesicles. Pretreatment of MCF-7 cells with colchicine, an inhibitor of microtubule polymerization, disrupts the cortex localization of SNX16 vesicles. On the other hand, inhibition of the actin filaments by cytochalasin B does not affect the cell cortex distribution of SNX16 (Figure 2D). So, the SNX23- and microtubule-dependent transport route is required for the cell cortex distribution of SNX16 vesicles.Figure 2

Bottom Line: Ectopic expression of SNX16 reduces the migration and the tumor formation activity of MCF-7 cells.Our results indicate that, in addition to the PI3P, there is a SNX23- and microtubule-dependent cargo transport pathway required for the proper subcellular distribution of SNX16.SNX16 plays a negative regulatory role during cell migration and tumorigenesis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Regenerative Biology, Chinese Academy of Sciences, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Guangzhou, 510530 China.

ABSTRACT

Background: Sorting nexins are a large family of proteins that are associated with various components of the endosome system and they play many roles in processes such as endocytosis, intracellular protein trafficking and cell signaling. The subcellular distribution patterns of many of them remain controversial and their in vivo functions have not been characterized yet.

Results: We investigated the subcellular distribution and function of SNX16 in this study. SNX16 is detected on Rab5-positive endosomes localized adjacent to focal adhesions at cell cortex. Inhibition of SNX23, polymerization of microtubule filaments as well as the PI3-kinase all disrupt the cell cortex distribution of SNX16. Ectopic expression of SNX16 reduces the migration and the tumor formation activity of MCF-7 cells.

Conclusion: Our results indicate that, in addition to the PI3P, there is a SNX23- and microtubule-dependent cargo transport pathway required for the proper subcellular distribution of SNX16. SNX16 plays a negative regulatory role during cell migration and tumorigenesis.

No MeSH data available.


Related in: MedlinePlus