Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice
Ryotaro Sakamori, Soumyashree Das, Shiyan Yu, Shanshan Feng, Ewa Stypulkowski, Yinzheng Guan, Veronique Douard, Waixing Tang, Ronaldo P. Ferraris, Akihiro Harada, Cord Brakebusch, Wei Guo, Nan Gao
Ryotaro Sakamori, Soumyashree Das, Shiyan Yu, Shanshan Feng, Ewa Stypulkowski, Yinzheng Guan, Veronique Douard, Waixing Tang, Ronaldo P. Ferraris, Akihiro Harada, Cord Brakebusch, Wei Guo, Nan Gao
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Research Article Gastroenterology

Cdc42 and Rab8a are critical for intestinal stem cell division, survival, and differentiation in mice

Abstract

The constant self renewal and differentiation of adult intestinal stem cells maintains a functional intestinal mucosa for a lifetime. However, the molecular mechanisms that regulate intestinal stem cell division and epithelial homeostasis are largely undefined. We report here that the small GTPases Cdc42 and Rab8a are critical regulators of these processes in mice. Conditional ablation of Cdc42 in the mouse intestinal epithelium resulted in the formation of large intracellular vacuolar structures containing microvilli (microvillus inclusion bodies) in epithelial enterocytes, a phenotype reminiscent of human microvillus inclusion disease (MVID), a devastating congenital intestinal disorder that results in severe nutrient deprivation. Further analysis revealed that Cdc42-deficient stem cells had cell division defects, reduced capacity for clonal expansion and differentiation into Paneth cells, and increased apoptosis. Cdc42 deficiency impaired Rab8a activation and its association with multiple effectors, and prevented trafficking of Rab8a vesicles to the midbody. This impeded cytokinesis, triggering crypt apoptosis and disrupting epithelial morphogenesis. Rab8a was also required for Cdc42-GTP activity in the intestinal epithelium, where continued cell division takes place. Furthermore, mice haploinsufficient for both Cdc42 and Rab8a in the intestine demonstrated abnormal crypt morphogenesis and epithelial transporter physiology, further supporting their functional interaction. These data suggest that defects of the stem cell niche can cause MVID. This hypothesis represents a conceptual departure from the conventional view of this disease, which has focused on the affected enterocytes, and suggests stem cell–based approaches could be beneficial to infants with this often lethal condition.

Authors

Ryotaro Sakamori, Soumyashree Das, Shiyan Yu, Shanshan Feng, Ewa Stypulkowski, Yinzheng Guan, Veronique Douard, Waixing Tang, Ronaldo P. Ferraris, Akihiro Harada, Cord Brakebusch, Wei Guo, Nan Gao

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Figure 7

Cdc42 depletion affects midbody trafficking of Rab8a vesicle and cytokinesis.

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Cdc42 depletion affects midbody trafficking of Rab8a vesicle and cytoki...
(A) Western blots confirmed Cdc42 knockdown by siRNA in Hela cells. (B and C) Live cell imaging showed that Rab8a-GFP (green) trafficked to the mcherry-tubulin–labeled midbody (red) during early cytokinesis in control cells but not in Cdc42-depleted cells. Arrows in B indicate enriched Rab8a at control midbody. Arrowhead in C indicates less Rab8a protein at midbody of Cdc42-depleted cells. (D and E) Rab8a-GFP localizes to the midbody (arrows in D) during late cytokinesis in control cells but not in Cdc42-depleted cells (arrowheads in E). (F) Quantification of cytokinesis in cells transfected with control siRNA, Cdc42 siRNA (Cdc42KD), and cells treated with 7 μM CASIN. (G) FACS cell cycle analyses of CASIN-treated cells showed accumulation at the G2/M phase. (H) Western blots confirmed Cdc42 knockdown in Caco2 cells by a lentiviral shRNA particle. (I and J) Rab8a and Pkcζ staining for control and Cdc42 knockdown Caco2 cysts. Arrowheads in J indicate large vacuoles where Rab8a was localized. Scale bars: 10 μm. *P < 0.05; **P < 0.01.