RAB11A and RAB11B regulate intestinal homeostasis by orchestrating mitotic spindle dynamics
Creators: name: Joseph, Ivor
Publisher: name: No Publisher Supplied
Year: 2023

description: The transportation and distribution of proteins to specific cellular compartments are vital for the proper functioning of eukaryotic cells and tissue homeostasis. Rab family of small GTPases are crucial regulators of vesicular and protein trafficking within the exo- and endocytic pathways. In the past decade, there has been a growing interest in understanding the biological functions of RAB11 small GTPase. Even though considerable progress has been made in terms of understanding its involvement in regulating the intracellular transport of important cargo proteins, the physiological relevance of these findings to mammalian tissue homeostasis and function remains largely unexplored. In addition, whether there is functional redundancy or divergence among different mammalian RAB11 protein family members are also not clear. To address these knowledge gaps, this thesis project has leveraged innovative Rab11a and Rab11b genetically engineered mouse models to investigate their functions in mouse intestinal epithelial cell homeostasis and renewal.The experimental approach involved genetic and tissue specific ablation of Rab11a, Rab11b, or both in mouse intestinal epithelial cells. Phenotypical analysis comparing the intestinal morphology and function was performed in neonatal mice with constitutive Rab11a deletion or in adult mice with inducible Rab11a deletion. Strikingly, none of the neonatal double knockout mice survive beyond the first day of birth, while adult double knockout mice died within 3 days of induced Rab11a deletion. A full phenotypic penetrance was observed for the lethality, and the results were replicated in different breeding pairs. Detailed histopathology and molecular analysis of the tissues revealed that Rab11a and Rab11b double knockout (DKO) intestinal epithelial cells had abnormal cell cycle entry, pronounced mitotic arrest, and an upsurge in apoptotic events. These cellular changes were echoed by a rapid loss of cycling intestinal progenitor or stem cells, ultimately leading to the death of the animals. Interestingly, enteriods derived from the DKO mice exhibited abnormal mitotic spindles and apoptosis upon induced Rab11 deletion, suggesting that the observed epithelial phenotypes were driven by an epithelial autonomous mechanism. To gain further insights into the underlying molecular mechanisms, we performed proteomics analysis for Rab11a and Rab11b using transfection, coimmunoprecipitation, and mass spectrometry analysis. These analyses uncovered a large set of mitotic spindle regulatory proteins that are shared by both Rab11a and Rab11b interactomes. Interestingly, the top candidate is a microtubule-associated kinesin motor protein (KIF11). Additional biochemical and cell biology experiments demonstrated a strong dependence of KIF11-associated bipolar spindle formation on Rab11a and Rab11b. Depletion of RAB11A in human Caco2 cells disrupted KIF11 interactome, notably the association of KIF11 and CLIP-1, a protein that links endosomes to mitotic spindle microtubules. These genetic and biochemical data strongly established an interdependent regulation of mitotic spindle formation by Rab11a and Rab11b in mouse gut. We envision such regulation may play a similar role in other mammalian tissues that rely on stem cell division to maintain tissue homeostasis.