Deciphering the interplay between autophagy and polarity in epithelial tubulogenesis.

The Metazoan complexity arises from a primary building block, the epithelium, which comprises a layer of polarized cells that divide the organism into compartments. Most of these body compartments are organs formed by epithelial tubes that enclose an internal hollow space or lumen. Over the last decades, multiple studies have unmasked the paramount events required to form this lumen de novo. In epithelial cells, these events mainly involve recognizing external clues, establishing and maintaining apicobasal polarity, endo-lysosomal trafficking, and expanding the created lumen. Although canonical autophagy has been classically considered a catabolic process needed for cell survival, multiple studies have also emphasized its crucial role in epithelial polarity, morphogenesis and cellular homeostasis. Furthermore, non-canonical autophagy pathways have been recently discovered as atypical secretory routes. Both canonical and non-canonical pathways play essential roles in epithelial polarity and lumen formation. This review addresses how the molecular machinery for epithelial polarity and autophagy interplay in different processes and how autophagy functions influence lumenogenesis, emphasizing its role in the lumen formation key events.

PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20.

Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase-promoting complex/cyclosome and its coactivator CDC20 (APC/CCDC20) form the main ubiquitin E3 ligase for these two proteins. APC/CCDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylation. Here, we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilizes cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1 phosphorylation-defective CDC206A mutants. These CDC206A cells show a normal spindle checkpoint response and rapidly destroy cyclin B once all chromosomes have aligned and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase transition by promoting APC/CCDC20-dependent destruction of cyclin B in human cells.

Checkpoint signaling and error correction require regulation of the MPS1 T-loop by PP2A-B56.

During mitosis, the formation of microtubule-kinetochore attachments is monitored by the serine/threonine kinase monopolar spindle 1 (MPS1). MPS1 is recruited to unattached kinetochores where it phosphorylates KNL1, BUB1, and MAD1 to initiate the spindle assembly checkpoint. This arrests the cell cycle until all kinetochores have been stably captured by microtubules. MPS1 also contributes to the error correction process rectifying incorrect kinetochore attachments. MPS1 activity at kinetochores requires autophosphorylation at multiple sites including threonine 676 in the activation segment or "T-loop." We now demonstrate that the BUBR1-bound pool of PP2A-B56 regulates MPS1 T-loop autophosphorylation and hence activation status in mammalian cells. Overriding this regulation using phosphomimetic mutations in the MPS1 T-loop to generate a constitutively active kinase results in a prolonged mitotic arrest with continuous turnover of microtubule-kinetochore attachments. Dynamic regulation of MPS1 catalytic activity by kinetochore-localized PP2A-B56 is thus critical for controlled MPS1 activity and timely cell cycle progression.

Orchestration of the spindle assembly checkpoint by CDK1-cyclin B1.

In mitosis, the spindle assembly checkpoint (SAC) monitors the formation of microtubule-kinetochore attachments during capture of chromosomes by the mitotic spindle. Spindle assembly is complete once there are no longer any unattached kinetochores. Here, we will discuss the mechanism and key components of spindle checkpoint signalling. Unattached kinetochores bind the principal spindle checkpoint kinase monopolar spindle 1 (MPS1). MPS1 triggers the recruitment of other spindle checkpoint proteins and the formation of a soluble inhibitor of anaphase, thus preventing exit from mitosis. On microtubule attachment, kinetochores become checkpoint silent due to the actions of PP2A-B56 and PP1. This SAC responsive period has to be coordinated with mitotic spindle formation to ensure timely mitotic exit and accurate chromosome segregation. We focus on the molecular mechanisms by which the SAC permissive state is created, describing a central role for CDK1-cyclin B1 and its counteracting phosphatase PP2A-B55. Furthermore, we discuss how CDK1-cyclin B1, through its interaction with MAD1, acts as an integral component of the SAC, and actively orchestrates checkpoint signalling and thus contributes to the faithful execution of mitosis.

CDK1-CCNB1 creates a spindle checkpoint-permissive state by enabling MPS1 kinetochore localization.

Spindle checkpoint signaling is initiated by recruitment of the kinase MPS1 to unattached kinetochores during mitosis. We show that CDK1-CCNB1 and a counteracting phosphatase PP2A-B55 regulate the engagement of human MPS1 with unattached kinetochores by controlling the phosphorylation status of S281 in the kinetochore-binding domain. This regulation is essential for checkpoint signaling, since MPS1S281A is not recruited to unattached kinetochores and fails to support the recruitment of other checkpoint proteins. Directly tethering MPS1S281A to the kinetochore protein Mis12 bypasses this regulation and hence the requirement for S281 phosphorylation in checkpoint signaling. At the metaphase-anaphase transition, MPS1 S281 dephosphorylation is delayed because PP2A-B55 is negatively regulated by CDK1-CCNB1 and only becomes fully active once CCNB1 concentration falls below a characteristic threshold. This mechanism prolongs the checkpoint-responsive period when MPS1 can localize to kinetochores and enables a response to late-stage spindle defects. By acting together, CDK1-CCNB1 and PP2A-B55 thus create a spindle checkpoint-permissive state and ensure the fidelity of mitosis.

MAD1-dependent recruitment of CDK1-CCNB1 to kinetochores promotes spindle checkpoint signaling.

Cyclin B-dependent kinase (CDK1-CCNB1) promotes entry into mitosis. Additionally, it inhibits mitotic exit by activating the spindle checkpoint. This latter role is mediated through phosphorylation of the checkpoint kinase MPS1 and other spindle checkpoint proteins. We find that CDK1-CCNB1 localizes to unattached kinetochores and like MPS1 is lost from these structures upon microtubule attachment. This suggests that CDK1-CCNB1 is an integral component and not only an upstream regulator of the spindle checkpoint pathway. Complementary proteomic and cell biological analysis demonstrate that the spindle checkpoint protein MAD1 is one of the major components of CCNB1 complexes, and that CCNB1 is recruited to unattached kinetochores in an MPS1-dependent fashion through interaction with the first 100 amino acids of MAD1. This MPS1 and MAD1-dependent pool of CDK1-CCNB1 creates a positive feedback loop necessary for timely recruitment of MPS1 to kinetochores during mitotic entry and for sustained spindle checkpoint arrest. CDK1-CCNB1 is therefore an integral component of the spindle checkpoint, ensuring the fidelity of mitosis.