Bfa1p and Bub2p are spindle checkpoint proteins that likely have GTPase activation activity and are associated with the budding yeast spindle pole body (SPB). Here, we show that Bfa1p and Bub2p bind the Ras-like GTPase Tem1p, a component of the mitotic exit network, to the cytoplasmic face of the SPB that enters the bud, whereas the GDP/GTP exchange factor Lte1p is associated with the cortex of the bud. Migration of the SPB into the bud probably allows activation of Tem1p through Lte1p, thereby linking nuclear migration with mitotic exit. Since components of the Bub2p checkpoint are conserved in other organisms, we propose that the position of the SPB or mammalian centrosome controls the timing of mitotic exit.

SummaryHere we show that Rab25 permits the sorting of ligand-occupied, active-conformation α5β1 integrin to late endosomes/lysosomes. Photoactivation and biochemical approaches show that lysosomally targeted integrins are not degraded but are retrogradely transported and recycled to the plasma membrane at the back of invading cells. This requires CLIC3, a protein upregulated in Rab25-expressing cells and tumors, which colocalizes with active α5β1 in late endosomes/lysosomes. CLIC3 is necessary for release of the cell rear during migration on 3D matrices and is required for invasion and maintenance of active Src signaling in organotypic microenvironments. CLIC3 expression predicts lymph node metastasis and poor prognosis in operable cases of pancreatic ductal adenocarcinoma (PDAC). The identification of CLIC3 as a regulator of a recycling pathway and as an independent prognostic indicator in PDAC highlights the importance of active integrin trafficking as a potential drive to cancer progression in vivo.

One-sentence summary: Analysis of the ERK circuitry suggests the most effective targets in the pathway for inhibition, which may aid in drug development Editor's Summary: Biological Circuits Inform Drug Development The mitogen-activated protein kinase (MAPK) pathway involves a three-tiered kinase module, which amplifies the signal. Many cells also have negative feedback loops from the last kinase in the module back to various points upstream in the pathway. Sturm et al. show that the MAPK module with negative feedback loops results in a system like that of a negative feedback amplifier (NFA), which is an engineering design that smoothens the output to changes in input and makes a system robust to change. These NFA-like properties may explain why some cells are sensitive to inhibition of the second kinase in the cascade (they lack the feedback loops); whereas other cells are resistant to inhibition at this point (their feedback loops are intact). These results also have implications for drug development, because inhibitors that target components that are outside the NFA are more effective at inhibiting the pathway. Abstract Three-tiered kinase modules, such as the Raf-MEK-ERK mitogen-activated protein kinase pathway, are widespread in biology suggesting that this structure conveys evolutionary advantageous properties. Here, we show that the three-tiered kinase amplifier module combined with negative feedback recapitulates the design principles of a negative feedback amplifier (NFA), which is used in electronic circuits to convey robustness, output stabilization, and linearization of nonlinear signal amplification. With mathematical modelling and experimental validation, we demonstrated that the ERK pathway has properties of a NFA that (i) converts intrinsic switch-like activation kinetics into graded linear responses; (ii) conveys robustness to changes in rates of reactions within the NFA module; and (iii) stabilizes outputs in response to drug-induced perturbations of the amplifier. These properties determine biological behavior, including activation kinetics and the response to drugs.

The budding yeast spindle pole body (SPB) not only organizes the astral and nuclear microtubules but is also associated with a number of cell-cycle regulators that control mitotic exit. Here, we describe that the core SPB component Nud1p is a key protein that functions in both processes. The astral microtubule organizing function of Nud1p is mediated by its interaction with the g-tubulin complex binding protein Spc72p. This function of Nud1p is distinct from its role in cellcycle control: Nud1p binds the spindle checkpoint control proteins Bfa1p and Bub2p to the SPB, and is part of the mitotic exit network (MEN) in which it functions upstream of CDC15 but downstream of LTE1. In conditional lethal nud1-2 cells, the MEN component Tem1p, a GTPase, is mislocalized, whereas the kinase Cdc15p is still associated with the SPB. Thus, in nud1-2 cells the failure of Tem1p to interact with Cdc15p at the SPB probably prevents mitotic exit. Keywords: Cdc15p/mitotic exit network/Nud1p/spindle pole body/Tem1p

The budding yeast mitotic exit network (MEN) is a GTPase-driven signal transduction cascade that controls the release of the phosphatase Cdc14p from the nucleolus in anaphase and thereby drives mitotic exit. We show that Cdc14p is partially released from the nucleolus in early anaphase independent of the action of the MEN components Cdc15p, Dbf2p, and Tem1p. Upon release, Cdc14p binds to the spindle pole body (SPB) via association with the Bfa1p–Bub2p GTPase activating protein complex, which is known to regulate the activity of the G protein Tem1p. Cdc14p also interacts with this GTPase. The association of the MEN component Mob1p with the SPB acts as a marker of MEN activation. The simultaneous binding of Cdc14p and Mob1p to the SPB in early anaphase suggests that Cdc14p initially activates the MEN. In a second, later step, which coincides with mitotic exit, Cdc14p reactivates the Bfa1p–Bub2p complex by dephosphorylating Bfa1p. This inactivates the MEN and displaces Mob1p from SPBs. These data indicate that Cdc14p activates the MEN in early anaphase but later inactivates it through Bfa1p dephosphorylation and so restricts MEN activity to a short period in anaphase.