EB1 Directly Regulates APC-Mediated Actin Nucleation.

EB1 was discovered 25 years ago as a binding partner of the tumor suppressor adenomatous polyposis coli (APC) [1]; however, the significance of EB1-APC interactions has remained poorly understood. EB1 functions at the center of a network of microtubule end-tracking proteins (+TIPs) [2-5], and APC binding to EB1 promotes EB1 association with microtubule ends and microtubule stabilization [6, 7]. Whether EB1 interactions govern functions of APC beyond microtubule regulation has not been explored. The C-terminal basic domain of APC (APC-B) directly nucleates actin assembly, and this activity is required in vivo for directed cell migration and for maintaining normal levels of F-actin [8-10]. Here, we show that EB1 binds APC-B and inhibits its actin nucleation function by blocking actin monomer recruitment. Consistent with these biochemical observations, knocking down EB1 increases F-actin levels in cells, and this can be rescued by disrupting APC-mediated actin nucleation. Conversely, overexpressing EB1 decreases F-actin levels and impairs directed cell migration without altering microtubule organization and independent of its direct binding interactions with microtubules. Overall, our results define a new function for EB1 in negatively regulating APC-mediated actin assembly. Combining these findings with other recent studies showing that APC interactions regulate EB1-dependent effects on microtubule dynamics [7], we propose that EB1-APC interactions govern bidirectional cytoskeletal crosstalk by coordinating microtubule and actin dynamics.

Recruitment of the mitotic exit network to yeast centrosomes couples septin displacement to actomyosin constriction.

In many eukaryotic organisms cytokinesis is driven by a contractile actomyosin ring (CAR) that guides membrane invagination. What triggers CAR constriction at a precise time of the cell cycle is a fundamental question. In budding yeast CAR is assembled via a septin scaffold at the division site. A Hippo-like kinase cascade, the Mitotic Exit Network (MEN), promotes mitotic exit and cytokinesis, but whether and how these two processes are independently controlled by MEN is poorly understood. Here we show that a critical function of MEN is to promote displacement of the septin ring from the division site, which in turn is essential for CAR constriction. This is independent of MEN control over mitotic exit and involves recruitment of MEN components to the spindle pole body (SPB). Ubiquitination of the SPB scaffold Nud1 inhibits MEN signaling at the end of mitosis and prevents septin ring splitting, thus silencing the cytokinetic machinery.

The final cut: cell polarity meets cytokinesis at the bud neck in S. cerevisiae.

Cell division is a fundamental but complex process that gives rise to two daughter cells. It includes an ordered set of events, altogether called "the cell cycle", that culminate with cytokinesis, the final stage of mitosis leading to the physical separation of the two daughter cells. Symmetric cell division equally partitions cellular components between the two daughter cells, which are therefore identical to one another and often share the same fate. In many cases, however, cell division is asymmetrical and generates two daughter cells that differ in specific protein inheritance, cell size, or developmental potential. The budding yeast Saccharomyces cerevisiae has proven to be an excellent system to investigate the molecular mechanisms governing asymmetric cell division and cytokinesis. Budding yeast is highly polarized during the cell cycle and divides asymmetrically, producing two cells with distinct sizes and fates. Many components of the machinery establishing cell polarization during budding are relocalized to the division site (i.e., the bud neck) for cytokinesis. In this review we recapitulate how budding yeast cells undergo polarized processes at the bud neck for cell division.

Rho1- and Pkc1-dependent phosphorylation of the F-BAR protein Syp1 contributes to septin ring assembly.

In many cell types, septins assemble into filaments and rings at the neck of cellular appendages and/or at the cleavage furrow to help compartmentalize the plasma membrane and support cytokinesis. How septin ring assembly is coordinated with membrane remodeling and controlled by mechanical stress at these sites is unclear. Through a genetic screen, we uncovered an unanticipated link between the conserved Rho1 GTPase and its effector protein kinase C (Pkc1) with septin ring stability in yeast. Both Rho1 and Pkc1 stabilize the septin ring, at least partly through phosphorylation of the membrane-associated F-BAR protein Syp1, which colocalizes asymmetrically with the septin ring at the bud neck. Syp1 is displaced from the bud neck upon Pkc1-dependent phosphorylation at two serines, thereby affecting the rigidity of the new-forming septin ring. We propose that Rho1 and Pkc1 coordinate septin ring assembly with membrane and cell wall remodeling partly by controlling Syp1 residence at the bud neck.

Budding yeast greatwall and endosulfines control activity and spatial regulation of PP2A(Cdc55) for timely mitotic progression.

Entry into mitosis is triggered by cyclinB/Cdk1, whose activity is abruptly raised by a positive feedback loop. The Greatwall kinase phosphorylates proteins of the endosulfine family and allows them to bind and inhibit the main Cdk1-counteracting PP2A-B55 phosphatase, thereby promoting mitotic entry. In contrast to most eukaryotic systems, Cdc14 is the main Cdk1-antagonizing phosphatase in budding yeast, while the PP2A(Cdc55) phosphatase promotes, instead of preventing, mitotic entry by participating to the positive feedback loop of Cdk1 activation. Here we show that budding yeast endosulfines (Igo1 and Igo2) bind to PP2A(Cdc55) in a cell cycle-regulated manner upon Greatwall (Rim15)-dependent phosphorylation. Phosphorylated Igo1 inhibits PP2A(Cdc55) activity in vitro and induces mitotic entry in Xenopus egg extracts, indicating that it bears a conserved PP2A-binding and -inhibitory activity. Surprisingly, deletion of IGO1 and IGO2 in yeast cells leads to a decrease in PP2A phosphatase activity, suggesting that endosulfines act also as positive regulators of PP2A in yeast. Consistently, RIM15 and IGO1/2 promote, like PP2A(Cdc55), timely entry into mitosis under temperature-stress, owing to the accumulation of Tyr-phosphorylated Cdk1. In addition, they contribute to the nuclear export of PP2A(Cdc55), which has recently been proposed to promote mitotic entry. Altogether, our data indicate that Igo proteins participate in the positive feedback loop for Cdk1 activation. We conclude that Greatwall, endosulfines, and PP2A are part of a regulatory module that has been conserved during evolution irrespective of PP2A function in the control of mitosis. However, this conserved module is adapted to account for differences in the regulation of mitotic entry in different organisms.

Targeting and membrane insertion into the endoplasmic reticulum membrane of Saccharomyces cerevisiae essential protein Rot1.

Rot1 is an essential yeast protein that has been related to cell wall biosynthesis, actin cytoskeleton dynamics and protein folding. Rot1 is an N-glycosylated protein anchored to the nuclear envelope-endoplasmic reticulum (ER) membrane by a transmembrane domain at its C-terminal end. Rot1 is translocated to the ER by a post-translational mechanism. Here, we investigate the protein domain required to target and translocate Rot1 to the ER membrane. We found that several deletions of the N-terminal region of Rot1 prevented neither membrane targeting nor the insertion of this protein. Interestingly, we obtained the same results when different truncated forms in the C-terminal transmembrane domain were analyzed, suggesting the presence of an internal topogenic element that is capable of translocating Rot1 to the ER. To identify this sequence, we generated a combination of N- and C-terminal deletion mutants of Rot1 and we investigated their insertion into the membrane. The results show that two regions, amino acids 26-60 and 200-228, are involved in the post-translational translocation of Rot1 across the ER membrane.