The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4.

The spindle position checkpoint (SPOC) is an essential surveillance mechanism that allows mitotic exit only when the spindle is correctly oriented along the cell axis. Key SPOC components are the kinase Kin4 and the Bub2-Bfa1 GAP complex that inhibit the mitotic exit-promoting GTPase Tem1. During an unperturbed cell cycle, Kin4 associates with the mother spindle pole body (mSPB), whereas Bub2-Bfa1 is at the daughter SPB (dSPB). When the spindle is mispositioned, Bub2-Bfa1 and Kin4 bind to both SPBs, which enables Kin4 to phosphorylate Bfa1 and thereby block mitotic exit. Here, we show that the daughter cell protein Lte1 physically interacts with Kin4 and inhibits Kin4 kinase activity. Specifically, Lte1 binds to catalytically active Kin4 and promotes Kin4 hyperphosphorylation, which restricts Kin4 binding to the mSPB. This Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB.

Targeted localization of Inn1, Cyk3 and Chs2 by the mitotic-exit network regulates cytokinesis in budding yeast.

The mitotic-exit network (MEN) is a signaling pathway that is essential for the coordination of mitotic exit and cytokinesis. Whereas the role of the MEN in mitotic exit is well established, the molecular mechanisms by which MEN components regulate cytokinesis remain poorly understood. Here, we show that the MEN controls components involved in septum formation, including Inn1, Cyk3 and Chs2. MEN-deficient mutants, forced to exit mitosis as a result of Cdk1 inactivation, show defects in targeting Cyk3 and Inn1 to the bud-neck region. In addition, we found that the chitin synthase Chs2 did not efficiently localize at the bud neck in the absence of MEN activity. Ultrastructural analysis of the bud neck revealed that low MEN activity led to unilateral, uncoordinated extension of the primary and secondary septa. This defect was partially suppressed by increased levels of Cyk3. We therefore propose that the MEN directly controls cytokinesis via targeting of Inn1, Cyk3 and Chs2 to the bud neck.

Activation of the complement system and leukocyte recruitment by Tityus serrulatus scorpion venom.

The scorpion Tityus serrulatus is considered one of the most dangerous species in Brazil. Its venom evokes an inflammatory response, although the exact mechanism of this effect is still unknown. The aim of the present study was to investigate the effect of Tityus serrulatus venom (TsV) on the complement system (CS) and on leukocyte recruitment. Complement consumption by TsV was evaluated using in vitro hemolytic assays, immunoelectrophoresis and two-dimensional immunoelectrophoresis of complement components (factor B and C3). In order to evaluate neutrophil migration induced in normal human serum (NHS) in the presence of TsV, in vitro chemotaxis assays were performed using the Boyden chamber model. In vitro TsV induced a concentration- and time-dependent reduction in hemolytic activity of the classical/lectin and alternative complement pathways, with samples of 43.0 microg and 43.4 microg, respectively, inhibiting 50% of the lytic activity. Alterations in C3 and factor B electrophoretic mobility after incubation of NHS with TsV, were identical to those obtained with zymosan (positive control). Incubation of NHS with TsV induced neutrophil chemotaxis similar to that observed with zymosan-activated serum. Our results show that TsV activates the CS, leading to factor B and C3 cleavage, to reduction of serum lytic activity and generation of complement chemotactic factors. Therefore, CS may play an important role in the inflammatory response observed upon scorpion envenomation.

Effect of Tityus serrulatus scorpion venom and its major toxin, TsTX-I, on the complement system in vivo.

The effects of Tityus serrulatus venom and TsTX-I (Ts1 or gamma-toxin) on the lytic activity of the complement system (CS) were investigated in vivo. Serum classical pathway (CP) and alternative pathway (AP) activities were determined in sera of rats (200+/-10 g) injected i.p. with soluble venom (150 microg/kg), TsTX-I (150 microg/kg) or saline (control). The animals were sacrificed 0.5, 1, 2, 4, 24 and 48 h after injection. The results showed an increase in serum lytic activity of animals injected with venom, reaching values up to 70% above controls in CP activity and 120% in AP activity. These effects were biphasic with maximum values 1 and 24 h after venom injection. Similar effects were obtained for TsTX-I, but with lower intensity. Hematocrit values of all tested animals were determined to evaluate the effect of hemoconcentration on the lytic activity of the CS. It was observed that the maximum hematocrit value was obtained 1 h after injection and returned to normal values within 24 h. These data indicate that hemoconcentration can play a relevant role in the first peak of complement activity, but we cannot discard a direct action of the venom on the system during this period, since the serum venom concentration is maximal 15-30 min after envenomation. The high lytic activity of the serum observed after 24 h, period in which the hematocrit values are normal and no venom can be detected, may be consequence of the inflammatory process induced by the venom or toxin. The lytic activity of the serum of rats injected with venom, TsTX-I or saline was abolished when the serum was previously adsorbed on zymosan. These data confirm that the increase of the lytic activity of the serum induced by the venom or toxin is dependent on CS. These results show that CS is involved in the inflammatory process induced by the venom or toxin and consequently in the lung edema, hemolysis, leukocytosis, among other clinical manifestations of severe envenomation.