Post-translational modifications regulate assembly of early spindle orientation complex in yeast

Mitosis begins with the tethering of chromosomes to the mitotic spindle and their orientation perpendicular to the axis of cell division. In budding yeast, mitotic spindle orientation and the subsequent chromosome segregation are two independent processes. Early spindle orientation is driven by the actin-bound myosin Myo2p, which interacts with the adapter Kar9p. The latter also binds to microtubule-associated Bim1p, thereby connecting both types of cytoskeleton. This study focuses on the interaction between Kar9p and Bim1p and its regulation. We solved the crystal structure of the previously reported Kar9p-binding motif of Bim1p and identified a second, novel Kar9p interaction domain. We further show that two independent post-translational modification events regulate their interaction. Whereas Kar9p sumoylation is required for efficient complex formation with Bim1p, Aurora B/Ipl1p-dependent phosphorylation of Bim1p down-regulates their interaction. The observed effects of these modifications allow us to propose a novel regulatory framework for the assembly and disassembly of the early spindle orientation complex.     

Non-redundant function of the MEK5-ERK5 pathway in thymocyte apoptosis

The mitogen-activated protein kinases (MAPKs) ERK1/2, p38, and JNK are thought to determine survival-versus-death fate in developing thymocytes. However, this view was challenged by studies using 'MEK1-ERK1/2-specific' pharmacological inhibitors, which block both positive and negative selection. Recently, these inhibitors were also shown to affect MEK5, an upstream activator of ERK5, another class of MAPK with homology to ERK1/2. To define the contribution of the MEK5-ERK5 pathway in T-cell development, we retrovirally expressed dominant-negative or constitutively activated form of MEK5 to inhibit or activate the MEK5-ERK5 pathway. We demonstrate that MEK5 regulates apoptosis of developing thymocytes but has no function in positive selection. ERK5 activity correlates with the levels of Nur77 family members but not that of Bim, two effector pathways of thymocyte apoptosis. These results illustrate the critical involvement of the MEK5-ERK5 pathway in thymocyte development distinct from that of ERK1/2 and highlight the importance of the MAPK network in mediating differential effects pertaining to T-cell differentiation and apoptosis.     

A novel cell death pathway that is partially caspase dependent, but morphologically non-apoptotic, elicited by proteasomal inhibition of rat sympathetic neurons

Proteasomal dysfunction has been linked to neurodegeneration. Pharmacological proteasomal inhibitors may have pro-survival or pro-death effects in neuronal cells. We have previously found that application of such agents to mouse sympathetic neurons leads to activation of the intrinsic apoptotic pathway. We show here that in rat sympathetic neurons proteasomal inhibition leads to a form of death that is morphologically non-apoptotic, with features of autophagy. The intrinsic apoptotic pathway is activated in a delayed fashion compared with mouse neurons, and is in part responsible for death, as evidenced by the partial protective effects of bcl-xL and the general caspase inhibitor Boc-aspartyl-fluoromethylketone. Death is accompanied by induction of Bim and caspase activation, but caspase 3 activation is lacking; 3-methyl-adenine inhibits macroautophagy, but has a relatively small pro-survival effect. We conclude that a complex array of pro- and anti-apoptotic effects elicited by proteasomal inhibition in rat sympathetic neurons leads to partial engagement of the intrinsic apoptotic pathway and a morphologically non-apoptotic, autophagic form of death. The species difference with mouse neurons is underscored by the fact that proteasomal inhibitors are protective against apoptosis elicited by nerve growth factor deprivation in rat, but not mouse, sympathetic neurons. The type of death described herein may be relevant to neurodegenerative diseases, where morphological evidence for apoptosis has been scant.     

Natural diterpenoid compound elevates expression of Bim protein, which interacts with antiapoptotic protein Bcl-2, converting it to proapoptotic Bax-like molecule

Overwhelming evidence indicates that Bax and Bak are indispensable for mediating cytochrome c release from mitochondria during apoptosis. Here we report a Bax/Bak-independent mechanism of cytochrome c release and apoptosis. We identified a natural diterpenoid compound that induced apoptosis in bax/bak double knock-out murine embryonic fibroblasts and substantially reduced the tumor growth from these cells implanted in mice. Treatment with the compound significantly increased expression of Bim, which migrated to mitochondria, altering the conformation of and forming oligomers with resident Bcl-2 to induce cytochrome c release and caspase activation. Importantly, purified Bim and Bcl-2 proteins cooperated to permeabilize a model mitochondrial outer membrane; this was accompanied by oligomerization of these proteins and deep embedding of Bcl-2 in the membrane. Therefore, the diterpenoid compound induces a structural and functional conversion of Bcl-2 through Bim to permeabilize the mitochondrial outer membrane, thereby inducing apoptosis independently of Bax and Bak. Because Bcl-2 family proteins play important roles in cancer development and relapse, this novel cell death mechanism can be explored for developing more effective anticancer therapeutics.     

The established and the predicted roles of dynein light chain in the regulation of mitochondrial apoptosis

The mitochondrial pathway of apoptosis is regulated by the interplay between the members of Bcl-2 family. Within this family, BH3-only proteins are the sensors of apoptotic stimuli and can trigger apoptosis either by inhibiting the anti-apoptotic Bcl-2-family proteins or by directly activating the effectors Bax and Bak. An expanding body of research suggests that a number of non-Bcl-2 proteins can also interact with Bcl-2 proteins and contribute to the decision of cell fate. Dynein light chain (LC8, DYNLL or DLC), a hub protein and a dimerizing engine has been proposed to regulate the pro-apoptotic activity of two BH3-only proteins, Bim and Bmf. Our recent work has provided insight into the mechanisms through which DLC1 (DYNLL1) modulates Bim activity. Here we discuss the present day understanding of Bim-DLC interaction and endeavor to evaluate this interaction in the light of information from studies of DLC with other binding partners.     

Detection of Bim and Puma in mouse hair follicles using immunofluorescence and TUNEL assay double staining

Apoptosis in hair follicles often is studied under pathological conditions; little is known about apoptotic mechanisms during normal hair follicle formation and maintenance. We investigated proteins of intrinsic apoptotic pathway, Bim and Puma, during hair follicle development and the first catagen stage using immunofluorescence to describe their expression patterns and to correlate them with apoptosis as determined by TUNEL assay. Both proteins were found in developing follicles. Bim and Puma overlapped apoptosis only partially during physiological apoptotic stage and they were present in non-apoptotic parts of the follicles. Our findings suggest that these primary apoptotic molecules participate in postnatal development and maintenance of hair follicles.     

Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9.

We have identified a pair of related yeast proteins, Sro7p and Sro77p, based on their ability to bind to the plasma membrane SNARE (SNARE) protein, Sec9p. These proteins show significant similarity to the Drosophila tumor suppressor, lethal giant larvae and to the neuronal syntaxin-binding protein, tomosyn. SRO7 and SRO77 have redundant functions as loss of both gene products leads to a severe cold-sensitive growth defect that correlates with a severe defect in exocytosis. We show that similar to Sec9, Sro7/77 functions in the docking and fusion of post-Golgi vesicles with the plasma membrane. In contrast to a previous report, we see no defect in actin polarity under conditions where we see a dramatic effect on secretion. This demonstrates that the primary function of Sro7/77, and likely all members of the lethal giant larvae family, is in exocytosis rather than in regulating the actin cytoskeleton. Analysis of the association of Sro7p and Sec9p demonstrates that Sro7p directly interacts with Sec9p both in the cytosol and in the plasma membrane and can associate with Sec9p in the context of a SNAP receptor complex. Genetic analysis suggests that Sro7 and Sec9 function together in a pathway downstream of the Rho3 GTPase. Taken together, our studies suggest that members of the lethal giant larvae/tomosyn/Sro7 family play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane.