Antagonistic Roles of PP2A-Pab1 and Etd1 in the Control of Cytokinesis in Fission Yeast

In Schizosaccharomyces pombe, Etd1 is a positive regulator of the septation initiation network (SIN), a conserved GTPase-regulated kinase cascade that triggers cytokinesis. Here we show that a mutation in the pab1 gene, which encodes the B-regulatory subunit of the protein phosphatase 2A (PP2A), suppresses mutations in the etd1 gene. Etd1 is required for the function of the GTPase Spg1, a key regulator of SIN signaling. Interestingly, the loss of Pab1 function restored the activity of Spg1 in Etd1-deficient cells. This result suggests that PP2A-Pab1–mediated dephosphorylation inhibits Spg1, thus antagonizing Etd1 function. The loss of pab1 function also rescues the lethality of mutants of other genes in the SIN cascade such as mob1, sid1, and cdc11. Two-hybrid assays indicate that Pab1 physically interacts with Mob1, Sid1, Sid2, and Cdc11, suggesting that the phosphatase 2A B-subunit is a component of the SIN complex. Together, our results indicate that PP2A-Pab1 plays a novel role in cytokinesis, regulating SIN activity at different levels. Pab1 is also required to activate polarized cell growth. Thus, PP2A-Pab1 may be involved in coordinating polar growth and cytokinesis.THE fission yeast Schizosaccharomyces pombe is a leading experimental model for eukaryotic cytokinesis (Bathe and Chang 2009; Pollard and Wu 2010). Fission yeast cells grow in a polarized manner by elongation at the cell ends and divide during cytokinesis by the action of a contractile actomyosin ring assembled in the middle of the cell (Snell and Nurse 1993). At the end of mitosis, when nuclear separation has been completed, actomyosin ring constriction is triggered by the septation initiation network (SIN). This signal transduction cascade is composed of the GTPase Spg1 and three protein kinases—Cdc7, GC-kinase Sid1, and NDR-kinase Sid2 in their presumed order of action—and the associated proteins Cdc14 with Sid1 and Mob1 with Sid2. These proteins are all located at the spindle pole body (SPB) during mitosis on a scaffold composed of the coiled-coil proteins Sid4 and Cdc11 (Krapp et al. 2004). The Sid2-Mob1 protein kinase complex is thought to transmit the division signal from the SPB to the actomyosin ring since it also associates at the division site during septation (Krapp and Simanis 2008). The SIN triggers actomyosin ring contraction coordinated with the synthesis of the primary and secondary septa that will form the new cell wall (Krapp et al. 2004; Wolfe and Gould 2005). The small GTPase Rho1 is known to promote cell-wall formation at the division site by stimulation of Cps1p/Drc1 1,3-β-glucan synthase (Le Goff et al. 1999), but the mechanism remains unclear.SIN activity is tightly regulated during the cell cycle to ensure proper coordination of mitosis and cytokinesis. Mutants that negatively affect SIN function undergo nuclear division in the absence of septation, while increased SIN activity induces septation in interphase cells (Krapp and Simanis 2008). Regulation of the SIN is complex, involving multiple, partially redundant mechanisms, but the nucleotide status of the Ras superfamily small GTPase, Spg1, represents a key step in SIN activity (Lattmann et al. 2009). Cdc16 and Byr4 form a two-component GTPase-activating protein (GAP) for Spg1 that inhibits its activity (Furge et al. 1998; Cerutti and Simanis 1999). Proteins acting as a guanine nucleotide-exchange factor (GEF) for this GTPase have not been identified. In the budding yeast Saccharomyces cerevisiae, the pathway analogous to the SIN is known as the mitotic exit network (MEN) (reviewed in Krapp and Simanis 2008). Contact between the SPB-localized GTPase Tem1 (the Spg1 homolog) with its putative GEF Lte1, which is present only within the bud, has been proposed as a mechanism to ensure that mitotic exit occurs only after the spindle has oriented correctly (Bardin et al. 2000; Pereira et al. 2000). Bfa1-Bub2 (the Cdc16-Byr4 equivalent) are negative regulators of the MEN, acting as a two-component GAP for Tem1 (Geymonat et al. 2002).Etd1 was identified in a genetic screen searching for new regulators of the S. pombe cell division cycle (Jimenez and Oballe 1994). Further characterization indicated that Etd1 acts as a positive regulator of the SIN (Daga et al. 2005). A recent study has established a key role for Etd1 in the timing of cytokinesis via the regulation of Spg1, acting as a potential homolog of budding yeast Lte1 (Garcia-Cortes and McCollum 2009). Loss of Etd1 function can be suppressed by mutations in a number of genes, some of which are involved in morphogenesis (Jimenez and Oballe 1994). Here we show that one of the mutations that bypass the requirement for etd1 in cytokinesis affects the activity of pab1, which encodes the protein phosphatase 2A (PP2A) regulatory subunit B. The characterization of Pab1 and pab1 mutants described in this study reveals a novel role for PP2A-Pab1 in SIN regulation and provides new insight into the mechanism by which Etd1 might regulate SIN signaling. We also show that Pab1 participates in activation of the morphological pathway, suggesting a role for PP2A-Pab1 in the coordination of cytokinesis and morphogenesis.     

The nuclease activity of DNA2 promotes exonuclease 1–independent mismatch repair

The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.

The mismatch repair (MMR) system has been conserved from bacteria to humans (1, 2). It promotes genome stability by suppressing spontaneous and DNA damage-induced mutations (1, 3, 4, 5, 6, 7, 8, 9, 10, 11). The key function of the MMR system is the correction of DNA replication errors that escape the proofreading activities of replicative DNA polymerases (1, 4, 5, 6, 7, 8, 9, 10, 12). In addition, the MMR system removes mismatches formed during strand exchange in homologous recombination, suppresses homeologous recombination, initiates apoptosis in response to irreparable DNA damage caused by several anticancer drugs, and contributes to instability of triplet repeats and alternative DNA structures (1, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18). The principal components of the eukaryotic MMR system are MutSα (MSH2-MSH6 heterodimer), MutLα (MLH1-PMS2 heterodimer in humans and Mlh1-Pms1 heterodimer in yeast), MutSβ (MSH2-MSH3 heterodimer), proliferating cell nuclear antigen (PCNA), replication factor C (RFC), exonuclease 1 (EXO1), RPA, and DNA polymerase δ (Pol δ). Loss-of-function mutations in the MSH2, MLH1, MSH6, and PMS2 genes of the human MMR system cause Lynch and Turcot syndromes, and hypermethylation of the MLH1 promoter is responsible for ∼15% of sporadic cancers in several organs (19, 20). MMR deficiency leads to cancer initiation and progression via a multistage process that involves the inactivation of tumor suppressor genes and action of oncogenes (21).MMR occurs behind the replication fork (22, 23) and is a major determinant of the replication fidelity (24). The correction of DNA replication errors by the MMR system increases the replication fidelity by ∼100 fold (25). Strand breaks in leading and lagging strands as well as ribonucleotides in leading strands serve as signals that direct the eukaryotic MMR system to remove DNA replication errors (26, 27, 28, 29, 30). MMR is more efficient on the lagging than the leading strand (31). The substrates for MMR are all six base–base mismatches and 1 to 13-nt insertion/deletion loops (25, 32, 33, 34). Eukaryotic MMR commences with recognition of the mismatch by MutSα or MutSβ (32, 34, 35, 36). MutSα is the primary mismatch-recognition factor that recognizes both base–base mismatches and small insertion/deletion loops whereas MutSβ recognizes small insertion/deletion loops (32, 34, 35, 36, 37). After recognizing the mismatch, MutSα or MutSβ cooperates with RFC-loaded PCNA to activate MutLα endonuclease (38, 39, 40, 41, 42, 43). The activated MutLα endonuclease incises the discontinuous daughter strand 5′ and 3′ to the mismatch. A 5'' strand break formed by MutLα endonuclease is utilized by EXO1 to enter the DNA and excise a discontinuous strand portion encompassing the mismatch in a 5''→3′ excision reaction stimulated by MutSα/MutSβ (38, 44, 45). The generated gap is filled in by the Pol δ holoenzyme, and the nick is ligated by a DNA ligase (44, 46, 47). DNA polymerase ε (Pol ε) can substitute for Pol δ in the EXO1-dependent MMR reaction, but its activity in this reaction is much lower than that of Pol δ (48). Although MutLα endonuclease is essential for MMR in vivo, 5′ nick-dependent MMR reactions reconstituted in the presence of EXO1 are MutLα-independent (44, 47, 49).EXO1 deficiency in humans does not seem to cause significant cancer predisposition (19). Nevertheless, it is known that Exo1^-/- mice are susceptible to the development of lymphomas (50). Genetic studies in yeast and mice demonstrated that EXO1 inactivation causes only a modest defect in MMR (50, 51, 52, 53). In agreement with these genetic studies, a defined human EXO1-independent MMR reaction that depends on the strand-displacement DNA synthesis activity of Pol δ holoenzyme to remove the mismatch was reconstituted (54). Furthermore, an EXO1-independent MMR reaction that occurred in a mammalian cell extract system without the formation of a gapped excision intermediate was observed (54). Together, these findings implicated the strand-displacement activity of Pol δ holoenzyme in EXO1-independent MMR.In this study, we investigated DNA2 in the context of MMR. DNA2 is an essential multifunctional protein that has nuclease, ATPase, and 5''→3′ helicase activities (55, 56, 57). Previous research ascertained that DNA2 removes long flaps during Okazaki fragment maturation (58, 59, 60), participates in the resection step of double-strand break repair (61, 62, 63), initiates the replication checkpoint (64), and suppresses the expansions of GAA repeats (65). We have found in vivo and in vitro evidence that DNA2 promotes EXO1-independent MMR. Our data have indicated that the nuclease activity of DNA2 enhances the strand-displacement activity of Pol δ holoenzyme in an EXO1-independent MMR reaction.     

Naturally Occurring Disseminated Group B Streptococcus Infections in Postnatal Rats

Group B Streptococcus (Streptococcus agalactiae, GBS) is a gram-positive commensal and occasional opportunistic pathogen of the human vaginal, respiratory, and intestinal tracts that can cause sepsis, pneumonia, or meningitis in human neonates, infants, and immunosuppressed persons. We report here on a spontaneous outbreak of postnatal GBS-associated disease in rats. Ten of 26 (38.5%) 21- to 24-d-old rat pups died or were euthanized due to a moribund state in a colony of rats transgenic for the human diphtheria toxin receptor on a Munich–Wistar–Frömter genetic background. Four pups had intralesional coccoid bacteria in various organs without accompanying inflammation. GBS was isolated from the liver of 2 of these pups and from skin abscesses in 3 littermates. A connection with the transgene could not be established. A treatment protocol was evaluated in the remaining breeding female rats. GBS is a potentially clinically significant spontaneous infection in various populations of research rats, with some features that resemble late-onset postnatal GBS infection in human infants.Abbreviations: GBS, Group B Streptococcus; MWF, Munich Wistar Frömter; hDTR, human diphtheria toxin receptorStreptococci are gram-positive, coccoid bacteria that typically are classified according to their hemolytic capacity. α-hemolytic streptococci produce a zone of partial hemolysis that appears greenish on blood agar, whereas β-hemolytic streptococci produce a zone of complete hemolysis, and γ-hemolytic organisms produce no hemolysis on blood agar.²⁴ The β-hemolytic streptococci are further subdivided into Lancefield groups (A through G), according to cell-wall carbohydrate antigens.^24,29,39 The group B β-hemolytic Streptococcus (GBS) have been speciated as Streptococcus agalactiae.^28,39 It was first isolated as a causative agent of mastitis in cattle.²⁹ This organism has since been recognized as a cause of severe infection in human neonates.^28,39 In humans, GBS is harbored asymptomatically in the maternal genitourinary tract.^24,28 Infants can be infected and present with serious systemic disease in the first week of life (early-onset GBS) or from 1 wk to 3 mo of age (late-onset GBS).³⁹ In laboratory animals, rats have been used experimentally as models for neonatal^{1,6,7,20,37,38,43,44,47,50,51} or adult⁴⁵ GBS infection, but to our knowledge, GBS has not been associated with spontaneous disease in rats.     

The Arabidopsis Callose Synthase Gene GSL8 Is Required for Cytokinesis and Cell Patterning

Cytokinesis is the division of the cytoplasm and its separation into two daughter cells. Cell plate growth and cytokinesis appear to require callose, but direct functional evidence is still lacking. To determine the role of callose and its synthesis during cytokinesis, we identified and characterized mutants in many members of the GLUCAN SYNTHASE-LIKE (GSL; or CALLOSE SYNTHASE) gene family in Arabidopsis (Arabidopsis thaliana). Most gsl mutants (gsl1–gsl7, gsl9, gsl11, and gsl12) exhibited roughly normal seedling growth and development. However, mutations in GSL8, which were previously reported to be gametophytic lethal, were found to produce seedlings with pleiotropic defects during embryogenesis and early vegetative growth. We found cell wall stubs, two nuclei in one cell, and other defects in cell division in homozygous gsl8 insertional alleles. In addition, gsl8 mutants and inducible RNA interference lines of GSL8 showed reduced callose deposition at cell plates and/or new cell walls. Together, these data show that the GSL8 gene encodes a putative callose synthase required for cytokinesis and seedling maturation. In addition, gsl8 mutants disrupt cellular and tissue-level patterning, as shown by the presence of clusters of stomata in direct contact and by islands of excessive cell proliferation in the developing epidermis. Thus, GSL8 is required for patterning as well as cytokinesis during Arabidopsis development.Cytokinesis divides the cytoplasm of a plant cell by the deposition of plasma membrane and a cell wall during late mitosis. This process requires the phragmoplast, a dynamic, plant-specific cytoskeletal and membranous array, which delivers vesicles containing lipids, proteins, and cell wall components to the division plane to construct the cell plate. Cell plate formation involves several stages: initiation through vesicle fusion, the formation of a tubular-vesicular network, a transition to a solely tubular phase, and then further fusion to form a fenestrated sheet (Samuels et al., 1995). The outward growth of the cell plate leads to its fusion with the parental cell wall (Jürgens, 2005a, 2005b; Backues et al., 2007).Key regulators of cytokinesis include KNOLLE, KEULE, KORRIGAN, and HINKEL, which when defective induce pleiotropic phenotypes and seedling lethality (Lukowitz et al., 1996; Nicol et al., 1998; Zuo et al., 2000; Assaad et al., 2001; Strompen et al., 2002). KNOLLE, a syntaxin homolog, is required for the fusion of exocytic vesicles via a SNARE/SNAP33 complex (Lukowitz et al., 1996; Heese et al., 2001). KEULE, a homolog of yeast Sec1p, regulates syntaxin function by interacting with KNOLLE (Waizenegger et al., 2000; Assaad et al., 2001). KORRIGAN is an endo-1,4-β-glucanase required for cell wall biogenesis during cytokinesis (Zuo et al., 2000). And HINKEL is a kinesin-related protein required for the reorganization of phragmoplast microtubules during cytokinesis (Strompen et al., 2002).Additional regulators include Formin5, TWO-IN-ONE (TIO), and Arabidopsis (Arabidopsis thaliana) dynamin-like proteins (ADLs; Kang et al., 2001, 2003; Hong et al., 2003; Collings et al., 2005; Ingouff et al., 2005; Oh et al., 2005). Formin5 localizes to the cell plate and is an actin-organizing protein involved in cytokinesis and cell polarity. TIO, a Ser/Thr protein kinase, functions in cytokinesis in plant meristems and in gametogenesis (Oh et al., 2005). Members of the Arabidopsis DRP family associate with the developing cell plate, whereas DRP1a (ADL1A) locally constricts tubular membranes, interacts with callose synthase, and may facilitate callose deposition into the lumen.Callose, a β-1,3-glucan polymer with β-1,6-branches (Stone and Clarke, 1992), is synthesized in both sporophytic and gametophytic tissues and appears to play various roles. Callose accumulates at the cell plate during cytokinesis, in plasmodesmata, where it regulates cell-to-cell communication, and in dormant phloem, where it seals sieve plates after mechanical injury, pathogen attack, and metal toxicity (Stone and Clarke, 1992; Samuels et al., 1995; Lucas and Lee, 2004).Twelve GLUCAN SYNYHASE-LIKE (GSL) genes (also known as CALLOSE SYNTHASE [CalS]) have been identified in the Arabidopsis genome based on sequence homology (Richmond and Somerville, 2000; Hong et al., 2001; Enns et al., 2005). A GSL that functions in callose deposition after injury and pathogen treatment is GSL5 (Jacobs et al., 2003). Five other members of the Arabidopsis GSL family are required for microgametogenesis. GSL1 and GSL5 act redundantly to produce a callosic wall that prevents microspore degeneration, and both are needed for fertilization (Enns et al., 2005). GSL2 is required for the callosic wall around pollen mother cells, for the patterning of the pollen exine (Dong et al., 2005), and for callose deposition in the wall and plugs of pollen tubes (Nishikawa et al., 2005). GSL8 and GSL10 are independently required for the asymmetric division of microspores and for the entry of microspores into mitosis (Töller et al., 2008; Huang et al., 2009).Callose is a major component of the cell plate, especially during later plate development (Kakimoto and Shibaoka, 1992; Samuels et al., 1995; Hong et al., 2001). Callose appears to structurally reinforce the developing cell plate after the breakdown of the phragmoplast microtubule array and during plate consolidation (Samuels and Staehelin, 1996; Rensing et al., 2002). It is likely that callose is synthesized at the cell plate rather than in the endoplasmic reticulum and in the Golgi (Kakimoto and Shibaoka, 1988). GSL6 (CalS1) appears to be involved in callose synthesis at the cell plate, since a 35S∷GFP-GSL6 fusion in transgenic BY-2 tobacco (Nicotiana tabacum) cells increases callose accumulation, and GFP fluorescence was found specifically at the cell plate (Hong et al., 2001). However, functional and genetic data on the role of any GSL in Arabidopsis sporophytic cytokinesis are still lacking.Here, we report that GSL8 (CalS10) is required for normal cytokinesis. In addition, gsl8 mutants exhibit excessive cell proliferation and abnormal cell patterning, phenotypes not previously reported for cytokinesis-defective mutants.     

Endomembrane Trafficking Protein SEC24A Regulates Cell Size Patterning in Arabidopsis

Size is a critical property of a cell, but how it is determined is still not well understood. The sepal epidermis of Arabidopsis (Arabidopsis thaliana) contains cells with a diversity of sizes ranging from giant cells to small cells. Giant cells have undergone endoreduplication, a specialized cell cycle in which cells replicate their DNA but fail to divide, becoming polyploid and enlarged. Through forward genetics, we have identified a new mutant with ectopic giant cells covering the sepal epidermis. Surprisingly, the mutated gene, SEC24A, encodes a coat protein complex II vesicle coat subunit involved in endoplasmic reticulum-to-Golgi trafficking in the early secretory pathway. We show that the ectopic giant cells of sec24a-2 are highly endoreduplicated and that their formation requires the activity of giant cell pathway genes LOSS OF GIANT CELLS FROM ORGANS, DEFECTIVE KERNEL1, and Arabidopsis CRINKLY4. In contrast to other trafficking mutants, cytokinesis appears to occur normally in sec24a-2. Our study reveals an unexpected yet specific role of SEC24A in endoreduplication and cell size patterning in the Arabidopsis sepal.Size is a fundamental characteristic of a cell, but how cell size is determined is still not well understood in most living organisms (Marshall et al., 2012). Cells of different types typically have characteristic sizes, indicating that size is carefully regulated to fit cell functions during differentiation. At the simplest level, cell size is determined by growth and division. Although many factors regulating these two processes have been studied, how they are comprehensively regulated to achieve specific size outcomes remains unclear.The sepal of Arabidopsis (Arabidopsis thaliana) is an excellent model to study the regulation of cell size because it exhibits a characteristic pattern of giant cells interspersed in between small cells. The giant cells are large cells that span about one-fifth the length of the sepal (approximately 360 μm), while the smallest cells only reach to about 10 μm (Roeder et al., 2010). Previously, we have shown that variability in cell division times is sufficient to produce the cell size pattern (Roeder et al., 2010). The giant cells stop dividing and enter endoreduplication, a specialized cell cycle in which the cell replicates its DNA but skips mitosis to continue growing (Edgar and Orr-Weaver, 2001; Sugimoto-Shirasu and Roberts, 2003; Inzé and De Veylder, 2006; Breuer et al., 2010). Alongside the giant cells, the smaller cells continue dividing mitotically. Giant cells and small cells are different cell types, as they can be distinguished by the expression pattern of two independent enhancers. Furthermore, mutant screens have shown that genes involved in epidermal specification and cell cycle regulation are crucial for sepal cell size patterning. DEFECTIVE KERNEL1 (DEK1), Arabidopsis thaliana MERISTEM LAYER1 (ATML1), Arabidopsis CRINKLY4 (ACR4), and HOMEODOMAIN GLABROUS11 first establish the identity of giant cells, and then the cyclin dependent kinase inhibitor LOSS OF GIANT CELLS FROM ORGANS (LGO) influences the probability with which cells enter endoreduplication. Endoreduplication can further suppress the identity of small cells through an unknown mechanism (Roeder et al., 2010, 2012). The number of giant cells influences the curvature of the sepal, which is important for protecting the flower (Roeder et al., 2012). Therefore, cell size patterning ensures the protective role of sepals at the physiological level.The secretory pathway in eukaryotes is crucial for cells to maintain membrane homeostasis and protein localization. Proteins destined for the cell surface are first translated on the rough endoplasmic reticulum (ER) and then incorporated into coat protein complex II (COPII) vesicles that bud from ER membranes on the way to the Golgi apparatus. COPII machinery is highly conserved in eukaryotes, and each COPII component acts sequentially on the surface of the ER (Bickford et al., 2004; Marti et al., 2010; Zanetti et al., 2012). Vesicle coat assembly is initiated by SEC12, an ER membrane-anchored guanine nucleotide exchange factor (Barlowe and Schekman, 1993). SEC12 exchanges GDP with GTP on the small GTPase Secretion-associated RAS-related protein1 (SAR1), which increases the membrane affinity of SAR1. The ER membrane-bound SAR1 subsequently brings the SEC23/SEC24 subunits to form the prebudding complex, and eventually SEC13/SEC31 are recruited to increase rigidity of the COPII vesicle coat (Nakano and Muramatsu, 1989; Barlowe et al., 1994; Shaywitz et al., 1997; Aridor et al., 1998; Kuehn et al., 1998; Stagg et al., 2006; Copic et al., 2012). For COPII vesicles to fuse with the target membrane, superfamily N-ethylmaleimide-sensitive factor adaptor protein receptors (SNAREs) must be incorporated by SEC24 (Mossessova et al., 2003; Lipka et al., 2007; Mancias and Goldberg, 2008). In addition to its role in SNARE packaging, SEC24 also binds and loads secretory cargo proteins (Miller et al., 2003). Both the cargo and SNARE specificities are determined by the correspondence between the SEC24 isoform and the various ER export signals of cargoes and SNAREs (Barlowe., 2003; Miller et al., 2003; Mossessova et al., 2003; Mancias and Goldberg, 2008). The Arabidopsis genome encodes four SEC24 isoforms, SEC24A to SEC24D; how they differentially regulate trafficking is unknown (Bassham et al., 2008). Likewise, SEC24-cargo/SNARE interactions remain elusive in plants.Secretion defects in plants often lead to cell division defects due to the unique mechanisms of plants cytokinesis (Sylvester, 2000; Jürgens, 2005). In many eukaryotes other than plants, cytokinesis is accomplished by contraction of the cleavage furrow at the division plane. By contrast, cytokinesis in plants requires de novo secretion of vesicles to the division plane, after formation of the phragmoplast as the scaffold for delivery. Homotypic vesicle fusion sets up the early cell plate, which then expands laterally by fusing with other arriving vesicles (Balasubramanian et al., 2004; Jürgens, 2005; Reichardt et al., 2007). Hence, disruption of secretion in plants can often result in cytokinesis defects. For instance, a mutation in the SNARE KNOLLE leads to enlarged embryo cells with multiple nuclei (Lukowitz et al., 1996).Another common phenotype observed in secretion-deficient plants is abnormal auxin responses. The phytohormone auxin acts as a prominent signal in Arabidopsis development, and auxin influx/efflux carriers are essential in directing auxin transport and creating local maxima in an auxin gradient (Reinhardt et al., 2003; Heisler et al., 2005; Jönsson et al., 2006; Smith et al., 2006; Vanneste and Friml, 2009). To maintain appropriate auxin gradients, the subcellular localization of auxin carriers must be delicately regulated. Thus, auxin responses are highly sensitive to trafficking perturbations in plants (Geldner et al., 2003; Grunewald and Friml, 2010).Here, we have identified a new mutant with ectopic giant cells. Through positional cloning, we determined that the mutation occurs in the SEC24A gene, which encodes the cargo-binding subunit of the COPII vesicle complex. In addition to altered cell size, this unique sec24a-2 allele shows pleiotropic defects, including dwarfism, which have not been reported previously for other SEC24A alleles (Faso et al., 2009; Nakano et al., 2009; Conger et al., 2011). Although the mutant is developmentally aberrant, both cytokinesis and auxin response appear normal in sec24a-2, unlike other transport mutants. Instead, we find SEC24A regulates cell size specifically via the giant cell development pathway. Thus, our data reveal an unexpected role of SEC24A in endoreduplication and cell size patterning in the Arabidopsis sepal.     

A Distinct Pathway for Polar Exocytosis in Plant Cell Wall Formation

Post-Golgi protein sorting and trafficking to the plasma membrane (PM) is generally believed to occur via the trans-Golgi network (TGN). In this study using Nicotiana tabacum pectin methylesterase (NtPPME1) as a marker, we have identified a TGN-independent polar exocytosis pathway that mediates cell wall formation during cell expansion and cytokinesis. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that Golgi-derived secretory vesicles (GDSVs) labeled by NtPPME1-GFP are distinct from those organelles belonging to the conventional post-Golgi exocytosis pathway. In addition, pharmaceutical treatments, superresolution imaging, and dynamic studies suggest that NtPPME1 follows a polar exocytic process from Golgi-GDSV-PM/cell plate (CP), which is distinct from the conventional Golgi-TGN-PM/CP secretion pathway. Further studies show that ROP1 regulates this specific polar exocytic pathway. Taken together, we have demonstrated an alternative TGN-independent Golgi-to-PM polar exocytic route, which mediates secretion of NtPPME1 for cell wall formation during cell expansion and cytokinesis and is ROP1-dependent.Plant development and growth require coordinated tissue and cell polarization. Two of the most essential cellular processes involved in polarization are cell expansion and cytokinesis, which determines cell morphology and functions (Jaillais and Gaude, 2008; Dettmer and Friml, 2011; Li et al., 2012). Pollen tube and root hair growth require highly polarized membrane trafficking (Libault et al., 2010; Kroeger and Geitmann, 2012). Cytokinesis, by which new cells are formed, separates daughter cells by forming a new structure within the cytoplasm termed the cell plate (CP). Made up of a cell wall (CW), surrounded by new plasma membrane (PM), the cell plate is generally considered to be an example of internal cell polarity in a nonpolarized plant cell (Bednarek and Falbel, 2002; Baluska et al., 2006).The conventional view of pollen tube tip growth and cell plate formation is supported by polar exocytic secretion of numerous vesicles (diameter of 60–100 nm) to the pollen tube tip and phragmoplast areas during cytokinesis. These polar exocytic vesicles, which are generally believed to originate from the Golgi apparatus, are delivered to the site of secretion via the cytoskeleton and fuse with the target membrane with the aid of fusion factors (Jurgens, 2005; Backues et al., 2007). However, whether these polar exocytic vesicles undergoing post-Golgi trafficking are part of the conventional Golgi-trans-Golgi network (TGN)-PM/CP exocytosis or are derived from some other unidentified exocytic secretion pathway remain unclear.Polar exocytosis is regulated and controlled by a conserved Rho GTPase signaling network in fungi, animals, and plants (Burkel et al., 2012; Ridley, 2013). Rho of plant (ROP), the sole subfamily of Rho GTPases in plant, participate in signaling pathways that regulate cytoskeleton organization and endomembrane trafficking, consequently determining cell polarization, polar growth and cell morphogenesis (Gu et al., 2005; Lee et al., 2008). In growing pollen tubes, ROP1 participates in regulating polar exocytosis in the tip region via two downstream pathways to regulate apical F-actin dynamics: RIC4-mediated F-actin polymerization and RIC3-mediated apical actin depolymerization. A constitutively active mutant of ROP1 (CA-rop1) prevents fusion of these vesicles with the PM and enhances the accumulation of exocytic vesicles in the apical cortex of pollen tubes (Lee et al., 2008). Although ROP GTPases have been extensively researched, their roles in polar membrane expansion in pollen tubes and epidermal pavement cells remains unclear (Xu et al., 2010; Yang and Lavagi, 2012), and there have been insufficient studies on the functions of ROPs in controlling cell plate formation during cytokinesis. Cell division requires precise regulation and spatial organization of the cytoskeleton for delivery of secretion vesicles to the expanding cell plate (Molendijk et al., 2001).In addition, newly made cell walls during cell expansion and cell plate formation require sufficient plasticity in order to integrate new membrane materials to support the polarized membrane extension. They also should be strong enough to withstand the internal turgor pressure and thereby maintain the shape of the cell (Zonia and Munnik, 2011; Hepler et al., 2013). Recent studies have demonstrated that pectins are important for both cytokinesis and cell expansion (Moore and Staehelin, 1988; Bosch et al., 2005; Chebli et al., 2012; Altartouri and Geitmann, 2015; Bidhendi and Geitmann, 2016). Pectins are one of the major cell wall components of the middle lamella and primary cell wall. They are polymerized and methylesterified in the Golgi and subsequently released into the apoplastic space as “soft” methylesterified polymers. The homogalacturonan components of pectin are later de-methylesterified by pectin methylesterases (PMEs). The demethylesterified pectins can be cross-linked, interact with Ca²⁺, and finally form the “hard” pectin matrix of the cell wall. Therefore, the enzymatic activity of PMEs determines the rigidity of the cell wall (Micheli, 2001; Peaucelle et al., 2011).In Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) pollen tubes, PMEs are found predominantly polar localized in the tip region and determine the rigidity of the apical cell wall (Bosch et al., 2005; Jiang et al., 2005; Fayant et al., 2010; Chebli et al., 2012; Wang et al., 2013). PME isoform knockout mutants in Arabidopsis (AtPPME1 or vanguard1) produce unstable pollen tubes which burst when germinated in vitro and have reduced fertilization abilities (Jiang et al., 2005; Rockel et al., 2008). Recent studies have shown that in growing tobacco pollen tubes, polar targeting of NtPPME1 to the pollen tube apex depends on an apical F-actin mesh network (Wang et al., 2013). Although the functions of PME in cell wall constriction are well documented, the intracellular secretion and regulation mechanism of the exocytic process of PME still remain largely unexplored. In addition, pectins are also found to be abundant in the forming cell plate, raising the possibility that PMEs may also function during cell plate formation (Moore and Staehelin, 1988; Dhonukshe et al., 2006).In our study, we have used NtPPME1 as a marker to identify a polar exocytic process which is distinct from the conventional Golgi-TGN-PM exocytosis pathway in both pollen tube tip growth and cell plate formation. We have identified a Golgi-derived secretory vesicle (GDSV) for the polar secretion and targeting of NtPPME1 to the cell wall that bypasses the TGN during cell polarization. Further investigations using ROP1 mutants have shown that this polar exocytosis is ROP1 dependent.     

Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity.Obesity, a medical condition predisposing to diabetes, cardiovascular diseases, cancer, and complicating other life-threatening diseases, is becoming an increasingly important social problem.^{1, 2, 3} Development of pharmacological approaches to reduction of body fat has remained a daunting task.⁴ Approved obesity treatments typically produce only moderate and temporary effects.^2,5 White adipocytes are the differentiated cells of white adipose tissue (WAT) that store triglycerides in lipid droplets.^6,7 In contrast, adipocytes of brown adipose tissue (BAT) dissipate excess energy through adaptive thermogenesis. Under certain conditions, white adipocytes can become partially replaced with brown-like ‘beige'' (‘brite'') adipocytes that simulate the thermogenic function of BAT adipocytes.^7,8 Obesity develops in the context of positive energy balance as a result of hypertrophy and hyperplasia of white adipocytes.⁹Expansion and renewal of the white adipocyte pool in WAT continues in adulthood.^10,11 This process is believed to rely on proliferation and self-renewal of mesenchymal precursor cells¹² that we term white adipocyte progenitors (WAPs). WAPs reside within the population of adipose stromal cells (ASCs)¹³ and are functionally similar to bone marrow mesenchymal stem cells (MSCs).^{14, 15, 16} ASCs can be isolated from the stromal/vascular fraction (SVF) of WAT based on negativity for hematopoietic (CD45) and endothelial (CD31) markers.^17,18 ASCs support vascularization as mural/adventitial cells secreting angiogenic factors^5,19 and, unlike bone marrow MSCs, express CD34.^19,20 WAPs have been identified within the ASC population based on expression of mesenchymal markers, such as platelet-derived growth factor receptor-β (PDGFRβ, aka CD140b) and pericyte markers.^17,18 Recently, a distinct ASC progenitor population capable of differentiating into both white and brown adipocytes has been identified in WAT based on PDGFRα (CD140a) expression and lack of PDGFRβ expression.^21,22 The physiological relevance of the two precursor populations residing in WAT has not been explored.We have previously established an approach to isolate peptide ligands binding to receptors selectively expressed on the surface of cell populations of interest.^{23, 24, 25, 26, 27} Such cell-targeted peptides can be used for targeted delivery of experimental therapeutic agents in vivo. A number of ‘hunter-killer'' peptides²⁸ composed of a cell-homing domain binding to a surface marker and of KLAKLAK₂ (sequence KLAKLAKKLAKLAK), a moiety inducing apoptosis upon receptor-mediated internalization, has been described by our group.^26,29 Such bimodal peptides have been used for depletion of malignant cells and organ-specific endothelial cells in preclinical animal models.^26,30,31 Recently, we isolated a cyclic peptide WAT7 (amino acid sequence CSWKYWFGEC) based on its specific binding to ASCs.²⁰ We identified Δ-decorin (ΔDCN), a proteolytic cleavage fragment of decorin, as the WAT7 receptor specifically expressed on the surface of CD34+PDGFRβ+CD31-CD45- WAPs and absent on MSCs in other organs.²⁰Here, we investigated whether WAPs are required for obesity development in adulthood. By designing a new hunter-killer peptide that directs KLAKLAK₂ to WAPs through WAT7/ΔDCN interaction, we depleted WAP in the mouse diet-induced obesity model. We demonstrate that WAP depletion suppresses WAT growth. We show that, in response to WAP deficiency, WAT becomes populated with beige adipocytes. Consistent with the reported thermogenic function of beige adipocytes,^32,33 the observed WAT remodeling is associated with increased energy expenditure. We identify a population of PDGFRα-positive, PDGFRβ-negative ASCs reported recently²² as a population surviving WAP depletion and responsible for WAT browning.     

Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells

Billions of inflammatory leukocytes die and are phagocytically cleared each day. This regular renewal facilitates the normal termination of inflammatory responses, suppressing pro-inflammatory mediators and inducing their anti-inflammatory counterparts. Here we investigate the role of the receptor tyrosine kinase (RTK) Mer and its ligands Protein S and Gas6 in the initial recognition and capture of apoptotic cells (ACs) by macrophages. We demonstrate extremely rapid binding kinetics of both ligands to phosphatidylserine (PtdSer)-displaying ACs, and show that ACs can be co-opsonized with multiple PtdSer opsonins. We further show that macrophage phagocytosis of ACs opsonized with Mer ligands can occur independently of a requirement for αV integrins. Finally, we demonstrate a novel role for Mer in the tethering of ACs to the macrophage surface, and show that Mer-mediated tethering and subsequent AC engulfment can be distinguished by their requirement for Mer kinase activity. Our results identify Mer as a receptor uniquely capable of both tethering ACs to the macrophage surface and driving their subsequent internalization.Many diseases, including rheumatoid arthritis, pulmonary fibrosis, adult respiratory distress syndrome, and inflammatory bowel disease,^{1, 2, 3, 4} are commonly marked by impaired resolution of inflammation that is linked to defects in the phagocytic clearance of apoptotic cells.^{5, 6, 7} Apoptotic cell (AC) clearance normally eliminates a plethora of pro-inflammatory stimuli,^{8, 9} and the recognition of ACs by phagocytes¹⁰ limits progression to necrosis,¹¹ suppresses pro-inflammatory mediator production, and induces IL-10 and TGF-β release.^{12, 13} As defective clearance of ACs is associated with the development of inflammatory disease and autoimmunity,^{14, 15} new therapeutic approaches designed to increase the capacity of phagocytes to remove ACs could effectively promote the resolution of inflammation.Phagocytosis of ACs can be regulated by soluble mediators, including cytokines,^{16, 17} prostaglandins and lipoxins,^{17, 18, 19} serum proteins,²⁰ agonists of Liver X receptors (LXRs),^{17, 21} and glucocorticoids (GC).^{17, 22} In particular, LXR agonists and GCs promote phagocytosis of ACs predominantly via a Tyro3/Axl/Mer (TAM) receptor tyrosine kinase (RTK)-dependent pathway.^{17, 21, 23} There are two established ligands for the TAM RTKs, Protein S (gene name Pros1), which activates Tyro3 and Mer, and Gas6, which activates all three TAMs,^{24, 25} although other ligands have been suggested.^{26, 27} The amino terminal Gla domains of Protein S and Gas6 bind to phosphatidylserine (PtdSer) on the plasma membrane of ACs,²⁸ a potent ‘eat-me'' signal by which ACs are recognized by phagocytes.²⁹ TAM receptors bind to the carboxy terminal domains of Protein S and Gas6, which effectively act as molecular ‘bridges'' between PtdSer on the AC and TAM receptors on the phagocyte.^{17, 30, 31} TAM receptor- and ligand-deficient mice exhibit defective phagocytic pruning of photoreceptor outer segments by retinal pigment epithelial (RPE) cells of the eye,^{32, 33, 34} defective clearance of apoptotic germ cells by Sertoli cells of the testis,³⁵ and defective clearance of ACs by macrophages/dendritic cells in lymphoid organs.³⁶ These phenotypes are also detectable in Mer (gene name Mertk) single knockouts.³⁷ In addition to phagocytic clearance, TAM signaling also has a pivotal role in controlling the innate immune response to pathogenic stimuli.^{13, 17, 38}Although the importance of Mer in the internalization of ACs by macrophages is now well-established, this receptor has been thought not to have a significant role in the initial ‘tethering'' of ACs to the macrophage surface.^{36, 39} In their studies, Scott et al.³⁶ used peritoneal macrophages for which tethering of ACs has now been shown to be mediated by T-cell immunoglobulin and mucin domain-containing molecule 4 (TIM4).³⁹ Subsequent internalization of tethered ACs is then mediated by either integrin αvβ3- or Mer-mediated signaling.^{39, 40} Similarly, for RPE cells, the initial capture of photoreceptor outer segments by RPE cells required the integrin αvβ5,⁴¹ with Mer-dependent signaling necessary for subsequent internalization. To further probe the mechanistic role of Mer in AC recognition and engulfment, we have now examined macrophages that predominantly use a Mer-dependent AC phagocytosis mechanism.^{17, 23} We show that in these cells, which do not express TIM4, Mer has the capacity to serve a unique dual role in mediating both tethering of ACs to the macrophage surface as well as subsequent AC engulfment.     

Distinct roles of RIP1–RIP3 hetero- and RIP3–RIP3 homo-interaction in mediating necroptosis

Necroptosis is mediated by a signaling complex called necrosome, containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL). It is known that RIP1 and RIP3 form heterodimeric filamentous scaffold in necrosomes through their RIP homotypic interaction motif (RHIM) domain-mediated oligomerization, but the signaling events based on this scaffold has not been fully addressed. By using inducible dimer systems we found that RIP1–RIP1 interaction is dispensable for necroptosis; RIP1–RIP3 interaction is required for necroptosis signaling, but there is no necroptosis if no additional RIP3 protein is recruited to the RIP1–RIP3 heterodimer, and the interaction with RIP1 promotes the RIP3 to recruit other RIP3; RIP3–RIP3 interaction is required for necroptosis and RIP3–RIP3 dimerization is sufficient to induce necroptosis; and RIP3 dimer-induced necroptosis requires MLKL. We further show that RIP3 oligomer is not more potent than RIP3 dimer in triggering necroptosis, suggesting that RIP3 homo-interaction in the complex, rather than whether RIP3 has formed homo polymer, is important for necroptosis. RIP3 dimerization leads to RIP3 intramolecule autophosphorylation, which is required for the recruitment of MLKL. Interestingly, phosphorylation of one of RIP3 in the dimer is sufficient to induce necroptosis. As RIP1–RIP3 heterodimer itself cannot induce necroptosis, the RIP1–RIP3 heterodimeric amyloid fibril is unlikely to directly propagate necroptosis. We propose that the signaling events after the RIP1–RIP3 amyloid complex assembly are the recruitment of free RIP3 by the RIP3 in the amyloid scaffold followed by autophosphorylation of RIP3 and subsequent recruitment of MLKL by RIP3 to execute necroptosis.Necroptosis is a type of programmed necrosis characterized by necrotic morphological changes, including cellular organelle swelling, cell membrane rupture,^{1, 2, 3} and dependence of receptor-interacting protein (RIP)1⁴ and RIP3.^{5, 6, 7} Physiological function of necroptosis has been illustrated in host defense,^{8, 9, 10, 11} inflammation,^{12, 13, 14, 15, 16} tissue injury,^{10, 17, 18} and development.^{19, 20, 21}Necroptosis can be induced by a number of different extracellular stimuli such as tumor necrosis factor (TNF). TNF stimulation leads to formation of TNF receptor 1 (TNFR1) signaling complex (named complex I), and complex II containing RIP1, TRADD, FAS-associated protein with a death domain (FADD), and caspase-8, of which the activation initiates apoptosis. If cells have high level of RIP3, RIP1 recruits RIP3 to form necrosome containing FADD,^{22, 23, 24} caspase-8, RIP1, and RIP3, and the cells undergo necroptosis.^{25, 26} Caspase-8 and FADD negatively regulates necroptosis,^{27, 28, 29, 30} because RIP1, RIP3, and CYLD are potential substrates of caspase-8.^{31, 32, 33, 34} Necrosome also suppresses apoptosis but the underlying mechanism has not been described yet. Mixed-lineage kinase domain-like (MLKL) is downstream of RIP3,^{35, 36} and phosphorylation of MLKL is required for necroptosis.^{37, 38, 39, 40, 41, 42}Apoptosis inducing complex (complex II) and necrosome are both supramolecular complexes.^{43, 44, 45} A recent study showed that RIP1 and RIP3 form amyloidal fibrils through their RIP homotypic interaction motif⁴⁶ (RHIM)-mediated polymerization, and suggested that amyloidal structure is essential for necroptosis signaling.⁴⁷ The RIP1–RIP3 heterodimeric amyloid complex is believed to function as a scaffold that brings signaling proteins into proximity to permit their activation. However, RIP1 and RIP3 also can each form fibrils on their own RHIM domains in vitro. It is unclear how the homo- and hetero-interactions are coordinated and organized on the amyloid scaffold to execute their functions in necroptosis. Here, we used inducible dimerization systems to study the roles of RIP1–RIP1, RIP1–RIP3, and RIP3–RIP3 interactions in necroptosis signaling. Our data suggested that it is the RIP1–RIP3 interaction in the RIP1–RIP3 heterodimeric amyloid complex that empowers to recruit other free RIP3; homodimerization of RIP3 triggers its autophosphorylation and only the phosphorylated RIP3 can recruit MLKL to execute necroptosis.     

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as ‘accidental cell death'' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. ‘Regulated cell death'' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.Defining life and death is more problematic than one would guess. In 1838, the work of several scientists including Matthias Jakob Schleiden, Theodor Schwann and Rudolf Carl Virchow culminated in the so-called ‘cell theory'', postulating that: (1) all living organisms are composed of one or more cells; (2) the cell is the basic unit of life; and (3) all cells arise from pre-existing, living cells.¹ Only a few decades later (in 1885), Walter Flemming described for the first time some of the morphologic features that have been largely (but often inappropriately) used to define apoptosis throughout the past four decades.^{2, 3, 4}A corollary of the cell theory is that viruses do not constitute bona fide living organisms.⁵ However, the discovery that the giant Acanthamoeba polyphaga mimivirus can itself be infected by other viral species has casted doubts on this point.^{6, 7, 8} Thus, the features that underlie the distinction between a living and an inert entity remain a matter of debate. Along similar lines, defining the transition between an organism''s life and death is complex, even when the organism under consideration is the basic unit of life, a cell. From a conceptual standpoint, cell death can obviously be defined as the permanent degeneration of vital cellular functions. Pragmatically speaking, however, the precise boundary between a reversible alteration in homeostasis and an irreversible loss of cellular activities appears to be virtually impossible to identify. To circumvent this issue, the Nomenclature Committee on Cell Death (NCCD) previously proposed three criteria for the identification of dead cells: (1) the permanent loss of the barrier function of the plasma membrane; (2) the breakdown of cells into discrete fragments, which are commonly referred to as apoptotic bodies; or (3) the engulfment of cells by professional phagocytes or other cells endowed with phagocytic activity.^{9, 10, 11}However, the fact that a cell is engulfed by another via phagocytosis does not imply that the cell-containing phagosome fuses with a lysosome and that the phagosomal cargo is degraded by lysosomal hydrolases.^{12, 13, 14} Indeed, it has been reported that engulfed cells can be released from phagosomes as they preserve their viability, at least under some circumstances.¹⁵ Thus, the NCCD recommends here to consider as dead only cells that either exhibit irreversible plasma membrane permeabilization or have undergone complete fragmentation. A compendium of techniques that can be used to quantify these two markers of end-stage cell death in vitro and in vivo goes beyond the scope of this review and can be found in several recent articles.^{16, 17, 18, 19, 20, 21, 22, 23, 24, 25}Importantly, cell death instances can be operationally classified into two broad, mutually exclusive categories: ‘accidental'' and ‘regulated''. Accidental cell death (ACD) is caused by severe insults, including physical (e.g., elevated temperatures or high pressures), chemical (e.g., potent detergents or extreme variations in pH) and mechanical (e.g., shearing) stimuli, is virtually immediate and is insensitive to pharmacologic or genetic interventions of any kind. The NCCD thinks that this reflects the structural disassembly of cells exposed to very harsh physicochemical conditions, which does not involve a specific molecular machinery. Although ACD can occur in vivo, for instance as a result of burns or traumatic injuries, it cannot be prevented or modulated and hence does not constitute a direct target for therapeutic interventions.^{23, 26, 27, 28} Nonetheless, cells exposed to extreme physicochemical or mechanical insults die while releasing elevated amounts of damage-associated molecular patterns (DAMPs), that is, endogenous molecules with immunomodulatory (and sometimes cytotoxic) activity. Some DAMPs can indeed propagate an unwarranted cytotoxic response (directly or upon the involvement of innate immune effectors) that promotes the demise of local cells surviving the primary insult.^{16, 19, 29, 30, 31} Intercepting DAMPs or blocking DAMP-ignited signaling pathways may mediate beneficial effects in a wide array of diseases involving accidental (as well as regulated) instances of cell death.^{19, 32}At odds with its accidental counterpart, regulated cell death (RCD) involves a genetically encoded molecular machinery.^{9, 33} Thus, the course of RCD can be altered by means of pharmacologic and/or genetic interventions targeting the key components of such a machinery. Moreover, RCD often occurs in a relatively delayed manner and is initiated in the context of adaptive responses that (unsuccessfully) attempt to restore cellular homeostasis.^{34, 35, 36, 37, 38} Depending on the initiating stimulus, such responses can preferentially involve an organelle, such as the reticular unfolded protein response, or operate at a cell-wide level, such as macroautophagy (hereafter referred to as autophagy).^{39, 40, 41, 42, 43, 44} Thus, while ACD is completely unpreventable, RCD can be modulated (at least to some extent, see below) not only by inhibiting the transduction of lethal signals but also by improving the capacity of cells to mount adaptive responses to stress.^{45, 46, 47, 48, 49, 50} Importantly, RCD occurs not only as a consequence of microenvironmental perturbations but also in the context of (post-)embryonic development, tissue homeostasis and immune responses.^{51, 52, 53, 54} Such completely physiologic instances of RCD are generally referred to as ‘programmed cell death'' (PCD) (Figure 1).^{9, 33}Open in a separate windowFigure 1Types of cell death. Cells exposed to extreme physical, chemical or mechanical stimuli succumb in a completely uncontrollable manner, reflecting the immediate loss of structural integrity. We refer to such instances of cellular demise with the term ‘accidental cell death'' (ACD). Alternatively, cell death can be initiated by a genetically encoded machinery. The course of such ‘regulated cell death'' (RCD) variants can be influenced, at least to some extent, by specific pharmacologic or genetic interventions. The term ‘programmed cell death'' (PCD) is used to indicate RCD instances that occur as part of a developmental program or to preserve physiologic adult tissue homeostasisFor the purpose of this discussion, it is useful to keep in mind the distinction that is currently made between the initiation of RCD and its execution. The term execution is generally used to indicate the ensemble of biochemical processes that truly cause the cellular demise. Conversely, initiation is commonly used to refer to the signal transduction events that activate executioner mechanisms. Thus, the activation of caspase-8 (CASP8) in the course of FAS ligand (FASL)-triggered apoptosis is widely considered as an initiator mechanism, whereas the consequent activation of caspase-3 (CASP3) is categorized as an executioner mechanism (see below).^{51, 55, 56, 57}Here, the NCCD formulates a set of recommendations to discriminate between essential and accessory aspects of RCD, that is, between those that etiologically mediate its occurrence and those that change its kinetics or morphologic and biochemical manifestations.     

Ret is essential to mediate GDNF's neuroprotective and neuroregenerative effect in a Parkinson disease mouse model

Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF''s neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF''s effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD.Glial cell line-derived neurotrophic factor (GDNF) is the founding member of the four ligands in the GDNF family, which belong to the transforming growth factor-β superfamily.¹ GDNF was characterized as a potent survival factor for many neurons in culture such as dopaminergic, motor, sympathetic, parasympathetic, sensory and enteric neurons.^{1, 2} In addition, in dopaminergic neuron cultures GDNF stimulates neuronal differentiation, neurite outgrowth, synapse formation and dopamine release.^{1, 2}As degeneration of midbrain dopaminergic neurons in the substantia nigra pars compacta (SNpc) represents a major hallmark of Parkinson disease (PD), the most common neurodegenerative movement disorder, GDNF has raised considerable interest as a therapeutic molecule for the treatment of PD.^{3, 4, 5} PD affects >2% of individuals over the age of 60 years, but no curative treatment is available to date, mainly due to a lack of understanding disease etiology.^{6, 7, 8} Preclinical studies in the established 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) rodent and primate models of PD demonstrated a substantial neuroprotection and regeneration effect by striatal provided GDNF or its close relative neurturin.^{3, 4, 9} However, clinical phase II trials on PD patients using GDNF or neurturin did so far not convincingly recapitulate their beneficial effects on the dopaminergic system in humans most likely due to technical problems and the selection of advanced PD patients.^{10, 11, 12, 13}GDNF signaling is highly complex as this neurotrophic factor can bind to a variety of receptors, thus being able to induce pleiotropic effects. GDNF efficiently binds to the GPI-linked GDNF family receptor α1 (GFRα1).^{1, 2} It has been shown that the GDNF/GFRα1 complex can activate not only the canonical GDNF receptor Ret, a receptor tyrosine kinase which signals through the sarcoma protein (Src)/rat sarcoma (Ras)/mitogen-activated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt, NF-κB (nuclear factor ''kappa-light-chain-enhancer'' of activated B cells), JNK (c-Jun N-terminal kinases) and PLCγ (phospholipase γ) pathway, but also with other signaling inducing receptors.^{1, 2, 4, 5, 13} So far, at least four alternative GDNF receptors have been described which are all expressed in midbrain dopaminergic neurons, NCAM,^{14, 15} the integrins αV and βI,^{14, 16} syndecan 3¹⁷ and N-cadherin.¹⁸ Interestingly, Ret is not essential during pre- and postnatal development of the mouse dopaminergic system,^{19, 20, 21, 22, 23} but specifically required for the maintenance of SNpc dopaminergic neurons and their striatal innervation in aged mice.^{23, 24, 25} In contrast, GDNF seems most likely under physiological conditions to be dispensable during development and maintenance of midbrain dopaminergic neurons in mice, although conflicting results exist.^{26, 27, 28} Thus, Ret might be activated by a GDNF-independent mechanism to stimulate SNpc dopaminergic neuron survival. In addition, the in vivo function of the alternative GDNF receptors in the dopaminergic system under physiological and pathophysiological conditions, like PD, and their dependence on GDNF has not yet been addressed in detail. This raised the important question which GDNF receptor might be required to mediate GDNF''s reported neuroprotective and regenerative effect in the dopaminergic system in PD animal models and potentially in PD patients.^{5, 29}Previously, we showed in dopaminergic neuron-specific Ret knockout mice that Ret receptor loss does not result in a higher vulnerability of midbrain dopaminergic neurons against MPTP but to less resprouting of left over dopaminergic neuron axons in the striatum after MPTP intoxication.³⁰ In adult mice endogenous GDNF levels are rather low.^{26, 31} Therefore, we could not rule out in that study the possibility, that higher levels of GDNF—as also used in the clinical GDNF trials in PD patients—might have neuroprotective and regenerating effects even in the absence of the Ret receptor. Here we addressed now this question by viral overexpression of GDNF in MPTP-treated mice lacking expression of Ret again specifically in dopaminergic neurons.^{23, 30} We found that in the absence of Ret in dopaminergic neurons even a substantial overexpression of GDNF in the striatum does not have a neuroprotective and regenerative effect. Thus, despite the expression of alternative GDNF receptors on midbrain dopaminergic neurons, the presence of the canonical GDNF receptor Ret seems to be mandatory for mediating GDNF''s beneficial survival and axonal resprouting effect in these neurons.     

Flexible open conformation of the AP-3 complex explains its role in cargo recruitment at the Golgi

Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding. The structural details that influence these processes, however, are still poorly understood. Here we present cryo-EM structures of the full-length stable 300 kDa yeast AP-3 complex. The structures reveal that AP-3 adopts an open conformation in solution, comparable to the membrane-bound conformations of AP-1 or AP-2. This open conformation appears to be far more flexible than AP-1 or AP-2, resulting in compact, intermediate, and stretched subconformations. Mass spectrometrical analysis of the cross-linked AP-3 complex further indicates that the ear domains are flexibly attached to the surface of the complex. Using biochemical reconstitution assays, we also show that efficient AP-3 recruitment to the membrane depends primarily on cargo binding. Once bound to cargo, AP-3 clustered and immobilized cargo molecules, as revealed by single-molecule imaging on polymer-supported membranes. We conclude that its flexible open state may enable AP-3 to bind and collect cargo at the Golgi and could thus allow coordinated vesicle formation at the trans-Golgi upon Arf1 activation.

Eukaryotic cells have membrane-enclosed organelles, which carry out specialized functions, including compartmentalized biochemical reactions, metabolic channeling, and regulated signaling, inside a single cell. The transport of proteins, lipids, and other molecules between these organelles is mediated largely by small vesicular carriers that bud off at a donor compartment and fuse with the target membrane to deliver their cargo. The generation of these vesicles has been subject to extensive studies and has led to the identification of numerous coat proteins that are required for their formation at different sites (1, 2). Coat proteins can be monomers, but in most cases, they consist of several proteins, which form a heteromeric complex.Heterotetrameric adapter protein (AP) complexes are required at several endomembranes for cargo binding. Five well-conserved AP-complexes with differing functions have been identified in mammalian cells, named AP-1–AP-5, of which three (AP-1–AP-3) are conserved from yeast to human (3, 4). The three conserved adapter complexes function at different membranes along the endomembrane system. AP-1 is required for cargo transport between the Golgi and the endosome, AP-2 is required for cargo recognition and transport between the plasma membrane and the early endosome. Finally, AP-3 functions between the trans Golgi and the vacuole in yeast, whereas mammalian AP-3 localizes to a tubular endosomal compartment, in addition to or instead of the TGN (2, 5, 6).Each of the complexes consists of four different subunits: two large adaptins (named α−ζ and β1-5 respectively), a medium-sized subunit (μ1-5), and a small subunit (σ1-5). While μ- and σ-subunits together with the N-termini of the large adaptins build the membrane-binding core of the complex, the C-termini of both adaptins contain the ear domains, which are connected via flexible linkers (2). The recruitment of these complexes to membranes is not entirely conserved. They all require cargo binding, yet AP-1 binds Arf1-GTP with the γ and β1 subunit and phosphatidylinositol-4-phosphate (PI4P) via a proposed conserved site on its γ-subunit (7, 8). AP-2, on the other hand, interacts with PI(4,5)P₂ at the plasma membrane via its α, β2, and μ2 subunits (9, 10, 11).Several studies have uncovered how AP-3 functions in cargo sorting in yeast. AP-3 recognizes cargo at the Golgi via two sorting motifs in the cytosolic segments of membrane proteins: a Yxxφ sorting motif, as found in yeast in the SNARE Nyv1 or the Yck3 casein kinase, which binds to a site in μ3, as shown for mammalian AP-3, which is similar to μ2 in AP-2 (12, 13, 14), and dileucine motifs as found in the yeast SNARE Vam3 or the alkaline phosphatase Pho8, potentially also at a site comparable to AP-1 and AP-2 (15, 16). Unlike AP-1 and AP-2-coated vesicles, which depend on clathrin for their formation (2, 17), AP-3 vesicle formation in yeast does not require clathrin or the HOPS subunit Vps41 (18), yet Vps41 is required at the vacuole to bind AP-3 vesicles prior to fusion (19, 20, 21, 22). Studies in metazoan cells revealed that Vps41 and AP-3 function in regulated secretion (23, 24, 25), and AP-3 is required for biogenesis of lysosome-related organelles (26). This suggests that the AP-3 complex has features that are quite different from AP-1 and AP-2 complexes, which cooperate with clathrin in vesicle formation (2).Among the three conserved AP complexes, the function of the AP-3 complex is the least understood. Arf1 is necessary for efficient AP-3 vesicle generation in mammalian cells and shows a direct interaction with the β3 and δ subunits of AP-3 (27, 28). In addition, in vitro experiments on mammalian AP-3 using liposomes or enriched Golgi membranes suggest Arf1 as an important factor in AP-3 recruitment, whereas acidic lipids do not have a major effect, in contrast to what was found for AP-1 and AP-2 (7, 11, 29, 30). Another study showed that membrane recruitment of AP-3 depends on the recognition of sorting signals in cargo tails and PI3P (31), similar to AP-1 recruitment via cargo tails, Arf1 and PI4P (32).However, since AP-1 and AP-3 are both recruited to the trans-Golgi network (TGN) in yeast (33), the mechanism of their recruitment likely differs. Even though Arf1 is required, yeast AP-3 seems to be present at the TGN before the arrival of the Arf1 guanine nucleotide exchange factor (GEF) Sec7 (33). This implies the necessity for additional factors at the TGN and a distinct mechanism to allow for spatial and temporal separation of AP-1 and AP-3 recruitment to membranes. Structural data on mammalian AP-1 and AP-2 “core” complexes without the hinge and ear domains of their large subunits revealed that both exist in at least two very defined conformational states: a “closed” cytosolic state, where the cargo-binding sites are buried within the complex, and an “open” state, where the same sites are available to bind cargo (7, 8, 10, 34, 35). Binding of Arf1 to AP-1 or PI(4,5)P₂ in case of AP-2 induces a conformational change in the complexes that enables them to bind cargo molecules carrying a conserved acidic di-Leucine or a Tyrosine-based motif, as for all three AP complexes in yeast (8, 34). Additional conformational states and intermediates have been reported for both, mammalian AP-1 and AP-2 complex. AP-1, for example, can be hijacked by the human immunodeficiency virus-1 (HIV-1) proteins viral protein u (Vpu) and negative factor (Nef), resulting in a hyper-open conformation of AP-1 (36, 37).An emerging model over the past years has suggested that APs have several binding sites that allow for the stabilization of membrane binding and the open conformation of the complexes, but there are initial interactions required that dictate their recruitment to the target membrane. Although these interaction sites for mammalian AP-1 and AP-2 have been identified in great detail based on interaction analyses and structural studies (8, 10, 11, 35, 36, 38, 39), structural data for AP-3 is largely missing. The C-terminal part of the μ-subunit of mammalian AP-3 has been crystallized together with a Yxxφ motif-containing a cargo peptide, which revealed a similar fold and cargo-binding site as shown for AP-1 and AP-2 (14). However, positively charged binding surfaces required for PIP-interaction were not well conserved. Although the “trunk” segment of AP-1 and AP-2 is known quite well by now, information on hinge and ear domains in context of these complexes is largely missing. Crystal structures of the isolated ear domains of α-, γ- and β2-adaptin have been published (40, 41, 42), and a study on mammalian AP-3 suggested a direct interaction between δ-ear and δ3 that interfered with Arf1-binding (43). Furthermore, during tethering of AP-3 vesicles with the yeast vacuole, the δ−subunit Apl5 of the yeast AP-3 complex binds to the Vps41 subunit of the HOPS complex as a prerequisite of fusion (18, 19, 21, 22).In this study, we applied single particle electron cryo-microscopy (cryo-EM) to analyze the purified full-length AP-3 complex from yeast and unraveled the factors required for AP-3 recruitment to membranes by biochemical reconstitution. Our data reveal that a surprisingly flexible AP-3 complex requires a combination of cargo, PI4P, and Arf1 for membrane binding, which explains its function in selective cargo sorting at the Golgi.     

Haploinsufficiency of cathepsin D leads to lysosomal dysfunction and promotes cell-to-cell transmission of α-synuclein aggregates

Lysosomal dysfunction has been implicated both pathologically and genetically in neurodegenerative disorders, such as Alzheimer''s disease and Parkinson''s disease (PD). Lysosomal gene deficiencies cause lysosomal storage disorders, many of which involve neurodegeneration. Heterozygous mutations of some of these genes, such as GBA1, are associated with PD. CTSD is the gene encoding Cathepsin D (CTSD), a lysosomal protein hydrolase, and homozygous CTSD deficiency results in neuronal ceroid-lipofuscinosis, which is characterized by the early onset, progressive neurodegeneration. CTSD deficiency was also associated with deposition of α-synuclein aggregates, the hallmark of PD. However, whether partial deficiency of CTSD has a role in the late onset progressive neurodegenerative disorders, including PD, remains unknown. Here, we generated cell lines harboring heterozygous nonsense mutations in CTSD with genomic editing using the zinc finger nucleases. Heterozygous mutation in CTSD resulted in partial loss of CTSD activity, leading to reduced lysosomal activity. The CTSD mutation also resulted in increased accumulation of intracellular α-synuclein aggregates and the secretion of the aggregates. When α-synuclein was introduced in the media, internalized α-synuclein aggregates accumulated at higher levels in CTSD+/− cells than in the wild-type cells. Consistent with these results, transcellular transmission of α-synuclein aggregates was increased in CTSD+/− cells. The increased transmission of α-synuclein aggregates sustained during the successive passages of CTSD+/− cells. These results suggest that partial loss of CTSD activity is sufficient to cause a reduction in lysosomal function, which in turn leads to α-synuclein aggregation and propagation of the aggregates.Maintaining protein homeostasis (proteostasis) is crucial in not only maintenance of physiological functions of cells, but survival of cells. Proteostasis is a particularly important issue for the survival of post-mitotic cells, such as neurons, while dividing cells can dilute aged and misfolded proteins during the mitosis process.^{1, 2} For the clearance of protein burden, cells utilize two major protein degradation systems, ubiquitin proteasome system and lysosomal degradation, the latter degrades endosomal and autophagosomal cargos.^{3, 4, 5, 6} Dysregulation of ubiquitin proteasome system and lysosome has been shown to cause protein conformational diseases, including neurodegenerative disorders and metabolic disorders.^{7, 8} Genetic studies have suggested that impairment of lysosomal functions has important roles in the pathogenesis of neurodegenerative diseases. Mutations in ATP13A2, GBA1 and VPS35 have been associated with PD.^{9, 10, 11, 12} Mutations in progranulin and charged multivesicular body protein 2B (CHMP2B) have been identified as genetic causes of amyotrophic lateral sclerosis and frontotemporal dementia.^{13, 14, 15} Postmortem brain tissues of neurodegenerative diseases have exhibited deposition of endosomal and autophagic vesicles.¹⁶ Therefore, neurodegenerative proteinopathies might be attributed to lysosomal dysfunction.Pathological examinations of patient tissues have exhibited that protein aggregates, such as amyloid beta (Aβ), tau and α-synuclein aggregates, spread to larger brain regions as disease progresses.¹⁷ In animal models, intracerebrally injected α-synuclein aggregates could spread into larger brain regions both in α-synuclein transgenic and non-transgenic mice.^{18, 19, 20, 21} Inoculation of Aβ or tau aggregates into either non-transgenic or transgenic models of AD also exhibited propagation of those aggregates.^{22, 23, 24, 25, 26, 27, 28} Studies have suggested that cell-to-cell transmission of protein aggregates is the underlying mechanism of the pathological propagation.^{29, 30}Mounting evidence have suggested that lysosomal function is important for the clearance of the transferred aggregates in recipient neurons during cell-to-cell aggregate transmission.³¹ This has been extensively studied in cell culture models for α-synuclein transmission. Previous studies showed α-synculein aggregates can be internalized and transported through the endolysosomal pathway.³² Lyososomal dysfunction led to increased accumulation of the internalized α-synuclein aggregates, suggesting that the lysosomal activity in recipient cells is critical in the clearance of the transmitted α-synuclein aggregates.^{32, 33}Lysosomal storage diseases (LSDs) are caused by defects in the lysosomal degradation process. Mutations in genes encoding lysosomal catabolic enzymes and transporters manifest excessive deposition of the enzyme substrates in various organs.³⁴ Though different LSDs show different symptoms, most of LSD patients exhibit neurological symptoms such as mental retardation, motor dysfunction and progressive neurodegeneration, as well as specific pathological changes in the nervous system.^{35, 36} In addition, some of progressive neurodegenerative disorders such as AD, PD and Huntington''s disease also show similar pathological features with LSD: accumulations of endosomal and autophagosomal vesicles and undegraded macromolecules, and inflammatory responses in brain.¹⁶Gaucher''s disease (GD) is the most common LSD, which is inherited in an autosomal recessive manner. Homozygous mutations of GBA1 gene, encoding β-glucocerebrosidase 1 (GCase 1), a lysosomal hydrolase, is responsible for GD.³⁷ Evidence has suggested that GD is closely related to PD. Patients with type-1 GD, the most common form of GD, frequently develop parkinsonism.³⁸ Heterozygous carriers of GBA1 mutations are at a higher risk for PD.^{39, 40} It has been shown that about 75% of Lewy bodies, a pathological hallmark of PD, colocalized with GCase 1 in brains of PD and DLB patients with heterozygous GBA1 mutations.⁴¹ These results suggest that lysosomal enzyme deficiency is associated with the development of PD.Cathepsin D (CTSD) is a major lysosomal endopeptidase, which is critical in the degradation of long-lived proteins.⁴² Genetic and clinical studies have shown that the homozygous deficiency of CTSD results in the early onset, progressive neurodegeneration, such as congenital neuronal ceroid-lipofuscinosis.⁴³ The heterozygous missense mutations in CTSD have been known to cause the early onset motor and visual problems, brain atrophy, and progressive psychomotor symptoms.⁴⁴ However, the effects of CTSD deficiency on the late onset progressive neurodegenerative disorders, including AD and PD, remain unclear. Nevertheless, it has become clear that CTSD activity is crucial in the degradation of pathogenic protein aggregates.^{45, 46}Herein, we generated a cell line with a heterozygous nonsense mutation in CTSD and investigated the roles of the CTSD activity in lysosomal function, α-synuclein aggregation and transcellular transmission of α-synuclein aggregates.     

Mps1 kinase regulates tumor cell viability via its novel role in mitochondria

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells.Massive chromosome missegregation induces cell death as observed by Theodor Boveri in the early 1900s.¹ However, the underlying mechanism remains elusive. The spindle assembly checkpoint (SAC) is a dominant machine monitoring chromosomal segregation during mitosis by delaying the onset of anaphase until all chromosomes are properly captured by microtubules. The SAC consists of kinetochore association sensors, including Mps1 (monopolar spindle 1), Bub1 (budding uninhibited by benzimidazole 1 homolog) and Aurora B; a signaling transducer termed the mitotic checkpoint complex (MCC), including CDC20 (cell division cycle 20), BubR1 (Bub1-related kinase), Bub3 (budding uninhibited by benzimidazole 3 homolog) and Mad2 (mitotic arrest deficient-like 2); and an effector APC/C (anaphase-promoting complex/cyclosome) that is inhibited by MCC in response to an active SAC.² Loss of SAC by inactivation of checkpoint sensors or signaling transducers elicits massive chromosome missegregation, induces severe gain or loss of chromosomes and eventually causes cell death.^{3, 4, 5, 6} Meanwhile, a weakened SAC due to the haploinsufficiency of the checkpoint proteins Mad1, Mad2, Bub1, BubR1 and CENP-E (centromere protein E) does not cause cell death but facilitates tumorigenesis.^{7, 8, 9, 10, 11} These studies suggest that the fate of these cells is dependent on their respective degree of SAC deficiency. Notably, in these studies SAC proteins were constitutively disturbed, raising the possibility that other signaling pathways could be affected as SAC proteins have functions beyond SAC regulation.^{12, 13, 14}Mps1 is an essential component of SAC that senses SAC signal by promoting MCC formation via kinetochore recruitment of Mad2, CENP-E and Knl1 (kinetochore-null protein 1).^{15, 16, 17, 18, 19} Recent studies show that Mps1 can discriminate between on or off SAC signaling by binding to NDC80c via the motif that associates microtubules.^{20, 21} Following SAC, Mps1 is involved in regulating chromosome alignment by phosphorylating Borealin, a component of chromosomal passenger complex (CPC).^{22, 23} In addition, Mps1 plays multiple roles beyond mitosis, including centrosome duplication, cytokinesis, ciliogenesis and DNA damage response.^{18, 24, 25, 26, 27, 28} Mps1 is indispensable for cell survival as loss of Mps1 function by specific siRNA or Mps1 kinase inhibitors causes significant cell death; it has been proposed that Mps1 regulates this process through its roles in SAC.^{29, 30, 31}Mps1 kinase is overexpressed in a variety of tumor types.^{32, 33, 34, 35} In breast cancer, high levels of Mps1 correlate with tumor grades; reducing Mps1 level induces massive apoptosis but allows a selective survival of tumor cells with less aneuploidy.³² Our recent results in colon cancer cells showed that overexpression of Mps1 facilitate the survival of tumor cells with higher aneuploidy by decreasing SAC threshold.³⁵ To further uncover the roles of high levels of Mps1 in tumorigenesis, we examined Mps1 levels in various stages of colon cancer tissues and found that Mps1 level peaks in tissues at stage II, at which stage tumor cells encounter various survival stresses, including genome instability. Aneuploid colon cancer cell lines bear higher levels of Mps1 than diploid cell lines and the amount of Mps1 required for cell survival is far more than that of maintaining SAC, suggesting that other functions of Mps1 are also employed to maintain cell viability. Short-term inhibition of Mps1 activity in mitosis with inhibitors at a dose of more than SAC depletion is sufficient to cause dividing cell death and increase mitochondrial fragmentation simultaneously. Finally, we found that Mps1 can regulate the release of cytochrome c by associating with mitochondrial protein VDAC1 (voltage-dependent anion channel 1). Based on these findings, we postulated that high levels of Mps1 contribute to survival of aneuploid cancer cells via its roles in SAC and mitochondria.