Septins modulate the autophagy response after nutrient starvation.

The pathways that induce macroautophagy (referred to as autophagy hereafter) in response to the stress of starvation are well conserved and essential under nutrient-limiting conditions. However, less is understood about the mechanisms that modulate the autophagy response. Here we present evidence that after induction of autophagy in budding yeast septin filaments rapidly assemble into discrete patches distributed along the cell cortex. These patches gradually mature over 12 h of nutrient deprivation to form extended structures around Atg9 membranes tethered at the cortical endoplasmic reticulum, a class of membranes that are limiting for autophagosome biogenesis. Loss of cortical septin structures alters the kinetics of autophagy activation and most dramatically extends the duration of the autophagy response. In wild-type cells, diffusion of Atg9 membranes at the cell cortex undergoes transient pauses that are dependent on septins, and septins at the bud neck block the diffusion of Atg9 membranes between mother and daughter cells. We conclude that septins reorganize at the cell cortex during autophagy to locally limit access of Atg9 membranes to autophagosome assembly sites, and thus modulate the autophagy response during nutrient deprivation.

APC mutations lead to cytokinetic failures in vitro and tetraploid genotypes in Min mice.

Previous research has proposed that genomic instability contributes to cancer progression, with its initiation linked to tetraploid cell formation (Duesberg, P., and R. Li. 2003. Cell Cycle. 2:202-210; Ganem, N.J., Z. Storchova, and D. Pellman. 2007. Curr. Opin. Genet. Dev. 17:157-162). However, there is little direct evidence linking cancer-causing mutations with such events, and it remains controversial whether genomic instability is a cause or an effect of cancer. In this study, we show that adenomatous polyposis coli (APC) mutations found in human colorectal cancers dominantly inhibit cytokinesis by preventing mitotic spindle anchoring at the anaphase cortex and, thus, blocking initiation of the cytokinetic furrow. We find that dividing crypt cells in the small intestines of APC(Min/+) mice exhibit similar mitotic defects, including misoriented spindles and misaligned chromosomes. These defects are observed in normal crypt cells with wild-type levels of beta-catenin and, importantly, are associated with tetraploid genotypes. We provide direct evidence that the dominant activity of APC mutants induces aneuploidy in vivo. Our data support a model whereby tetraploid cells represent a first step in the onset of genomic instability and colorectal cancer.

A novel role for the CBF3 kinetochore-scaffold complex in regulating septin dynamics and cytokinesis.

In budding yeast, the kinetochore scaffold complex centromere binding factor 3 (CBF3) is required to form kinetochores on centromere DNA and to allow proper chromosome segregation. We have previously shown that SKP1 and SGT1 balance the assembly and turnover of CBF3 complexes, a cycle that we suggest is independent of its role in chromosome segregation (Rodrigo-Brenni, M.C., S. Thomas, D.C. Bouck, and K.B. Kaplan. 2004. Mol. Biol. Cell. 15:3366-3378). We provide evidence that this cycle contributes to a second, kinetochore-independent function of CBF3. In this study, we show that inhibiting the assembly of CBF3 causes disorganized septins and defects in cell polarity that give rise to cytokinesis failures. Specifically, we show that septin ring separation and disassembly is delayed in anaphase, suggesting that CBF3 regulates septin dynamics. Only mutations that affect the CBF3 cycle, and not mutants in outer kinetochore subunits, cause defects in septins. These results demonstrate a novel role for CBF3 in regulating cytokinesis, a role that is reminiscent of passenger proteins. Consistent with this possibility, we find that CBF3 interacts with Bir1p, the homologue of the passenger protein Survivin. Mutants in Bir1p similarly affect septin organization, leading us to propose that CBF3 and Bir1p act as passenger proteins to coordinate chromosome segregation with cytokinesis.

A Bir1p Sli15p kinetochore passenger complex regulates septin organization during anaphase.

Kinetochore-passenger complexes in metazoans have been proposed to coordinate the segregation of chromosomes in anaphase with the induction of cytokinesis. Passenger protein homologues in the budding yeast Saccharomyces cerevisiae play a critical role early in mitosis, ensuring proper biorientation of kinetochore-microtubule attachments. Our recent work has implicated the passenger protein Bir1p (Survivin) and the inner kinetochore complex centromere binding factor 3 (CBF3) in the regulation of septin dynamics during anaphase. Here, we present data that is consistent with there being multiple passenger protein complexes. Our data show that Bir1p links together a large passenger complex containing Ndc10p, Sli15p (INCENP), and Ipl1p (Aurora B) and that the interaction between Bir1p and Sli15p is specifically involved in regulating septin dynamics during anaphase. Neither conditional alleles nor mutants of BIR1 that disrupt the interaction between Bir1p and Sli15p resulted in mono-attached kinetochores, suggesting that the Bir1p-Sli15p complex functions in anaphase and independently from Sli15p-Ipl1p complexes. We present a model for how discrete passenger complexes coordinate distinct aspects of mitosis.

Chromosome instability in colorectal tumor cells is associated with defects in microtubule plus-end attachments caused by a dominant mutation in APC.

The attachment of microtubule plus ends to kinetochores and to the cell cortex is essential for the fidelity of chromosome segregation. Here, we characterize the causes underlying the high rates of chromosome instability (CIN+) observed in colorectal tumor cells. We show that CIN+ tumor cells exhibit inefficient microtubule plus-end attachments during mitosis, accompanied by impairment of chromosome alignment in metaphase. The mitotic abnormalities associated with CIN+ tumor cells correlated with status of adenomatous polyposis coli (APC). Importantly, we have shown that a single truncating mutation in APC, similar to mutations found in tumor cells, acts dominantly to interfere with microtubule plus-end attachments and to cause a dramatic increase in mitotic abnormalities. We propose that APC functions to modulate microtubule plus-end attachments during mitosis, and that a single mutant APC allele predisposes cells to increased mitotic abnormalities, which may contribute to tumor progression.

The role of APC in mitosis and in chromosome instability.

The established role of APC in regulating microtubules and actin in polarized epithelia naturally raises the possibility that APC similarly influences the mitotic cytoskeleton. The recent accumulation of experimental evidence in mitotic cells supports this supposition. APC associates with mitotic spindle microtubules, most notably at the plus-ends of microtubules that interact with kinetochores. Genetic experiments implicate APC in the regulation of spindle microtubule dynamics, probably through its interaction with the microtubule plus-end binding protein, EB1. Moreover, functional data show that APC modulates kinetochore-microtubule attachments and is required for the spindle checkpoint to detect transiently misaligned chromosomes. Together this evidence points to a role for APC in maintaining mitotic fidelity. Such a role is particularly significant when considered in the context of the chromosome instability observed in colorectal tumors bearing mutations in APC. The prevalence of APC truncation mutants in colorectal tumors and the ability of these alleles to act dominantly to inhibit the mitotic spindle place chromosome instability at the earliest stage of colorectal cancer progression (i.e., prior to deregulation of beta-catenin). This may contribute to the autosomal dominant predisposition of patients with familial adenomatous polyposis to develop colon cancer. In this chapter, we will review the literature linking APC to regulation of mitotic fidelity and discuss the implications for dividing epithelial cells in the intestine.

APC and EB1 function together in mitosis to regulate spindle dynamics and chromosome alignment.

Recently, we have shown that a cancer causing truncation in adenomatous polyposis coli (APC) (APC(1-1450)) dominantly interferes with mitotic spindle function, suggesting APC regulates microtubule dynamics during mitosis. Here, we examine the possibility that APC mutants interfere with the function of EB1, a plus-end microtubule-binding protein that interacts with APC and is required for normal microtubule dynamics. We show that siRNA-mediated inhibition of APC, EB1, or APC and EB1 together give rise to similar defects in mitotic spindles and chromosome alignment without arresting cells in mitosis; in contrast inhibition of CLIP170 or LIS1 cause distinct spindle defects and mitotic arrest. We show that APC(1-1450) acts as a dominant negative by forming a hetero-oligomer with the full-length APC and preventing it from interacting with EB1, which is consistent with a functional relationship between APC and EB1. Live-imaging of mitotic cells expressing EB1-GFP demonstrates that APC(1-1450) compromises the dynamics of EB1-comets, increasing the frequency of EB1-GFP pausing. Together these data provide novel insight into how APC may regulate mitotic spindle function and how errors in chromosome segregation are tolerated in tumor cells.

The interaction between Sgt1p and Skp1p is regulated by HSP90 chaperones and is required for proper CBF3 assembly.

Sgt1p is a well-conserved protein proposed to be involved in a number of cellular processes. Genetic studies of budding yeast suggest a role for SGT1 in signal transduction, cell cycle advance, and chromosome segregation. Recent evidence has linked Sgt1p to HSP90 chaperones, although the precise relationship between these proteins is unclear. To further explore the role of Sgt1p in these processes, we have characterized the interactions among Sgt1p, the inner kinetochore complex CBF3, and HSP90 chaperones. We show that the amino terminus of Sgt1p interacts with CBF3 subunits Skp1p and Ctf13p. HSP90 interacts with Sgt1p and, in combination with the carboxy terminus of Sgt1p, regulates the interaction between Sgt1p and Skp1p in a nucleotide-dependent manner. While the Sgt1p-Skp1p interaction is required for CBF3 assembly, mutations that stabilize this interaction prevent the turnover of protein complexes important for CBF3 assembly. We propose that HSP90 and Sgt1p act together as a molecular switch, maintaining transient interactions required to balance protein complex assembly with turnover.

Sgt1p and Skp1p modulate the assembly and turnover of CBF3 complexes required for proper kinetochore function.

Kinetochores are composed of a large number of protein complexes that must be properly assembled on DNA to attach chromosomes to the mitotic spindle and to coordinate their segregation with the advance of the cell cycle. CBF3 is an inner kinetochore complex in the budding yeast Saccharomyces cerevisiae that nucleates the recruitment of all other kinetochore proteins to centromeric DNA. Skp1p and Sgt1p act through the core CBF3 subunit, Ctf13p, and are required for CBF3 to associate with centromeric DNA. To investigate the contribution of Skp1p and Sgt1p to CBF3 function, we have used a combination of in vitro binding assays and a unique protocol for synchronizing the assembly of kinetochores in cells. We have found that the interaction between Skp1p and Sgt1p is critical for the assembly of CBF3 complexes. CBF3 assembly is not restricted during the cell cycle and occurs in discrete steps; Skp1p and Sgt1p contribute to a final, rate-limiting step in assembly, the binding of the core CBF3 subunit Ctf13p to Ndc10p. The assembly of CBF3 is opposed by its turnover and disruption of this balance compromises kinetochore function without affecting kinetochore formation on centromeric DNA.

Adenomatous polyposis coli mutants dominantly activate Hsf1-dependent cell stress pathways through inhibition of microtubule dynamics.

Cancer cells up-regulate cell stress pathways, including the protein chaperone Hsp90. Increases in Hsp90 are believed "buffer" mutant protein activities necessary for cancer phenotypes. Activation of the cell stress pathway also alters the transcriptional landscape of cells in ways that are critical for cancer progression. However, it is unclear when and how the cell stress pathway is de-regulated during cancer progression. Here we report that mutations in adenomatous polyposis coli (APC) found in colorectal cancer activate cell stress pathways in mouse intestinal crypt cells, prior to loss of heterozygosity at APC or to the appearance of canonical intestinal cancer markers. Hsp90 levels are elevated in normal APC heterozygote crypt cells and further elevated in non-cancer cells adjacent to dysplasias, suggesting that the Hsp90 stress pathway marks the "cancer-field" effect. Expression of mutant APC in normal human epithelial cells is sufficient to activate a cell stress pathway via perturbations in microtubule dynamics. Inhibition of microtubule dynamics is sufficient to activate an Hsf1-dependent increase in gene transcription and protein levels. We suggest that the early activation of this Hsf1 dependent cell stress pathway by mono-allelic mutations in APC can affect cell programming in a way that contributes to cancer onset.

A prescription for 'stress'--the role of Hsp90 in genome stability and cellular adaptation.

Changes in cell homeostasis, or cell 'stress', are thought to tax the ability of the Hsp90 chaperone to facilitate an array of processes critical for genome maintenance. Here, we review the current understanding of how the Hsp90 chaperone machinery ensures the function of proteins important for DNA repair, recombination, and chromosome segregation. We discuss the idea that cell stress can overload Hsp90, resulting in genomic instability that may have important implications for stress adaptation and selection. The importance of Hsp90 in genome maintenance and its limited capacity to buffer the proteome may underlie the initiation or progression of diseases such as cancer.

Chromosome passenger complexes control anaphase duration and spindle elongation via a kinesin-5 brake.

During mitosis, chromosome passenger complexes (CPCs) exhibit a well-conserved association with the anaphase spindle and have been implicated in spindle stability. However, their precise effect on the spindle is not clear. In this paper, we show, in budding yeast, that a CPC consisting of CBF3, Bir1, and Sli15, but not Ipl1, is required for normal spindle elongation. CPC mutants slow spindle elongation through the action of the bipolar kinesins Cin8 and Kip1. The same CPC mutants that slow spindle elongation also result in the enrichment of Cin8 and Kip1 at the spindle midzone. Together, these findings argue that CPCs function to organize the spindle midzone and potentially switch motors between force generators and molecular brakes. We also find that slowing spindle elongation delays the mitotic exit network (MEN)-dependent release of Cdc14, thus delaying spindle breakdown until a minimal spindle size is reached. We propose that these CPC- and MEN-dependent mechanisms are important for coordinating chromosome segregation with spindle breakdown and mitotic exit.

Hsp90-Sgt1 and Skp1 target human Mis12 complexes to ensure efficient formation of kinetochore-microtubule binding sites.

The formation of functional kinetochores requires the accurate assembly of a large number of protein complexes. The Hsp90-Sgt1 chaperone complex is important for this process; however, its targets are not conserved and its exact contribution to kinetochore assembly is unclear. Here, we show that human Hsp90-Sgt1 interacts with the Mis12 complex, a so-called keystone complex required to assemble a large fraction of the kinetochore. Inhibition of Hsp90 or Sgt1 destabilizes the Mis12 complex and delays proper chromosome alignment due to inefficient formation of microtubule-binding sites. Interestingly, coinhibition of Sgt1 and the SCF subunit, Skp1, increases Mis12 complexes at kinetochores and restores timely chromosome alignment but forms less-robust microtubule-binding sites. We propose that a balance of Mis12 complex assembly and turnover is required for the efficient and accurate assembly of kinetochore-microtubule binding sites. These findings support a novel role for Hsp90-Sgt1 chaperones in ensuring the fidelity of multiprotein complex assembly.

Sgt1p is a unique co-chaperone that acts as a client adaptor to link Hsp90 to Skp1p.

Sgt1p is a conserved, essential protein required for kinetochore assembly in both yeast and animal cells. Sgt1p has homology to both TPR and p23 domains, sequences often found in proteins that interact with and regulate the molecular chaperone, Hsp90. The presence of these domains and the recent findings that Sgt1p interacts with Hsp90 has led to the speculation that Sgt1p and Hsp90 form a co-chaperone complex. To test this possibility, we have used purified recombinant proteins to characterize the in vitro interactions between yeast Sgt1p and Hsp82p (an Hsp90 homologue in yeast). We show that Sgt1p interacts directly with Hsp82p via its p23 homology region in a nucleotide-dependent manner. However, Sgt1p binding does not alter the enzymatic activity of Hsp82p, suggesting that it is distinct from other co-chaperones. We find that Sgt1p can form a ternary chaperone complex with Hsp82p and Sti1p, a well characterized Hsp90 co-chaperone. Sgt1p interacts with its binding partner Skp1p through its TPR domains and links Skp1p to the core Hsp82p-Sti1p co-chaperone complex. The multidomain nature of Sgt1p and its ability to bridge the interaction between Skp1p and Hsp82p argue that Sgt1p acts as a "client adaptor" recruiting specific clients to Hsp82p co-chaperone complexes.