Brian Haarer

Immunofluorescence methods for yeast

Publisher Summary This chapter provides protocols for the application of immunofluorescence procedures to yeast. It should perhaps be stressed that immunofluorescence and other light microscopic techniques play a role that is separate from but equal to the role of electron microscopy. Although in some situations the greater resolving power of the electron microscope is clearly essential to obtain the needed structural information, in other situations the necessary information can be obtained more easily, more reliably, or both, by light microscopy. The potential advantages of light microscopic approaches derive from various facts: (1) they can be applied to lightly processed or (in some cases) living cells, (2) Much larger numbers of cells can be examined than by electron microscopy (note especially the great labor involved in visualizing the structure of whole cells by serial-section methods), and (3) Some structures (for example, the cytoplasmic microtubules) have simply been easier to see by light microscopy than by electron microscopy.

Fluorescence microscopy methods for yeast.

Publisher Summary This chapter reviews and provides detailed protocols for the application of immunofluorescence and other fluorescence-microscopic procedures to yeast. These procedures play a role that is separate from but equal to the role of electron microscopy. Although in some situations the greater resolving power of the electron microscope is clearly essential to obtain the needed structural information, in other situations the necessary information can be obtained more easily, more reliably, or both, by light (including fluorescence) microscopy. The potential advantages of light-microscopic approaches derive from the facts (1) that they can be applied to lightly processed or living cells, (2) that much larger numbers of cells can be examined than by electron microscopy (note especially the great labor involved in visualizing the structure of whole cells by serial-section methods), and (3) that some structures have simply been easier to see by light microscopy than by electron microscopy. The methods are also effective with other yeasts such as Schizosaccharomyces pombe and Candida albicans .

Immunofluorescence localization of the Saccharomyces cerevisiae CDC12 gene product to the vicinity of the 10-nm filaments in the mother-bud neck.

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud (B. Byers and L. Goetsch, J. Cell Biol. 69:717-721, 1976). Mutants defective in any of four genes (CDC3, CDC10, CDC11, and CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and cell-wall deposition and an inability to complete cytokinesis. We fused the cloned CDC12 gene to the Escherichia coli lacZ and trpE genes and used the resulting fusion proteins to raise polyclonal antibodies specific for the CDC12 gene product. In immunofluorescence experiments with affinity-purified antibodies, the neck region of wild-type and mutant cells stained in patterns consistent with the hypothesis that the CDC12 gene product is a constituent of the ring of 10-nm filaments. Without careful affinity purification of the CDC12-specific antibodies, these staining patterns were completely obscured by the staining of residual cell wall components in the neck by antibodies present even in the preimmune sera of all rabbits tested.

Evidence for a functional link between profilin and CAP in the yeast S. cerevisiae

CAP is a component of the S. cerevisiae adenylyl cyclase complex. The N-terminal domain is required for cellular RAS responsiveness. Loss of the C-terminal domain is associated with morphological and nutritional defects. Here we report that cap- cells bud randomly and are defective in actin distribution. The morphological and nutritional defects associated with loss of the CAP C-terminal domain are suppressed by over-expression of PFY, the gene encoding profilin, an actin- and polyphosphoinositide-binding protein. The phenotype of cells lacking PFY resembles that of cells lacking the CAP C-terminal domain. Study of mutated yeast profilins and profilins from Acanthamoeba suggests that the ability of profilin to suppress cap- cells is dependent upon a property other than, or in addition to, its ability to bind actin. This property may be its ability to bind polyphosphoinositides. We propose that CAP and profilin provide a link between growth signals and remodeling of the cellular cytoskeleton.

Mutational analysis of yeast profilin.

We have mutated two regions within the yeast profilin gene in an effort to functionally dissect the roles of actin and phosphatidylinositol 4,5-bisphosphate (PIP2) binding in profilin function. A series of truncations was carried out at the C terminus of profilin, a region that has been implicated in actin binding. Removal of the last three amino acids nearly eliminated the ability of profilin to bind polyproline in vitro but had no dramatic in vivo effects. Thus, the extreme C terminus is implicated in polyproline binding, but the physiological relevance of this interaction is called into question. More extensive truncation, of up to eight amino acids, had in vivo effects of increasing severity and resulted in changes in conformation and expression level of the mutant profilins. However, the ability of these mutants to bind actin in vitro was not eliminated, suggesting that this region cannot be solely responsible for actin binding. We also mutagenized a region of profilin that we hypothesized might be involved in PIP2 binding. Alteration of basic amino acids in this region produced mutant profilins that functioned well in vivo. Many of these mutants, however, were unable to suppress the loss of adenylate cyclase-associated protein (Cap/Srv2p [A. Vojtek, B. Haarer, J. Field, J. Gerst, T. D. Pollard, S. S. Brown, and M. Wigler, Cell 66:497-505, 1991]), indicating that a defect could be demonstrated in vivo. In vitro assays demonstrated that the inability to suppress loss of Cap/Srv2p correlated with a defect in the interaction with actin, independently of whether PIP2 binding was reduced. Since our earlier studies of Acanthamoeba profilins suggested the importance of PIP2 binding for suppression, we conclude that both activities are implicated and that an interplay between PIP2 binding and actin binding may be important for profilin function.

Diverse protective roles of the actin cytoskeleton during oxidative stress

Actin oxidation is known to result in changes in cytoskeleton organization and dynamics. Actin oxidation is clinically relevant since it occurs in the erythrocytes of sickle cell patients and may be the direct cause of the lack of morphological plasticity observed in irreversibly sickled red blood cells (ISCs). During episodes of crisis, ISCs accumulate C284‐C373 intramolecularly disulfide bonded actin, which reduces actin filament dynamics. Actin cysteines 284 and 373 (285 and 374 in yeast) are conserved, suggesting that they play an important functional role. We have been investigating the physiological roles of these cysteines using the model eukaryote Saccharomyces cerevisiae in response to oxidative stress load. During acute oxidative stress, all of the F‐actin in wild‐type cells collapses into a few puncta that we call oxidation‐induced actin bodies (OABs). In contrast, during acute oxidative stress the actin cytoskeleton in Cys‐to‐Ala actin mutants remains polarized longer, OABs are slower to form, and the cells recover more slowly than wild‐type cells, suggesting that the OABs play a protective role. Live cell imaging revealed that OABs are large, immobile structures that contain actin‐binding proteins and that can form by the fusion of actin cortical patches. We propose that actins C285 and C374 may help to protect the cell from oxidative stress arising from normal oxidative metabolism and contribute to the cells general adaptive response to oxidative stress.

F1-ATPase of Escherichia coli THE ϵ-INHIBITED STATE FORMS AFTER ATP HYDROLYSIS, IS DISTINCT FROM THE ADP-INHIBITED STATE, AND RESPONDS DYNAMICALLY TO CATALYTIC SITE LIGANDS

Background: Bacterial ATP synthases are autoinhibited by the subunit ϵ C-terminal domain. Results: Nucleotide hydrolysis is required to form the ϵ-inhibited state, which also responds dynamically to different ligand conditions. Conclusion: ϵ inhibition initiates at the catalytic dwell angle, but reversible rotation over ∼40° is probably involved in nucleotide effects on the inhibitory state of ϵ. Significance: ϵ inhibition may provide a new target for antimicrobial discovery. F1-ATPase is the catalytic complex of rotary nanomotor ATP synthases. Bacterial ATP synthases can be autoinhibited by the C-terminal domain of subunit ϵ, which partially inserts into the enzymes central rotor cavity to block functional subunit rotation. Using a kinetic, optical assay of F1·ϵ binding and dissociation, we show that formation of the extended, inhibitory conformation of ϵ (ϵX) initiates after ATP hydrolysis at the catalytic dwell step. Prehydrolysis conditions prevent formation of the ϵX state, and post-hydrolysis conditions stabilize it. We also show that ϵ inhibition and ADP inhibition are distinct, competing processes that can follow the catalytic dwell. We show that the N-terminal domain of ϵ is responsible for initial binding to F1 and provides most of the binding energy. Without the C-terminal domain, partial inhibition by the ϵ N-terminal domain is due to enhanced ADP inhibition. The rapid effects of catalytic site ligands on conformational changes of F1-bound ϵ suggest dynamic conformational and rotational mobility in F1 that is paused near the catalytic dwell position.

Break-seq reveals hydroxyurea-induced chromosome fragility as a result of unscheduled conflict between DNA replication and transcription

We have previously demonstrated that in Saccharomyces cerevisiae replication, checkpoint inactivation via a mec1 mutation leads to chromosome breakage at replication forks initiated from virtually all origins after transient exposure to hydroxyurea (HU), an inhibitor of ribonucleotide reductase. Here we sought to determine whether all replication forks containing single-stranded DNA gaps have equal probability of producing double-strand breaks (DSBs) when cells attempt to recover from HU exposure. We devised a new methodology, Break-seq, that combines our previously described DSB labeling with next generation sequencing to map chromosome breaks with improved sensitivity and resolution. We show that DSBs preferentially occur at genes transcriptionally induced by HU. Notably, different subsets of the HU-induced genes produced DSBs in MEC1 and mec1 cells as replication forks traversed a greater distance in MEC1 cells than in mec1 cells during recovery from HU. Specifically, while MEC1 cells exhibited chromosome breakage at stress-response transcription factors, mec1 cells predominantly suffered chromosome breakage at transporter genes, many of which are the substrates of those transcription factors. We propose that HU-induced chromosome fragility arises at higher frequency near HU-induced genes as a result of destabilized replication forks encountering transcription factor binding and/or the act of transcription. We further propose that replication inhibitors can induce unscheduled encounters between replication and transcription and give rise to distinct patterns of chromosome fragile sites.

Novel interactions between actin and the proteasome revealed by complex haploinsufficiency.

Saccharomyces cerevisiae has been a powerful model for uncovering the landscape of binary gene interactions through whole-genome screening. Complex heterozygous interactions are potentially important to human genetic disease as loss-of-function alleles are common in human genomes. We have been using complex haploinsufficiency (CHI) screening with the actin gene to identify genes related to actin function and as a model to determine the prevalence of CHI interactions in eukaryotic genomes. Previous CHI screening between actin and null alleles for non-essential genes uncovered ∼240 deleterious CHI interactions. In this report, we have extended CHI screening to null alleles for essential genes by mating a query strain to sporulations of heterozygous knock-out strains. Using an act1Δ query, knock-outs of 60 essential genes were found to be CHI with actin. Enriched in this collection were functional categories found in the previous screen against non-essential genes, including genes involved in cytoskeleton function and chaperone complexes that fold actin and tubulin. Novel to this screen was the identification of genes for components of the TFIID transcription complex and for the proteasome. We investigated a potential role for the proteasome in regulating the actin cytoskeleton and found that the proteasome physically associates with actin filaments in vitro and that some conditional mutations in proteasome genes have gross defects in actin organization. Whole-genome screening with actin as a query has confirmed that CHI interactions are important phenotypic drivers. Furthermore, CHI screening is another genetic tool to uncover novel functional connections. Here we report a previously unappreciated role for the proteasome in affecting actin organization and function.

Stable Preanaphase Spindle Positioning Requires Bud6p and an Apparent Interaction between the Spindle Pole Bodies and the Neck

ABSTRACT Faithful partitioning of genetic material during cell division requires accurate spatial and temporal positioning of nuclei within dividing cells. In Saccharomyces cerevisiae, nuclear positioning is regulated by an elegant interplay between components of the actin and microtubule cytoskeletons. Regulators of this process include Bud6p (also referred to as the actin-interacting protein Aip3p) and Kar9p, which function to promote contacts between cytoplasmic microtubule ends and actin-delimited cortical attachment points. Here, we present the previously undetected association of Bud6p with the cytoplasmic face of yeast spindle pole bodies, the functional equivalent of metazoan centrosomes. Cells lacking Bud6p show exaggerated movements of the nucleus between mother and daughter cells and display reduced amounts of time a given spindle pole body spends in close association with the neck region of budding cells. Furthermore, overexpression of BUD6 greatly enhances interactions between the spindle pole body and mother-bud neck in a spindle alignment-defective dynactin mutant. These results suggest that association of either spindle pole body with neck components, rather than simply entry of a spindle pole body into the daughter cell, provides a positive signal for the progression of mitosis. We propose that Bud6p, through its localization at both spindle pole bodies and at the mother-bud neck, supports this positive signal and provides a regulatory mechanism to prevent excessive oscillations of preanaphase nuclei, thus reducing the likelihood of mitotic delays and nuclear missegregation.