Protein interactions regulate the systems-level behavior of cells; thus, deciphering the structure and dynamics of protein interaction networks in their cellular context is a central goal in biology. We have performed a genome-wide in vivo screen for protein-protein interactions in Saccharomyces cerevisiae by means of a protein-fragment complementation assay (PCA). We identified 2770 interactions among 1124 endogenously expressed proteins. Comparison with previous studies confirmed known interactions, but most were not known, revealing a previously unexplored subspace of the yeast protein interactome. The PCA detected structural and topological relationships between proteins, providing an 8-nanometer-resolution map of dynamically interacting complexes in vivo and extended networks that provide insights into fundamental cellular processes, including cell polarization and autophagy, pathways that are evolutionarily conserved and central to both development and human health.T he elucidation of protein-protein interaction networks (PINs, or interactomes) holds the promise of answering fundamental questions about how the biochemical machinery of cells organizes matter, information, and energy transformations to perform specific functions (1). An essential and rarely addressed question is whether protein complexes and PINs that are reconstructed or reconstituted in vitro or removed from the normal context in which they are expressed reflect their organization in living cells. For eukaryotes, the test bed for large-scale analysis of PINs is the yeast Saccharomyces cerevisiae, where several PIN analyses have been performed using yeast two-hybrid screens (Y2H) (2-4) or tandem affinity purification followed by massspectrometric analyses (TAP-MSs) (5-8). Each approach captures specific features of protein interactions; two-hybrid methods are best at measuring direct binary interactions between pairs of proteins, whereas affinity purification techniques best capture stable protein complexes. However, neither approach measures interactions between proteins in their natural cellular context, and are not easily amenable to studying protein complexes that are transiently associated or dynamic under different conditions, that do not survive in vitro purification, or that cannot be transported to the nucleus and form interactions in the absence of other

Spindle alignment is the process in which the two spindle poles are directed toward preselected and opposite cell ends. In budding yeast, the APC-related molecule Kar9 is required for proper alignment of the spindle with the mother-bud axis. We find that Kar9 localizes to the prospective daughter cell spindle pole. Kar9 is transferred from the pole to cytoplasmic microtubules, which are then guided in a myosin-dependent manner to the bud. Clb4/Cdc28 kinase phosphorylates Kar9 and accumulates on the pole destined to the mother cell. Mutations that block phosphorylation at Cdc28 consensus sites result in localization of Kar9 to both poles and target them both to the bud. Thus, Clb4/Cdc28 prevents Kar9 loading on the mother bound pole. In turn, asymmetric distribution of Kar9 ensures that only one pole orients toward the bud. Our results indicate that Cdk1-dependent spindle asymmetry ensures proper alignment of the mitotic spindle with the cell division axis.

Centrosomes organize the bipolar mitotic spindle, and centrosomal defects cause chromosome instability. Protein phosphorylation modulates centrosome function, and we provide a comprehensive map of phosphorylation on intact yeast centrosomes (18 proteins). Mass spectrometry was used to identify 297 phosphorylation sites on centrosomes from different cell cycle stages. We observed different modes of phosphoregulation via specific protein kinases, phosphorylation site clustering, and conserved phosphorylated residues. Mutating all eight cyclin-dependent kinase (Cdk)–directed sites within the core component, Spc42, resulted in lethality and reduced centrosomal assembly. Alternatively, mutation of one conserved Cdk site within γ-tubulin (Tub4-S360D) caused mitotic delay and aberrant anaphase spindle elongation. Our work establishes the extent and complexity of this prominent posttranslational modification in centrosome biology and provides specific examples of phosphorylation control in centrosome function.

Often the time derivative of a measured variable is of as much interest as the variable itself. For a growing population of biological cells, for example, the population's growth rate is typically more important than its size. Here we introduce a non-parametric method to infer first and second time derivatives as a function of time from time-series data. Our approach is based on Gaussian processes and applies to a wide range of data. In tests, the method is at least as accurate as others, but has several advantages: it estimates errors both in the inference and in any summary statistics, such as lag times, and allows interpolation with the corresponding error estimation. As illustrations, we infer growth rates of microbial cells, the rate of assembly of an amyloid fibril and both the speed and acceleration of two separating spindle pole bodies. Our algorithm should thus be broadly applicable.

Like many asymmetrically dividing cells, budding yeast segregates mitotic spindle poles nonrandomly between mother and daughter cells. During metaphase, the spindle positioning protein Kar9 accumulates asymmetrically, localizing specifically to astral microtubules emanating from the old spindle pole body (SPB) and driving its segregation to the bud. Here, we show that the SPB component Nud1/centriolin acts through the mitotic exit network (MEN) to specify asymmetric SPB inheritance. In the absence of MEN signaling, Kar9 asymmetry is unstable and its preference for the old SPB is disrupted. Consistent with this, phosphorylation of Kar9 by the MEN kinases Dbf2 and Dbf20 is not required to break Kar9 symmetry but is instead required to maintain stable association of Kar9 with the old SPB throughout metaphase. We propose that MEN signaling links Kar9 regulation to SPB identity through biasing and stabilizing the age-insensitive, cyclin-B-dependent mechanism of symmetry breaking.

Correlation of spindle architecture with dynamic behavior shows that pairs of antiparallel microtubules are sufficient to form a bipolar spindle, whereas interpolar microtubules maintain the speed of pole displacement during spindle assembly. The number of interpolar microtubules formed is controlled in part through γ-tubulin phosphorylation.

Microtubule plus-end-interacting proteins (؉TIPs) promote the dynamic interactions between the plus ends (؉ends) of astral microtubules and cortical actin that are required for preanaphase spindle positioning. Paradoxically, ؉TIPs such as the EB1 orthologue Bim1 and Kar9 also associate with spindle pole bodies (SPBs), the centrosome equivalent in budding yeast. Here, we show that deletion of four C-terminal residues of the budding yeast ␥-tubulin Tub4 (tub4-⌬dsyl) perturbs Bim1 and Kar9 localization to SPBs and Kar9-dependant spindle positioning. Surprisingly, we find Kar9 localizes to microtubule ؉ends in tub4-⌬dsyl cells, but these microtubules fail to position the spindle when targeted to the bud. Using cofluorescence and coaffinity purification, we show Kar9 complexes in tub4-⌬dsyl cells contain reduced levels of Bim1. Astral microtubule dynamics is suppressed in tub4-⌬dsyl cells, but it are restored by deletion of Kar9. Moreover, Myo2-and F-actin-dependent dwelling of Kar9 in the bud is observed in tub4-⌬dsyl cells, suggesting defective Kar9 complexes tether microtubule ؉ends to the cortex. Overproduction of Bim1, but not Kar9, restores Kar9-dependent spindle positioning in the tub4-⌬dsyl mutant, reduces cortical dwelling, and promotes Bim1-Kar9 interactions. We propose that SPBs, via the tail of Tub4, promote the assembly of functional ؉TIP complexes before their deployment to microtubule ؉ends.

Protein phosphatase 2A (PP2A) has been implicated in cell cycle progression and mitosis; however, the complexity of PP2A regulation via multiple B subunits makes its functional characterization a significant challenge. The human adenovirus protein E4orf4 has been found to induce both high Cdk1 activity and the accumulation of cells in G 2 /M in both mammalian and yeast cells, effects which are largely dependent on the B55/Cdc55 regulatory subunit of PP2A. Thus, E4orf4 represents a unique means by which the function of a specific form of PP2A can be delineated in vivo. In Saccharomyces cerevisiae, only two PP2A regulatory subunits exist, Cdc55 and Rts1. Here, we show that E4orf4-induced toxicity depends on a functional interaction with Cdc55. E4orf4 expression correlates with the inappropriate reduction of Pds1 and Scc1 in S-phase-arrested cells. The unscheduled loss of these proteins suggests the involvement of PP2A Cdc55 in the regulation of the Cdc20 form of the anaphase-promoting complex (APC). Contrastingly, activity of the Hct1 form of the APC is not induced by E4orf4, as demonstrated by the observed stability of its substrates. We propose that E4orf4, being a Cdc55-specific inhibitor of PP2A, demonstrates the role of PP2A Cdc55 in regulating APC Cdc20 activity.Protein phosphatase 2A (PP2A) represents a major class of serine/threonine phosphatases that is evolutionally conserved across eukaryotes and plays a regulatory role in numerous cellular processes, including signal transduction, cell morphology, and cell cycle control (12,15,26,43). The diversity of PP2A functions is due primarily to the existence of several PP2A holoenzyme variants. PP2A generally exists as a heterotrimer, composed of a catalytic C subunit, a structural A subunit, and a crucial regulatory B subunit that not only confers substrate specificity to the enzyme but also directs its cellular localization (13). In mammalian cells, there exist at least 18 known B regulatory subunit isoforms categorized into three classes (B, BЈ, and BЈЈ) and a related fourth class (sometimes referred to as BЈЈЈ). These classes share very little or no homology, despite their common abilities to bind overlapping sites within the A subunit (12). The situation is simpler in yeast, in which the catalytic C subunit is encoded redundantly by two duplicated genes, PPH21 and PPH22; a single A subunit is encoded by TPD3; and only two B-type subunits exist, encoded by CDC55 (corresponding to mammalian B/B55 subunits) and RTS1 (corresponding to the BЈ/B56 family) (51). Nevertheless, it is difficult to determine what particular form of PP2A actually functions in specific PP2A-regulated processes in all eukaryotic cells. PP2A has previously been implicated in the control of mitotic events in yeast and higher eukaryotes that are essential for cell survival (12, 15); however, the precise role of PP2A regarding mitotic progression and cell cycle regulation has yet to be fully defined, especially with regard to the specific form of PP2A that is involved.The E4orf4 (early regi...