For high-fidelity chromosome segregation, kinetochores must be properly captured by spindle microtubules, but the mechanisms underlying initial kinetochore capture have remained elusive. Here we visualized individual kinetochore-microtubule interactions in Saccharomyces cerevisiae by regulating the activity of a centromere. Kinetochores are captured by the side of microtubules extending from spindle poles, and are subsequently transported poleward along them. The microtubule extension from spindle poles requires microtubule plus-end-tracking proteins and the Ran GDP/GTP exchange factor. Distinct kinetochore components are used for kinetochore capture by microtubules and for ensuring subsequent sister kinetochore bi-orientation on the spindle. Kar3, a kinesin-14 family member, is one of the regulators that promote transport of captured kinetochores along microtubules. During such transport, kinetochores ensure that they do not slide off their associated microtubules by facilitating the conversion of microtubule dynamics from shrinkage to growth at the plus ends. This conversion is promoted by the transport of Stu2 from the captured kinetochores to the plus ends of microtubules.

Any which way but loose back-to-back arrangement of kinetochores is not needed for correct alignment on the mitotic spindle, as shown by Hilary Dewar, Tomoyuki Tanaka (University of Dundee, UK), and colleagues. Sister chromatids are glued together by cohesin such that their kinetochores face opposing poles. This arrangement might thus prevent both chromatids from attaching to spindle microtubules from the same pole. But Tanaka's group shows that even when geometry fails, a tension-sensitive mechanism fixes any mistakes. The authors messed with the usual geometry in two ways. First, they confronted yeast cells with a nonreplicating dicentric minichromosome. Its two kinetochores are not held in any fixed relative orientation , yet were efficiently attached to opposing poles. Second, normal chromosomes in cohesin mutants (which have attachment defects), were roughly linked by inhibiting topoisomerase II. This restored bipolar attachments. Thus, any connection that can produce tension is enough to ensure biorientation. The tension-sensitive correction depends on the Ipl1 kinase, whose mammalian homo-logue, Aurora B, prevents monopolar attachments. This suggests that Ipl1 activity knocks off attachments until tension somehow stops it-perhaps by turning off the kinase, turning on a counteracting phosphatase, or pulling substrates away from the kinase. A dynamite intercellular highway ong, delicate tubules are a new trade route for the intercellular exchange of goods, as shown by Amin Rustom, Hans-Hermann Gerdes (University

SLA1 was identified previously in budding yeast in a genetic screen for mutations that caused a requirement for the actin-binding protein Abp1p and was shown to be required for normal cortical actin patch structure and organization. Here, we show that Sla1p, like Abp1p, localizes to cortical actin patches. Furthermore, Sla1p is required for the correct localization of Sla2p, an actin-binding protein with homology to talin implicated in endocytosis, and the Rho1p-GTPase, which is associated with the cell wall biosynthesis enzyme ␤-1,3-glucan synthase. Mislocalization of Rho1p in sla1 null cells is consistent with our observation that these cells possess aberrantly thick cell walls.Expression of mutant forms of Sla1p in which specific domains were deleted showed that the phenotypes associated with the full deletion are functionally separable. In particular, a region of Sla1p encompassing the third SH3 domain is important for growth at high temperatures, for the organization of cortical actin patches, and for nucleated actin assembly in a permeabilized yeast cell assay. The apparent redundancy between Sla1p and Abp1p resides in the C-terminal repeat region of Sla1p. A homologue of SLA1 was identified in Schizosaccharomyces pombe. Despite relatively low overall sequence homology, this gene was able to rescue the temperature sensitivity associated with a deletion of SLA1 in Saccharomyces cerevisiae.

The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified and characterized. Here we demonstrate the importance of two additional cortical components, Ysc84p and Lsb5p, which together are essential for the organization of the actin cytoskeleton and for fluid phase endocytosis. Both Ysc84p and Lsb5p were identified through two-hybrid screens with different domains of the adaptor protein Sla1p. Ysc84p colocalizes with cortical actin and requires the presence of an intact actin cytoskeleton for its cortical localization. Ycl034w/Lsb5p localizes to the cell cortex but does not colocalize with actin. The Lsb5 protein contains putative VHS and GAT domains as well as an NPF motif, which are all domains characteristic of proteins involved in membrane trafficking. Deletion of either gene alone does not confer any dramatic phenotype on cells. However, deletion of both genes is lethal at elevated temperatures. Furthermore, at all temperatures this double mutant has depolarized actin and an almost undetectable level of fluid phase endocytosis. Our data demonstrate that Ysc84p and Lsb5p are important components of complexes involved in overlapping pathways coupling endocytosis with the actin cytoskeleton in yeast.

The importance of a dynamic actin cytoskeleton for facilitating endocytosis has been recognised for many years in budding yeast and is increasingly recognised in mammalian cells. However, the mechanism for actin recruitment and the role it plays in endocytosis is unclear. Here we show the importance of two yeast proteins in this process. We demonstrate that Sla1p and Sla2p interact in vitro and in vivo and that this interaction is mediated by the central domain of Sla2p, which includes its coiled-coil region, and by a domain of Sla1p between residues 118 and 361. Overexpression of the interacting fragment of Sla1p causes reduced fluid-phase endocytosis and,interestingly, defects in subsequent trafficking to vacuoles. We show that Sla2p is required for the polarised localisation of Sla1p in cells but not for its cortical localisation or for its overlapping localisation with actin. Generation of an Δsla1Δsla2 double mutant demonstrates that Sla2p is likely to act upstream of Sla1p in endocytosis,whereas sensitivity to latrunculin-A suggests that the proteins have opposite effects on actin dynamics. We propose that Sla2p recruits Sla1p to endocytic sites. Sla1p and its associated protein Pan1p then regulate actin assembly through interactions with Arp2/3 and Arp2/3-activating proteins Abp1p and Las17/Bee1p.

No abstract

Regulation of the Saccharomyces cerevisiae HO promoter has been shown to require the recruitment of chromatin-modifying and -remodeling enzymes. Despite this, relatively little is known about what changes to chromatin structure occur during the course of regulation at HO. Here, we used indirect end labeling in synchronized cultures to show that the chromatin structure is disrupted in a region that spans bp ؊600 to ؊1800 relative to the transcriptional start site. Across this region, there is a loss of canonical nucleosomes and a reduction in histone DNA cross-linking, as monitored by chromatin immunoprecipitation. The ATPase Snf2 is required for these alterations, but the histone acetyltransferase Gcn5 is not. This suggests that the SWI/SNF complex is directly involved in nucleosome removal at HO. We also present evidence indicating that the histone chaperone Asf1 assists in this. These observations suggest that SWI/SNF-related complexes in concert with histone chaperones act to remove histone octamers from DNA during the course of gene regulation.The Saccharomyces cerevisiae HO gene encodes an endonuclease that generates a double-stranded break at the matingtype locus that allows the yeast to switch between a and ␣ mating types (53). HO is transcribed transiently during the late G 1 phase only in mother cells, but not in daughter cells (40). As such, it has been considered a paradigm for a developmentally and cell cycle-regulated gene in a relatively simple eukaryote.Considerable progress has been made in defining the order of events that result in HO transcription. They are triggered by the dephosphorylation of the transcription factor Swi5 during late anaphase, which allows it to enter the nucleus (56). The HO promoter contains two binding sites for Swi5 1,300 and 1,800 bp upstream of the transcriptional start site within a region referred to as URS1. Binding of Swi5 and Pho2 to URS1 (8,38) results in the recruitment of the SWI/SNF complex in mother cells (14). In daughter cells, the presence of the repressor Ash1 prevents SWI/SNF recruitment and subsequent stages in the activation of HO (14; reviewed in reference 12). The SAGA histone acetyltransferase complex is also recruited to URS1 and is required for HO transcription (14, 34). Chromatin immunoprecipitation (ChIP) studies suggest that both the SAGA complex itself and histone acetylation spread from URS1 to a second regulatory region,URS2, where the transcription factor SBF is recruited to a series of binding sites in the region from 100 to 700 bp upstream of the transcription start site (14,34). SBF is in turn required for recruitment of the mediator complex and, subsequently, following reactivation of Cdk1, RNA polymerase II (13).The involvement of chromatin-modifying and -remodeling enzymes in the activation of HO raises the possibility that the chromatin structure may be altered. In vitro, SWI/SNF-related complexes have been found to be capable of generating a range of different transitions in chromatin structure. These can involve nucleosome sliding,...