The cortical localization of the microtubule orientation protein, Kar9p, is dependent upon actin and proteins required for polarization

In the yeast Saccharomyces cerevisiae, positioning of the mitotic spindle requires both the cytoplasmic microtubules and actin. Kar9p is a novel cortical protein that is required for the correct position of the mitotic spindle and the orientation of the cytoplasmic microtubules. Green fluorescent protein (GFP)- Kar9p localizes to a single spot at the tip of the growing bud and the mating projection. However, the cortical localization of Kar9p does not require microtubules (Miller, R.K., and M.D. Rose. 1998. J. Cell Biol. 140: 377), suggesting that Kar9p interacts with other proteins at the cortex. To investigate Kar9p's cortical interactions, we treated cells with the actin-depolymerizing drug, latrunculin-A. In both shmoos and mitotic cells, Kar9p's cortical localization was completely dependent on polymerized actin. Kar9p localization was also altered by mutations in four genes, spa2Delta, pea2Delta, bud6Delta, and bni1Delta, required for normal polarization and actin cytoskeleton functions and, of these, bni1Delta affected Kar9p localization most severely. Like kar9Delta, bni1Delta mutants exhibited nuclear positioning defects during mitosis and in shmoos. Furthermore, like kar9Delta, the bni1Delta mutant exhibited misoriented cytoplasmic microtubules in shmoos. Genetic analysis placed BNI1 in the KAR9 pathway for nuclear migration. However, analysis of kar9Delta bni1Delta double mutants suggested that Kar9p retained some function in bni1Delta mitotic cells. Unlike the polarization mutants, kar9Delta shmoos had a normal morphology and diploids budded in the correct bipolar pattern. Furthermore, Bni1p localized normally in kar9Delta. We conclude that Kar9p's function is specific for cytoplasmic microtubule orientation and that Kar9p's role in nuclear positioning is to coordinate the interactions between the actin and microtubule networks.     

Coordinated spindle assembly and orientation requires Clb5p-dependent kinase in budding yeast

The orientation of the mitotic spindle along a polarity axis is critical in asymmetric cell divisions. In the budding yeast, Saccharomyces cerevisiae, loss of the S-phase B-type cyclin Clb5p under conditions of limited cyclin-dependent kinase activity (cdc28-4 clb5Delta cells) causes a spindle positioning defect that results in an undivided nucleus entering the bud. Based on time-lapse digital imaging microscopy of microtubules labeled with green fluorescent protein fusions to either tubulin or dynein, we observed that the asymmetric behavior of the spindle pole bodies during spindle assembly was lost in the cdc28-4 clb5Delta cells. As soon as a spindle formed, both poles were equally likely to interact with the bud cell cortex. Persistent dynamic interactions with the bud ultimately led to spindle translocation across the bud neck. Thus, the mutant failed to assign one spindle pole body the task of organizing astral microtubules towards the mother cell. Our data suggest that Clb5p-associated kinase is required to confer mother-bound behavior to one pole in order to establish correct spindle polarity. In contrast, B-type cyclins, Clb3p and Clb4p, though partially redundant with Clb5p for an early role in spindle morphogenesis, preferentially promote spindle assembly.     

Pheromone-induced polarization is dependent on the Fus3p MAPK acting through the formin Bni1p

During mating, budding yeast cells reorient growth toward the highest concentration of pheromone. Bni1p, a formin homologue, is required for this polarized growth by facilitating cortical actin cable assembly. Fus3p, a pheromone-activated MAP kinase, is required for pheromone signaling and cell fusion. We show that Fus3p phosphorylates Bni1p in vitro, and phosphorylation of Bni1p in vivo during the pheromone response is dependent on Fus3p. fus3 mutants exhibited multiple phenotypes similar to bni1 mutants, including defects in actin and cell polarization, as well as Kar9p and cytoplasmic microtubule localization. Disruption of the interaction between Fus3p and the receptor-associated Galpha subunit caused similar mutant phenotypes. After pheromone treatment, Bni1p-GFP and Spa2p failed to localize to the cortex of fus3 mutants, and cell wall growth became completely unpolarized. Bni1p overexpression suppressed the actin assembly, cell polarization, and cell fusion defects. These data suggest a model wherein activated Fus3p is recruited back to the cortex, where it activates Bni1p to promote polarization and cell fusion.     

Dynamic localization and function of Bni1p at the sites of directed growth in Saccharomyces cerevisiae

Formin homology (FH) proteins are implicated in cell polarization and cytokinesis through actin organization. There are two FH proteins in the yeast Saccharomyces cerevisiae, Bni1p and Bnr1p. Bni1p physically interacts with Rho family small G proteins (Rho1p and Cdc42p), actin, two actin-binding proteins (profilin and Bud6p), and a polarity protein (Spa2p). Here we analyzed the in vivo localization of Bni1p by using a time-lapse imaging system and investigated the regulatory mechanisms of Bni1p localization and function in relation to these interacting proteins. Bni1p fused with green fluorescent protein localized to the sites of cell growth throughout the cell cycle. In a small-budded cell, Bni1p moved along the bud cortex. This dynamic localization of Bni1p coincided with the apparent site of bud growth. A bni1-disrupted cell showed a defect in directed growth to the pre-bud site and to the bud tip (apical growth), causing its abnormally spherical cell shape and thick bud neck. Bni1p localization at the bud tips was absolutely dependent on Cdc42p, largely dependent on Spa2p and actin filaments, and partly dependent on Bud6p, but scarcely dependent on polarized cortical actin patches or Rho1p. These results indicate that Bni1p regulates polarized growth within the bud through its unique and dynamic pattern of localization, dependent on multiple factors, including Cdc42p, Spa2p, Bud6p, and the actin cytoskeleton.     

Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton in Saccharomyces cerevisiae

The RHO1 gene encodes a yeast homolog of the mammalian RhoA protein. Rho1p is localized to the growth sites and is required for bud formation. We have recently shown that Bni1p is one of the potential downstream target molecules of Rho1p. The BNI1 gene is implicated in cytokinesis and the establishment of cell polarity in Saccharomyces cerevisiae but is not essential for cell viability. In this study, we screened for mutations that were synthetically lethal in combination with a bni1 mutation and isolated two genes. They were the previously identified PAC1 and NIP100 genes, both of which are implicated in nuclear migration in S. cerevisiae. Pac1p is a homolog of human LIS1, which is required for brain development, whereas Nip100p is a homolog of rat p150(Glued), a component of the dynein-activated dynactin complex. Disruption of BNI1 in either the pac1 or nip100 mutant resulted in an enhanced defect in nuclear migration, leading to the formation of binucleate mother cells. The arp1 bni1 mutant showed a synthetic lethal phenotype while the cin8 bni1 mutant did not, suggesting that Bni1p functions in a kinesin pathway but not in the dynein pathway. Cells of the pac1 bni1 and nip100 bni1 mutants exhibited a random distribution of cortical actin patches. Cells of the pac1 act1-4 mutant showed temperature-sensitive growth and a nuclear migration defect. These results indicate that Bni1p regulates microtubule-dependent nuclear migration through the actin cytoskeleton. Bni1p lacking the Rho-binding region did not suppress the pac1 bni1 growth defect, suggesting a requirement for the Rho1p-Bni1p interaction in microtubule function.     

Microtubule interactions with the cell cortex causing nuclear movements in Saccharomyces cerevisiae

During mitosis in budding yeast the nucleus first moves to the mother-bud neck and then into the neck. Both movements depend on interactions of cytoplasmic microtubules with the cortex. We investigated the mechanism of these movements in living cells using video analysis of GFP-labeled microtubules in wild-type cells and in EB1 and Arp1 mutants, which are defective in the first and second steps, respectively. We found that nuclear movement to the neck is largely mediated by the capture of microtubule ends at one cortical region at the incipient bud site or bud tip, followed by microtubule depolymerization. Efficient microtubule interactions with the capture site require that microtubules be sufficiently long and dynamic to probe the cortex. In contrast, spindle movement into the neck is mediated by microtubule sliding along the bud cortex, which requires dynein and dynactin. Free microtubules can also slide along the cortex of both bud and mother. Capture/shrinkage of microtubule ends also contributes to nuclear movement into the neck and can serve as a backup mechanism to move the nucleus into the neck when microtubule sliding is impaired. Conversely, microtubule sliding can move the nucleus into the neck even when capture/shrinkage is impaired.     

The cortical protein Num1p is essential for dynein-dependent interactions of microtubules with the cortex

In budding yeast, the mitotic spindle moves into the neck between the mother and bud via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. How dynein and microtubules interact with the cortex is unknown. We found that cells lacking Num1p failed to exhibit dynein-dependent microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Num1p localized to the bud cortex, and that localization was independent of microtubules, dynein, or dynactin. These data are consistent with Num1p being an essential element of the cortical attachment mechanism for dynein-dependent sliding of microtubules in the bud.     

Dynamic positioning of mitotic spindles in yeast: role of microtubule motors and cortical determinants

In the budding yeast Saccharomyces cerevisiae, movement of the mitotic spindle to a predetermined cleavage plane at the bud neck is essential for partitioning chromosomes into the mother and daughter cells. Astral microtubule dynamics are critical to the mechanism that ensures nuclear migration to the bud neck. The nucleus moves in the opposite direction of astral microtubule growth in the mother cell, apparently being "pushed" by microtubule contacts at the cortex. In contrast, microtubules growing toward the neck and within the bud promote nuclear movement in the same direction of microtubule growth, thus "pulling" the nucleus toward the bud neck. Failure of "pulling" is evident in cells lacking Bud6p, Bni1p, Kar9p, or the kinesin homolog, Kip3p. As a consequence, there is a loss of asymmetry in spindle pole body segregation into the bud. The cytoplasmic motor protein, dynein, is not required for nuclear movement to the neck; rather, it has been postulated to contribute to spindle elongation through the neck. In the absence of KAR9, dynein-dependent spindle oscillations are evident before anaphase onset, as are postanaphase dynein-dependent pulling forces that exceed the velocity of wild-type spindle elongation threefold. In addition, dynein-mediated forces on astral microtubules are sufficient to segregate a 2N chromosome set through the neck in the absence of spindle elongation, but cytoplasmic kinesins are not. These observations support a model in which spindle polarity determinants (BUD6, BNI1, KAR9) and cytoplasmic kinesin (KIP3) provide directional cues for spindle orientation to the bud while restraining the spindle to the neck. Cytoplasmic dynein is attenuated by these spindle polarity determinants and kinesin until anaphase onset, when dynein directs spindle elongation to distal points in the mother and bud.     

Rho1p-Bni1p-Spa2p Interactions: Implication in Localization of Bni1p at the Bud Site and Regulation of the Actin Cytoskeleton in Saccharomyces cerevisiae

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213–1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826–987). Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.     

Phosphorylation-dependent septin interaction of Bni5 is important for cytokinesis

In budding yeast, septin plays as a scaffold to recruits protein components and regulates crucial cellular events including bud site selection, bud morphogenesis, Cdc28 activation pathway, and cytokinesis. Phosphorylation of Bni5 isolated as a suppressor for septin defect is essential to Swe1-dependent regulation of bud morphogenesis and mitotic entry. The mechanism by which Bni5 regulates normal septin function is not completely understood. Here, we provide evidence that Bni5 phosphorylation is important for interaction with septin component Cdc11 and for timely delocalization from septin filament at late mitosis. Phosphorylation-deficient bni5-4A was synthetically lethal with hof1Delta. bni5-4A cells had defective structure of septin ring and connected cell morphology, indicative of defects in cytokinesis. Two-hybrid analysis revealed that bni5-4A has a defect in direct interaction with Cdc11 and Cdc12. GFP-tagged bni5-4A was normally localized at mother-bud neck of budded cells before middle of mitosis. In contrast, at large-budded telophase cells, bni5-4A-GFP was defective in localization and disappeared from the neck approximately 2 min earlier than that of wild type, as evidenced by time-lapse analysis. Therefore, earlier delocalization of bni5-4A from septin filament is consistent with phosphorylation-dependent interaction with the septin component. These results suggest that timely delocalization of Bni5 by phosphorylation is important for septin function and regulation of cytokinesis.     

Bni1p and Bnr1p: downstream targets of the Rho family small G-proteins which interact with profilin and regulate actin cytoskeleton in Saccharomyces cerevisiae.

The RHO1 gene encodes a homologue of mammalian RhoA small G-protein in the yeast Saccharomyces cerevisiae. Rho1p is required for bud formation and is localized at a bud tip or a cytokinesis site. We have recently shown that Bni1p is a potential target of Rho1p. Bni1p shares the FH1 and FH2 domains with proteins involved in cytokinesis or establishment of cell polarity. In S. cerevisiae, there is an open reading frame (YIL159W) which encodes another protein having the FH1 and FH2 domains and we have named this gene BNR1 (BNI1 Related). Bnr1p interacts with another Rho family member, Rho4p, but not with Rho1p. Disruption of BNI1 or BNR1 does not show any deleterious effect on cell growth, but the bni1 bnr1 mutant shows a severe temperature-sensitive growth phenotype. Cells of the bni1 bnr1 mutant arrested at the restrictive temperature are deficient in bud emergence, exhibit a random distribution of cortical actin patches and often become multinucleate. These phenotypes are similar to those of the mutant of PFY1, which encodes profilin, an actin-binding protein. Moreover, yeast two-hybrid and biochemical studies demonstrate that Bni1p and Bnr1p interact directly with profilin at the FH1 domains. These results indicate that Bni1p and Bnr1p are potential targets of the Rho family members, interact with profilin and regulate the reorganization of actin cytoskeleton.     

Differential contribution of Bud6p and Kar9p to microtubule capture and spindle orientation in S. cerevisiae

In Saccharomyces cerevisiae, spindle orientation is controlled by a temporal and spatial program of microtubule (MT)-cortex interactions. This program requires Bud6p/Aip3p to direct the old pole to the bud and confine the new pole to the mother cell. Bud6p function has been linked to Kar9p, a protein guiding MTs along actin cables. Here, we show that Kar9p does not mediate Bud6p functions in spindle orientation. Based on live microscopy analysis, kar9Delta cells maintained Bud6p-dependent MT capture. Conversely, bud6Delta cells supported Kar9p-associated MT delivery to the bud. Moreover, additive phenotypes in bud6Delta kar9Delta or bud6Delta dyn1Delta mutants underscored the separate contributions of Bud6p, Kar9p, and dynein to spindle positioning. Finally, tub2C354S, a mutation decreasing MT dynamics, suppressed a kar9Delta mutation in a BUD6-dependent manner. Thus, Kar9p-independent capture at Bud6p sites can effect spindle orientation provided MT turnover is reduced. Together, these results demonstrate Bud6p function in MT capture at the cell cortex, independent of Kar9p-mediated MT delivery along actin cables.     

A Bni4-Glc7 phosphatase complex that recruits chitin synthase to the site of bud emergence

Bni4 is a scaffold protein in the yeast Saccharomyces cerevisiae that tethers chitin synthase III to the bud neck by interacting with septin neck filaments and with Chs4, a regulatory subunit of chitin synthase III. We show herein that Bni4 is also a limiting determinant for the targeting of the type 1 serine/threonine phosphatase (Glc7) to the bud neck. Yeast cells containing a Bni4 variant that fails to associate with Glc7 fail to tether Chs4 to the neck, due in part to the failure of Bni4(V831A/F833A) to localize properly. Conversely, the Glc7-129 mutant protein fails to bind Bni4 properly and glc7-129 mutants exhibit reduced levels of Bni4 at the bud neck. Bni4 is phosphorylated in a cell cycle-dependent manner and Bni4(V831A/F833A) is both hyperphosphorylated and mislocalized in vivo. Yeast cells lacking the protein kinase Hsl1 exhibit increased levels of Bni4-GFP at the bud neck. GFP-Chs4 does not accumulate at the incipient bud site in either a bni4::TRP1 or a bni4(V831A/F833A) mutant but does mobilize to the neck at cytokinesis. Together, these results indicate that the formation of the Bni4-Glc7 complex is required for localization to the site of bud emergence and for subsequent targeting of chitin synthase.     

Kar9p-independent microtubule capture at Bud6p cortical sites primes spindle polarity before bud emergence in Saccharomyces cerevisiae

Spindle orientation is critical for accurate chromosomal segregation in eukaryotic cells. In the yeast Saccharomyces cerevisiae, orientation of the mitotic spindle is achieved by a program of microtubule-cortex interactions coupled to spindle morphogenesis. We previously implicated Bud6p in directing microtubule capture throughout this program. Herein, we have analyzed cells coexpressing GFP:Bud6 and GFP:Tub1 fusions, providing a kinetic view of Bud6p-microtubule interactions in live cells. Surprisingly, even during the G1 phase, microtubule capture at the recent division site and the incipient bud is dictated by Bud6p. These contacts are eliminated in bud6 delta cells but are proficient in kar9 delta cells. Thus, Bud6p cues microtubule capture, as soon as a new cell polarity axis is established independent of Kar9p. Bud6p increases the duration of interactions and promotes distinct modes of cortical association within the bud and neck regions. In particular, microtubule shrinkage and growth at the cortex rarely occur away from Bud6p sites. These are the interactions selectively impaired at the bud cortex in bud6 delta cells. Finally, interactions away from Bud6p sites within the bud differ from those occurring at the mother cell cortex, pointing to the existence of an independent factor controlling cortical contacts in mother cells after bud emergence.     

Requirement of Bni5 phosphorylation for bud morphogenesis in Saccharomyces cerevisiae

In budding yeast, G2/M transition is tightly correlated with bud morphogenesis regulated by Swe1 and septin that plays as a scaffold to recruits protein components. BNI5 isolated as a suppressor for septin defect is implicated in septin organization and cytokinesis. The mechanism by which Bni5 regulates normal septin function is not completely understood. Here, we show that Bni5 phosphorylation is required for mitotic entry regulated by Swe1 pathway. Bni5 modification was evident from late mitosis to G1 phase, and CIP treatment in vitro of affinity-purified Bni5 removed the modification, indicative of phosphorylation on Bni5. The phosphorylation-deficient mutant of BNI5 (bni5-4A) was defective in both growth at semi-restrictive temperature and suppression of septin defect. Loss of Bni5 phosphorylation resulted in abnormal bud morphology and cell cycle delay at G2 phase, as evidenced by the formation of elongated cells with multinuclei. However, deletion of Swe1 completely eliminated the elongated-bud phenotypes of both bni5 deletion and bni5-4A mutants. These results suggest that the bud morphogenesis and mitotic entry are positively regulated by phosphorylation-dependent function of Bni5 which is under the control of Swe1 morphogenesis pathway.     

Actin-mediated Delivery of Astral Microtubules Instructs Kar9p Asymmetric Loading to the Bud-Ward Spindle Pole

In Saccharomyces cerevisiae, Kar9p, one player in spindle alignment, guides the bud-ward spindle pole by linking astral microtubule plus ends to Myo2p-based transport along actin cables generated by the formins Bni1p and Bnr1p and the polarity determinant Bud6p. Initially, Kar9p labels both poles but progressively singles out the bud-ward pole. Here, we show that this polarization requires cell polarity determinants, actin cables, and microtubules. Indeed, in a bud6Δ bni1Δ mutant or upon direct depolymerization of actin cables Kar9p symmetry increased. Furthermore, symmetry was selectively induced by myo2 alleles, preventing Kar9p binding to the Myo2p cargo domain. Kar9p polarity was rebuilt after transient disruption of microtubules, dependent on cell polarity and actin cables. Symmetry breaking also occurred after transient depolymerization of actin cables, with Kar9p increasing at the spindle pole engaging in repeated cycles of Kar9p-mediated transport. Kar9p returning to the spindle pole on shrinking astral microtubules may contribute toward this bias. Thus, Myo2p transport along actin cables may support a feedback loop by which delivery of astral microtubule plus ends sustains Kar9p polarized recruitment to the bud-ward spindle pole. Our findings also explain the link between Kar9p polarity and the choice setting aside the old spindle pole for daughter-bound fate.     

Yeast formin Bni1p has multiple localization regions that function in polarized growth and spindle orientation

Formins are conserved proteins that assemble unbranched actin filaments in a regulated, localized manner. Budding yeast's two formins, Bni1p and Bnr1p, assemble actin cables necessary for polarized cell growth and organelle segregation. Here we define four regions in Bni1p that contribute to its localization to the bud and at the bud neck. The first (residues 1-333) requires dimerization for its localization and encompasses the Rho-binding domain. The second (residues 334-821) covers the Diaphanous inhibitory-dimerization-coiled coil domains, and the third is the Spa2p-binding domain. The fourth region encompasses the formin homology 1-formin homology 2-COOH region of the protein. These four regions can each localize to the bud cortex and bud neck at the right stage of the cell cycle independent of both F-actin and endogenous Bni1p. The first three regions contribute cumulatively to the proper localization of Bni1p, as revealed by the effects of progressive loss of these regions on the actin cytoskeleton and fidelity of spindle orientation. The fourth region contributes to the localization of Bni1p in tiny budded cells. Expression of mislocalized Bni1p constructs has a dominant-negative effect on both growth and nuclear segregation due to mislocalized actin assembly. These results define an unexpected complexity in the mechanism of formin localization and function.     

The role of the proteins Kar9 and Myo2 in orienting the mitotic spindle of budding yeast

BACKGROUND: Two genetic 'pathways' contribute to the fidelity of nuclear segregation during the process of budding in the yeast Saccharomyces cerevisiae. An early pathway, involving Kar9p and other proteins, orients the mitotic spindle along the mother-bud axis. Upon the onset of anaphase, cytoplasmic dynein provides the motive force for nuclear movement into the bud. Loss of either pathway results in nuclear-migration defects; loss of both is lethal. Here, to visualize the functional steps leading to correct spindle orientation along the mother-bud axis, we imaged live yeast cells expressing Kar9p and dynein as green fluorescent protein fusions. RESULTS: Transport of Kar9p into the bud was found to require the myosin Myo2p. Kar9p interacted with microtubules through the microtubule-binding protein Bim1p and facilitated microtubule penetration into the bud. Once microtubules entered the bud, Kar9p provided a platform for microtubule capture at the bud cortex. Kar9p was also observed at sites of microtubule shortening in the bud, suggesting that Kar9p couples microtubule shortening to nuclear migration. CONCLUSIONS: Thus, Kar9p provides a key link between the actin cytoskeleton and microtubules early in the cell cycle. A cooperative mechanism between Kar9p and Myo2p facilitates the pre-anaphase orientation of the spindle. Later, Kar9p couples microtubule disassembly with nuclear migration.     

Roles of Hof1p, Bni1p, Bnr1p, and myo1p in cytokinesis in Saccharomyces cerevisiae

Cytokinesis in Saccharomyces cerevisiae occurs by the concerted action of the actomyosin system and septum formation. Here we report on the roles of HOF1, BNI1, and BNR1 in cytokinesis, focusing on Hof1p. Deletion of HOF1 causes a temperature-sensitive defect in septum formation. A Hof1p ring forms on the mother side of the bud neck in G2/M, followed by the formation of a daughter-side ring. Around telophase, Hof1p is phosphorylated and the double rings merge into a single ring that contracts slightly and may colocalize with the actomyosin structure. Upon septum formation, Hof1p splits into two rings, disappearing upon cell separation. Hof1p localization is dependent on septins but not Myo1p. Synthetic lethality suggests that Bni1p and Myo1p belong to one functional pathway, whereas Hof1p and Bnr1p belong to another. These results suggest that Hof1p may function as an adapter linking the primary septum synthesis machinery to the actomyosin system. The formation of the actomyosin ring is not affected by bni1Delta, hof1Delta, or bnr1Delta. However, Myo1p contraction is affected by bni1Delta but not by hof1Delta or bnr1Delta. In bni1Delta cells that lack the actomyosin contraction, septum formation is often slow and asymmetric, suggesting that actomyosin contraction may provide directionality for efficient septum formation.     

Microtubules Orient the Mitotic Spindle in Yeast through Dynein-dependent Interactions with the Cell Cortex

Proper orientation of the mitotic spindle is critical for successful cell division in budding yeast. To investigate the mechanism of spindle orientation, we used a green fluorescent protein (GFP)–tubulin fusion protein to observe microtubules in living yeast cells. GFP–tubulin is incorporated into microtubules, allowing visualization of both cytoplasmic and spindle microtubules, and does not interfere with normal microtubule function. Microtubules in yeast cells exhibit dynamic instability, although they grow and shrink more slowly than microtubules in animal cells. The dynamic properties of yeast microtubules are modulated during the cell cycle. The behavior of cytoplasmic microtubules revealed distinct interactions with the cell cortex that result in associated spindle movement and orientation. Dynein-mutant cells had defects in these cortical interactions, resulting in misoriented spindles. In addition, microtubule dynamics were altered in the absence of dynein. These results indicate that microtubules and dynein interact to produce dynamic cortical interactions, and that these interactions result in the force driving spindle orientation.