M. Angeles Juanes

Mechanism for Astral Microtubule Capture by Cortical Bud6p Priming Spindle Polarity in S. cerevisiae

BACKGROUND Budding yeast is a unique model to dissect spindle orientation in a cell dividing asymmetrically. In yeast, this process begins with the capture of pole-derived astral microtubules (MTs) by the polarity determinant Bud6p at the cortex of the bud in G(1). Bud6p couples MT growth and shrinkage with spindle pole movement relative to the contact site. This activity resides in N-terminal sequences away from a domain linked to actin organization. Kip3p (kinesin-8), a MT depolymerase, may be implicated, but other molecular details are essentially unknown. RESULTS We show that Bud6p and Kip3p play antagonistic roles in controlling the length of MTs contacting the bud. The stabilizing role of Bud6p required the plus-end-tracking protein Bim1p (yeast EB1). Bim1p bound Bud6p N terminus, an interaction that proved essential for cortical capture of MTs in vivo. Moreover, Bud6p influenced Kip3p dynamic distribution through its effect on MT stability during cortical contacts via Bim1p. Coupling between Kip3p-driven depolymerization and shrinkage at the cell cortex required Bud6p, Bim1p, and dynein, a minus-end-directed motor helping tether the receding plus ends to the cell cortex. Validating these findings, live imaging of the interplay between dynein and Kip3p demonstrated that both motors decorated single astral MTs with dynein persisting at the plus end in association with the site of cortical contact during shrinkage at the cell cortex. CONCLUSIONS Astral MT shrinkage linked to Bud6p involves its direct interaction with Bim1p and the concerted action of two MT motors-Kip3p and dynein.

Spindle pole body history intrinsically links pole identity with asymmetric fate in budding yeast.

BACKGROUND Budding yeast is a unique model for exploring differential fate in a cell dividing asymmetrically. In yeast, spindle orientation begins with the old spindle pole body (SPB) (from the preceding cell cycle) contacting the bud by its existing astral microtubules (aMTs) while the new pole delays astral microtubule organization. This appears to prime the inheritance of the old pole by the bud. The basis for this asymmetry and the discrimination of the poles by virtue of their history remain a mystery. RESULTS Here, we report that asymmetric aMT organization stems from an outstanding structural asymmetry linked to the SPB cycle. We show that the γ-tubulin nucleation complex (γTC) favors the old spindle pole, an asymmetry inherent to the outer plaque (the cytoplasmic face of the SPB). Indeed, Spc72 (the receptor for the γTC) is acquired by the new SPB outer plaque partway through spindle assembly. The significance of this asymmetry was explored in cells expressing an Spc72(1-276)-Cnm67 fusion that forced symmetric nucleation at the SPB outer plaques. This manipulation triggered simultaneous aMT organization by both spindle poles from the outset and led to symmetric contacts between poles and the bud, effectively disrupting the program for spindle polarity. Temporally symmetric aMT organization perturbed Kar9 polarization by randomizing the choice of the pole to be guided toward the bud. Accordingly, the pattern of SPB inheritance was also randomized. CONCLUSIONS Spc72 differential recruitment imparting asymmetric aMT organization represents the most upstream determinant linking SPB historical identity and fate.

Adenomatous polyposis coli nucleates actin assembly to drive cell migration and microtubule-induced focal adhesion turnover

Cell motility depends on tight coordination between the microtubule (MT) and actin cytoskeletons, but the mechanisms underlying this MT–actin cross talk have remained poorly understood. Here, we show that the tumor suppressor protein adenomatous polyposis coli (APC), which is a known MT-associated protein, directly nucleates actin assembly to promote directed cell migration. By changing only two residues in APC, we generated a separation-of-function mutant, APC (m4), that abolishes actin nucleation activity without affecting MT interactions. Expression of full-length APC carrying the m4 mutation (APC (m4)) rescued cellular defects in MT organization, MT dynamics, and mitochondrial distribution caused by depletion of endogenous APC but failed to restore cell migration. Wild-type APC and APC (m4) localized to focal adhesions (FAs), and APC (m4) was defective in promoting actin assembly at FAs to facilitate MT-induced FA turnover. These results provide the first direct evidence for APC-mediated actin assembly in vivo and establish a role for APC in coordinating MTs and actin at FAs to direct cell migration.

Rot1 plays an antagonistic role to Clb2 in actin cytoskeleton dynamics throughout the cell cycle.

ROT1 is an essential gene whose inactivation causes defects in cell cycle progression and morphogenesis in budding yeast. Rot1 affects the actin cytoskeleton during the cell cycle at two levels. First, it is required for the maintenance of apical growth during bud growth. Second, Rot1 is necessary to polarize actin cytoskeleton to the neck region at the end of mitosis; because of this defect, rot1 cells do not properly form a septum to complete cell division. The inability to polarize the actin cytoskeleton at the end of mitosis is not due to a defect in the recruitment of the polarisome scaffold protein Spa2 or the actin cytoskeleton regulators Cdc42 and Cdc24 in the neck region. Previous results indicate a connection between Rot1 and the cyclin Clb2. In fact, overexpression of CLB2 is toxic when ROT1 is partially inactivated, and reciprocally, deletion of CLB2 suppresses the lethality of the rot1 mutant, which indicates a functional antagonism between Clb2 and Rot1. Several genetic interactions suggest a link between Rot1 and the ubiquitin-proteasome system and we show that the Clb2 cyclin is not properly degraded in rot1 cells.

Membrane topology and post-translational modification of the Saccharomyces cerevisiae essential protein Rot1

ROT1 is an essential gene that has been related to cell wall biosynthesis, the actin cytoskeleton and protein folding. In order to help to understand its molecular function, we carried out a characterization of the Rot1 protein. It is primarily located at the endoplasmic reticulum–nuclear membrane facing the lumen. Rot1 migrates more slowly than expected, which might suggest post‐translational modification. Our results indicate that Rot1 is a protein that is neither GPI‐anchored nor O‐glycosylated. In contrast, it is N‐glycosylated. By a directed mutagenesis of several Asn residues, we identified that the protein is simultaneously glycosylated at N103, N107 and N139. Although the mutation of these three N sites is not lethal, cellular growth is impaired. Sequence analysis predicts a transmembrane domain at the C‐terminus. This fragment affects neither the targeting of the Rot1 protein to the ER nor its N‐glycosylation, although it is important for the anchoring of the protein to the membrane and for its functionality. The existence of a signal sequence at the N‐terminus has been suggested. However, deletion of this fragment impedes neither translocation to the ER nor N‐glycosylation, but it is required for cell viability. Finally, we found that Rot1 is translocated to the ER by an SRP‐independent post‐translational mechanism which depends on Sec62. Copyright

Control of Formin Distribution and Actin Cable Assembly by the E3 Ubiquitin Ligases Dma1 and Dma2.

Formins are widespread actin-polymerizing proteins that play pivotal roles in a number of processes, such as cell polarity, morphogenesis, cytokinesis, and cell migration. In agreement with their crucial function, formins are prone to a variety of regulatory mechanisms that include autoinhibition, post-translational modifications, and interaction with formin modulators. Furthermore, activation and function of formins is intimately linked to their ability to interact with membranes. In the budding yeast Saccharomyces cerevisiae, the two formins Bni1 and Bnr1 play both separate and overlapping functions in the organization of the actin cytoskeleton. In addition, they are controlled by both common and different regulatory mechanisms. Here we show that proper localization of both formins requires the redundant E3 ubiquitin ligases Dma1 and Dma2, which were previously involved in spindle positioning and septin organization. In dma1 dma2 double mutants, formin distribution at polarity sites is impaired, thus causing defects in the organization of the actin cable network and hypersensitivity to the actin depolymerizer latrunculin B. Expression of a hyperactive variant of Bni1 (Bni1-V360D) rescues these defects and partially restores proper spindle positioning in the mutant, suggesting that the failure of dma1 dma2 mutant cells to position the spindle is partly due to faulty formin activity. Strikingly, Dma1/2 interact physically with both formins, while their ubiquitin-ligase activity is required for formin function and polarized localization. Thus, ubiquitylation of formin or a formin interactor(s) could promote formin binding to membrane and its ability to nucleate actin. Altogether, our data highlight a novel level of formin regulation that further expands our knowledge of the complex and multilayered controls of these key cytoskeleton organizers.

A transmembrane serine residue in the Rot1 protein is essential for yeast cell viability

Polar residues are present in TM (transmembrane) helices and may influence the folding or association of membrane proteins. In the present study, we use an in vivo approach to analyse the functional and structural roles for amino acids in membrane-spanning motifs using the Rot1 (reversal of Tor2 lethality 1) protein as a model. Rot1 is an essential membrane protein in Saccharomyces cerevisiae and it contains a single TM domain. An alanine insertion scanning analysis of this TM helix revealed that the integrity of the central domain is essential for protein function. We identified a critical serine residue inside the helix that plays an essential role in maintaining cell viability in S. cerevisiae. Replacement of the serine residue at position 250 with a broad variety of amino acids did not affect protein targeting and location, but completely disrupted protein function causing cell death. Interestingly, substitution of the serine residue by threonine resulted in sustained cell viability, demonstrating that the hydroxy group of the TM serine side chain plays a critical role in protein function. The results of the present study indicate that Rot1 needs the TM Ser250 to interact with other membrane components and exert its functional role, avoiding exposure of the serine hydrogen-bonding group at the lipid-exposed surface.

Ase1p phosphorylation by cyclin-dependent kinase promotes correct spindle assembly in S. cerevisiae.

Spindle morphogenesis and dynamics follow an orderly sequence of events coupled to the oscillatory activation of cyclin-dependent kinase (CDK). Using S. cerevisiae, we have addressed the requirement of CDK for phosphorylation of the spindle midzone component Ase1p and its significance to spindle assembly. Ase1p is related to human PRC1, a protein negatively regulated by CDK until late mitosis, when it is required for central spindle organization and cytokinesis. By contrast, we show here that Ase1p phosphorylation by CDK promotes correct spindle assembly. Indeed, Ase1p phosphorylation coincident with spindle assembly requires Clb5p, Clb3p and Clb4p. Moreover, in clb5Δ cells, Ase1p recruitment and the kinetics of spindle formation were perturbed. These phenotypes were enhanced in a cdc28-4 clb5Δ mutant to the extent that midzone disruption resulted in transient breaks of the short spindle. By contrast, clb3Δ clb4Δ cells delayed spindle assembly downstream to Ase1p recruitment. Expression of Ase17Dp that mimics the phosphorylated state restored timely recruitment in clb5Δ cells and fully rescued the corresponding spindle phenotypes. Finally, Ase17Dp partially suppressed the spindle assembly delay in clb3Δ clb4Δ cells. Thus, Ase1p phosphorylation by CDK promotes the assembly and stability of the mitotic spindle. It follows that CDK may differentially alter the functionality of members of the Ase1p/PRC1 family to place their distinct roles in their respective stage-specific contexts, a further factor of complexity in the organization of pathways promoting spindle assembly and dynamics.

Rot1, an essential yeast protein, is degraded through the ER-associated protein degradation system (ERAD)

S. cerevisiae ROT1 is an essential gene which has been related to cell wall biosynthesis, the actin cytoskeleton and protein folding. Rot1 protein is primarily located at the endoplasmic reticulumnuclear membrane facing the lumen where it is translocated through two internal topogenic elements by an SRP-independent posttranslational mechanism which depends on Sec62 to then be N-glycosylated at the lumen of the ER. Despite Rot1 protein levels are critical for yeast survival, proper cell cycle progression and morphogenesis, Rot1 protein levels has not been investigated so far. Here we carry out an in vivo study to analyse the Rot1 protein levels and show that Rot1 is a short-lived protein and its turnover is mediated by the ubiquitin proteasome system (UPS), dependent on the main degradation pathway located at the ER, the ER-associated degradation system (ERAD). This finding correlates with genetic interactions we previously found and that suggested a link between Rot1 and the ubiquitin-proteasome system. In addition, by using mutant cells components of the ERAD pathway, we demonstrated that Rot1 is degraded through the ubiquitin conjugating enzymes (E2) components of ERAD, Ubc6 and Ubc7, and it seems to require only one of the E3 ubiquitin ligases involved in ERAD, Hrd1 but not Doa1.

Methods of Synchronization of Yeast Cells for the Analysis of Cell Cycle Progression

Cell division is a fascinating and fundamental process that sustains life. By this process, unicellular organisms reproduce and multicellular organisms sustain development, growth, and tissue repair. Division of a mother cell gives rise to two daughter cells according to an ordered set of events within four successive phases called G1 (gap1), S (DNA Synthesis), G2 (gap2), and M (Mitosis) phase. How these different phases are orchestrated to ensure the physical separation of the two daughter cells is a tightly regulated process. Indeed, inappropriate cell division could lead to uncontrolled cell proliferation and ultimately to cancer. Saccharomyces cerevisiae is an excellent model system for unraveling the secrets of cell division. A large community of researchers has chosen budding yeast as a model because of its advantages: rapid growth in simple and economical media, tractable genetics, powerful biochemistry, cell biology, and proteomics approaches. Furthermore, the cell cycle mechanisms, as elucidated in yeast, are conserved in higher eukaryotes. The ability to synchronize and get large numbers of cells in a particular stage of the cell cycle is crucial to properly explore the mechanisms of the cell cycle. An overview of the most common yeast synchronization techniques has been compiled in this chapter.