Cortical dynein is critical for proper spindle positioning in human cells

Correct spindle positioning is fundamental for proper cell division during development and in stem cell lineages. Dynein and an evolutionarily conserved ternary complex (nuclear mitotic apparatus protein [NuMA]–LGN–Gα in human cells and LIN-5–GPR-1/2–Gα in Caenorhabditis elegans) are required for correct spindle positioning, but their relationship remains incompletely understood. By analyzing fixed specimens and conducting live-imaging experiments, we uncovered that appropriate levels of ternary complex components are critical for dynein-dependent spindle positioning in HeLa cells and C. elegans embryos. Moreover, using mutant versions of Gα in both systems, we established that dynein acts at the membrane to direct spindle positioning. Importantly, we identified a region within NuMA that mediates association with dynein. By using this region to target dynein to the plasma membrane, we demonstrated that the mere presence of dynein at that location is sufficient to direct spindle positioning in HeLa cells. Overall, we propose a model in which the ternary complex serves to anchor dynein at the plasma membrane to ensure correct spindle positioning.     

Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning

Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, we used Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis. We report that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. We identify two Dyn2-binding sites in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function.     

Changes in the localization of the Saccharomyces cerevisiae anaphase-promoting complex upon microtubule depolymerization and spindle checkpoint activation

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase in the ubiquitin-mediated proteolysis pathway (UMP). To understand how the APC/C was targeted to its substrates, we performed a detailed analysis of one of the APC/C components, Cdc23p. In live cells, Cdc23-GFP localized to punctate nuclear spots surrounded by homogenous nuclear signal throughout the cell cycle. These punctate spots colocalized with two outer kinetochore proteins, Slk19p and Okp1p, but not with the spindle pole body protein, Spc42p. In late anaphase, the Cdc23-GFP was also visualized along the length of the mitotic spindle. We hypothesized that spindle checkpoint activation may affect the APC/C nuclear spot localization. Localization of Cdc23-GFP was disrupted upon nocodazole treatment in the kinetochore mutant okp1-5 and in the cdc20-1 mutant. Cdc23-GFP nuclear spot localization was not affected in the ndc10-1 mutant, which is defective in spindle checkpoint function. Additional studies using a mad2Delta strain revealed a microtubule dependency of Cdc23-GFP spot localization, whether or not the checkpoint response was activated. On the basis of these data, we conclude that Cdc23p localization was dependent on microtubules and was affected by specific types of kinetochore disruption.     

Coupling of cortical dynein and G alpha proteins mediates spindle positioning in Caenorhabditis elegans

Despite being essential for spatial cell division control, the mechanisms governing spindle positioning remain incompletely understood. In the Caenorhabditis elegans one-cell stage embryo, the spindle becomes asymmetrically positioned during anaphase through the action of as-yet unidentified cortical force generators that pull on astral microtubules and that depend on two G alpha proteins and associated proteins. We performed spindle-severing experiments following temporally restricted gene inactivation and drug exposure, and established that microtubule dynamics and dynein are both required for generating efficient pulling forces. We found that the G alpha-associated proteins GPR-1/2 and LIN-5 interact in vivo with LIS-1, a component of the dynein complex. Moreover, we discovered that the LIN-5, GPR-1/2 and the G alpha proteins promote the presence of the dynein complex at the cell cortex. Our findings suggest a mechanism by which the G alpha proteins enable GPR-1/2 and LIN-5 recruitment to the cortex, thus ensuring the presence of cortical dynein. Together with microtubule dynamics, this allows pulling forces to be exerted and proper cell division to be achieved.     

Regulation of dynein localization and centrosome positioning by Lis-1 and asunder during Drosophila spermatogenesis

Dynein, a microtubule motor complex, plays crucial roles in cell-cycle progression in many systems. The LIS1 accessory protein directly binds dynein, although its precise role in regulating dynein remains unclear. Mutation of human LIS1 causes lissencephaly, a developmental brain disorder. To gain insight into the in vivo functions of LIS1, we characterized a male-sterile allele of the Drosophila homolog of human LIS1. We found that centrosomes do not properly detach from the cell cortex at the onset of meiosis in most Lis-1 spermatocytes; centrosomes that do break cortical associations fail to attach to the nucleus. In Lis-1 spermatids, we observed loss of attachments between the nucleus, basal body and mitochondria. The localization pattern of LIS-1 protein throughout Drosophila spermatogenesis mirrors that of dynein. We show that dynein recruitment to the nuclear surface and spindle poles is severely reduced in Lis-1 male germ cells. We propose that Lis-1 spermatogenesis phenotypes are due to loss of dynein regulation, as we observed similar phenotypes in flies null for Tctex-1, a dynein light chain. We have previously identified asunder (asun) as another regulator of dynein localization and centrosome positioning during Drosophila spermatogenesis. We now report that Lis-1 is a strong dominant enhancer of asun and that localization of LIS-1 in male germ cells is ASUN dependent. We found that Drosophila LIS-1 and ASUN colocalize and coimmunoprecipitate from transfected cells, suggesting that they function within a common complex. We present a model in which Lis-1 and asun cooperate to regulate dynein localization and centrosome positioning during Drosophila spermatogenesis.     

Asymmetric division: a kinesin for spindle positioning

The meiotic spindles of animal eggs move to extremely asymmetric positions, close to the cell cortex. A recent paper has identified a motor complex that may move the meiotic spindle toward the cortex in Caenorhabditis elegans eggs.     

A novel patch assembly domain in Num1 mediates dynein anchoring at the cortex during spindle positioning

During mitosis in budding yeast, cortically anchored dynein generates pulling forces on astral microtubules to position the mitotic spindle across the mother-bud neck. The attachment molecule Num1 is required for dynein anchoring at the cell membrane, but how Num1 assembles into stationary cortical patches and interacts with dynein is unknown. We show that an N-terminal Bin/Amphiphysin/Rvs (BAR)-like domain in Num1 mediates the assembly of morphologically distinct patches and its interaction with dynein for spindle translocation into the bud. We name this domain patch assembly domain (PA; residues 1-303), as it was both necessary and sufficient for the formation of functional dynein-anchoring patches when it was attached to a pleckstrin homology domain or a CAAX motif. Distinct point mutations targeting the predicted BAR-like PA domain differentially disrupted patch assembly, dynein anchoring, and mitochondrial attachment functions of Num1. We also show that the PA domain is an elongated dimer and discuss the mechanism by which it drives patch assembly.     

Cyclin B in Xenopus oocytes: implications for the mechanism of pre-MPF activation.

Using a polyclonal antibody raised against B2 cyclin from Xenopus laevis, we show that prophase-arrested Xenopus oocytes contain a stockpile of cyclin B2 protein. During progesterone-induced maturation, an increase in the synthesis of cyclin B2 is observed, although Western blotting experiments show that this new synthesis does not significantly increase the mass of cyclin over the maternal stockpile. In the oocyte cyclin B2 is already present in two forms which differ in the extent of phosphorylation, but the phosphorylated form becomes predominant as oocytes progress towards germinal vesicle breakdown (GVBD), coincident with cdc2 protein kinase activation. These two events do not depend upon formation of a new complex between cyclin and cdc2 protein kinase, since these two proteins are already found associated in resting oocytes, prior to activation of the kinase.     

Reversibility of the activation of soluble phospholipase A2 on lipid bilayers: implications for the activation mechanism.

The time-courses of hydrolysis of large vesicles of dipalmitoylphosphatidylcholine were compared using four species of phospholipase A2 (Agkistrodon piscivorus piscivorus, Crotalus adamanteus and Naja naja venoms and porcine pancreatic). In all four cases, the hydrolysis rate suddenly increases 10 to 100-fold at the time (tau) when a specific mole fraction of reaction products has accumulated. The intrinsic fluorescence emission of the three venom enzymes also increases suddenly at time tau. Both the activation and the fluorescence change are reversible with a half-time of about 50 s for the activity and 2 to 6 s for the fluorescence. These reversal rates and the vesicle concentration dependence of tau are considered for monomer and dimer enzyme activation models. Apparently, at least three states of the enzyme exist beyond the initial unbound state: (1) inactive and bound, (2) inactive with high fluorescence and (3) active. The dimer model already contains the necessary number of states but requires that the activation rate be much lower than the reversal rate to account for the vesicle concentration dependence of tau. Success of the monomer model requires an enzyme state additional to those proposed previously. Although these results do not exclude either the monomer or dimer models conclusively, they do impose important constraints on each model.     

Immunological relation between 14 S dynein and 30 S dynein from the cilia of Tetrahymena pyriformis.

The immunological relation between 14 S dynein and 30 S dynein obtained from Tetrahymena cilia was investigated by using antisera specific for each dynein subunit or some dynein subunits separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although 14 and 30 S dynein main subunits have different electrophoretic mobilities, our immunodiffusion tests showed that there exists a close immunological relation between them. At least three immunologically different polypeptides designated polypeptides A, B and C are included in the 30 S dynein main band which has been recognized as a single component by electrophoresis, and that the polypeptides designated A',B' and C' are included in the 14 S dynein main bands. Polypeptides A and A',B and B', or C and C' appeared to have a certain common antigenic determinant(s). Polypeptide C of 30 S dynein was shown to possess a certain antigenic determinant(s) specific for 30 S dynein, besides the determinant common with that of polypeptide C' of 14S dynein. The second main component of 30 S dynein proved to be a specific polypeptide of 30 S dynein but not to be a degraded product of the main polypedtide. All antisera reacted with native dynein molecules to some extent, but did not inhibit dynein ATPase (ATP phosphohydrase, EC 3.6.1.3) activity significantly.     

Determinants of human glucokinase activation and implications for small molecule allosteric control

Glucokinase (GK) is an enzyme that catalyzes the ATP-dependent phosphorylation of glucose to form glucose-6-phosphate, and it is a tightly regulated checkpoint in glucose homeostasis. GK is known to undergo substantial conformational changes upon glucose binding. The monomeric enzyme possesses a highly exotic kinetic activity profile with an unusual sigmoidal dependence on glucose concentration. In this interdisciplinary study, which draws on small angle X-ray scattering (SAXS) integrated with 250?ns of atomistic molecular dynamics (MD) simulations and experimental glucose binding thermodynamics, we reveal that the critical regulation of this glucose sensor is due to a solvent controlled “switch”. We demonstrate that the “solvent switch” is driven by specific protein structural dynamics, which leads to an enzyme structure that has a much more favorable solvation energy than most of the protein ensemble. These findings uncover the physical workings of an agile and flexible protein scaffold, which derives its long-range allosteric control through specific regions with favorable solvation energy. The physiological framework presented herein provides insights that have direct implications for the design of small molecule GK activators as anti-diabetes therapeutics as well as for understanding how proteins can be designed to have built-in regulatory functions via solvation energy dynamics.     

ACT3: a putative centractin homologue in S. cerevisiae is required for proper orientation of the mitotic spindle

As part of our ongoing efforts to understand the functional role of vertebrate centractins, we have identified a new member of the actin- related family of proteins in the yeast Saccharomyces cerevisiae using a PCR-based approach. Consistent with the current nomenclature for actin-related proteins in yeast, we propose to denote this locus ACT3. The primary amino acid sequence of Act3p is most similar to canine and human alpha-centractin (73% similarity/54% identity). The sequence of a genomic clone indicates ACT3 lies adjacent to and is transcribed convergently with respect to FUR1 on chromosome VIII. Molecular genetic analysis indicates ACT3 is represented by a single gene from which the corresponding mRNA is expressed at a low level compared to ACT1. Tetrad analysis of heterozygotes harboring a TRP1 replacement of the ACT3- coding region indicates ACT3 is nonessential for growth under normal conditions and at extremes of temperature and osmolarity. However, growth at 14 degrees C indicates a spindle orientation defect similar to phenotypes recently described for yeast harboring mutations in actin, tubulin, or cytoplasmic dynein. Taken together, our data suggest that ACT3 is the S. cerevisiae homologue of vertebrate centractins.     

A novel roll-and-slide mechanism of DNA folding in chromatin: implications for nucleosome positioning

How eukaryotic genomes encode the folding of DNA into nucleosomes and how this intrinsic organization of chromatin guides biological function are questions of wide interest. The physical basis of nucleosome positioning lies in the sequence-dependent propensity of DNA to adopt the tightly bent configuration imposed by the binding of the histone proteins. Traditionally, only DNA bending and twisting deformations are considered, while the effects of the lateral displacements of adjacent base pairs are neglected. We demonstrate, however, that these displacements have a much more important structural role than ever imagined. Specifically, the lateral Slide deformations observed at sites of local anisotropic bending of DNA define its superhelical trajectory in chromatin. Furthermore, the computed cost of deforming DNA on the nucleosome is sequence-specific: in optimally positioned sequences the most easily deformed base-pair steps (CA:TG and TA) occur at sites of large positive Slide and negative Roll (where the DNA bends into the minor groove). These conclusions rest upon a treatment of DNA that goes beyond the conventional ribbon model, incorporating all essential degrees of freedom of "real" duplexes in the estimation of DNA deformation energies. Indeed, only after lateral Slide displacements are considered are we able to account for the sequence-specific folding of DNA found in nucleosome structures. The close correspondence between the predicted and observed nucleosome locations demonstrates the potential advantage of our "structural" approach in the computer mapping of nucleosome positioning.     

The spindle positioning protein Kar9p interacts with the sumoylation machinery in Saccharomyces cerevisiae

Accurate positioning of the mitotic spindle is important for the genetic material to be distributed evenly in dividing cells, but little is known about the mechanisms that regulate this process. Here we report that two microtubule-associated proteins important for spindle positioning interact with several proteins in the sumoylation pathway. By two-hybrid analysis, Kar9p and Bim1p interact with the yeast SUMO Smt3p, the E2 enzyme Ubc9p, an E3 Nfi1p, as well as Wss1p, a weak suppressor of a temperature-sensitive smt3 allele. The physical interaction between Kar9p and Ubc9p was confirmed by in vitro binding assays. A single-amino-acid substitution in Kar9p, L304P disrupted its two-hybrid interaction with proteins in the sumoylation pathway, but retained its interactions with the spindle positioning proteins Bim1p, Stu2p, Bik1p, and Myo2p. The kar9-L304P mutant showed defects in positioning the mitotic spindle, with the spindle located more distally than normal. Whereas wild-type Kar9p-3GFP normally localizes to only the bud-directed spindle pole body (SPB), Kar9p-L304P-3GFP was mislocalized to both SPBs. Using a reconstitution assay, Kar9p was sumoylated in vitro. We propose a model in which sumoylation regulates spindle positioning by restricting Kar9p to one SPB. These findings raise the possibility that sumoylation could regulate other microtubule-dependent processes.     

Structure of the catalytic core of S. cerevisiae DNA polymerase eta: implications for translesion DNA synthesis.

DNA polymerase eta is unique among eukaryotic polymerases in its proficient ability to replicate through a variety of distorting DNA lesions. We report here the crystal structure of the catalytic core of S. cerevisiae DNA polymerase eta, determined at 2.25A resolution. The structure reveals a novel polydactyl right hand-shaped molecule with a unique polymerase-associated domain. We identify the catalytic residues and show that the fingers and thumb domains are unusually small and stubby. In particular, the unexpected absence of helices "O" and "O1" in the fingers domain suggests that openness of the active site is the critical feature which enables DNA polymerase eta to replicate through DNA lesions such as a UV-induced cis-syn thymine-thymine dimer.     

Novel roles for saccharomyces cerevisiae mitotic spindle motors.

The single cytoplasmic dynein and five of the six kinesin-related proteins encoded by Saccharomyces cerevisiae participate in mitotic spindle function. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle. Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell. This study reveals that kinesin-related Kar3p and Kip3p are unique in that they perform roles both inside and outside the nucleus. Kar3p, like Kip3p, was found to be required for spindle positioning in the absence of dynein. The spindle positioning role of Kar3p is performed in concert with the Cik1p accessory factor, but not the homologous Vik1p. Kar3p and Kip3p were also found to overlap for a function essential for the structural integrity of the bipolar spindle. The cytoplasmic and nuclear roles of both these motors could be partially substituted for by the microtubule-destabilizing agent benomyl, suggesting that these motors perform an essential microtubule-destabilizing function. In addition, we found that yeast cell viability could be supported by as few as two microtubule-based motors: the BimC-type kinesin Cin8p, required for spindle structure, paired with either Kar3p or Kip3p, required for both spindle structure and positioning.