PGRMC1 participates in late events of bovine granulosa cells mitosis and oocyte meiosis

Progesterone Receptor Membrane Component 1 (PGRMC1) is expressed in both oocyte and ovarian somatic cells, where it is found in multiple cellular sub-compartments including the mitotic spindle apparatus. PGRMC1 localization in the maturing bovine oocytes mirrors its localization in mitotic cells, suggesting a possible common action in mitosis and meiosis. To test the hypothesis that altering PGRMC1 activity leads to similar defects in mitosis and meiosis, PGRMC1 function was perturbed in cultured bovine granulosa cells (bGC) and maturing oocytes and the effect on mitotic and meiotic progression assessed. RNA interference-mediated PGRMC1 silencing in bGC significantly reduced cell proliferation, with a concomitant increase in the percentage of cells arrested at G2/M phase, which is consistent with an arrested or prolonged M-phase. This observation was confirmed by time-lapse imaging that revealed defects in late karyokinesis. In agreement with a role during late mitotic events, a direct interaction between PGRMC1 and Aurora Kinase B (AURKB) was observed in the central spindle at of dividing cells. Similarly, treatment with the PGRMC1 inhibitor AG205 or PGRMC1 silencing in the oocyte impaired completion of meiosis I. Specifically the ability of the oocyte to extrude the first polar body was significantly impaired while meiotic figures aberration and chromatin scattering within the ooplasm increased. Finally, analysis of PGRMC1 and AURKB localization in AG205-treated oocytes confirmed an altered localization of both proteins when meiotic errors occur. The present findings demonstrate that PGRMC1 participates in late events of both mammalian mitosis and oocyte meiosis, consistent with PGRMC1's localization at the mid-zone and mid-body of the mitotic and meiotic spindle.     

Progesterone signaling mediated through progesterone receptor membrane component-1 in ovarian cells with special emphasis on ovarian cancer

Various ovarian cell types including granulosa cells and ovarian surface epithelial cells express the progesterone (P4) binding protein, progesterone receptor membrane component-1 (PGRMC1). PGRMC1 is also expressed in ovarian tumors. PGRMC1 plays an essential role in promoting the survival of both normal and cancerous ovarian cell in vitro. Given the clinical significance of factors that regulate the viability of ovarian cancer, this review will focus on the role of PGRMC1 in ovarian cancer, while drawing insights into the mechanism of PGRMC1's action from cell lines derived from healthy ovaries as well as ovarian tumors.Studies using PGRMC1siRNA demonstrated that P4's ability to inhibit ovarian cells from undergoing apoptosis in vitro is dependent on PGRMC1. To confirm the importance of PGRMC1, the ability of PGRMC1-deplete ovarian cancer cell lines to form tumors in intact nude mice was assessed. Compared to PGRMC1-expressing ovarian cancer cells, PGRMC1-deplete ovarian cancer cells formed tumors in fewer mice (80% compared to 100% for controls). Moreover, the number of tumors derived from PGRMC1-deplete ovarian cancer cells was 50% of that observed in controls. Finally, the tumors that formed from PGRMC1-deplete ovarian cancer cells were about a fourth the size of tumors derived from ovarian cancer cells with normal levels of PGRMC1. One reason for PGRMC1-deplete tumors being smaller is that they had a poorly developed microvasculature system. How PGRMC1 regulates cell viability and in turn tumor growth is not known but part of the mechanism likely involves the regulation of genes that promote cell survival and inhibit apoptosis.     

Consequences of defective tubulin folding on heterodimer levels, mitosis and spindle morphology in Saccharomyces cerevisiae

In budding yeast, the essential roles of microtubules include segregating chromosomes and positioning the nucleus during mitosis. Defects in these functions can lead to aneuploidy and cell death. To ensure proper mitotic spindle and cytoplasmic microtubule formation, the cell must maintain appropriate stoichiometries of alpha- and beta-tubulin, the basic subunits of microtubules. The experiments described here investigate the minimal levels of tubulin heterodimers needed for mitotic function. We have found a triple-mutant strain, pac10Delta plp1Delta yap4Delta, which has only 20% of wild-type tubulin heterodimer levels due to synthesis and folding defects. The anaphase spindles in these cells are approximately 64% the length of wild-type spindles. The mutant cells are viable and accurately segregate chromosomes in mitosis, but they do have specific defects in mitosis such as abnormal nuclear positioning. The results establish that cells with 20% of wild-type levels of tubulin heterodimers can perform essential cellular functions with a short spindle, but require higher tubulin heterodimer concentrations to attain normal spindle length and prevent mitotic defects.     

BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin

BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.     

JMJD5 (Jumonji Domain-containing 5) Associates with Spindle Microtubules and Is Required for Proper Mitosis

Precise mitotic spindle assembly is a guarantee of proper chromosome segregation during mitosis. Chromosome instability caused by disturbed mitosis is one of the major features of various types of cancer. JMJD5 has been reported to be involved in epigenetic regulation of gene expression in the nucleus, but little is known about its function in mitotic process. Here we report the unexpected localization and function of JMJD5 in mitotic progression. JMJD5 partially accumulates on mitotic spindles during mitosis, and depletion of JMJD5 results in significant mitotic arrest, spindle assembly defects, and sustained activation of the spindle assembly checkpoint (SAC). Inactivating SAC can efficiently reverse the mitotic arrest caused by JMJD5 depletion. Moreover, JMJD5 is found to interact with tubulin proteins and associate with microtubules during mitosis. JMJD5-depleted cells show a significant reduction of α-tubulin acetylation level on mitotic spindles and fail to generate enough interkinetochore tension to satisfy the SAC. Further, JMJD5 depletion also increases the susceptibility of HeLa cells to the antimicrotubule agent. Taken together, these results suggest that JMJD5 plays an important role in regulating mitotic progression, probably by modulating the stability of spindle microtubules.     

Effects of mitotic and tubulin mutations on microtubule architecture in actively growing protoplasts of Aspergillus nidulans

We used immunofluorescent microscopy to characterize microtubule (MT) architecture in wild-type and mutant protoplasts of Aspergillus nidulans at interphase and at mitosis. Because the visualization of MTs by immunofluorescence is technically difficult in intact hyphae of A. nidulans, we developed a method for removing the cell wall under conditions that do not perturb cell physiology, as evidenced by the fact that the resulting protoplasts undergo nuclear division at a normal rate and that cell cycle mutant phenotypes are expressed at restrictive temperature. Interphase cells exhibited an extensive network of cytoplasmic MTs. During mitosis the cytoplasmic MTs mostly disappeared and an intranuclear mitotic spindle appeared. We have previously shown that the benA 33 beta-tubulin mutation causes hyperstabilization of the mitotic spindle, and we have presented additional indirect evidence that suggested that the tubA1 and tubA4 alpha-tubulin mutations destabilize spindle MTs. In this paper, we show that the benA33 mutation increases the stability of cytoplasmic MTs as well as spindle MTs and that the tubA1 and tubA4 mutations destabilize both spindle and cytoplasmic MTs.     

Pleckstrin homology domains of phospholipase C-gamma1 directly interact with beta-tubulin for activation of phospholipase C-gamma1 and reciprocal modulation of beta-tubulin function in microtubule assembly

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains, an N-terminal domain and a split PH domain. Here we show that pull down of NIH3T3 cell extracts with PLC-gamma1 PH domain-glutathione S-transferase fusion proteins, followed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry, identified beta-tubulin as a binding protein of both PLC-gamma1 PH domains. Tubulin is a main component of microtubules and mitotic spindle fibers, which are composed of alpha- and beta-tubulin heterodimers in all eukaryotic cells. PLC-gamma1 and beta-tubulin colocalized in the perinuclear region in COS-7 cells and cotranslocated to the plasma membrane upon agonist stimulation. Membrane-targeted translocation of depolymerized tubulin by agonist stimulation was also supported by immunoprecipitation analyses. The phosphatidylinositol 4,5-bisphosphate (PIP(2)) hydrolyzing activity of PLC-gamma1 was substantially increased in the presence of purified tubulin in vitro, whereas the activity was not promoted by bovine serum albumin, suggesting that beta-tubulin activates PLC-gamma1. Furthermore, indirect immunofluorescent microscopy showed that PLC-gamma1 was highly concentrated in mitotic spindle fibers, suggesting that PLC-gamma1 is involved in spindle fiber formation. The effect of PLC-gamma1 in microtubule formation was assessed by overexpression and silencing PLC-gamma1 in COS-7 cells, which resulted in altered microtubule dynamics in vivo. Cells overexpressing PLC-gamma1 showed higher microtubule densities than controls, whereas PLC-gamma1 silencing with small interfering RNAs led to decreased microtubule network densities as compared with control cells. Taken together, our results suggest that PLC-gamma1 and beta-tubulin transmodulate each other, i.e. that PLC-gamma1 modulates microtubule assembly by beta-tubulin, and beta-tubulin promotes PLC-gamma1 activity.     

Fission yeast cells undergo nuclear division in the absence of spindle microtubules

Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.     

Binding of E-MAP-115 to microtubules is regulated by cell cycle- dependent phosphorylation

Expression levels of E-MAP-115, a microtubule-associated protein that stabilizes microtubules, increase with epithelial cell polarization and differentiation (Masson and Kreis, 1993). Although polarizing cells contain significant amounts of this protein, they can still divide and thus all stabilized microtubules must disassemble at the onset of mitosis to allow formation of the dynamic mitotic spindle. We show here that binding of E-MAP-115 to microtubules is regulated by phosphorylation during the cell cycle. Immunolabeling of HeLa cells for E-MAP-115 indicates that the protein is absent from microtubules during early prophase and progressively reassociates with microtubules after late prophase. A fraction of E-MAP-115 from HeLa cells released from a block at the G1/S boundary runs with higher apparent molecular weight on SDS-PAGE, with a peak correlating with the maximal number of cells in early stages of mitosis. E-MAP-115 from nocodazole-arrested mitotic cells, which can be obtained in larger amounts, displays identical modifications and was used for further biochemical characterization. The level of incorporation of 32P into mitotic E-MAP-115 is about 15- fold higher than into the interphase protein. Specific threonine phosphorylation occurs in mitosis, and the amount of phosphate associated with serine also increases. Hyperphosphorylated E-MAP-115 from mitotic cells cannot bind stably to microtubules in vitro. These results suggest that phosphorylation of E-MAP-115 is a prerequisite for increasing the dynamic properties of the interphase microtubules which leads to the assembly of the mitotic spindle at the onset of mitosis. Microtubule-associated proteins are thus most likely key targets for kinases which control changes in microtubule dynamic properties at the G2- to M-phase transition.     

Microtubule regulation in mitosis: tubulin phosphorylation by the cyclin-dependent kinase Cdk1

The activation of the cyclin-dependent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates beta-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of beta-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-beta3-tubulin(S172D/E) mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to beta-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.     

Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells

The protein kinase inhibitor 2-aminopurine induces checkpoint override and mitotic exit in BHK cells which have been arrested in mitosis by inhibitors of microtubule function (Andreassen, P. R., and R. L. Margolis. 1991. J. Cell Sci. 100:299-310). Mitotic exit is monitored by loss of MPM-2 antigen, by the reformation of nuclei, and by the extinction of p34cdc2-dependent H1 kinase activity. 2-AP-induced inactivation of p34cdc2 and mitotic exit depend on the assembly state of microtubules. During mitotic arrest generated by the microtubule assembly inhibitor nocodazole, the rate of mitotic exit induced by 2-AP decreases proportionally with increasing nocodazole concentrations. At nocodazole concentrations of 0.12 microgram/ml or greater, 2-AP induces no apparent exit through 75 min of treatment. In contrast, 2-AP brings about a rapid exit (t1/2 = 20 min) from mitotic arrest by taxol, a drug which causes inappropriate overassembly of microtubules. In control mitotic cells, p34cdc2 localizes to kinetochores, centrosomes, and spindle microtubules. We find that efficient exit from mitosis occurs under conditions where p34cdc2 remains associated with centrosomal microtubules, suggesting it must be present on these microtubules in order to be inactivated. Mitotic slippage, the natural reentry of cells into G1 during prolonged mitotic block, is also microtubule dependent. At high nocodazole concentrations slippage is prevented and mitotic arrest approaches 100%. We conclude that essential components of the machinery for exit from mitosis are present on the mitotic spindle, and that normal mitotic exit thereby may be regulated by the microtubule assembly state.     

The role of stathmin in the regulation of the cell cycle

Stathmin is the founding member of a family of proteins that play critically important roles in the regulation of the microtubule cytoskeleton. Stathmin regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers. Upon entry into mitosis, microtubules polymerize to form the mitotic spindle, a cellular structure that is essential for accurate chromosome segregation and cell division. The microtubule-depolymerizing activity of stathmin is switched off at the onset of mitosis by phosphorylation to allow microtubule polymerization and assembly of the mitotic spindle. Phosphorylated stathmin has to be reactivated by dephosphorylation before cells exit mitosis and enter a new interphase. Interfering with stathmin function by forced expression or inhibition of expression results in reduced cellular proliferation and accumulation of cells in the G2/M phases of the cell cycle. Forced expression of stathmin leads to abnormalities in or a total lack of mitotic spindle assembly and arrest of cells in the early stages of mitosis. On the other hand, inhibition of stathmin expression leads to accumulation of cells in the G2/M phases and is associated with severe mitotic spindle abnormalities and difficulty in the exit from mitosis. Thus, stathmin is critically important not only for the formation of a normal mitotic spindle upon entry into mitosis but also for the regulation of the function of the mitotic spindle in the later stages of mitosis and for the timely exit from mitosis. In this review, we summarize the early studies that led to the identification of the important mitotic function of stathmin and discuss the present understanding of its role in the regulation of microtubules dynamics during cell-cycle progression. We also describe briefly other less mature avenues of investigation which suggest that stathmin may participate in other important biological functions and speculate about the future directions that research in this rapidly developing field may take.     

GSK3 regulates mitotic chromosomal alignment through CRMP4

Background

Glycogen Synthase Kinase 3 (GSK3) has been implicated in regulating chromosomal alignment and mitotic progression but the physiological substrates mediating these GSK3-dependent effects have not been identified. Collapsin Response Mediator Protein 4 (CRMP4) is a cytosolic phosphoprotein known to regulate cytoskeletal dynamics and is a known physiological substrate of GSK3. In this study, we investigate the role of CRMP4 during mitosis.

Methodology and Principal Findings

Here we demonstrate that during mitosis CRMP4 phosphorylation is regulated in a GSK3-dependent manner. We show that CRMP4 localizes to spindle microtubules during mitosis and loss of CRMP4 disrupts chromosomal alignment and mitotic progression. The effect of CRMP4 on chromosomal alignment is dependent on phosphorylation by GSK3 identifying CRMP4 as a critical GSK3 substrate during mitotic progression. We also provide mechanistic data demonstrating that CRMP4 regulates spindle microtubules consistent with its known role in the regulation of the microtubule cytoskeleton.

Conclusion and Significance

Our findings identify CRMP4 as a key physiological substrate of GSK3 in regulating chromosomal alignment and mitotic progression through its effect on spindle microtubules.     

Okadaic acid inhibits a protein phosphatase activity involved in formation of the mitotic spindle of GH4 rat pituitary cells.

Okadaic acid, a selective inhibitor of serine/threonine protein phosphatases, was utilized to investigate the requirement for phosphatases in cell cycle progression of GH4 rat pituitary cells. Okadaic acid inhibited GH4 cell proliferation in a concentration-dependent manner with a half-maximal inhibition (IC50) of approximately 5 nM. Treatment of GH4 cells with 10 nM okadaic acid resulted in a 40-60% decrease in phosphatase activity and an increase in the proportion of phosphorylated retinoblastoma (RB) protein. Cell cycle analysis indicated that okadaic acid increased the percentage of cells in G2-M, decreased proportionally the percentage of cells in G1 phase, and had little effect on the percentage of cells in S-phase. The absence of a change in the proportion of S-phase cells indicates that G1-specific phosphatases responsible for dephosphorylation of RB protein were not inhibited by 10 mM okadaic acid. Mitotic index revealed that 10 nM okadaic acid decreased proliferation of GH4 cells specifically by slowing the progression through mitosis. Immunostaining with anti-tubulin demonstrated that 10 nM okadaic acid-treated mitotic cells contained mitotic spindles; however, the spindle apparatus in these cells frequently contained multiple poles. These results suggest that the organization of spindle microtubules during prometaphase requires a protein phosphatase that is sensitive to nanomolar concentrations of okadaic acid. Chromosomes in 10 nM okadaic acid-treated cells appear to be attached to spindle microtubules and the nuclear envelope is absent. The effects of okadaic acid on the spindle differ from those elicited by the calcium channel blocker, nimodipine, indicating that this okadaic acid sensitive phosphatase is not part of the calcium signalling events which participate in mitotic progression.     

Stu1p is physically associated with beta-tubulin and is required for structural integrity of the mitotic spindle

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1-5 mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1-5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin-related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with beta-tubulin and identify the domains required for this interaction on both Stu1p and beta-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.     

Dual functions of Nicotiana benthamiana Rae1 in interphase and mitosis

Rae1 performs multiple functions in animal systems, acting in interphase as an mRNA export factor and during mitosis as a mitotic checkpoint and spindle assembly regulator. In this study we characterized multiple functions of Rae1 in plants. Virus-induced gene silencing of Nicotiana benthamiana Rae1 , NbRae1 , which encodes a protein with four WD40 repeats, resulted in growth arrest and abnormal leaf development. NbRae1 was mainly associated with the nuclear envelope during interphase, and NbRae1 deficiency caused accumulation of poly(A) RNA in the nuclei of leaf cells, suggesting defective mRNA export. In the shoot apex, depletion of NbRae1 led to reduced mitotic activities, accompanied by reduced cyclin-dependent kinase (CDK) activity and decreased expression of cyclin B1, CDKB1-1, and histones H3 and H4. The secondary growth of stem vasculature was also inhibited, indicating reduced cambial activities. Differentiated leaf cells of NbRae1 -silenced plants exhibited elevated ploidy levels. Immunolabeling in BY-2 cells showed that NbRae1 protein localized to mitotic microtubules and the cell plate-forming zone during mitosis, and recombinant NbRae1 directly bound to microtubules in vitro . Inhibition of NbRae1 expression in BY-2 cells using a β-estradiol-inducible RNAi system resulted in severe defects in spindle organization and chromosome alignment and segregation, which correlated with delays in cell cycle progression. Together, these results suggest that NbRae1 plays a dual role in mRNA export in interphase and in spindle assembly in mitosis.     

Expression and function of PAIRBP1 within gonadotropin-primed immature rat ovaries: PAIRBP1 regulation of granulosa and luteal cell viability

The protein PAIRBP1, which was initially referred to as RDA288, is involved in mediating the antiapoptotic action of progesterone (P4) in spontaneously immortalized granulosa cells (SIGCs). The present studies were designed to assess the expression and function of PAIRBP1 in the different cell types within the immature rat ovary. Western blot analysis detected PAIRBP1 within whole-cell lysates of immature rat ovaries. Equine gonadotropin (eCG) induced a 3-fold increase in ovarian levels of PAIRBP1. Moreover, human chorionic gonadotropin (hCG), given 48 h after eCG, maintained these elevated levels for up to 4 days. Immunohistochemical analysis confirmed this and further demonstrated that interstitial, thecal, and surface epithelial cells also expressed PAIRBP1. The level of PAIRBP1 in these cells was not influenced by gonadotropin treatment. In contrast, eCG stimulated an increase in PAIRBP1 within the granulosa cells of the developing follicles. Treatment with hCG induced ovulation and ultimately the formation of corpora lutea (CL). High levels of PAIRBP1 expression were also observed within the luteal cells. Immunocytochemical studies on living, nonpermeabilized granulosa and luteal cells revealed that some PAIRBP1 localized to the extracellular surface of these cells. The presence of PAIRBP1 on the extracellular surface was consistent with the observation that an antibody to PAIRBP1 attenuated P4's antiapoptotic action in both granulosa and luteal cells. Although the PAIRBP1 antibody attenuated P4's action, it did not reduce the capacity of cells to specifically bind (3)H-P4. Immunoprecipitation with the PAIRBP1 antibody pulled down the membrane P4 binding protein known as progesterone receptor membrane complex-1 (PGRMC1; rat homolog accession number AJ005837). Taken together, these findings suggest that gonadotropins regulate the expression of PAIRBP1 in granulosa and luteal cells and that PAIRBP1 plays an important role in mediating P4's antiapoptotic action in these ovarian cell types. The exact mechanism of PAIRBP1's action remains to be elucidated, but it may involve an interaction with PGRMC1.     

The Caenorhabditis elegans microtubule-severing complex MEI-1/MEI-2 katanin interacts differently with two superficially redundant beta-tubulin isotypes

The microtubule-severing protein complex katanin is required for a variety of important microtubule-base morphological changes in both animals and plants. Caenorhabditis elegans katanin is encoded by the mei-1 and mei-2 genes and is required for oocyte meiotic spindle formation and must be inactivated before the first mitotic cleavage. We identified a mutation, sb26, in the tbb-2 beta-tubulin gene that partially inhibits MEI-1/MEI-2 activity: sb26 rescues lethality caused by ectopic MEI-1/MEI-2 expression during mitosis, and sb26 increases meiotic defects in a genetic background where MEI-1/MEI-2 activity is lower than normal. sb26 does not interfere with MEI-1/MEI-2 microtubule localization, suggesting that this mutation likely interferes with severing. Tubulin deletion alleles and RNA-mediated interference revealed that TBB-2 and the other germline enriched beta-tubulin isotype, TBB-1, are redundant for embryonic viability. However, limiting MEI-1/MEI-2 activity in these experiments revealed that MEI-1/MEI-2 preferentially interacts with TBB-2-containing microtubules. Our results demonstrate that these two superficially redundant beta-tubulin isotypes have functionally distinct roles in vivo.