Microtubule Nucleation at Non-Spindle Pole Body Microtubule-Organizing Centers Requires Fission Yeast Centrosomin-Related Protein mod20p

AbstractBackground: Many types of differentiated eukaryotic cells display microtubule distributions consistent with nucleation from noncentrosomal intracellular microtubule organizing centers (MTOCs), although such structures remain poorly characterized. In fission yeast, two types of MTOCs exist in addition to the spindle pole body, the yeast centrosome equivalent. These are the equatorial MTOC, which nucleates microtubules from the cell division site at the end of mitosis, and interphase MTOCs, which nucleate microtubules from multiple sites near the cell nucleus during interphase.Results: From an insertional mutagenesis screen we identified a novel gene, mod20+, which is required for microtubule nucleation from non-spindle pole body MTOCs in fission yeast. Mod20p is not required for intranuclear mitotic spindle assembly, although it is required for cytoplasmic astral microtubule growth during mitosis. Mod20p localizes to MTOCs throughout the cell cycle and is also dynamically distributed along microtubules themselves. We find that mod20p is required for the localization of components of the γ-tubulin complex to non-spindle pole body MTOCs and physically interacts with the γ-tubulin complex in vivo. Database searches reveal a family of eukaryotic proteins distantly related to mod20p; these are found in organisms ranging from fungi to mammals and include Drosophila centrosomin.Conclusions: Mod20p appears to act by recruiting components of the γ-tubulin complex to non-spindle pole body MTOCs. The identification of mod20p-related proteins in higher eukaryotes suggests that this may represent a general mechanism for the organization of noncentrosomal MTOCs in eukaryotic cells

Fission yeast mod5p regulates polarized growth through anchoring of tea1p at cell tips

Microtubules have a central role in eukaryotic cell polarity1, in part through interactions between microtubule end-binding proteins and the cell cortex2,3. In the fission yeast Schizosaccharomyces pombe, microtubules and the polarity modulator tea1p maintain cylindrical cell shape and strictly antipodal cell growth4–7. The tea1p protein is transported to cell tips by association with growing microtubule plus ends8; once at cell tips, tea1p releases from microtubule ends and associates with the cell cortex, where it coordinates polarized growth4,6. Here we describe a cortical protein, mod5p, that regulates the dynamic behaviour of tea1p. In mod5D cells, tea1p is efficiently transported on microtubules to cell tips but fails to anchor properly at the cortex and thus fails to accumulate to normal levels. mod5p contains a signal for carboxy-terminal prenylation and in wildtype cells is associated with the plasma membrane at cell tips. However, in tea1D cells, although mod5p remains localized to the plasma membrane, mod5p is no longer restricted to the cell tips. We propose that tea1p and mod5p act in a positive-feedback loop in the microtubule-mediated regulation of cell polarity

Double-blind, 12 month follow-up, placebo-controlled trial of mifepristone on cognition in alcoholics: the MIFCOG trial protocol

Background: Increased levels of cortisol during acute alcohol withdrawal have been linked to cognitive deficits and depression. Preclinical research found that the glucocorticoid Type II receptor antagonist, mifepristone, prevented some of the neurotoxic effects of withdrawal and memory loss. Clinical trials have shown mifepristone effective in the treatment of depression. This study aims to examine the extent to which the glucocorticoid Type II receptor antagonist, mifepristone, when given to alcohol dependent males during the acute phase of alcohol withdrawal, will protect against the subsequent memory loss and depressive symptoms during abstinence from alcohol. Methods/Design: The study is a Phase 4 therapeutic use, “Proof of Concept” trial. The trial is a double-blind randomised controlled clinical trial of mifepristone versus inactive placebo. The trial aims to recruit 120 participants referred for an inpatient alcohol detoxification from community alcohol teams, who meet the inclusion criteria; 1) Male, 2) Aged 18–60 inclusive, 3) alcohol dependent for 5 or more years. A screening appointment will take place prior to admission to inpatient alcohol treatment units to ensure that the individual is suitable for inclusion in the trial in accordance with the inclusion and exclusion criteria. On admission participants are randomised to receive 600 mg a day of mifepristone (200 mg morning, afternoon and evening) for 7 days and 400 mg for the subsequent 7 days (200 mg morning and evening) or the equivalent number of placebo tablets for 14 days. Participants will remain in the trial for 4 weeks (at least 2 weeks as an inpatient) and will be followed up at 3, 6 and 12 months post randomisation. Primary outcome measures are cognitive function at week 3 and 4 after cessation of drinking and symptoms of depression over the 4 weeks after cession of drinking, measured using the Cambridge Neuropsychological Test Automated battery and Beck Depression Inventory, respectively. Secondary outcome measures are severity of the acute phase of alcohol withdrawal, alcohol craving, symptoms of protracted withdrawal and maintenance of abstinence and levels of relapse drinking at follow-up. Discussion: The current trial will provide evidence concerning the role of glucocorticoid Type II receptor activation in cognitive function and depression during acute alcohol withdrawal and the efficacy of treatment with mifepristone

Supplementary information for Snaith, H. A. et al. Fission yeast mod5p regulates polarized growth through anchoring of tea1p at cell tips. Nature 423, 647-651 (2003)

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Fission Yeast mto2p Regulates Microtubule Nucleation by the Centrosomin-related Protein mto1p

From an insertional mutagenesis screen, we isolated a novel gene, mto2+, involved in microtubule organization in fission yeast. mto2Δ strains are viable but exhibit defects in interphase microtubule nucleation and in formation of the postanaphase microtubule array at the end of mitosis. The mto2Δ defects represent a subset of the defects displayed by cells deleted for mto1+ (also known as mod20+ and mbo1+), a centrosomin-related protein required to recruit the γ-tubulin complex to cytoplasmic microtubule-organizing centers (MTOCs). We show that mto2p colocalizes with mto1p at MTOCs throughout the cell cycle and that mto1p and mto2p coimmunoprecipitate from cytoplasmic extracts. In vitro studies suggest that mto2p binds directly to mto1p. In mto2Δ mutants, although some aspects of mto1p localization are perturbed, mto1p can still localize to spindle pole bodies and the cell division site and to “satellite” particles on interphase microtubules. In mto1Δ mutants, localization of mto2p to all of these MTOCs is strongly reduced or absent. We also find that in mto2Δ mutants, cytoplasmic forms of the γ-tubulin complex are mislocalized, and the γ-tubulin complex no longer coimmunoprecipitates with mto1p from cell extracts. These experiments establish mto2p as a major regulator of mto1p-mediated microtubule nucleation by the γ-tubulin complex

Multistep and multimode cortical anchoring of tea1p at cell tips in fission yeast

The fission yeast cell-polarity regulator tea1p is targeted to cell tips by association with growing microtubule ends. Tea1p is subsequently anchored at the cell cortex at cell tips via an unknown mechanism that requires both the tea1p carboxy-terminus and the membrane protein mod5p. Here, we show that a tea1p-related protein, tea3p, binds independently to both mod5p and tea1p, and that tea1p and mod5p can also interact directly, independent of tea3p. Despite their related structures, different regions of tea1p and tea3p are required for their respective interactions with an essential central region of mod5p. We demonstrate that tea3p is required for proper cortical localization of tea1p, specifically at nongrowing cell tips, and that tea1p and mod5p are independently required for tea3p localization. Further, we find that tea3p fused to GFP or mCherry is cotransported with tea1p by microtubules to cell tips, but this occurs only in the absence of mod5p. These results suggest that independent protein–protein interactions among tea1p, tea3p and mod5p collectively contribute to tea1p anchoring at cell tips via a multistep and multimode mechanism

A Catalytic Role for Mod5 in the Formation of the Tea1 Cell Polarity Landmark

Many systems regulating cell polarity involve stable landmarks defined by internal cues [1–5]. In the rod-shaped fission yeast Schizosaccharomyces pombe, microtubules regulate polarized vegetative growth via a landmark involving the protein Tea1 [6–9]. Tea1 is delivered to cell tips as packets of molecules associated with growing microtubule ends [10] and anchored at the plasma membrane via a mechanism involving interaction with the membrane protein Mod5 [11, 12]. Tea1 and Mod5 are highly concentrated in clusters at cell tips in a mutually dependent manner, but how the Tea1-Mod5 interaction contributes mechanistically to generating a stable landmark is not understood. Here, we use live-cell imaging, FRAP, and computational modeling to dissect dynamics of the Tea1-Mod5 interaction. Surprisingly, we find that Tea1 and Mod5 exhibit distinctly different turnover rates at cell tips. Our data and modeling suggest that rather than acting simply as a Tea1 receptor or as a molecular “glue” to retain Tea1, Mod5 functions catalytically to stimulate incorporation of Tea1 into a stable tip-associated cluster network. The model also suggests an emergent self-focusing property of the Tea1-Mod5 cluster network, which can increase the fidelity of polarized growth