Polo-like kinase acts as a molecular timer that safeguards the asymmetric fate of spindle microtubule-organizing centers.

The microtubules that form the mitotic spindle originate from microtubule-organizing centers (MTOCs) located at either pole. After duplication, spindle MTOCs can be differentially inherited during asymmetric cell division in organisms ranging from yeast to humans. Problems with establishing predetermined spindle MTOC inheritance patterns during stem cell division have been associated with accelerated cellular aging and the development of both cancer and neurodegenerative disorders. Here, we expand the repertoire of functions Polo-like kinase family members fulfill in regulating pivotal cell cycle processes. We demonstrate that the Plk1 homolog Cdc5 acts as a molecular timer that facilitates the timely and sequential recruitment of two key determinants of spindle MTOCs distribution, that is the γ-tubulin complex receptor Spc72 and the protein Kar9, and establishes the fate of these structures, safeguarding their asymmetric inheritance during Saccharomyces cerevisiae mitosis.

Asymmetric cell division and replicative aging: a new perspective from the spindle poles.

Although cell division is usually portrayed as an equitable process by which a progenitor cell originates two identical daughter cells, there are multiple examples of asymmetric divisions that generate two cells that differ in their content, morphology and/or proliferative potential. The capacity of the cells to generate asymmetry during their division is of paramount biological relevance, playing essential roles during embryonic development, cellular regeneration and tissue morphogenesis. Problems with the proper establishment of asymmetry and polarity during cell division can give rise to cancer and neurodevelopmental disorders, as well as to also accelerate cellular aging. Interestingly, the microtubule organizing centers that orchestrate the formation of the mitotic spindle have been described among the cellular structures that can be differentially allocated during asymmetric cell divisions. This mini-review focuses on recent research from our group and others uncovering a role for the non-random distribution of the spindle-associated microtubule organizing centers in the differential distribution of aging factors during asymmetric mitoses and therefore in the maintenance of the replicative lifespan of the cells.

Nuclear proteasomal degradation of Saccharomyces cerevisiae inorganic pyrophosphatase Ipp1p, a nucleocytoplasmic protein whose stability depends on its subcellular localization.

Inorganic pyrophosphate (PPi) is an abundant by-product of cellular metabolism. PPi-producing reactions take place in the nucleus concurrently with reactions that use PPi as a substrate. Saccharomyces cerevisiae possesses two soluble pyrophosphatases (sPPases): Ipp1p, an essential and allegedly cytosolic protein, and Ipp2p, a mitochondrial isoenzyme. However, no sPPase has yet been unambiguously described in the nucleus. In vivo studies with fluorescent fusions together with activity and immunodetection analyses demonstrated that Ipp1p is a nucleocytoplasmic protein. Mutagenesis analysis showed that this sPPase possesses a nuclear localization signal which participates in its nuclear targeting. Enforced nucleocytoplasmic targeting by fusion to heterologous nuclear import and export signals caused changes in polypeptide abundance and activity levels, indicating that Ipp1p is less stable in the nucleus that in the cytoplasm. Low nuclear levels of this sPPase are physiologically relevant and may be related to its catalytic activity, since cells expressing a functional nuclear-targeted chimaera showed impaired growth and reduced chronological lifespan, while a nuclear-targeted catalytically inactive protein was not degraded and accumulated in the nucleus. Moreover, nuclear proteasome inhibition stabilized Ipp1p whereas nuclear targeting promoted its ubiquitination and interaction with Ubp3p, a component of the ubiquitin-proteasome system. Overall, our results indicate that Ipp1p is nucleocytoplasmic, that its stability depends on its subcellular localization and that sPPase catalytic competence drives its nuclear degradation through the ubiquitin-proteasome system. This suggests a new scenario for PPi homeostasis where both nucleocytoplasmic transport and nuclear proteasome degradation of the sPPase should contribute to control nuclear levels of this ubiquitous metabolite.

Manganese redistribution by calcium-stimulated vesicle trafficking bypasses the need for P-type ATPase function.

Regulation of intracellular ion homeostasis is essential for eukaryotic cell physiology. An example is provided by loss of ATP2C1 function, which leads to skin ulceration, improper keratinocyte adhesion, and cancer formation in Hailey-Hailey patients. The yeast ATP2C1 orthologue PMR1 codes for a Mn(2+)/Ca(2+) transporter that is crucial for cis-Golgi manganese supply. Here, we present evidence that calcium overcomes the lack of Pmr1 through vesicle trafficking-stimulated manganese delivery and requires the endoplasmic reticulum Mn(2+) transporter Spf1 and the late endosome/trans-Golgi Nramp metal transporter Smf2. Smf2 co-localizes with the putative Mn(2+) transporter Atx2, and ATX2 overexpression counteracts the beneficial impact of calcium treatment. Our findings suggest that vesicle trafficking promotes organelle-specific ion interchange and cytoplasmic metal detoxification independent of calcineurin signaling or metal transporter re-localization. Our study identifies an alternative mode for cis-Golgi manganese supply in yeast and provides new perspectives for Hailey-Hailey disease treatment.