Background Cytokinesis in many eukaryotes involves the function of an actomyosin-based contractile ring. In fission yeast, actomyosin ring maturation and stability require a conserved signaling pathway termed the SIN (septation initiation network). The SIN consists of a GTPase (Spg1p) and three protein kinases, all of which localize to the mitotic spindle pole bodies (SPBs). Two of the SIN kinases, Cdc7p and Sid1p, localize asymmetrically to the newly duplicated SPB in late anaphase. How this asymmetry is achieved is not understood, although it is known that their symmetric localization impairs cytokinesis. Results Here we characterize a new Forkhead-domain-associated protein, Csc1p, and identify SIN-Inhibitory PP2A-complex (SIP), which is crucial for the establishment of SIN asymmetry. Csc1p localizes to both SPBs early in mitosis, is lost from the SPB that accumulates Cdc7p, and instead accumulates at the SPB lacking Cdc7p. Csc1p is required for the dephosphorylation of the SIN scaffolding protein Cdc11p and is thereby required for the recruitment of Byr4p, a component of the GTPase-activating subunit for Spg1p, to the SPB. Conclusions Since Cdc7p does not bind to GDP-Spg1p, we propose that the SIP-mediated Cdc11p-dephosphorylation and the resulting recruitment of Byr4p are amongst the earliest steps in the establishment of SIN asymmetry.

Proteomic, localization, and enzymatic activity screens in fission yeast reveal how deubiquitinating enzyme localization and function are tuned.

Transition between epithelial and mesenchymal states is a feature of both normal development and tumor progression. We report that expression of chloride channel accessory protein hCLCA2 is a characteristic of epithelial differentiation in the immortalized MCF10A and HMLE models, while induction of EMT by cell dilution, TGFbeta, or mesenchymal transcription factors sharply reduces hCLCA2 levels. Attenuation of hCLCA2 expression by lentiviral shRNA caused cell overgrowth and focus formation, enhanced migration and invasion, and increased mammosphere formation in methylcellulose. These changes were accompanied by downregulation of E-cadherin and upregulation of mesenchymal markers such as vimentin and fibronectin. Moreover, hCLCA2 expression is greatly downregulated in breast cancer cells with a mesenchymal or claudin-low profile. These observations suggest that loss of hCLCA2 may promote metastasis. We find that higher-than-median expression of hCLCA2 is associated with a one-third lower rate of metastasis over an 18 year period among breast cancer patients compared to lower-than-median (n=344, unfiltered for subtype). Thus, hCLCA2 is required for epithelial differentiation, and its loss during tumor progression contributes to metastasis. Overexpression of hCLCA2 has been reported to inhibit cell proliferation and is accompanied by increases in chloride current at the plasma membrane and reduced intracellular pH (pHi). We found that knockdown cells have sharply reduced chloride current and higher pHi, both characteristics of tumor cells. These results suggest a mechanism for the effects on differentiation. Loss of hCLCA2 may allow escape from pHi homeostatic mechanisms, permitting the higher intracellular and lower extracellular pH that are characteristic of aggressive tumor cells.