The yeast P5 type ATPase, Spf1, regulates manganese transport into the endoplasmic reticulum

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn2+ in ∆spf1 cells and an increase following it’s overexpression. In agreement with the observed loss of luminal Mn2+ we could observe concurrent reduction in many Mn2+-related process in the ER lumen. Conversely, cytosolic Mn2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn2+-dependent neurological disorders

Whole-genome single-cell copy number profiling from formalin-fixed paraffin-embedded samples

A substantial proportion of tumors consist of genotypically distinct subpopulations of cancer cells. This intratumor genetic heterogeneity poses a substantial challenge for the implementation of precision medicine. Single-cell genomics constitutes a powerful approach to resolve complex mixtures of cancer cells by tracing cell lineages and discovering cryptic genetic variations that would otherwise be obscured in tumor bulk analyses. Because of the chemical alterations that result from formalin fixation, single-cell genomic approaches have largely remained limited to fresh or rapidly frozen specimens. Here we describe the development and validation of a robust and accurate methodology to perform whole-genome copy-number profiling of single nuclei obtained from formalin-fixed paraffin-embedded clinical tumor samples. We applied the single-cell sequencing approach described here to study the progression from in situ to invasive breast cancer, which revealed that ductal carcinomas in situ show intratumor genetic heterogeneity at diagnosis and that these lesions may progress to invasive breast cancer through a variety of evolutionary processes