Zinc plays both physiological and pathological roles in biology, making it of increasing interest. To date, intracellular free zinc has been measured in cell types supplemented with or enriched in zinc, such as hippocampal neurons. Here we quantitatively image intracellular exchangeable zinc in an ordinary resting cell culture line (PC-12), using an excitation ratiometric fluorescent biosensor based on carbonic anhydrase (CA). Human CA II has a K d of 4 pM for zinc and suffers no interference from millimolar calcium or magnesium ions. The CA-based biosensor was readily introduced into the cell by a novel approach: fusing a transactivator of transcription (TAT)-derived cell penetrating peptide to the CA molecule and adding it to the cells. Our results indicate that the resting concentration is approximately 5-10 pM in cytoplasm and nucleus. Interestingly, the tetrakis(2-pyridylmethyl)ethylenediamine (TPEN)-Zn complex and TPEN are both apoptogenic for this cell line.

The zinc(II) ion has recently been implicated in a number of novel functions and pathologies in loci as diverse as the brain, retina, small intestine, prostate, heart, pancreas and immune system. Zinc ions are a required nutrient but elevated concentrations are known to kill cells in vitro. Paradoxical observations regarding zinc's effects have appeared frequently in the literature, and often their physiological relevance is unclear. We found that for PC-12, HeLa and HT-29 cell lines as well as primary cultures of cardiac myocytes and neurons in vitro in differing media, approximately 5 nmol/ L free zinc (pZn = 8.3, where pZn is defined as − log 10 [free Zn 2+ ]) produced apparently healthy cells, but 20-fold higher or (in one case) lower concentrations were usually harmful as judged by multiple criteria. These results indicate that (1) the free zinc ion levels of media should be controlled with a metal ion buffer; (2) adding zinc or strong zinc ligands to an insufficiently buffered medium may lead to unpredictably low or high free zinc levels that are often harmful to cells; and (3) it is generally desirable to measure free zinc ion levels due to the presence of contaminating zinc in many biochemicals and unknown buffering capacity of many media.

Group B coxsackieviruses (CVB) are associated with viral-induced heart disease and are among the leading causes of aseptic meningitis worldwide. Here we show that CVB entry into polarized brain microvasculature and aortic endothelial cells triggers a depletion of intracellular calcium stores initiated through viral attachment to the apical attachment factor decay-accelerating factor. Calcium release was dependent upon a signaling cascade that required the activity of the Src family of tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3. CVB-mediated calcium release was required for the activation of calpain-2, a calcium-dependent cysteine protease, which controlled the vesicular trafficking of internalized CVB particles. These data point to a specific role for calcium signaling in CVB entry into polarized endothelial monolayers and highlight the unique signaling mechanisms used by these viruses to cross endothelial barriers.

Summary Enteroviruses, including coxsackievirus B (CVB) and poliovirus (PV), can access the CNS through the blood brain barrier (BBB) endothelium to cause aseptic meningitis. To identify cellular components required for CVB and PV infection of human brain microvascular endothelial cells, an in vitro BBB model, we performed comparative RNAi screens and identified 117 genes that influenced infection. Whereas a large proportion of genes whose depletion enhanced infection (17 of 22) were broadly anti-enteroviral, only 46 of the 95 genes whose depletion inhibited infection were required by both CVB and PV and included components of cell signaling pathways such as adenylate cyclases. Downregulation of genes including Rab GTPases, Src tyrosine kinases, and tyrosine phosphatases, displayed specificity in their requirement for either CVB or PV infection. These findings highlight the pathways hijacked by enteroviruses for entry and replication in the BBB endothelium, a specialized and clinically relevant cell type for these viruses.