Validation of high-throughput single cell analysis methodology.

High-throughput quantitative polymerase chain reaction (qPCR) approaches enable profiling of multiple genes in single cells, bringing new insights to complex biological processes and offering opportunities for single cell-based monitoring of cancer cells and stem cell-based therapies. However, workflows with well-defined sources of variation are required for clinical diagnostics and testing of tissue-engineered products. In a study of neural stem cell lines, we investigated the performance of lysis, reverse transcription (RT), preamplification (PA), and nanofluidic qPCR steps at the single cell level in terms of efficiency, precision, and limit of detection. We compared protocols using a separate lysis buffer with cell capture directly in RT-PA reagent. The two methods were found to have similar lysis efficiencies, whereas the direct RT-PA approach showed improved precision. Digital PCR was used to relate preamplified template copy numbers to Cq values and reveal where low-quality signals may affect the analysis. We investigated the impact of calibration and data normalization strategies as a means of minimizing the impact of inter-experimental variation on gene expression values and found that both approaches can improve data comparability. This study provides validation and guidance for the application of high-throughput qPCR workflows for gene expression profiling of single cells.

Instability of 8E5 calibration standard revealed by digital PCR risks inaccurate quantification of HIV DNA in clinical samples by qPCR.

ABTRACT: Establishing a cure for HIV is hindered by the persistence of latently infected cells which constitute the viral reservoir. Real-time qPCR, used for quantification of this reservoir by measuring HIV DNA, requires external calibration; a common choice of calibrator is the 8E5 cell line, which is assumed to be stable and to contain one HIV provirus per cell. In contrast, digital PCR requires no external calibration and potentially provides 'absolute' quantification. We compared the performance of qPCR and dPCR in quantifying HIV DNA in 18 patient samples. HIV DNA was detected in 18 by qPCR and in 15 by dPCR, the difference being due to the smaller sample volume analysed by dPCR. There was good quantitative correlation (R2 = 0.86) between the techniques but on average dPCR values were only 60% of qPCR values. Surprisingly, investigation revealed that this discrepancy was due to loss of HIV DNA from the 8E5 cell calibrant. 8E5 extracts from two other sources were also shown to have significantly less than one HIV DNA copy per cell and progressive loss of HIV from 8E5 cells during culture was demonstrated. We therefore suggest that the copy number of HIV in 8E5 extracts be established by dPCR prior to use as calibrator.

Recruitment of Cdc20 to the kinetochore requires BubR1 but not Mad2 in Drosophila melanogaster.

To prevent aneuploidy, cells require a mitotic surveillance mechanism, the spindle assembly checkpoint (SAC). The SAC prevents metaphase/anaphase transition by blocking the ubiquitylation and destruction of cyclin B and securin via the Cdc20-activated anaphase-promoting complex or cyclosome (APC/C)-mediated proteolysis pathway. This checkpoint involves the kinetochore proteins Mad2, BubR1, and Cdc20. Mad2 and BubR1 are inhibitors of the APC/C, but Cdc20 is an activator. Exactly how the SAC regulates Cdc20 via unattached kinetochores remains unclear; in vertebrates, most current models suggest that kinetochore-bound Mad2 is required for initial binding to Cdc20 to form a stable complex that includes BubR1. Here, we show that the Mad2 kinetochore dimerization recruitment mechanism is conserved and that the recruitment of Cdc20 to kinetochores in Drosophila requires BubR1 but not Mad2. BubR1 and Mad2 can bind to Cdc20 independently, and the interactions are enhanced after cells are arrested at mitosis by the depletion of Cdc27 using RNA interference (RNAi) in S2 cells or by MG132 treatment in syncytial embryos. These findings offer an explanation of why BubR1 is more important than Mad2 for SAC function in flies. These findings could lead to a better understanding of vertebrate SAC mechanisms.

Cdk1 phosphorylation sites on Cdc27 are required for correct chromosomal localisation and APC/C function in syncytial Drosophila embryos.

Anaphase-promoting complex or cyclosome (APC/C) controls the metaphase-to-anaphase transition and mitosis exit by triggering the degradation of key cell cycle regulators such as securin and B-type cyclins. However, little is known about the functions of individual APC/C subunits and how they might regulate APC/C activity in space and time. Here, we report that two potential Cdk1 kinase phosphorylation sites are required for the chromosomal localisation of GFP::Cdc27 during mitosis. Either or both of the highly conserved proline residues in the Cdk1 phosphorylation consensus sequence motifs were mutated to alanine (Cdc27 P304A or P456A). The singly mutated fusion proteins, GFP::Cdc27P304A and GFP::Cdc27P456A, can still localise to mitotic chromosomes in a manner identical to wild-type GFP::Cdc27 and are functional in that they can rescue the phenotype of the cdc27L7123 mutant in vivo. However, when both of the Cdk1 phosphorylation sequence motifs were mutated, the resulting GFP::Cdc27P304A,P456A construct was not localised to the chromosomes during mitosis and was no longer functional, as it failed to rescue mutant phenotypes of the cdc27L7123 gene. High levels of cyclin B and cyclin A were detected in mutant third instar larvae brain samples compared with its wild-type control. These results show for the first time that the two potential Cdk1 phosphorylation sites on Drosophila Cdc27 are required for its chromosomal localisation during mitosis and imply that these localisations specific to Cdc27 are crucial for APC/C functions.

The Src-selective kinase inhibitor PP1 also inhibits Kit and Bcr-Abl tyrosine kinases.

4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]- pyrimidine (PP1) was identified as an Src-selective tyrosine kinase inhibitor and has been used extensively to investigate signaling pathways involving Src kinases, including events downstream of the stem cell factor (SCF) receptor c-Kit. While investigating the role of Src kinases in SCF signaling, we found that PP1 completely abrogated the proliferation of M07e cells in response to SCF. PP1 inhibited SCF-induced c-Kit autophosphorylation in intact cells and blocked the activation of mitogen-activated protein kinase and Akt. In vitro kinase assays using immunoprecipitated c-Kit confirmed direct inhibition by PP1. SCF-induced c-Kit phosphorylation was also inhibited by the related inhibitor 4-amino-5- (4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP2) and by STI571 but not by the Src inhibitor SU6656. PP1 inhibited the activity of mutant constitutively active forms of c-Kit (D814V and D814Y) found in mast cell disorders, and triggered apoptosis in the rat basophilic leukemia cell line RBL-2H3 that expresses mutant c-Kit. In addition, PP1 (and PP2) inhibited the in vitro kinase activity and autophosphorylation in whole cells of p210 Bcr-Abl. PP1 reduced the constitutive activation of signal transducer and activators of transcription 5 and mitogen-activated protein kinase and triggered apoptosis in FDCP1 cells expressing Bcr-Abl. These results have implications for the use of PP1 in investigating intracellular signaling and suggest that PP1 or related compounds may be useful in the treatment of malignant diseases associated with dysregulated c-Kit or Abl tyrosine kinase activity.