PDH-HT dots in PC somata expressing WT cPKC-GFP. These results suggest that mutant cPKC inhibits both basal and induced CMA activity in primary cultured PCs. February 2012 | Volume 7 | Issue 2 | e31232 A Novel Method to Evaluate CMA in a Single Neuron Discussion In contrast to macroautophagy, it remains unclear how CMA activity is regulated and how CMA relates to various physiological functions, even though several molecules involved in CMA have been discovered. While macroautophagy activity can be easily assessed by the amount and localization of LC3-II, existing methods for evaluating CMA activity require special lysosome isolation techniques or radioisotopic analysis. In addition, these methods only reflect CMA activity for a large cell population, which has hampered a detailed understanding of CMA. In the present study, we succeeded in establishing a novel method to monitor CMA at a single-cell level. Our method enables simple and real-time observation of CMA activity in single cells and may represent a breakthrough for elucidating the regulation of CMA and the role of CMA in normal cellular functions and in disease pathogenesis. We believe that GAPDH-HT accumulates in lysosome since dots of GAPDH-HT colocalize with LAMP2 immunoreactivity and LysoTracker red fluorescence. February 2012 | Volume 7 | Issue 2 | e31232 A Novel Method to Evaluate CMA in a Single Neuron However, LAMP2A is frequently used as a marker of both late endosomes and lysosomes, and LysoTracker stains acidic organelles including late endosomes and lysosomes. Therefore, we cannot exclude the possibility that GAPDH-HT accumulates in late endosomes as well as lysosomes. Recently, it has been demonstrated that late endosomes are involved in mammalian microautophagy, in which the delivery of cytosolic substrates to late endosomes is mediated by Hsc70, but not by LAMP2A. Therefore, GAPDH-HT might partly accumulate in late endosomes via microautophagy, which is supported by the result that siRNA-mediated knockdown of LAMP2A did not completely inhibit the dot-like accumulation of GAPDH-HT. However, because this dot-like accumulation was highly sensitive to chemicals that affect 17062696” CMA activity, CMA is likely more responsible for this accumulation than Hsc70mediated microautophagy. Reciprocal crosstalk has been reported between CMA and macroautophagy; when one is impaired, the other is Elesclomol activated to compensate. Similarly, in ” the present study siRNAmediated LAMP2A knockdown activated macroautophagy. However, it remains unclear how CMA blockage activates macroautophagy. Ubiquilin was identified as a candidate to regulate this crosstalk; it is involved in the formation of autophagosomes in macroautophagy and is degraded both by macroautophagy and by CMA. Blockage of CMA would increase the amount of ubiquilin, leading to the activation of macroautophagy. In the present study, we showed 
that the amount of the Atg5-Atg12 complex, which is essential for autophagosome formation, was elevated by siRNA-mediated LAMP2A knockdown, suggesting that this complex is involved in the crosstalk between macroautophagy and CMA. Unlike ubiquilin, Atg5 and Atg12 are not substrates for CMA since they do not have the KFERQ-like motif required for recognition by Hsc70 and degradation through CMA. In this scenario, CMA would affect macroautophagy by regulating other proteins involved in Atg5 and Atg12 expression, degradation or complex formation. In the present study, we evaluated CMA activity in HeLa c
