The Road from Marja: Wounded in Afghanistan, Capt. Erik Quist '99, USMC, sees another side of combat

Marine Corps Capt. Erik Quist ’99 had fought in two wars. Now he fights again as he recovers from severe wounds suffered in Afghanistan.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.

The Cytoplasmic Chaperone Hsp104 Is Required for Conformational Repair of Heat-denatured Proteins in the Yeast Endoplasmic Reticulum

Severe heat stress causes protein denaturation in various cellular compartments. If Saccharomyces cerevisiae cells grown at 24°C are preconditioned at 37°C, proteins denatured by subsequent exposure to 48–50°C can be renatured when the cells are allowed to recover at 24°C. Conformational repair of vital proteins is essential for survival, because gene expression is transiently blocked after the thermal insult. Refolding of cytoplasmic proteins requires the Hsp104 chaperone, and refolding of lumenal endoplasmic reticulum (ER) proteins requires the Hsp70 homologue Lhs1p. We show here that conformational repair of heat-damaged glycoproteins in the ER of living yeast cells required functional Hsp104. A heterologous enzyme and a number of natural yeast proteins, previously translocated and folded in the ER and thereafter denatured by severe heat stress, failed to be refolded to active and secretion-competent structures in the absence of Hsp104 or when an ATP-binding site of Hsp104 was mutated. During recovery at 24°C, the misfolded proteins persisted in the ER, although the secretory apparatus was fully functional. Hsp104 appears to control conformational repair of heat-damaged proteins even beyond the ER membrane.