Autophagy plays a central role in regulating important cellular functions such as cell survival during starvation and control of infectious pathogens. Recently, it has been shown that autophagy can induce cells to die; however, the mechanism of the autophagic cell death program is unclear. We now show that caspase inhibition leading to cell death by means of autophagy involves reactive oxygen species (ROS) accumulation, membrane lipid oxidation, and loss of plasma membrane integrity. Inhibition of autophagy by chemical compounds or knocking down the expression of key autophagy proteins such as ATG7, ATG8, and receptor interacting protein (RIP) blocks ROS accumulation and cell death. The cause of abnormal ROS accumulation is the selective autophagic degradation of the major enzymatic ROS scavenger, catalase. Caspase inhibition directly induces catalase degradation and ROS accumulation, which can be blocked by autophagy inhibitors. These findings unveil a molecular mechanism for the role of autophagy in cell death and provide insight into the complex relationship between ROS and nonapoptotic programmed cell death.apoptosis ͉ necrosis ͉ nonapoptotic ͉ reactive oxygen species P rogrammed cell death plays an important role in embryonic development, immune regulation, and general cellular homeostasis (1-3). Apoptosis, the most thoroughly studied form of programmed cell death is defined by dependence on a family of proteases known as caspases. Activation of caspases leads to distinct morphological features in the cell, such as nuclear condensation, membrane blebbing, and cell shrinkage (4). Several studies have identified cell death programs that are clearly distinct from apoptosis (5, 6). These nonapoptotic cell death programs are genetically regulated and often have morphological features resembling necrosis, yet their underlying molecular mechanisms are unclear. Molecular insights into alternative cell death programs could lead to a better understanding of their role in normal cellular homeostasis as well as disease processes.Autophagy is a cellular process that causes degradation of long-lived proteins and recycling of cellular components to ensure survival during starvation. During autophagy, cells exhibit extensive internal membrane remodeling, engulfing portions of the cytoplasm in large double-membrane vesicles. These autophagosomes dock and fuse with lysosomes, and the contents of the fusion vacuoles are eventually degraded (7). A group of genes known as ATG genes, which are conserved from yeast to humans, have been found to regulate autophagy (8). Genetic analyses in yeast, and recently in higher eukaryotes, have shown that autophagy plays a central role in the regulation of cell survival during nutritional deprivation (9, 10). In addition, autophagy has been shown to play a role in tumor suppression (11, 12), pathogen control (13), antigen presentation (14), and the regulation of organismal lifespan (15).The involvement of autophagy in programmed cell death has been controversial. Autophagy has long been obs...

We describe quantitative imaging of the sheet resistance of metallic thin films by monitoring frequency shift and quality factor in a resonant scanning near-field microwave microscope. This technique allows fast acquisition of images at approximately 10 ms per pixel over a frequency range from 0.1 to 50 GHz. In its current configuration, the system can resolve changes in sheet resistance as small as 0.6 Ω/✷ for 100 Ω/✷ films. We demonstrate its use at 7.5 GHz by generating a quantitative sheet resistance image of a YBa2Cu3O 7−δ thin film on a 5 cm-diameter sapphire wafer.

We describe near-field imaging of sample sheet resistance via frequency shifts in a resonant coaxial scanning microwave microscope. The frequency shifts are related to local sample properties, such as surface resistance and dielectric constant. We use a feedback circuit to track a given resonant frequency, allowing measurements with a sensitivity to frequency shifts as small as two parts in 10 6 for a 30 ms sampling time. The frequency shifts can be converted to sheet resistance based on a simple model of the system.There is a growing need to develop non-destructive microscopy techniques to quantitatively measure the microwave properties of materials on a length scale much less than the free space wavelength. For example, spatially resolved measurements of complex conductivity would be of significant utility for evaluating oxide superconducting and ferroelectric thin film samples. Sensitivity to microwave and millimeter wave surface resistance and dielectric constant have been previously demonstrated; for example, Bryant and Gunn 1 used a coaxial resonator to measure semiconductor resistivities on a 1 mm length scale. Waveguides 2 and coaxial geometries 3−7 have also been used to image conductivity and dielectric constant contrast. In this letter, we describe the use of a microwave microscope with an open-ended coaxial probe to quantitatively map the surface resistance of a metallic thin film.The key element in our system 7,8 is a 2 meter-long resonant coaxial transmission line (see Fig. 1). One end of the line is connected to an open-ended coaxial probe and the other end is weakly coupled to a microwave source via a capacitor C D . Near-field microwave energy at the exposed tip of the probe center conductor is coupled to the sample. As the sample is scanned beneath the probe tip, the resonant frequencies and quality factor Q of the open transmission line shift depending on the surface properties of the region of the sample closest to the probe's center conductor. 7,8 We measure the microwave power reflected back up the transmission line with a diode detector. 9 By using a fixed frequency source near one of the resonances f R , and scanning a sample underneath the probe, one can map the reflected power and generate an image. 7,8 However, this results in a convolution of two distinct contrast mechanisms: the frequency shift of the standing wave resonances and the change in Q.To disentangle these effects, we have developed a frequency-following feedback circuit, as shown in Fig. 1. We frequency modulate the source at a rate f F M ≈ 3 kHz with a deviation of about ±3 MHz and use a feedback loop to keep the average microwave source frequency locked to a specific resonant frequency f R (t) of the microscope. To accomplish this, the diode detector output voltage is amplified and sent to a lock-in amplifier referenced at the frequency f F M . The lock-in output is time-integrated; this voltage signal V out is added to the f F M oscillator signal and fed to the frequency-control input of the microwave source, (see Fig. 1...

Blindness due to retinal degeneration affects millions of people worldwide, but many disease-causing mutations remain unknown. PNPLA6 encodes the patatin-like phospholipase domain containing protein 6, also known as neuropathy target esterase (NTE), which is the target of toxic organophosphates that induce human paralysis due to severe axonopathy of large neurons. Mutations in PNPLA6 also cause human spastic paraplegia characterized by motor neuron degeneration. Here we identify PNPLA6 mutations in childhood blindness in seven families with retinal degeneration, including Leber congenital amaurosis and Oliver McFarlane syndrome. PNPLA6 localizes mostly at the inner segment plasma membrane in photo-receptors and mutations in Drosophila PNPLA6 lead to photoreceptor cell death. We also report that lysophosphatidylcholine and lysophosphatidic acid levels are elevated in mutant Drosophila. These findings show a role for PNPLA6 in photoreceptor survival and identify phospholipid metabolism as a potential therapeutic target for some forms of blindness.