Following the Behavior of the Flagellar Rotary Motor Near Zero Load

At room temperature at stall, the flagellar motor of the bacterium Escherichia coli exerts a torque of ~1300 pN nm. At zero external load, it spins ~330 Hz. Techniques for studying the motor near zero load are novel and are summarized here.Molecular and Cellular Biolog

Live imaging and single-cell analysis reveal differential dynamics of autophagy and apoptosis

Autophagy is induced by many cytotoxic stimuli but it is often unclear whether, under specific conditions, autophagy plays a prosurvival or a prodeath role. To answer this critical question we developed a novel methodology that employs automated live microscopy and image analysis to measure autophagy and apoptosis simultaneously in single cells. We used this approach to perform a systems-level analysis of pathway dynamics for both autophagy and apoptosis. We found that induction of autophagy in response to different stimuli is uniformly unimodal; in contrast, cells induce apoptosis in an all-or-none bimodal fashion. By tracking the fate of single cells we found that autophagy precedes apoptosis, and that within the same population apoptosis is delayed in cells that mount a stronger autophagy response. Inhibition of autophagy by knocking down ATG5 promoted apoptosis, thus confirming that autophagy plays a protective role. We anticipate that our single-cell approach will be a powerful tool for gaining a quantitative understanding of the complex regulation of autophagy, its influence on cell fate decisions and its relationship with other cellular pathways

The channel of death

The proapoptotic members of the Bcl-2 family have been proposed to participate in the formation of a channel that releases these apoptogenic factors when mitochondria receive apoptotic signals. A recent study provides the first direct, biophysical measurement of a potentially apoptosis-specific mitochondrial channel, which is regulated by Bcl-2 family members and may play a primary role in the release of the proapoptotic factors

Flightless-I regulates proinflammatory caspases by selectively modulating intracellular localization and caspase activity

Caspase-1 and caspase-11 are proinflammatory caspases that regulate cytokine production and leukocyte migration during pathogen infection. In an attempt to identify new intracellular regulators of caspase-11, we found that Flightless-I, a member of the gelsolin superfamily of actin-remodeling proteins, interacts and regulates both caspase-11 and caspase-1. Flightless-I targets caspase-11 to the Triton X-100–insoluble cytoskeleton fraction and the cell leading edge. In addition, Flightless-I inhibits caspase-1 activation and caspase-1–mediated interleukine-1β (IL-1β) maturation. The physiological relevance of these findings is supported by the opposing effects of Flightless-I overexpression and knockdown on caspase-1 activity and IL-1β maturation. Our results suggest that Flightless-I may be a bona fide caspase-1 inhibitor that acts through a mechanism similar to that of cytokine response modifier A, a potent caspase-1 inhibitor from the cowpox virus. Our study provides a new mechanism controlling the localization and activation of proinflammatory caspases

Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade

AbstractBackground: Fas (APO-1/CD95) is a member of the tumor necrosis factor receptor (TNF-R) family and induces apoptosis when crosslinked with either Fas ligand or agonistic antibody (Fas antibody). The Fas–Fas ligand system has an important role in the immune system where it is involved in the downregulation of immune responses and the deletion of peripheral autoreactive T lymphocytes. The intracellular domain of Fas interacts with several proteins including FADD (MORT-1), DAXX, RIP, FAF-1, FAP-1 and Sentrin. The adaptor protein FADD can, in turn, interact with the cysteine protease caspase-8 (FLICE/MACH/Mch5).Results: In a genetic screen for essential components of the Fas-mediated apoptotic cascade, we isolated a Jurkat T lymphocyte cell line deficient in caspase-8 that was completely resistant to Fas-induced apoptosis. Complementation of this cell line with wild-type caspase-8 restored Fas-mediated apoptosis. Fas activation of multiple caspases and of the stress kinases p38 and c-Jun NH2-terminal kinase (JNK) was completely blocked in the caspase-8-deficient cell line. Furthermore, the cell line was severely deficient in cell death induced by TNF-α and was partially deficient in cell death induced by ultraviolet irradiation, adriamycin and etoposide.Conclusions: This study provides the first genetic evidence that caspase-8 occupies an essential and apical position in the Fas signaling pathway and suggests that caspase-8 may participate broadly in multiple apoptotic pathways

Structure–activity relationship and liver microsome stability studies of pyrrole necroptosis inhibitors

Necroptosis is a regulated caspase-independent cell death pathway resulting in morphology reminiscent of passive non-regulated necrosis. Several diverse structure classes of necroptosis inhibitors have been reported to date, including a series of [1,2,3]thiadiazole benzylamide derivatives. However, initial evaluation of mouse liver microsome stability indicated that this series of compounds was rapidly degraded. A structure–activity relationship (SAR) study of the [1,2,3]thiadiazole benzylamide series revealed that increased mouse liver microsome stability and increased necroptosis inhibitory activity could be accomplished by replacement of the 4-cyclopropyl-[1,2,3]thiadiazole with a 5-cyano-1-methylpyrrole. In addition, the SAR and the cellular activity profiles, utilizing different cell types and necroptosis-inducing stimuli, of representative [1,2,3]thiadiazole and pyrrole derivatives were very similar suggesting that the two compound series inhibit necroptosis in the same manner

Nematic topological superconducting phase in Nb-doped Bi2Se3

A nematic topological superconductor has an order parameter symmetry, which spontaneously breaks the crystalline symmetry in its superconducting state. This state can be observed, for example, by thermodynamic or upper critical field experiments in which a magnetic field is rotated with respect to the crystalline axes. The corresponding physical quantity then directly reflects the symmetry of the order parameter. We present a study on the superconducting upper critical field of the Nb-doped topological insulator NbxBi2Se3 for various magnetic field orientations parallel and perpendicular to the basal plane of the Bi2Se3 layers. The data were obtained by two complementary experimental techniques, magnetoresistance and DC magnetization, on three different single crystalline samples of the same batch. Both methods and all samples show with perfect agreement that the in-plane upper critical fields clearly demonstrate a two-fold symmetry that breaks the three-fold crystal symmetry. The two-fold symmetry is also found in the absolute value of the magnetization of the initial zero-field-cooled branch of the hysteresis loop and in the value of the thermodynamic contribution above the irreversibility field, but also in the irreversible properties such as the value of the characteristic irreversibility field and in the width of the hysteresis loop. This provides strong experimental evidence that Nb-doped Bi2Se3 is a nematic topological superconductor similar to the Cu- and Sr-doped Bi2Se3