The mechanism of apoptosis has been extensively characterized over the past decade, but little is known about alternative forms of regulated cell death. Although stimulation of the Fas/TNFR receptor family triggers a canonical 'extrinsic' apoptosis pathway, we demonstrated that in the absence of intracellular apoptotic signaling it is capable of activating a common nonapoptotic death pathway, which we term necroptosis. We showed that necroptosis is characterized by necrotic cell death morphology and activation of autophagy. We identified a specific and potent small-molecule inhibitor of necroptosis, necrostatin-1, which blocks a critical step in necroptosis. We demonstrated that necroptosis contributes to delayed mouse ischemic brain injury in vivo through a mechanism distinct from that of apoptosis and offers a new therapeutic target for stroke with an extended window for neuroprotection. Our study identifies a previously undescribed basic cell-death pathway with potentially broad relevance to human pathologies.

The polymerization dynamics of microtubules are central to their biological functions. Polymerization dynamics allow microtubules to adopt spatial arrangements that can change rapidly in response to cellular needs and, in some cases, to perform mechanical work. Microtubules utilize the energy of GTP hydrolysis to fuel a unique polymerization mechanism termed dynamic instability. In this review, we first describe progress toward understanding the mechanism of dynamic instability of pure tubulin and then discuss the function and regulation of microtubule dynamic instability in living cells.

We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 ؉ 2] cycloaddition reaction (''click'' chemistry). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount preparations of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.BrdU ͉ click chemistry ͉ DNA replication ͉ EdU ͉ microscopy D etection of DNA synthesis in proliferating cells relies on the incorporation of labeled DNA precursors into cellular DNA during the S phase of the cell cycle (1-3). The labeled DNA precursors, usually pyrimidine deoxynucleosides, are added to cells during replication, and their incorporation into genomic DNA is quantified or visualized after incubation and sample staining. The same labeled deoxynucleosides can be injected into experimental animals to assay cellular proliferation in specific organs and tissues. The most common deoxynucleosides used for assaying DNA replication are [ 3 H]thymidine and 5-bromo-2Ј-deoxyuridine (BrdU). [ 3 H]Thymidine incorporated into DNA is usually detected by autoradiography, whereas detection of BrdU is accomplished immunologically, through specific anti-BrdU antibodies.Although [ 3 H]thymidine and BrdU labeling methods have been very useful for studying cell cycle kinetics, DNA replication, and sister chromatid exchange and for assessing cell proliferation of normal or pathological cells or tissues under different conditions, these methods exhibit several limitations. Working with [ 3 H]thymidine is cumbersome because of its radioactivity. Autoradiography is also labor-intensive and slow (detection often lasts several months), and thus not suitable for rapid high-throughput studies. Finally, microscopic images of [ 3 H]thymidine-labeled DNA suffer from poor resolution and low signal-to-noise ratios. In contrast to [ 3 H]thymidine autoradiography, BrdU immunostaining is both faster (although it still lasts several hours) and allows better microscopic imaging of the labeled DNA. One major disadvantage of BrdU staining is that the complementary base pairing in double-stranded DNA blocks the access of the anti-BrdU antibody to BrdU subunits. To expose the BrdU epitope, cells and tissue samples are subjected to strong denaturing conditions such as concentrated hydrochloric acid or mixtures of methanol and acetic acid. These harsh staining conditions invariably degrade the structure of the specimen while also making the intensity of BrdU staining highly dependent on the conditions used for detection by each investigator (4). Finally, as with any immunohistologic...