Bax and Bak promote apoptosis by perturbing the permeability of the mitochondrial outer membrane and facilitating the release of cytochrome c by a mechanism that is still poorly defined. During apoptosis, Bax and Bak also promote fragmentation of the mitochondrial network, possibly by activating the mitochondrial fission machinery. It has been proposed that Bax/Bak-induced mitochondrial fission may be required for release of cytochrome c from the mitochondrial intermembrane space, although this has been a subject of debate. Here we show that Bcl-xL, as well as other members of the apoptosis-inhibitory subset of the Bcl-2 family, antagonized Bax and/or Bak-induced cytochrome c release but failed to block mitochondrial fragmentation associated with Bax/Bak activation. These data suggest that Bax/Bak-initiated remodeling of mitochondrial networks and cytochrome c release are separable events and that Bcl-2 family proteins can influence mitochondrial fission-fusion dynamics independent of apoptosis.

Bcl-2 family proteins play central roles in apoptosis by regulating the release of mitochondrial intermembrane space proteins such as cytochrome c. Deathpromoting Bcl-2 family members, such as Bax, can promote cytochrome c release and fragmentation of the mitochondrial network, whereas apoptosis-inhibitory members, such as Bcl-2 and Bcl-xL, can antagonize these events. It remains unclear whether CED-9, the worm Bcl-2 relative, can regulate mitochondrial fission/fusion dynamics or the release of proteins from the mitochondrial intermembrane space. Here, we show that CED-9 interacts with Mitofusin-2/fuzzy onions and can promote mitochondrial clustering and dramatic reorganization of mitochondrial networks. Consistent with its ability to neutralize CED-9 function, EGL-1 antagonized CED-9-dependent remodeling of the mitochondrial network. However, CED-9 failed to inhibit mitochondrial cytochrome c release or apoptosis induced by diverse triggers in mammalian cells. These data suggest that the ability to regulate mitochondrial fission/fusion dynamics is an evolutionarily conserved property of the Bcl-2 family.

During cell division, spindle microtubules attach to chromosomes through kinetochores, protein complexes on the chromosome. The central question is how microtubules find kinetochores. According to the pioneering idea termed search-and-capture, numerous microtubules grow from a centrosome in all directions and by chance capture kinetochores. The efficiency of search-and-capture can be improved by a bias in microtubule growth towards the kinetochores, by nucleation of microtubules at the kinetochores and at spindle microtubules, by kinetochore movement, or by a combination of these processes. Here we show in fission yeast that kinetochores are captured by microtubules pivoting around the spindle pole, instead of growing towards the kinetochores. This pivoting motion of microtubules is random and independent of ATP-driven motor activity. By introducing a theoretical model, we show that the measured random movement of microtubules and kinetochores is sufficient to explain the process of kinetochore capture. Our theory predicts that the speed of capture depends mainly on how fast microtubules pivot, which was confirmed experimentally by speeding up and slowing down microtubule pivoting. Thus, pivoting motion allows microtubules to explore space laterally, as they search for targets such as kinetochores.

Granzyme B, a serine protease derived from cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell granules, plays an important role in coordinating apoptosis of CTL and NK target cells. Here, we report that granzyme B targets the cytoskeleton by cleaving and removing the acidic C-terminal tail of ␣-tubulin. Consistent with this, Granzyme B markedly enhanced rates of microtubule polymerization in vitro, most likely by removal of an autoinhibitory domain within the tubulin C terminus. Moreover, delivery of Granzyme B into HeLa target cells promoted dramatic reorganization of the microtubule network in a caspase-independent manner. These data reveal that granzyme B directly attacks a major component of the cell cytoskeleton, which may contribute to the incapacitation of target cells during CTL/NK-mediated killing.

No abstract