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Mitochondrial morphology is dynamically controlled by a balance between fusion and fission. The physiological importance of mitochondrial fission in vertebrates is less clearly defined than that of mitochondrial fusion. Here we show that mice lacking the mitochondrial fission GTPase Drp1 have developmental abnormalities, particularly in the forebrain, and die after embryonic day 12.5. Neural cell-specific (NS) Drp1(-/-) mice die shortly after birth as a result of brain hypoplasia with apoptosis. Primary culture of NS-Drp1(-/-) mouse forebrain showed a decreased number of neurites and defective synapse formation, thought to be due to aggregated mitochondria that failed to distribute properly within the cell processes. These defects were reflected by abnormal forebrain development and highlight the importance of Drp1-dependent mitochondrial fission within highly polarized cells such as neurons. Moreover, Drp1(-/-) murine embryonic fibroblasts and embryonic stem cells revealed that Drp1 is required for a normal rate of cytochrome c release and caspase activation during apoptosis, although mitochondrial outer membrane permeabilization, as examined by the release of Smac/Diablo and Tim8a, may occur independently of Drp1 activity.

Organelles are inherited to daughter cells beyond dynamic changes of the membrane structure during mitosis. Mitochondria are dynamic entities, frequently dividing and fusing with each other, during which dynamin-related GTPase Drp1 is required for the fission reaction. In this study, we analyzed mitochondrial dynamics in mitotic mammalian cells. Although mitochondria in interphase HeLa cells are long tubular network structures, they are fragmented in early mitotic phase, and the filamentous network structures are subsequently reformed in the daughter cells. In marked contrast, tubular mitochondrial structures are maintained during mitosis in Drp1 knockdown cells, indicating that the mitochondrial fragmentation in mitosis requires mitochondrial fission by Drp1. Drp1 was specifically phosphorylated in mitosis by Cdk1/cyclin B on Ser-585. Exogenous expression of unphosphorylated mutant Drp1S585A led to reduced mitotic mitochondrial fragmentation. These results suggest that phosphorylation of Drp1 on Ser-585 promotes mitochondrial fission in mitotic cells.

In yeast, mitochondrial-fission is regulated by the cytosolic dynamin-like GTPase (Dnm1p) in conjunction with a peripheral protein, Mdv1p, and a C-tail-anchored outer membrane protein, Fis1p. In mammals, a dynamin-related protein (Drp1) and Fis1 are involved in the mitochondrial-fission reaction as Dnm1 and Fis1 orthologues, respectively. The involvement of other component(s), such as the Mdv1 homologue, and the mechanisms regulating mitochondrial-fission remain unclear. Here, we identified rat Fis1 (rFis1) and analyzed its structure-function relationship. Blue-native-polyacrylamide gel electrophoresis revealed that rFis1 formed a approximately 200-kDa complex in the outer mitochondrial membrane. Its expression in HeLa cells promoted extensive mitochondrial fragmentation, and gene knock-down by RNAi induced extension of the mitochondrial networks. Taking advantage of these properties, we analyzed functional domains of rFis1. These experiments revealed that the N-terminal and C-terminal segments are both essential for oligomeric rFis1 interaction, and the middle TPR-like domains regulate proper oligomer assembly. Any mutations that disturb the proper oligomeric assembly compromise mitochondrial division-stimulating activity of rFis1.

Little is known about the signals that govern the network of meristem and organ identity genes that control flower development. In Arabidopsis , we can induce a heterochronic switch from flower to shoot development, a process known as floral meristem reversion, by manipulating photoperiod in the floral homeotic mutant agamous and in plants heterozygous for the meristem identity gene leafy . The transformation from flower to shoot meristem is suppressed by hy1 , a mutation blocking phytochrome activity, by spindly , a mutation that activates basal gibberellin signal transduction in a hormone independent manner, or by the exogenous application of gibberellins. We propose that LFY and AG play an important role in the maintenance of flower meristem identity and that floral meristem reversion in heterozygous lfy and in ag flowers is regulated by a phytochrome and gibberellin signal transduction cascade.

Summary In yeast, C-tail-anchored mitochondrial outer membrane protein Fis1 recruits the mitochondrial-fission-regulating GTPase Dnm1 to mitochondrial fission sites. However, the function of its mammalian homologue remains enigmatic because it has been reported to be dispensable for the mitochondrial recruitment of Drp1, a mammalian homologue of Dnm1. We identified TBC1D15 as a Fis1-binding protein in HeLa cell extracts. Immunoprecipitation revealed that Fis1 efficiently interacts with TBC1D15 but not with Drp1. Bacterially expressed Fis1 and TBC1D15 formed a direct and stable complex. Exogenously expressed TBC1D15 localized mainly in cytoplasm in HeLa cells, but when coexpressed with Fis1 it localized to mitochondria. Knockdown of TBC1D15 induced highly developed mitochondrial network structures similar to the effect of Fis1 knockdown, suggesting that the TBC1D15 and Fis1 are associated with the regulation of mitochondrial morphology independently of Drp1. These data suggest that Fis1 acts as a mitochondrial receptor in the recruitment of mitochondrial morphology protein in mammalian cells.

APETALA2 ( AP2 ) is best known for its role in the regulation of flower meristem and flower organ identity and development in Arabidopsis . We show here that AP2 also plays an important role in determining seed size, seed weight, and the accumulation of seed oil and protein. We demonstrate genetically that AP2 acts through the maternal sporophyte and endosperm genomes to control seed weight and seed yield. Thus, AP2 functions outside the boundaries of flower meristem and flower organ development to affect agronomically relevant traits in Arabidopsis .

Mitochondria are dynamic organelles that undergo frequent fission and fusion or branching. To analyze the mitochondrial fusion reaction, mitochondria were separately labeled with green or red fluorescent protein (GFP and RFP, respectively) in HeLa cells, and the cells were fused using hemagglutinating virus of Japan (HVJ). The resulting mixing of the fluorescent reporters was then followed using fluorescence microscopy. This system revealed that mitochondria fuse frequently in mammalian cells, and the fusion depends on the membrane potential across the inner membrane. The protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), led to fragmentation of the mitochondria and inhibited the fusion reaction. Removal of CCCP recovered the fusion activity to reform filamentous mitochondrial networks. Analysis of the effects of GTP-binding proteins, DRP1 and two FZO1 isoforms, and the GTPase-domain mutants on the CCCP-induced mitochondrial morphologic changes revealed that DRP1 and FZO1 are involved in membrane budding and fusion, respectively. Furthermore, a HVJ-dependent cell fusion assay combined with RNA interference (RNAi) demonstrated that both FZO1 isoforms are essential and must be acting in cis for the mitochondrial fusion reaction to occur.

APETALA2 ( AP2 ) plays an important role in the control of Arabidopsis flower and seed development and encodes a putative transcription factor that is distinguished by a novel DNA binding motif referred to as the AP2 domain. In this study we show that the AP2 domain containing or RAP2 (related to AP2) family of proteins is encoded by a minimum of 12 genes in Arabidopsis . The RAP2 genes encode two classes of proteins, AP2-like and EREBP-like, that are defined by the number of AP2 domains in each polypeptide as well as by two sequence motifs referred to as the YRG and RAYD elements that are located within each AP2 domain. RAP2 genes are differentially expressed in flower, leaf, inflorescence stem, and root. Moreover, the expression of at least three RAP2 genes in vegetative tissues are controlled by AP2 . Thus, unlike other floral homeotic genes, AP2 is active during both reproductive and vegetative development.

We have analyzed the contributions of phytochrome and gibberellin signal transduction to the control of flower meristem identity in the Arabidopsis mutants apetala1 (ap1) and apetala2 (ap2). ap1 flowers are partially defective for the establishment of flower meristem identity and are characterized by the production of ectopic secondary or axillary flowers and by branching. Axillary flower production is also induced in ap2-1 flowers by short-day photoperiod and is suppressed by hy1, a mutation blocking phytochrome activity. The production of axillary flower by ap2-1 is also suppressed by exogenous gibberellins and by spindly (spy), a mutation that activates basal gibberellin signal transduction in hormone-independent manner. Ectopic axillary flower production and floral branching by ap1 flowers are also suppressed by spy. We conclude that gibberellins promote flower meristem identity and that the inflorescence-like traits of ap2-1 and ap1-1 flowers are due in part to SPY gene activity.

There have been few reports on cerebral arteriovenous malformation (AVM) of newborns. We present here an interesting case of occult AVM diagnosed 17 years after an episode of acute subdural hematoma in the neonatal period. The cause of subdural hematoma had remained unclear and the patient had suffered from an intractable epilepsy of 17 years duration. Seizures were mainly characterized by drop attacks and included other seizure types such as complex partial seizure and generalized tonic clonic seizure. The symptoms had gradually become worse and the intervals between the occurrences of symptoms had become shorter. An interictal scalp EEG showed a focal spike in the left temporoparietal lobe. CT and MRI of that region demonstrated a porencephalic cyst which was supposed to have resulted from an old hematoma. There were no vascular abnormalities in angiography. Temporoparietal craniotomy and a corticogram were performed. The cortex with a focus was resected and the disappearance of a focal spike in the corticogram was confirmed during operation. Microscopically the cortex included AVM with gliosis. The initial postoperative course was good and seizures disappeared immediately after the operation. However, the symptoms of drop attacks observed before operation began to occur again 3 months later. The fact of postoperative recurrence suggests that the long history of the patient's seizures originating from AVM may have produced secondary epileptic foci.

Plant development is governed by intrinsic and environmen- tal factors that regulate the identity and activity of meristems, organized tissues of pluripotent "stem" cells, that together de- termine plant form and architecture. However, little is known about how these factors act at the molecular leve1 to affect meristem identity and function. Genetic studies in Arabidop- sis and other plant species such as snapdragon, petunia, and maize have revealed a hierarchy of regulatory genes that func- tion together to promote the formation of the floral meristem and to regulate floral organogenesis. Mutations in these genes result in dramatic defects in flower development that can af- fect both meristem identity and organ development. Table 1 lists the Arabidopsis genes that are known to control meristem and organ identity and their snapdragon equivalents. One class of regulatory genes, the homeotic organ identity genes, have provided important insights into the genetic and molecular mechanisms that govern floral organ identity and development (Bowman et al., 1991; Coen and Meyerowitz, 1991; Coen and Carpenter, 1993, this issue; van der Krol and Chua, 1993, this issue). In Arabidopsis, these genes include