Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.
The GTPase dynamin has been clearly implicated in clathrin-mediated endocytosis of synaptic vesicle membranes at the presynaptic nerve terminal. Here we describe a novel 52-kDa protein in rat brain that binds the proline-rich C terminus of dynamin. Syndapin I (synaptic, dynamin-associated protein I) is highly enriched in brain where it exists in a high molecular weight complex. Syndapin I can be involved in multiple protein-protein interactions via a src homology 3 (SH3) domain at the C terminus and two predicted coiled-coil stretches. Coprecipitation studies and blot overlay analyses revealed that syndapin I binds the brain-specific proteins dynamin I, synaptojanin, and synapsin I via an SH3 domain-specific interaction. Coimmunoprecipitation of dynamin I with antibodies recognizing syndapin I and colocalization of syndapin I with dynamin I at vesicular structures in primary neurons indicate that syndapin I associates with dynamin I in vivo and may play a role in synaptic vesicle endocytosis. Furthermore, syndapin I associates with the neural Wiskott-Aldrich syndrome protein, an actin-depolymerizing protein that regulates cytoskeletal rearrangement. These characteristics of syndapin I suggest a molecular link between cytoskeletal dynamics and synaptic vesicle recycling in the nerve terminal. INTRODUCTIONNeurotransmitter release requires that synaptic vesicles fuse with the plasma membrane when intraterminal calcium rises, after which the synaptic vesicle membrane is rapidly recycled and refilled with neurotransmitter. Recovery of plasma membrane after stimulated exocytosis is commonly referred to as compensatory endocytosis. Compensatory endocytosis of synaptic vesicle membrane proteins from the plasma membrane was originally attributed to only two cytoplasmic proteins, clathrin and the heterotetrameric adaptor complex, adaptor protein 2 (AP2) 1 (for review, see Schmid, 1997). A more complex model of endocytosis became necessary when the Drosophila shibire mutant that could not recycle synaptic vesicle membranes was shown to be defective in a GTPase, dynamin (Kosaka and Ikeda, 1983;. Dynamin is now known to form tightly wound helical structures that participate in pinching off the constricted neck of a clathrincoated pit (Hinshaw and Schmid, 1995;Takei et al., 1995Takei et al., , 1998Sweitzer and Hinshaw, 1998). It is likely that dynamin is normally part of a much larger molecular machine that is responsible for compensatory endocytosis after exocytosis. Dynamin binds with high affinity via a proline-rich domain (PRD) to four brain-specific proteins: amphiphysin I , amphiphysin II (Leprince et al., 1997;Ramjaun et al., 1997;Wigge et al., 1997a) , 1997;Ringstad et al., 1997), and an undescribed protein of ϳ52 kDa (Roos and Kelly, 1998). Amphiphysin I, amphiphysin II, and endophilin bind to dynamin and other PRD-containing proteins such as synapsin I (De Camilli et al., 1983) and synaptojanin via their src homology 3 (SH3) domains.Interaction of dynamin via its PRD with SH3 domain-containing proteins is essential...
Summary Alzheimer's disease (AD) is characterized pathologically by the abundance of senile plaques and neurofibrillary tangles in the brain. We synthesized over 1200 novel gamma-secretase modulator (GSM) compounds that reduced Abeta42 levels without inhibiting epsilon-site cleavage of APP and Notch, the generation of the APP and Notch intracellular domains, respectively. These compounds also reduced Abeta40 levels while concomitantly elevating levels of Abeta38 and Abeta37. Immobilization of a potent GSM onto an agarose matrix quantitatively recovered Pen-2 and to a lesser degree PS-1 NTFs from cellular extracts. Moreover, oral administration (once daily) of another potent GSM to Tg 2576 transgenic AD mice displayed dose-responsive lowering of plasma and brain Abeta42; chronic daily administration led to significant reductions in both diffuse and neuritic plaques. These effects were observed in the absence of Notch-related changes (e.g. intestinal proliferation of goblet cells), which are commonly associated with repeated exposure to functional gamma-secretase inhibitors (GSIs).
The cyclin proteolysis that accompanies the exit from mitosis in diverse systems appears to be essential for restoration of interphase. The early syncytial divisions of Drosophila embryos, however, occur without detectable oscillations in the total cyclin level or Cdk1 activity. Nonetheless, we found that injection of an established inhibitor of cyclin proteolysis, a cyclin B amino-terminal peptide, prevents exit from mitosis in syncytial embryos. Similarly, injection of a version of Drosophila cyclin B that is refractory to proteolysis results in mitotic arrest. We infer that proteolysis of cyclins is required for exit from syncytial mitoses. This inference can be reconciled with the failure to observe oscillations in total cyclin levels if only a small pool of cyclins is destroyed in each cycle. We find that antibody detection of histone H3 phosphorylation (PH3) acts as a reporter for Cdk1 activity. A gradient of PH3 along anaphase chromosomes suggests local Cdk1 inactivation near the spindle poles in syncytial embryos. This pattern of Cdk1 inactivation would be consistent with local cyclin destruction at centrosomes or kinetochores. The local loss of PH3 during anaphase is specific to the syncytial divisions and is not observed after cellularization. We suggest that exit from mitosis in syncytial cycles is modified to allow nuclear autonomy within a common cytoplasm.
Cells can respond to reductions in oxygen (hypoxia) by metabolic adaptations, quiescence or cell death (1). The nuclear division cycles of syncytial stage Drosophila melanogaster embryos reversibly arrest upon hypoxia. We examined this rapid arrest in real time using a fusion of green fluorescent protein and histone 2A. In addition to an interphase arrest, mitosis was specifically blocked in metaphase, much like a checkpoint arrest. Nitric oxide, recently proposed as a hypoxia signal in Drosophila, induced a reversible arrest of the nuclear divisions comparable with that induced by hypoxia. Syncytial stage embryos die during prolonged hypoxia, whereas post-gastrulation embryos (cellularized) survive (2, 3). We examined ATP levels and morphology of syncytial and cellularized embryos arrested by hypoxia, nitric oxide, or cyanide. Upon oxygen deprivation, the ATP levels declined only slightly in cellularized embryos and more substantially in syncytial embryos. Reversal of hypoxia restored ATP levels and relieved the cell cycle and developmental arrests. However, morphological abnormalities suggested that syncytial embryos suffered irreversible disruption of developmental programs. Our results suggest that nitric oxide plays a role in the response of the syncytial embryo to hypoxia but that it is not the sole mediator of these responses.Oxygen is essential for the life of most multi-cellular organisms. Limitations in oxygen have profound physiological and health consequences in humans. Prolonged or severe decreases in physiological oxygen resulting from ischemia are a major contributor to morbidity and mortality in stroke and cardiac infarction (4). Cells in pretumorous growths are often deprived of oxygen as a result of insufficient and inefficient vascularization, and this hypoxia (low oxygen) is postulated to play a role in limiting tumor progression (5). The outcome of hypoxia can differ dramatically. For example, a turtle can survive months of hypoxic conditions, whereas the cells of the human brain begin to die following a few minutes of hypoxia (6). These outcomes presumably depend on the cellular responses to hypoxia, which range from metabolic adaptation to quiescence to apoptosis (1). Among the cellular responses to hypoxia is an arrest of the cell cycle (1, 5). Hypoxia is reported to arrest mammalian cells during G 1 , mid-S phase, and G 2 /M. Analysis of the response to hypoxia in Drosophila has similarly revealed an arrest of the cell cycle in mid-S phase and mitosis (2, 3). Despite the importance of these cellular responses, the signals mediating responses to hypoxia are ill defined, and the factors influencing the type of response are poorly understood. What allows some cells to survive when others die?In this study, we examined the responses to hypoxia during Drosophila embryogenesis. The development of Drosophila from egg to larva is remarkably fast and dynamic. Development begins with 13 rapid mitotic divisions in a common cytoplasm, referred to as the syncytial stage. Next, cell membranes invagina...
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