Micro-Meta App: an interactive tool for collecting microscopy metadata based on community specifications.

For quality, interpretation, reproducibility and sharing value, microscopy images should be accompanied by detailed descriptions of the conditions that were used to produce them. Micro-Meta App is an intuitive, highly interoperable, open-source software tool that was developed in the context of the 4D Nucleome (4DN) consortium and is designed to facilitate the extraction and collection of relevant microscopy metadata as specified by the recent 4DN-BINA-OME tiered-system of Microscopy Metadata specifications. In addition to substantially lowering the burden of quality assurance, the visual nature of Micro-Meta App makes it particularly suited for training purposes.

Oscillating calcium signals in smooth muscle cells underlie the persistent basal tone of internal anal sphincter.

A persistent basal tone in the internal anal sphincter (IAS) is essential for keeping the anal canal closed and fecal continence; its inhibition via the rectoanal inhibitory reflex (RAIR) is required for successful defecation. However, cellular signals underlying the IAS basal tone remain enigmatic. Here we report the origin and molecular mechanisms of calcium signals that control the IAS basal tone, using a combination approach including a novel IAS slice preparation that retains cell arrangement and architecture as in vivo, 2-photon imaging, and cell-specific gene-modified mice. We found that IAS smooth muscle cells generate two forms of contractions (i.e., phasic and sustained contraction) and Ca2+ signals (i.e., synchronized Ca2+ oscillations [SCaOs] and asynchronized Ca2+ oscillations [ACaOs]) that last for hours. RyRs, TMEM16A, L-type Ca2+ channels, and gap junctions are required for SCaOs, which account for phasic contraction and 75% of sustained contraction. Nevertheless, only RyRs are required for ACaOs, which contribute 25% of sustained contraction. Nitric oxide, the primary neurotransmitter mediating the RAIR, blocks both types of Ca2+ signals, leading to IAS's full relaxation. Our results show that the oscillating nature of Ca2+ signals generates and maintains the basal tone without causing cytotoxicity to IAS. Our study provides insight into fecal continence and normal defecation.

The Dopamine Transporter Recycles via a Retromer-Dependent Postendocytic Mechanism: Tracking Studies Using a Novel Fluorophore-Coupling Approach.

Presynaptic reuptake, mediated by the dopamine (DA) transporter (DAT), terminates DAergic neurotransmission and constrains extracellular DA levels. Addictive and therapeutic psychostimulants inhibit DA reuptake and multiple DAT coding variants have been reported in patients with neuropsychiatric disorders. These findings underscore that DAT is critical for DA neurotransmission and homeostasis. DAT surface availability is regulated acutely by endocytic trafficking, and considerable effort has been directed toward understanding mechanisms that govern DAT's plasma membrane expression and postendocytic fate. Multiple studies have demonstrated DAT endocytic recycling and enhanced surface delivery in response to various stimuli. Paradoxically, imaging studies have not detected DAT targeting to classic recycling endosomes, suggesting that internalized DAT targets to either degradation or an undefined recycling compartment. Here, we leveraged PRIME (PRobe Incorporation Mediated by Enzyme) labeling to couple surface DAT directly to fluorophore, and tracked DAT's postendocytic itinerary in immortalized mesencephalic cells. Following internalization, DAT robustly targeted to retromer-positive endosomes, and DAT/retromer colocalization was observed in male mouse dopaminergic somatodendritic and terminal regions. Short hairpin RNA-mediated Vps35 knockdown revealed that DAT endocytic recycling requires intact retromer. DAT also targeted rab7-positive endosomes with slow, linear kinetics that were unaffected by either accelerating DAT internalization or binding a high-affinity cocaine analog. However, cocaine increased DAT exit from retromer-positive endosomes significantly. Finally, we found that the DAT carboxy-terminal PDZ-binding motif was required for DAT recycling and exit from retromer. These results define the DAT recycling mechanism and provide a unifying explanation for previous, seemingly disparate, DAT endocytic trafficking findings.SIGNIFICANCE STATEMENT The neuronal dopamine (DA) transporter (DAT) recaptures released DA and modulates DAergic neurotransmission, and a number of DAT coding variants have been reported in several DA-related disorders, including infantile parkinsonism, attention-deficit/hyperactivity disorder and autism spectrum disorder. DAT is also competitively inhibited by psychostimulants with high abuse potential. Therefore, mechanisms that acutely affect DAT availability will likely exert significant impact on both normal and pathological DAergic homeostasis. Here, we explore the cellular mechanisms that acutely control DAT surface expression. Our results reveal the intracellular mechanisms that mediate DAT endocytic recycling following constitutive and regulated internalization. In addition to shedding light on this critical process, these findings resolve conflict among multiple, seemingly disparate, previous reports on DAT's postendocytic fate.

Ack1 is a dopamine transporter endocytic brake that rescues a trafficking-dysregulated ADHD coding variant.

The dopamine (DA) transporter (DAT) facilitates high-affinity presynaptic DA reuptake that temporally and spatially constrains DA neurotransmission. Aberrant DAT function is implicated in attention-deficit/hyperactivity disorder and autism spectrum disorder. DAT is a major psychostimulant target, and psychostimulant reward strictly requires binding to DAT. DAT function is acutely modulated by dynamic membrane trafficking at the presynaptic terminal and a PKC-sensitive negative endocytic mechanism, or "endocytic brake," controls DAT plasma membrane stability. However, the molecular basis for the DAT endocytic brake is unknown, and it is unknown whether this braking mechanism is unique to DAT or common to monoamine transporters. Here, we report that the cdc42-activated, nonreceptor tyrosine kinase, Ack1, is a DAT endocytic brake that stabilizes DAT at the plasma membrane and is released in response to PKC activation. Pharmacologic and shRNA-mediated Ack1 silencing enhanced basal DAT internalization and blocked PKC-stimulated DAT internalization, but had no effects on SERT endocytosis. Both cdc42 activation and PKC stimulation converge on Ack1 to control Ack1 activity and DAT endocytic capacity, and Ack1 inactivation is required for stimulated DAT internalization downstream of PKC activation. Moreover, constitutive Ack1 activation is sufficient to rescue the gain-of-function endocytic phenotype exhibited by the ADHD DAT coding variant, R615C. These findings reveal a unique endocytic control switch that is highly specific for DAT. Moreover, the ability to rescue the DAT(R615C) coding variant suggests that manipulating DAT trafficking mechanisms may be a potential therapeutic approach to correct DAT coding variants that exhibit trafficking dysregulation.

Chemokine signaling guides regional patterning of the first embryonic artery.

The aorta traverses the body, yet little is known about how it is patterned in different anatomical locations. Here, we show that the aorta develops from genetically distinct endothelial cells originating from diverse locations within the embryo. Furthermore, chemokine (C-X-C motif) receptor 4a (cxcr4a) is restricted to endothelial cells derived from anterior mesoderm, and is required specifically for formation of the lateral aortae. Cxcl12b, a cxcr4a ligand, is expressed in endoderm underlying the lateral aortae, and loss of cxcl12b phenocopies cxcr4a deficiency. These studies reveal unexpected endothelial diversity within the aorta that is necessary to facilitate its regional patterning by local cues.

NPHP4 controls ciliary trafficking of membrane proteins and large soluble proteins at the transition zone.

The protein nephrocystin-4 (NPHP4) is widespread in ciliated organisms, and defects in NPHP4 cause nephronophthisis and blindness in humans. To learn more about the function of NPHP4, we have studied it in Chlamydomonas reinhardtii. NPHP4 is stably incorporated into the distal part of the flagellar transition zone, close to the membrane and distal to CEP290, another transition zone protein. Therefore, these two proteins, which are incorporated into the transition zone independently of each other, define different domains of the transition zone. An nphp4-null mutant forms flagella with nearly normal length, ultrastructure and intraflagellar transport. When fractions from isolated wild-type and nphp4 flagella were compared, few differences were observed between the axonemes, but the amounts of certain membrane proteins were greatly reduced in the mutant flagella, and cellular housekeeping proteins >50 kDa were no longer excluded from mutant flagella. Therefore, NPHP4 functions at the transition zone as an essential part of a barrier that regulates both membrane and soluble protein composition of flagella. The phenotypic consequences of NPHP4 mutations in humans likely follow from protein mislocalization due to defects in the transition zone barrier.

The cellular and molecular basis of bitter tastant-induced bronchodilation.

Bronchodilators are a standard medicine for treating airway obstructive diseases, and ß2 adrenergic receptor agonists have been the most commonly used bronchodilators since their discovery. Strikingly, activation of G-protein-coupled bitter taste receptors (TAS2Rs) in airway smooth muscle (ASM) causes a stronger bronchodilation in vitro and in vivo than ß2 agonists, implying that new and better bronchodilators could be developed. A critical step towards realizing this potential is to understand the mechanisms underlying this bronchodilation, which remain ill-defined. An influential hypothesis argues that bitter tastants generate localized Ca(2+) signals, as revealed in cultured ASM cells, to activate large-conductance Ca(2+)-activated K(+) channels, which in turn hyperpolarize the membrane, leading to relaxation. Here we report that in mouse primary ASM cells bitter tastants neither evoke localized Ca(2+) events nor alter spontaneous local Ca(2+) transients. Interestingly, they increase global intracellular [Ca(2+)]i, although to a much lower level than bronchoconstrictors. We show that these Ca(2+) changes in cells at rest are mediated via activation of the canonical bitter taste signaling cascade (i.e., TAS2R-gustducin-phospholipase Cß [PLCß]- inositol 1,4,5-triphosphate receptor [IP3R]), and are not sufficient to impact airway contractility. But activation of TAS2Rs fully reverses the increase in [Ca(2+)]i induced by bronchoconstrictors, and this lowering of the [Ca(2+)]i is necessary for bitter tastant-induced ASM cell relaxation. We further show that bitter tastants inhibit L-type voltage-dependent Ca(2+) channels (VDCCs), resulting in reversal in [Ca(2+)]i, and this inhibition can be prevented by pertussis toxin and G-protein ßγ subunit inhibitors, but not by the blockers of PLCß and IP3R. Together, we suggest that TAS2R stimulation activates two opposing Ca(2+) signaling pathways via Gßγ to increase [Ca(2+)]i at rest while blocking activated L-type VDCCs to induce bronchodilation of contracted ASM. We propose that the large decrease in [Ca(2+)]i caused by effective tastant bronchodilators provides an efficient cell-based screening method for identifying potent dilators from among the many thousands of available bitter tastants.

MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis.

Within the circulatory system, blood flow regulates vascular remodelling, stimulates blood stem cell formation, and has a role in the pathology of vascular disease. During vertebrate embryogenesis, vascular patterning is initially guided by conserved genetic pathways that act before circulation. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system. However, few details are known about how these signals are integrated during development. To investigate this process, we focused on the aortic arch (AA) blood vessels, which are known to remodel in response to blood flow. By using two-photon imaging of live zebrafish embryos, we observe that flow is essential for angiogenesis during AA development. We further find that angiogenic sprouting of AA vessels requires a flow-induced genetic pathway in which the mechano-sensitive zinc finger transcription factor klf2a induces expression of an endothelial-specific microRNA, mir-126, to activate Vegf signalling. Taken together, our work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.

Rabenosyn-5 defines the fate of the transferrin receptor following clathrin-mediated endocytosis.

Cell surface receptors and other proteins internalize through diverse mechanisms at the plasma membrane and are sorted to different destinations. Different subpopulations of early endosomes have been described, raising the question of whether different internalization mechanisms deliver cargo into different subsets of early endosomes. To address this fundamental question, we developed a microscopy platform to detect the precise position of endosomes relative to the plasma membrane during the uptake of ligands. Axial resolution is maximized by concurrently applied total internal reflection fluorescence and epifluorescence-structured light. We found that transferrin receptors are delivered selectively from clathrin-coated pits on the plasma membrane into a specific subpopulation of endosomes enriched in the multivalent Rab GTPase and phosphoinositide-binding protein Rabenosyn-5. Depletion of Rabenosyn-5, but not of other early endosomal proteins such as early endosome antigen 1, resulted in impaired transferrin uptake and lysosomal degradation of transferrin receptors. These studies reveal a critical role for Rabenosyn-5 in determining the fate of transferrin receptors internalized by clathrin-mediated endocytosis and, more broadly, a mechanism whereby the delivery of cargo from the plasma membrane into specific early endosome subpopulations is required for its appropriate intracellular traffic.

Type 1 ryanodine receptor knock-in mutation causing central core disease of skeletal muscle also displays a neuronal phenotype.

The type 1 ryanodine receptor (RyR1) is expressed widely in the brain, with high levels in the cerebellum, hippocampus, and hypothalamus. We have shown that L-type Ca(2+) channels in terminals of hypothalamic magnocellular neurons are coupled to RyRs, as they are in skeletal muscle, allowing voltage-induced Ca(2+) release (VICaR) from internal Ca(2+) stores without Ca(2+) influx. Here we demonstrate that RyR1 plays a role in VICaR in nerve terminals. Furthermore, in heterozygotes from the Ryr1(I4895T/WT) (IT/+) mouse line, carrying a knock-in mutation corresponding to one that causes a severe form of human central core disease, VICaR is absent, demonstrating that type 1 RyR mediates VICaR and that these mice have a neuronal phenotype. The absence of VICaR was shown in two ways: first, depolarization in the absence of Ca(2+) influx elicited Ca(2+)syntillas (scintilla, spark, in a nerve terminal, a SYNaptic structure) in WT, but not in mutant terminals; second, in the presence of extracellular Ca(2+), IT/+ terminals showed a twofold decrease in global Ca(2+) transients, with no change in plasmalemmal Ca(2+) current. From these studies we draw two conclusions: (i) RyR1 plays a role in VICaR in hypothalamic nerve terminals; and (ii) a neuronal alteration accompanies the myopathy in IT/+ mice, and, possibly in humans carrying the corresponding RyR1 mutation.

Dopamine transporter endocytic trafficking in striatal dopaminergic neurons: differential dependence on dynamin and the actin cytoskeleton.

Dopaminergic signaling profoundly impacts rewarding behaviors, movement, and executive function. The presynaptic dopamine (DA) transporter (DAT) recaptures released DA, thereby limiting synaptic DA availability and maintaining dopaminergic tone. DAT constitutively internalizes and PKC activation rapidly accelerates DAT endocytosis, resulting in DAT surface loss. Longstanding evidence supports PKC-stimulated DAT trafficking in heterologous expression studies. However, PKC-stimulated DAT internalization is not readily observed in cultured dopaminergic neurons. Moreover, conflicting reports implicate both classic and nonclassic endocytic mechanisms mediating DAT trafficking. Prior DAT trafficking studies relied primarily upon chronic gene disruption and dominant-negative protein expression, or were performed in cell lines and cultured neurons, yielding results difficult to translate to adult dopaminergic neurons. Here, we use newly described dynamin inhibitors to test whether constitutive and PKC-stimulated DAT internalization are dynamin-dependent in adult dopaminergic neurons. Ex vivo biotinylation studies in mouse striatal slices demonstrate that acute PKC activation drives native DAT surface loss, and that surface DAT surprisingly partitions between endocytic-willing and endocytic-resistant populations. Acute dynamin inhibition reveals that constitutive DAT internalization is dynamin-independent, whereas PKC-stimulated DAT internalization is dynamin-dependent. Moreover, total internal reflection fluorescence microscopy experiments demonstrate that constitutive DAT internalization occurs equivalently from lipid raft and nonraft microdomains, whereas PKC-stimulated DAT internalization arises exclusively from lipid rafts. Finally, DAT endocytic recycling relies on a dynamin-dependent mechanism that acts in concert with the actin cytoskeleton. These studies are the first comprehensive investigation of native DAT trafficking in ex vivo adult neurons, and reveal that DAT surface dynamics are governed by complex multimodal mechanisms.

Catecholamine exocytosis during low frequency stimulation in mouse adrenal chromaffin cells is primarily asynchronous and controlled by the novel mechanism of Ca2+ syntilla suppression.

Adrenal chromaffin cells (ACCs), stimulated by the splanchnic nerve, generate action potentials (APs) at a frequency near 0.5 Hz in the resting physiological state, at times described as 'rest and digest'. How such low frequency stimulation in turn elicits sufficient catecholamine exocytosis to set basal sympathetic tone is not readily explained by the classical mechanism of stimulus-secretion coupling, where exocytosis is synchronized to AP-induced Ca(2+) influx. By using simulated action potentials (sAPs) at 0.5 Hz in isolated patch-clamped mouse ACCs, we show here that less than 10% of all catecholaminergic exocytosis, measured by carbon fibre amperometry, is synchronized to an AP. The asynchronous phase, the dominant phase, of exocytosis does not require Ca(2+) influx. Furthermore, increased asynchronous exocytosis is accompanied by an AP-dependent decrease in frequency of Ca(2+) syntillas (i.e. transient, focal Ca(2+) release from internal stores) and is ryanodine sensitive. We propose a mechanism of disinhibition, wherein APs suppress Ca(2+) syntillas, which themselves inhibit exocytosis as they do in the case of spontaneous catecholaminergic exocytosis.

Ca2+ sparks act as potent regulators of excitation-contraction coupling in airway smooth muscle.

Ca2+ sparks are short lived and localized Ca2+ transients resulting from the opening of ryanodine receptors in sarcoplasmic reticulum. These events relax certain types of smooth muscle by activating big conductance Ca2+-activated K+ channels to produce spontaneous transient outward currents (STOCs) and the resultant closure of voltage-dependent Ca2+ channels. But in many smooth muscles from a variety of organs, Ca2+ sparks can additionally activate Ca2+-activated Cl(-) channels to generate spontaneous transient inward current (STICs). To date, the physiological roles of Ca2+ sparks in this latter group of smooth muscle remain elusive. Here, we show that in airway smooth muscle, Ca2+ sparks under physiological conditions, activating STOCs and STICs, induce biphasic membrane potential transients (BiMPTs), leading to membrane potential oscillations. Paradoxically, BiMPTs stabilize the membrane potential by clamping it within a negative range and prevent the generation of action potentials. Moreover, blocking either Ca2+ sparks or hyperpolarization components of BiMPTs activates voltage-dependent Ca2+ channels, resulting in an increase in global [Ca2+](i) and cell contraction. Therefore, Ca2+ sparks in smooth muscle presenting both STICs and STOCs act as a stabilizer of membrane potential, and altering the balance can profoundly alter the status of excitability and contractility. These results reveal a novel mechanism underlying the control of excitability and contractility in smooth muscle.

A novel pleckstrin homology domain-containing protein enhances insulin-stimulated Akt phosphorylation and GLUT4 translocation in adipocytes.

Protein kinase B/Akt protein kinases control an array of diverse functions, including cell growth, survival, proliferation, and metabolism. We report here the identification of pleckstrin homology-like domain family B member 1 (PHLDB1) as an insulin-responsive protein that enhances Akt activation. PHLDB1 contains a pleckstrin homology domain, which we show binds phosphatidylinositol PI(3,4)P(2), PI(3,5)P(2), and PI(3,4,5)P(3), as well as a Forkhead-associated domain and coiled coil regions. PHLDB1 expression is increased during adipocyte differentiation, and it is abundant in many mouse tissues. Both endogenous and HA- or GFP-tagged PHLDB1 displayed a cytoplasmic disposition in unstimulated cultured adipocytes but translocated to the plasma membrane in response to insulin. Depletion of PHLDB1 by siRNA inhibited insulin stimulation of Akt phosphorylation but not tyrosine phosphorylation of IRS-1. RNAi-based silencing of PHLDB1 in cultured adipocytes also attenuated insulin-stimulated deoxyglucose transport and Myc-GLUT4-EGFP translocation to the plasma membrane, whereas knockdown of the PHLDB1 isoform PHLDB2 failed to attenuate insulin-stimulated deoxyglucose transport. Furthermore, adenovirus-mediated expression of PHLDB1 in adipocytes enhanced insulin-stimulated Akt and p70 S6 kinase phosphorylation, as well as GLUT4 translocation. These results indicate that PHLDB1 is a novel modulator of Akt protein kinase activation by insulin.

Individual calcium syntillas do not trigger spontaneous exocytosis from nerve terminals of the neurohypophysis.

Recently, highly localized Ca(2+) release events, similar to Ca(2+) sparks in muscle, have been observed in neuronal preparations. Specifically, in murine neurohypophysial terminals (NHT), these events, termed Ca(2+) syntillas, emanate from a ryanodine-sensitive intracellular Ca(2+) pool and increase in frequency with depolarization in the absence of Ca(2+) influx. Despite such knowledge of the nature of these Ca(2+) release events, their physiological role in this system has yet to be defined. Such localized Ca(2+) release events, if they occur in the precise location of the final exocytotic event(s), may directly trigger exocytosis. However, directly addressing this hypothesis has not been possible, since no method capable of visualizing individual release events in these CNS terminals has been available. Here, we have adapted an amperometric method for studying vesicle fusion to this system which relies on loading the secretory granules with the false transmitter dopamine, thus allowing, for the first time, the recording of individual exocytotic events from peptidergic NHT. Simultaneous use of this technique along with high-speed Ca(2+) imaging has enabled us to establish that spontaneous neuropeptide release and Ca(2+) syntillas do not display any observable temporal or spatial correlation, confirming similar findings in chromaffin cells. Although these results indicate that syntillas do not play a direct role in eliciting spontaneous release, they do not rule out indirect modulatory effects of syntillas on secretion.

Insulin stimulates membrane fusion and GLUT4 accumulation in clathrin coats on adipocyte plasma membranes.

Total internal reflection fluorescence (TIRF) microscopy reveals highly mobile structures containing enhanced green fluorescent protein-tagged glucose transporter 4 (GLUT4) within a zone about 100 nm beneath the plasma membrane of 3T3-L1 adipocytes. We developed a computer program (Fusion Assistant) that enables direct analysis of the docking/fusion kinetics of hundreds of exocytic fusion events. Insulin stimulation increases the fusion frequency of exocytic GLUT4 vesicles by approximately 4-fold, increasing GLUT4 content in the plasma membrane. Remarkably, insulin signaling modulates the kinetics of the fusion process, decreasing the vesicle tethering/docking duration prior to membrane fusion. In contrast, the kinetics of GLUT4 molecules spreading out in the plasma membrane from exocytic fusion sites is unchanged by insulin. As GLUT4 accumulates in the plasma membrane, it is also immobilized in punctate structures on the cell surface. A previous report suggested these structures are exocytic fusion sites (Lizunov et al., J. Cell Biol. 169:481-489, 2005). However, two-color TIRF microscopy using fluorescent proteins fused to clathrin light chain or GLUT4 reveals these structures are clathrin-coated patches. Taken together, these data show that insulin signaling accelerates the transition from docking of GLUT4-containing vesicles to their fusion with the plasma membrane and promotes GLUT4 accumulation in clathrin-based endocytic structures on the plasma membrane.

Wheat germ agglutinin-conjugated fluorescent pH sensors for visualizing proton fluxes.

Small-molecule fluorescent wheat germ agglutinin (WGA) conjugates are routinely used to demarcate mammalian plasma membranes, because they bind to the cell's glycocalyx. Here, we describe the derivatization of WGA with a pH-sensitive rhodamine fluorophore (pHRho; pKa = 7) to detect proton channel fluxes and extracellular proton accumulation and depletion from primary cells. We found that WGA-pHRho labeling was uniform and did not appreciably alter the voltage gating of glycosylated ion channels, and the extracellular changes in pH correlated with proton channel activity. Using single-plane illumination techniques, WGA-pHRho was used to detect spatiotemporal differences in proton accumulation and depletion over the extracellular surface of cardiomyocytes, astrocytes, and neurons. Because WGA can be derivatized with any small-molecule fluorescent ion sensor, WGA conjugates should prove useful to visualize most electrogenic and nonelectrogenic events on the extracellular side of the plasma membrane.

Recombinant expression of the voltage-dependent anion channel enhances the transfer of Ca2+ microdomains to mitochondria.

Although the physiological relevance of mitochondrial Ca2+ homeostasis is widely accepted, no information is yet available on the molecular identity of the proteins involved in this process. Here we analyzed the role of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane in the transmission of Ca2+ signals between the ER and mitochondria by measuring cytosolic and organelle [Ca2+] with targeted aequorins and Ca2+-sensitive GFPs. In HeLa cells and skeletal myotubes, the transient expression of VDAC enhanced the amplitude of the agonist-dependent increases in mitochondrial matrix Ca2+ concentration by allowing the fast diffusion of Ca2+ from ER release sites to the inner mitochondrial membrane. Indeed, high speed imaging of mitochondrial and cytosolic [Ca2+] changes showed that the delay between the rises occurring in the two compartments is significantly shorter in VDAC-overexpressing cells. As to the functional consequences, VDAC-overexpressing cells are more susceptible to ceramide-induced cell death, thus confirming that mitochondrial Ca2+ uptake plays a key role in the process of apoptosis. These results reveal a novel function for the widely expressed VDAC channel, identifying it as a molecular component of the routes for Ca2+ transport across the mitochondrial membranes.

The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly.

Eukaryotic cilia are assembled via intraflagellar transport (IFT) in which large protein particles are motored along ciliary microtubules. The IFT particles are composed of at least 17 polypeptides that are thought to contain binding sites for various cargos that need to be transported from their site of synthesis in the cell body to the site of assembly in the cilium. We show here that the IFT20 subunit of the particle is localized to the Golgi complex in addition to the basal body and cilia where all previous IFT particle proteins had been found. In living cells, fluorescently tagged IFT20 is highly dynamic and moves between the Golgi complex and the cilium as well as along ciliary microtubules. Strong knock down of IFT20 in mammalian cells blocks ciliary assembly but does not affect Golgi structure. Moderate knockdown does not block cilia assembly but reduces the amount of polycystin-2 that is localized to the cilia. This work suggests that IFT20 functions in the delivery of ciliary membrane proteins from the Golgi complex to the cilium.