Richard A. Tuft

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.

Movement of nuclear poly(A) RNA throughout the interchromatin space in living cells

BACKGROUND Messenger RNA (mRNA) is transcribed and processed in the nucleus of eucaryotic cells and then exported to the cytoplasm through nuclear pores. It is not known whether the movement of mRNA from its site of synthesis to the nuclear pore is directed or random. Directed movement would suggest that there is an energy-requiring step in addition to the step required for active transport through the pore, whereas random movement would indicate that mRNAs can make their way to the nuclear envelope by diffusion. RESULTS We devised a method to visualize movement of endogenous polymerase II transcripts in the nuclei of living cells. Oligo(dT) labeled with chemically masked (caged) fluorescein was allowed to penetrate cells and hybridize to nuclear poly(A) RNA. Laser spot photolysis then uncaged the oligo(dT) at a given intranuclear site and the resultant fluorescent, hybridized oligo(dT) was tracked using high-speed imaging microscopy. Poly(A) RNA moved away from the uncaging spot in all directions with a mean square displacement that varied linearly with time, and the same apparent diffusion coefficient was measured for the movement at both 37 degrees C and 23 degrees C. These properties are characteristic of a random diffusive process. High resolution three-dimensional imaging of live cells containing both Hoechst-labeled chromosomes and uncaged oligo(dT) showed that, excluding nucleoli, the poly(A) RNA could access most, if not all, of the non-chromosomal space in the nucleus. CONCLUSIONS Poly(A) RNA can move freely throughout the interchromatin space of the nucleus with properties characteristic of diffusion.

Ca2+ sparks activate K+ and Cl− channels, resulting in spontaneous transient currents in guinea‐pig tracheal myocytes

1 Local changes in cytosolic [Ca2+] were imaged with a wide‐field, high‐speed, digital imaging system while membrane currents were simultaneously recorded using whole‐cell, perforated patch recording in freshly dissociated guinea‐pig tracheal myocytes. 2 Depending on membrane potential, Ca2+ sparks triggered ‘spontaneous’ transient inward currents (STICs), ‘spontaneous’ transient outward currents (STOCs) and biphasic currents in which the outward phase always preceded the inward (STOICs). The outward currents resulted from the opening of large‐conductance Ca2+‐activated K+ (BK) channels and the inward currents from Ca2+‐activated Cl− (ClCa) channels. 3 A single Ca2+ spark elicited both phases of a STOIC, and sparks originating from the same site triggered STOCs, STICs and STOICs, depending on membrane potential. 4 STOCs had a shorter time to peak (TTP) than Ca2+ sparks and a much shorter half‐time of decay. In contrast, STICs had a somewhat longer TTP than sparks but the same half‐time of decay. Thus, the STIC, not the STOC, more closely reflected the time course of cytosolic Ca2+ elevation during a Ca2+ spark. 5 These findings suggest that ClCa channels and BK channels may be organized spatially in quite different ways in relation to points of Ca2+ release from intracellular Ca2+ stores. The results also suggest that Ca2+ sparks may have functions in smooth muscle not previously suggested, such as a stabilizing effect on membrane potential and hence on the contractile state of the cell, or as activators of voltage‐gated Ca2+ channels due to depolarization mediated by STICs.

Release of Ca2+ from the sarcoplasmic reticulum increases mitochondrial [Ca2+] in rat pulmonary artery smooth muscle cells

1 The Ca2+‐sensitive fluorescent indicator rhod‐2 was used to measure mitochondrial [Ca2+] ([Ca2+]m) in single smooth muscle cells from the rat pulmonary artery, while simultaneously monitoring cytosolic [Ca2+] ([Ca2+]i) with fura‐2. 2 Application of caffeine produced an increase in [Ca2+]i and also increased [Ca2+]m. The increase in [Ca2+]m occurred after the increase in [Ca2+]i, and remained elevated for a considerable time after [Ca2+]i had returned to resting values. 3 The protonophore carbonyl cyanide p‐(trifluoromethoxy)phenylhydrazone (FCCP), which causes the mitochondrial membrane potential to collapse, markedly attenuated the increase in [Ca2+]m following caffeine application and also increased the half‐time for recovery of [Ca2+]i to resting values. 4 Activation of purinoceptors with ATP also produced increases in both [Ca2+]i and [Ca2+]m in these smooth muscle cells. In some cells, oscillations in [Ca2+]i were observed during ATP application, which produced corresponding oscillations in [Ca2+]m and membrane currents. 5 This study provides direct evidence that Ca2+ release from the sarcoplasmic reticulum, either through ryanodine or inositol 1,4,5‐trisphosphate (InsP3) receptors, increases both cytosolic and mitochondrial [Ca2+] in smooth muscle cells. These results have potential implications both for the role of mitochondria in Ca2+ regulation in smooth muscle, and for understanding how cellular metabolism is regulated.

Effects of the Regulatory Light Chain Phosphorylation of Myosin II on Mitosis and Cytokinesis of Mammalian Cells

Myosin plays an important role in mitosis, especially during cytokinesis. Although it has been assumed that phosphorylation of regulatory light chain of myosin (RLC) controls motility of mammalian non-muscle cells, the functional significance of RLC phosphorylation remains uninvestigated. To address this problem, we have produced unphosphorylatable RLC (T18A/S19A RLC) and overexpressed it in COS-7 cells and normal rat kidney cells. Overexpression of T18A/S19A RLC but not wild type RLC almost completely abolished concanavalin A-induced receptor cap formation. The results indicate that myosin phosphorylation is critical for concanavalin A-induced gathering of surface receptors. T18A/S19A RLC overexpression resulted in the production of multinucleated cells, suggesting the failure of proper cell division in these cells. Video microscopic observation revealed that cells expressing T18A/S19A RLC showed abnormalities during mitosis in two respects. One is that the cells produced abnormal cleavage furrows, resulting in incomplete cytokinesis, which suggests that myosin phosphorylation is important for the normal recruitment of myosin molecules into the contractile ring structure. The other is that separation of chromosomes from the metaphase plate is disrupted in T18A/S19A RLC expressing cells, thus preventing proper transition from metaphase to anaphase. These results suggest that, in addition to cytokinesis, myosin and myosin phosphorylation play a role in the karyokinetic process.

Spontaneous Transient Outward Currents Arise from Microdomains Where BK Channels Are Exposed to a Mean Ca2+ Concentration on the Order of 10 μM during a Ca2+ Spark

Ca2+ sparks are small, localized cytosolic Ca2+ transients due to Ca2+ release from sarcoplasmic reticulum through ryanodine receptors. In smooth muscle, Ca2+ sparks activate large conductance Ca2+-activated K+ channels (BK channels) in the spark microdomain, thus generating spontaneous transient outward currents (STOCs). The purpose of the present study is to determine experimentally the level of Ca2+ to which the BK channels are exposed during a spark. Using tight seal, whole-cell recording, we have analyzed the voltage-dependence of the STOC conductance (g(STOC)), and compared it to the voltage-dependence of BK channel activation in excised patches in the presence of different [Ca2+]s. The Ca2+ sparks did not change in amplitude over the range of potentials of interest. In contrast, the magnitude of g(STOC) remained roughly constant from 20 to −40 mV and then declined steeply at more negative potentials. From this and the voltage dependence of BK channel activation, we conclude that the BK channels underlying STOCs are exposed to a mean [Ca2+] on the order of 10 μM during a Ca2+ spark. The membrane area over which a concentration ≥10 μM is reached has an estimated radius of 150–300 nm, corresponding to an area which is a fraction of one square micron. Moreover, given the constraints imposed by the estimated channel density and the Ca2+ current during a spark, the BK channels do not appear to be uniformly distributed over the membrane but instead are found at higher density at the spark site.

Quantitative Analysis of Spontaneous Mitochondrial Depolarizations

Spontaneous transient depolarizations in mitochondrial membrane potential (DeltaPsi(m)), mitochondrial flickers, have been observed in isolated mitochondria and intact cells using the fluorescent probe, tetramethylrhodamine ethyl ester (TMRE). In theory, the ratio of [TMRE] in cytosol and mitochondrion allows DeltaPsi(m) to be calculated with the Nernst equation, but this has proven difficult in practice due to fluorescence quenching and binding of dye to mitochondrial membranes. We developed a new method to determine the amplitude of flickers in terms of millivolts of depolarization. TMRE fluorescence was monitored using high-speed, high-sensitivity three-dimensional imaging to track individual mitochondria in freshly dissociated smooth muscle cells. Resting mitochondrial fluorescence, an exponential function of resting DeltaPsi(m), varied among mitochondria and was approximately normally distributed. Spontaneous changes in mitochondrial fluorescence, indicating depolarizations and repolarizations in DeltaPsi(m), were observed. The depolarizations were reversible and did not result in permanent depolarization of the mitochondria. The magnitude of the flickers ranged from <10 mV to >100 mV with a mean of 17.6 +/- 1.0 mV (n = 360) and a distribution skewed to smaller values. Nearly all mitochondria flickered, and they did so independently of one another, indicating that mitochondria function as independent units in the myocytes employed here.

Visualization of Ca2+ entry through single stretch-activated cation channels

Stretch-activated channels (SACs) have been found in smooth muscle and are thought to be involved in myogenic responses. Although SACs have been shown to be Ca2+ permeable when Ca2+ is the only charge carrier, it has not been clearly demonstrated that significant Ca2+ passes through SACs in physiological solutions. By imaging at high temporal and spatial resolution the single-channel Ca2+ fluorescence transient (SCCaFT) arising from Ca2+ entry through a single SAC opening, we provide direct evidence that significant Ca2+ can indeed pass through SACs and increase the local [Ca2+]. Results were obtained under conditions where the only source of Ca2+ was the physiological salt solution in the patch pipette containing 2 mM Ca2+. Single smooth muscle cells were loaded with fluo-3 acetoxymethyl ester, and the fluorescence was recorded by using a wide-field digital imaging microscope while SAC currents were simultaneously recorded from cell-attached patches. Fluorescence increases at the cell-attached patch were clearly visualized before the simultaneous global Ca2+ increase that occurred because of Ca2+ influx through voltage-gated Ca2+ channels when the membrane was depolarized by inward SAC current. From measurements of total fluorescence (“signal mass”) we determined that about 18% of the SAC current is carried by Ca2+ at membrane potentials more negative than the resting level. This would translate into at least a 0.35-pA unitary Ca2+ current at the resting potential. Such Ca2+ currents passing through SACs are sufficient to activate large-conductance Ca2+-activated K+ channels and, as shown previously, to trigger Ca2+ release from intracellular stores.

Digital imaging microscopy of living cells

Fluorescence microscopy has undergone a resurgence in interest following the discovery of green-fluorescent protein (GFP) and its increasing use in live-cell imaging. This article describes an enhanced form of epifluorescence microscopy, digital imaging microscopy, that can be used to produce high-resolution three-dimensional images of samples labelled with GFP, or other fluorochromes, using simple instrumentation and image-restoration software.

Phosphatidylinositol-4,5-bisphosphate-rich plasma membrane patches organize active zones of endocytosis and ruffling in cultured adipocytes

ABSTRACT A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cδ1 (PLCδ1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCδ1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.