Host cell invasion by trypanosomes requires lysosomes and microtubule/kinesin-mediated transport.

Invasion of mammalian cells by the protozoan parasite Trypanosoma cruzi occurs by an actin-independent mechanism distinct from phagocytosis. Clusters of host lysosomes are observed at the site of parasite attachment, and lysosomal markers are detected in the vacuolar membrane at early stages of the entry process. These observations led to the hypothesis that the trypanosomes recruit host lysosomes to their attachment site, and that lysosomal fusion serves as a source of membrane to form the parasitophorous vacuole. Here we directly demonstrate directional migration of lysosomes to the parasite entry site, using time-lapse video-enhanced microscopy of L6E9 myoblasts exposed to T. cruzi trypomastigotes. BSA-gold-loaded lysosomes moved towards the cell periphery, in the direction of the parasite attachment site, but only when their original position was less than 11-12 microns from the invasion site. Lysosomes more distant from the invasion area exhibited only the short multi-directional saltatory movements previously described for lysosomes, regardless of their proximity to the cell margins. Specific depletion of peripheral lysosomes was obtained by microinjection of NRK cells with antibodies against the cytoplasmic domain of lgp 120, a treatment that aggregated lysosomes in the perinuclear area and inhibited T. cruzi entry. The microtubule-binding drugs nocodazole, colchicine, vinblastine, and taxol also inhibited invasion, in both NRK and L6E9 cells. Furthermore, microinjection of antibodies to the heavy chain of kinesin blocked the acidification-induced, microtubule-dependent redistribution of lysosomes to the host cell periphery, and reduced trypomastigote entry. Our results therefore demonstrate that during T. cruzi invasion of host cells lysosomes are mobilized from the immediately surrounding area, and that availability of lysosomes at the cell periphery and microtubule/kinesin-mediated transport are requirements for parasite entry.

Propagation of action potentials in the dendrites of neurons from rat spinal cord slice cultures.

1. We examined the propagation of action potentials in the dendrites of ventrally located presumed motoneurons of organotypic rat spinal cord cultures. Simultaneous patch electrode recordings were made from the dendrites and somata of individual cells. In other experiments we visualized the membrane voltage over all the proximal dendrites simultaneously using a voltage-sensitive dye and an array of photodiodes. Calcium imaging was used to measure the dendritic rise in Ca2+ accompanying the propagating action potentials. 2. Spontaneous and evoked action potentials were recorded using high-resistance patch electrodes with separations of 30-423 microm between the somatic and dendritic electrodes. 3. Action potentials recorded in the dendrites varied considerably in amplitude but were larger than would be expected if the dendrites were to behave as passive cables (sometimes little or no decrement was seen for distances of > 100 microm). Because the amplitude of the action potentials in different dendrites was not a simple function of distance from the soma, we suggest that the conductance responsible for the boosting of the action potential amplitude varied in density from dendrite to dendrite and possibly along each dendrite. 4. The dendritic action potentials were usually smaller and broader and arrived later at the dendritic electrode than at the somatic electrode irrespective of whether stimulation occurred at the dendrite or soma or as a result of spontaneous synaptic activity. This is clear evidence that the action potential is initiated at or near the soma and spreads out into the dendrites. The conduction velocity of the propagating action potential was estimated to be 0.5 m/s. 5. The voltage time courses of previously recorded action potentials were generated at the soma using voltage clamp before and after applying 1 microM tetrodotoxin (TTX) over the soma and dendrites. TTX reduced the amplitude of the action potential at the dendritic electrode to a value in the range expected for dendrites that behave as passive cables. This indicates that the conductance responsible for the actively propagating action potentials is a Na+ conductance. 6. The amplitude of the dendritic action potential could also be initially reduced more than the somatic action potential using 1-10 mM QX-314 (an intracellular sodium channel blocker) in the dendritic electrode as the drug diffused from the dendritic electrode toward the soma. Furthermore, in some cases the action potential elicited by current injection into the dendrite had two components. The first component was blocked by QX-314 in the first few seconds of the diffusion of the blocker. 7. In some cells, an afterdepolarizing potential (ADP) was more prominent in the dendrite than in the soma. This ADP could be reversibly blocked by 1 mM Ni2+ or by perfusion of a nominally Ca2+-free solution over the soma and dendrites. This suggests that the back-propagating action potential caused an influx of Ca2+ predominantly in the dendrites. 8. With the use of a voltage-sensitive dye (di-8-ANEPPS) and an array of photodiodes, the action potential was tracked along all the proximal dendrites simultaneously. The results confirmed that the action potential propagated actively, in contrast to similarly measured hyperpolarizing pulses that spread passively. There were also indications that the action potential was not uniformly propagated in all the dendrites, suggesting the possibility that the distribution of Na+ channels over the dendritic membrane is not uniform. 9. Calcium imaging with the Ca2+ fluorescent indicator Fluo-3 showed a larger percentage change in fluorescence in the dendrites than in the soma. Both bursts and single action potentials elicited sharp rises in fluorescence in the proximal dendrites, suggesting that the back-propagating action potential causes a concomitant rise in intracellular calcium concentration...

Octanol, a gap junction uncoupling agent, changes intracellular [H+] in rat astrocytes.

Octanol rapidly closes gap junction channels but its mechanism of action is not known. Because intracellular [H+], pHi, also affects the conductance of gap junctions, we studied octanol's effects on pHi in cultured rat astrocytes, which are highly coupled cells. Octanol (1 mM) caused an acid shift in the pHi of 90% of rat hippocampal astrocytes which averaged -0.19 +/- 0.09 pH units in magnitude. In 58% of the cells tested, a biphasic change in pHi was seen; octanol produced an initial acidification lasting approximately 10 min that was followed by a persistent alkalinization. The related gap junction uncoupling agent, heptanol, had similar effects on pHi. Octanol-induced changes in pHi were similar in nominally HCO(3-)-free and HCO(3-)-containing solutions, although the rate of initial acidification was significantly greater in the presence of HCO3-. The initial acidification was inhibited in the presence of the stilbene DIDS, an inhibitor of Na+/HCO3- cotransport, indicating that octanol caused acidification by blocking this powerful acid extruder. The alkalinization was inhibited by amiloride which blocks the Na+/H+ exchanger (NHE), an acid extruder, suggesting that the alkaline shift induced by octanol was caused by stimulation of NHE. As expected, octanol's effects on astrocytic pHi were prevented by removal of external Na+, which blocks both Na+/HCO3- cotransport and NHE. Octanol had only small effects on intracellular Ca2+ (Ca2+i) in astrocytes. Hepatocytes which, like astrocytes, are strongly coupled to one another, showed no change in pHi with octanol application. Fluorescence recovery after photobleaching (FRAP) was used to study the effect of changes in astrocyte pHi on degree of coupling in hippocampal astrocytes. Coupling was decreased by intracellular acid shifts approximately -0.2 pH units in size. Octanol's effects on astrocyte pHi were complex but a prompt initial acidification was nearly always seen and could contribute to the uncoupling action of this drug in astrocytes. Because octanol uncouples hepatocytes without changing their pHi, this compound clearly can influence gap junctional conductance independent of changes in pHi.

A trypanosome-soluble factor induces IP3 formation, intracellular Ca2+ mobilization and microfilament rearrangement in host cells.

Lysosomes are recruited to the invasion site during host cell entry by Trypanosoma cruzi, an unusual process suggestive of the triggering of signal transduction mechanisms. Previous studies showed that trypomastigotes, but not the noninfective epimastigotes, contain a proteolytically generated trypomastigote factor (PGTF) that induces intracellular free Ca2+ transients in several mammalian cell types. Using confocal time-lapse imaging of normal rat kidney (NRK) fibroblasts loaded with the Ca(2+)-sensitive dye fluo-3, we show that the initial intracellular free Ca(2+) concentration ([Ca2+]i) transient detected a few seconds after exposure to trypomastigote extracts is a result of Ca2+ release from intracellular stores. Removal of Ca2+ from the extracellular medium or inhibition of Ca2+ channels with NiCl2 did not affect the response to PGTF, while depletion of intracellular stores with thapsigargin abolished it. [Ca2+]i transients induced by PGTF were shown to be coupled to the activity of phospholipase C (PLC), since the specific inhibitor U73122 completely blocked the response, while its inactive analogue U73343 had no effect. In addition, polyphosphoinositide hydrolysis and inositol 1,4,5-trisphosphate (IP3) were detected upon cell stimulation with PGTF, suggesting the participation of IP3-sensitive intracellular Ca2+ channels. An immediate effect of the signaling induced by PGTF and live trypomastigotes was a rapid and transient reorganization of host cell microfilaments. The redistribution of F-actin appeared to be a direct consequence of increased [Ca2+]i, since thrombin and the Ca2+ ionophore ionomycin produced a similar effect, with a time course that corresponded to the kinetics of the elevation in [Ca2+]i. These observations support the hypothesis that PGTF-induced disassembly of the cortical actin cytoskeleton may play a role in T. cruzi invasion, by facilitating lysosome access to the invasion site. Taken together, our findings suggest that the proteolytically generated trypomastigote factor PGTF is a novel agonist that acts through the PLC/phosphoinositide signaling pathway of mammalian cells.

Glutamate-induced calcium signaling in astrocytes.

Astrocytes respond to the excitatory neurotransmitter glutamate with dynamic spatio-temporal changes in intracellular calcium [Ca2+]i. Although they share a common wave-like appearance, the different [Ca2+]i changes--an initial spike, sustained elevation, oscillatory intracellular waves, and regenerative intercellular waves--are actually separate and distinct phenomena. These separate components of the astrocytic Ca2+ response appear to be generated by two different signal transduction pathways. The metabotropic response evokes an initial spatial Ca2+ spike that can propagate rapidly from cell to cell and appears to involve IP3. The metabotropic response can also produce oscillatory intracellular waves of various amplitudes and frequencies that propagate within cells and are sustained only in the presence of external Ca2+. The ionotropic response, however, evokes a sustained elevation in [Ca2+]i associated with receptor-mediated Na+ and Ca2+ influx, depolarization, and voltage-dependent Ca2+ influx. In addition, the ionotropic response can lead to regenerative intercellular waves that propagate smoothly and nondecrementally from cell to cell, possibly involving Na+/Ca2+ exchange. All these astrocytic [Ca2+]i changes tend to appear wave-like, traveling from region to region as a transient rise in [Ca2+]i. Nevertheless, as our understanding of the cellular events that underlie these [Ca2+]i changes grows, it becomes increasingly clear that glutamate-induced Ca2+ signaling is a composite of separate and distinct phenomena, which may be distinguished not based on appearance alone, but rather on their underlying mechanisms.